triethylenetetramine synergizes with pharmacologic
TRANSCRIPT
Research ArticleTriethylenetetramine Synergizes with PharmacologicAscorbic Acid in Hydrogen Peroxide Mediated SelectiveToxicity to Breast Cancer Cell
Lianlian Wang123 Xiaofang Luo23 Cong Li2 Yubing Huang4 Ping Xu23
Laetitia H Lloyd-Davies5 Thibaut Delplancke3 Chuan Peng6 Rufei Gao7
Hongbo Qi23 Chao Tong23 and Philip Baker35
1Department of Reproduction Health and Infertility The First Affiliated Hospital of Chongqing Medical UniversityChongqing 400016 China2Department of Obstetrics and Gynecology The First Affiliated Hospital of Chongqing Medical University Chongqing 400016 China3Canada-China-New Zealand Joint Laboratory of Maternal and Fetal Medicine Chongqing Medical UniversityChongqing 400016 China4Molecular Medicine and Cancer Research Center Department of Biochemistry and Molecular BiologyChongqing Medical University Chongqing 400016 China5College of Medicine University of Leicester Leicester LE1 7RH UK6Laboratory of Lipid amp Glucose Metabolism The First Affiliated Hospital of Chongqing Medical University Chongqing 400016 China7Laboratory of Reproductive Biology School of Public Health and Management Chongqing Medical UniversityChongqing 400016 China
Correspondence should be addressed to Chao Tong chaotongcqmu163com
Received 12 October 2016 Accepted 5 January 2017 Published 8 February 2017
Academic Editor Lynne Postovit
Copyright copy 2017 Lianlian Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Breast cancer is characterized by overexpression of superoxide dismutase (SOD) and downregulation of catalase and moreresistance to hydrogen peroxide (H
2O2) than normal cells Thus relatively high H
2O2promotes breast cancer cell growth and
proliferation However excessive intracellular H2O2leads to death of breast cancer cells In cancer cells high level ascorbic acid
(Asc) is able to be autoxidized and thus provides an electron to oxygen to generate H2O2 In the present study we demonstrated
that triethylenetetramine (TETA) enhances Asc autoxidation and thus elevates H2O2production in MCF-7 cells Furthermore
AscTETA combination significantly impaired cancer cell viability while having much milder effects on normal cells indicatingAscTETA could be a promising therapy for breast cancer Moreover SOD1 and N-acetyl-L-cysteine failed to improve MCF-7 cellsviability in the presence of AscTETA while catalase significantly inhibited the cytotoxicity of AscTETA to breast cancer cellsstrongly suggesting that the selective cytotoxicity of AscTETA to cancer cells is H
2O2-dependent In addition AscTETA induces
RASERK downregulation in breast cancer cells Animal studies confirmed that AscTETA effectively suppressed tumor growthin vivo In conclusion TETA synergizes pharmacologic Asc autoxidation and H
2O2overproduction in breast cancer cells which
suppresses RASERK pathway and results in apoptosis
1 Introduction
Hydrogen peroxide plays an integral role in cancer cellbiology Cancer cells produce more H
2O2than normal
cells [1] firstly due to an overreaction of enzymes in theelectron transport chain that produces excessive reactive
oxygen species (ROS) [2] and secondly as a consequence ofthe overexpression of superoxide dismutase (SOD) whichconverts superoxide (O
2
minus) to hydrogen peroxide (H2O2) [3]
Breast cancer is the leading cause of cancer-relateddeaths in females worldwide [4] Like many malignanciesit is characterized by overexpression of SOD along with
HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 3481710 13 pageshttpsdoiorg10115520173481710
2 Oxidative Medicine and Cellular Longevity
downregulation of catalase (CAT) which converts H2O2to
H2O and O
2 Thus breast cancer cells maintain a higher
intracellular H2O2than normal cells [5] suggesting breast
cancer cells are able to accumulate and tolerate H2O2within
certain range However mild elevating of H2O2in cancer
cells has been shown to arrest the cell cycle and induceapoptosis and has proven beneficial [6 7] this indicatesselective overload of H
2O2in cancer cells could be a thera-
peutic strategy for breast cancer Indeed hydrogen peroxideinducible agents have shownpotential as anticancer drugs [8]However most chemotherapeutic agents for cancer are toxicto the host Therefore existing medicine or natural productsthat selectively promote H
2O2production in cancer cells
sparing normal cells are promising candidates for achievingtherapeutic activity and selectivity
Ascorbic acid (Asc) also known as vitamin C is a well-known natural antioxidant It has been long assumed to beessential for free radical clearance [9] Previous studies havereported that high concentrations of Asc are able to induceautoxidation and thus reveal anticancer effects [7] whilelower concentrations of Asc failed to show similar effects [10]
In sequential one-electron oxidations the high concen-tration of Asc donates 2 electrons to oxygen resulting information of dehydroascorbic acid (DHA) and H
2O2 The
sequential one-electron oxidation of Asc can occur via thedianionAsc2minus which autoxidizes in the presence of dioxide toproduce the Ascminus dehydroascorbic acid and H
2O2[11] This
process is shown in the following formulas
Ascorbate +H2O 997888rarr AscHminus + Asc∙minus
Asc2minus +O2997888rarr Asc∙minus +O
2
∙minus
2Asc∙minus +H+ 997888rarr AscHminus + DHA
2O2
∙minus + 2H+ 997888rarr H2O2+O2
(1)
Therefore it is important to investigate whether a highconcentration of Asc associated autoxidation is critical for itsanticancer effects Asc is very stable and barely autoxidizesalone However in the presence of oxidative metal activatorssuch as iron copper and manganese Asc autoxidation canbe dramatically promoted as evidenced by the accumulationof ascorbic acid ion (Asc∙minus) and ultimately result in elevatedO2
∙minus andH2O2production [12] In addition O
2
∙minus can furtherbe reduced to H
2O2by accepting electron from Asc [13]
The function of catalysts for Asc autoxidation in aqueoussolution mainly relies on their component groups [7] espe-cially the amino groups which are considered prooxidative[14] Triethylenetetramine (TETA) is a telomerase inhibitorthat has been clinically used to treat cancer for decades [15]Previous studies demonstrate that TETA could overcomecisplatin resistance in human ovarian cancer cell culturesvia inhibition of CuZn superoxide dismutase namely SOD1[16 17] TETA is an alkali compound containing two aminogroups and has a tendency to undergo redox as well asacid-base reactions in aqueous solution Moreover it pos-sesses 4 nitrogen atoms which have 1s22s22p3 electronicarrangement Each nitrogen atom has a lone pair of electronswhich is able to pair with a proton from Asc Therefore we
AscTETAAscTETA
Catalase
[O2]
(120583m
olL
)
0
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Figure 1 The effects of TETA on Asc oxidation Oxygen consump-tion rate (OCR) of Asc in aqueous solution was determined bythe use of an electrode oxygen monitor 1mM Asc alone 30 120583MTETA1mM Asc or 30120583M TETA alone was added to DMEM with10 FBS 600UmL catalase was then given into each reaction todetermine the return of O
2
hypothesized that TETA could enhance Asc autoxidation inbreast cancer cells and thus elevate intracellular H
2O2 which
will further boost Asc derived selective cytotoxicity to breastcancer cells (Figure 1) In the present study we investigatedthe effect TETA has on H
2O2production from Asc solution
We then used in vitro models to study its role in regulatingbreast cancer cell apoptosis as well as underlying molecularmechanisms by the use of an in vivo animal model
2 Materials and Methods
21 Cell Culture All the cell lines were purchased from TheCell Bank of Chinese Academy of Sciences (Shanghai)Cells were cultured in Dulbeccorsquos modified Eaglersquos medium(DMEM HyClone SH3002201B USA) supplemented with10 Fetal Bovine Serum (FBS) (Gibco USA) at 37∘C in ahumidified atmosphere with 5 CO
2
22 Oxygen Consumption Assay The rate of oxygen con-sumption (OCR 119889[O
2]119889119905) was determined as previously
described [7] Briefly a Clark electrode oxygen monitor (YSIInc) was connected to an ESA Biostat multielectrode systemin DMEM (10 FBS) The effect of TETA on the OCR of Ascwas thenmeasured and recorded Accumulation ofH
2O2was
determined by adding catalase (Sigma C9322-1G Germany)
23MTTAssay MCF-7 cells were seeded in 96-well plates (3times 103 cellswell) followed by 12 h or 24 h of treatments Themedia containing Asc was removed before being subjected toMTT assay because the oxidative products of Asc interferewith the MTT assay 100 120583L of serum-free DMEM medium
Oxidative Medicine and Cellular Longevity 3
was applied into each well and then 20 120583L of 3-(45)-dime-thylthiahiazo(-z-y1)-35-diphenytetrazoliumromide (MTT)(Sigma Germany) was added to each well Followed by 4 h ofincubation at 37∘C all media were removed and then 150 120583Lof dimethyl sulfoxide (DMSO) (Sigma Germany) was addedto each well after 10min of shaking the value of OD
490was
recorded by a Varioskan Flash (Thermo Fisher Finland)
24 ROS Measurement The intracellular ROS was detectedby using H
2DCF (Sigma Germany) as previously reported
[18] Briefly 10 120583MofH2DCFwas added onto cells for 30min
and then cells were harvested and analyzed by a fluorescencemicroscope (KEYENCE Corporation)
25 Western Blotting MCF-7 cells were harvested by theuse of ice-cold RIPA lysis buffer (Beyotime BiotechnologyShanghai China) and protein concentration was determinedusing the BCA protein quantification kit (Beyotime Biotech-nology Shanghai China) western blotting was performed aspreviously described [19] Primary antibodies of anti-Ac-H3anti-SOD1 anti-CAT anti-ERK anti-p-ERK anti-Cyt-C andanti-caspase 9 were purchased from Bioworld Technology(Nanjing China) anti-PARP rabbit anti-caspase 3 and anti-Ac-H3 were purchased from Cell Signaling TechnologyDanvers USA anti-RAS was purchased from BD USAand anti-GAPDH was purchased from Earthox MillbraeUSA The secondary antibodies were purchased from BiogotBiotechnology (Nanjing China) and ECL SuperSignal WestFemto Maximum Sensitivity Substrate was purchased fromThermo Fisher
26 Transient Transfection 1times105 cells were plated into eachwell of a 24-well plate 24 hours before transfection shRNA(600 ngwell) or Plasmids (800 ngwell) were transfectedwith Lipofectamine 3000 (Invitrogen USA) according to themanufacturerrsquos protocol
SOD1 NM 0004543-582s1c1 CCGGGCTGTAGA-AATGTATCCTGATCTCGAGATCAGGATACA-TTTCTACAGCTTTTTGCAT NM 0017522-1371s1c1 CCGGCGGAGATT-CAACACTGCCAATCTCGAGATTGGCAGTGT-TGAATCTCCGTTTTTG
27 Xenograft Study 6-week-old female nudemice (BALBc-nu) were purchased from Vital River Laboratories (BeijingChina) the animal experiments were approved by the Medi-cal Ethics Committee of Chongqing Medical University andall of the procedures were in accordance with the NationalInstitutes of Health guide for the care and use of Laboratoryanimals In short MCF-7 cells (5 times 106 in 200120583L) weresubcutaneously delivered into the hind leg of mice anda 072mg 90 daysrsquo release 17120573-estradiol pellet (InnovativeResearch of America USA) was implanted subcutaneouslyinto the front-back area to facilitate optimal tumor growthThe tumors were allowed to grow 14 days to reach the greatestdimension of about 3ndash5mm and treatments were initiatedon the 14th day Mice were randomly divided into 4 groups
including control (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) and Asc (3 gkg body weight)plus TETA (30mgkg body weight) 10 mice in each groupTreatments were given via intraperitoneal injection daily for25 consecutive days Tumor size was measured every 2 daysusing a vernier caliper while tumor volume was estimatedbased on the following formula tumor volume (mm3) = 119871 times11988222 where 119871 is the greatest dimension of the tumor and119882 means the dimension of the tumor in the perpendiculardirection Animals were sacrificed by CO
2euthanasia when
the tumor size reached 1000mm3
28 Statistical Analysis Data are expressed as mean plusmn SD Avariety of statistical tests using GraphPad Prism 5 softwarewere used on the basis of the design required for the specificquestion being asked This meant using 119905-tests and 2-wayANOVA A value of 119901 lt 005 was considered statisticallysignificant
3 Results
31 TETA Synergizes Ascorbic Acid Oxidation To investigatethe effect of TETA on promoting H
2O2generation from Asc
oxygen consumption of Asc in the presence and absence ofTETA has been measured respectively As shown in Figure 11mM Asc in DMEM with 10 FBS resulted in an OCRof 55 nmolLs and additional 30 120583M of TETA increasedOCR to 110 nmolLs while 30 120583M of TETA alone barelyconsumes O
2and generates H
2O2 However in the presence
of catalase (600UmL) H2O2accumulation in AscTETA
was dramatically suppressed compared toAsc or TETA aloneTaken together this evidence strongly suggested that TETAenhanced Asc-dependent H
2O2generation
32 AscTETA Combination Promotes Apoptosis in MCF-7Cells To further examine the cytotoxicity of AscTETAderived H
2O2to breast cancer cells MCF-7 cells were
treated with Asc (1mM) along with different doses of TETA(10 120583M 30 120583M and 50 120583M) for 12 hours MTT assay resultsdemonstrated that Asc alone resulted in a 40 reduction inMCF-7 proliferation while TETA alone did not show anyeffect on cell viability Intriguingly the combinations of Ascand TETA significantly decreased cell viability compared toAsc alone (Figure 2(a)) Moreover a prolonged incubationof AscTETA for 24 hours led to further reduction in cellviability indicating that the anticancer effect of AscTETAcombination is in a time-dose manner (Figure 2(b)) Furtherwestern blotting unveiled that AscTETA suppresses MCF-7 cell viability by elevating apoptotic signaling of caspase 9and caspase 3 (Figure 2(c)) Morphology and cell cloningexperiments confirmed that TETA synergizes Asc mediatedcytotoxicity on MCF-7 cells (Figures 2(d) and 2(e)) Takentogether these results strongly suggest that TETA synergizesthe anti-breast cancer effect of Asc in vitro
33 Asc and TETA Synergize to Enhance Cytotoxicity In VitroTo further validate whether the synergistic effects of Asc andTETA on cell death are specific for cancer cells in addition to
4 Oxidative Medicine and Cellular Longevity
1m
M A
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ETA
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ETA
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ETA
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ETA
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(d)
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Figure 2 AscTETA combination induces apoptosis in MCF-7 cells MCF-7 cells were treated with 1mM Asc along with 10120583M 30 120583Mor 50 120583M of TETA for 12 hours (a) MTT assay was performed to assess MCF-7 viability lowast119901 lt 0005 119901 lt 00001 119899 = 6 (b) viability ofMCF-7 cells was measured by MTT assay after 6 12 and 24 hours of 1mM Asc10 120583M TETA treatment 119899 = 6 (c) Effects of different dosageof AscTETA (1 100) on proapoptotic signaling were examined by western blotting (d) MCF-7 cells cloning formation experiments wereperformed after 12 hours of 1mM Asc10120583M TETA treatment (e) Statistic analysis of 3 independent experiments lowast119901 lt 005 versus control119901 lt 001 versus Asc 119899 = 3
various cancer cell lines such asMCF-7MDA-MB-157MDA-MB-231 U87 HCC-9204 and H1299 multiple normal celllines including Hs578Bst HUVEC and V79 were incubatedwith different concentrations of TETA (5120583M 10 120583M 30 120583Mand 50 120583M) and corresponding doses of Asc (05mM 1mM3mM and 5mM) in accordance with the 1 100 TETAAscratio MTT assay results demonstrated that Asc and TETAhave synergistic cytotoxicity on cancer cells but much mildereffects on the viability of normal cells (Figure 3)
34 H2O2 Is Critical for AscTETA Induced Cytotoxicity toMCF-7 Cell We then assessed the ROS level in MCF-7 cellsAfter 4 hours of incubation Asc alone moderately elevatedROS level inMCF-7 cells while TETA alone did not show anyeffect on ROS generation However AscTETA coincubationresulted in a dramatic increase of ROS compared to the othergroups while in the presence of N-acetyl-L-cysteine (NACSigma V900429 Germany) which degrade ROS exceptH2O2 AscTETA showed much less fluorescence staining
Oxidative Medicine and Cellular Longevity 5
Normal cell
Cancer cell0
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cont
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321 4 50Asc concentration (mM)
MCF-7MDA-MB-157MDA-MB-231
U87HCC-9204H1299
HS578BstHVVecsV79
Figure 3 Selective cytotoxicity of AscTETA on various cell lines MCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 H1299 Hs578BstHUVEC or V79 cells were incubated with 1mM 3mM or 5mMofAsc respectively alongwith 10120583M 30120583M or 50 120583Mof TETA tomaintain1 100 TETA-to-Asc ratio MTT assay was performed to assess the cytotoxicity of AscTETA on different cell lines after 12 hours of incubationexperiment was repeated three times
(Figure 4(a)) Taken together these data suggested TETApotentiated not only H
2O2but also other types of ROS
production from Asc in MCF-7 cellsNevertheless ROS include hydrogen peroxide (H
2O2)
superoxide anions (O2
∙minus) and hydroxyl radical to ascertainwhether the cytotoxicity of AscTETA specifically resultsfrom H
2O2 NAC was applied to MCF-7 cells along with
AscTETAWestern blotting showed that AscTETA combination
treatment resulted in inhibition of RAS expression whichwere not rescued by extraNAC treatment Inversely althoughAscTETA also lead to elevation of H3 acetylation in dose-dependent manner such effect was totally reversed by 5mMNAC (Figure 4(b)) Moreover the effects of AscTETA onRAS expression and H3 acetylation demonstrated a time-dependent manner (Figure 4(c)) The data suggests thatTETAAsc suppresses RAS expression in MCF-7 cells prin-cipally through H
2O2 while it upregulates H3 acetylation
mainly through the other types of ROS Most importantly5mM NAC failed to eliminate the cytotoxicity caused byAscTETA Taken together these results indicate that H
2O2
is the primary cytotoxic ROS induced by AscTETA inMCF-7 cells and possibly through the inhibition of RAS expression
35 The Selective Cytotoxicity of AscTETA Derived H2O2to MCF-7 Cells due to Compromised CAT Expression Wesubsequently investigated the mechanism underlying thediscrepancy of the cytotoxicity of AscTETA combinationin cancer cells and normal cells The effect of catalaseon AscTETA induced cell death was examined by cloneformation experiment the data (Figure 5(a)) shows thatAsc alone moderately reduced plating efficiency of MCF-7cells while neither TETA nor catalase shows similar effectHowever AscTETA combination significantly suppressedplating efficiency of MCF-7 cells but the reduction was
almost fully restored in the presence of catalase We thendetermined the expression levels of CAT in MCF-7 cellsand normal cells by immunoblotting It was shown thatCAT expression level is significantly compromised in MCF-7 cells compared to the normal cells (Figure 5(b)) Tofurther determine the importance of CAT in resistance toAscTETA induced cell death shCATwas transfected into thenormal breast epithelial HS578Bst cells it only dramaticallyrepressed CAT expression in HS578Bst cells (Figure 5(c))but also significantly impaired cell viability (Figure 5(d))This strongly suggests that the selective toxicity to cancercells could be attributed to CAT downregulation and thusthe cytotoxicity of combined TETA and Asc use is primarilymediated by H
2O2
36 AscTETA Induced Cytotoxicity to MCF-7 Cells Is NotMediated by O2
∙minus Although H2O2has been found critical
for TETAAsc mediated cytotoxicity the possibility of O2
∙minus
being involved in such anticancer effects has yet to beruled out To distinguish the roles of O
2
∙minus and H2O2in
TETAAsc combination derived cytotoxicity 100UmL SODwas applied onto MCF-7 cells with 1mMAsc and 10120583MTETA In contrast to CAT SOD failed to rescue cell survivalin the presence of Asc and TETA but exacerbated Asc andTETA combination induced cell death (Figure 6(a)) Thisindicates that O
2
∙minus does not contribute to TETA and Ascmediated cytotoxicity to cancer cells Not surprisingly SODprotein expression in MCF-7 cells was significantly higherthan normal cells (Figure 6(b)) To further confirm thisfinding SOD1 in MCF-7 cells was knocked down by shRNA(Figure 6(c)) In combined AscTETA treatment shSOD1 didnot result in enhanced viability of MCF-7 cell but led to morecell death (Figure 6(d)) This data suggests that the selectivecytotoxicity of AscTETA to MCF-7 cells is not mediated byO2
∙minus
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
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Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
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(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
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(c)Vector CAT shRNA
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Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
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(c)
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lowast
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Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
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Cyt-C
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trol
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ETA
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Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
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Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Oxidative Medicine and Cellular Longevity
downregulation of catalase (CAT) which converts H2O2to
H2O and O
2 Thus breast cancer cells maintain a higher
intracellular H2O2than normal cells [5] suggesting breast
cancer cells are able to accumulate and tolerate H2O2within
certain range However mild elevating of H2O2in cancer
cells has been shown to arrest the cell cycle and induceapoptosis and has proven beneficial [6 7] this indicatesselective overload of H
2O2in cancer cells could be a thera-
peutic strategy for breast cancer Indeed hydrogen peroxideinducible agents have shownpotential as anticancer drugs [8]However most chemotherapeutic agents for cancer are toxicto the host Therefore existing medicine or natural productsthat selectively promote H
2O2production in cancer cells
sparing normal cells are promising candidates for achievingtherapeutic activity and selectivity
Ascorbic acid (Asc) also known as vitamin C is a well-known natural antioxidant It has been long assumed to beessential for free radical clearance [9] Previous studies havereported that high concentrations of Asc are able to induceautoxidation and thus reveal anticancer effects [7] whilelower concentrations of Asc failed to show similar effects [10]
In sequential one-electron oxidations the high concen-tration of Asc donates 2 electrons to oxygen resulting information of dehydroascorbic acid (DHA) and H
2O2 The
sequential one-electron oxidation of Asc can occur via thedianionAsc2minus which autoxidizes in the presence of dioxide toproduce the Ascminus dehydroascorbic acid and H
2O2[11] This
process is shown in the following formulas
Ascorbate +H2O 997888rarr AscHminus + Asc∙minus
Asc2minus +O2997888rarr Asc∙minus +O
2
∙minus
2Asc∙minus +H+ 997888rarr AscHminus + DHA
2O2
∙minus + 2H+ 997888rarr H2O2+O2
(1)
Therefore it is important to investigate whether a highconcentration of Asc associated autoxidation is critical for itsanticancer effects Asc is very stable and barely autoxidizesalone However in the presence of oxidative metal activatorssuch as iron copper and manganese Asc autoxidation canbe dramatically promoted as evidenced by the accumulationof ascorbic acid ion (Asc∙minus) and ultimately result in elevatedO2
∙minus andH2O2production [12] In addition O
2
∙minus can furtherbe reduced to H
2O2by accepting electron from Asc [13]
The function of catalysts for Asc autoxidation in aqueoussolution mainly relies on their component groups [7] espe-cially the amino groups which are considered prooxidative[14] Triethylenetetramine (TETA) is a telomerase inhibitorthat has been clinically used to treat cancer for decades [15]Previous studies demonstrate that TETA could overcomecisplatin resistance in human ovarian cancer cell culturesvia inhibition of CuZn superoxide dismutase namely SOD1[16 17] TETA is an alkali compound containing two aminogroups and has a tendency to undergo redox as well asacid-base reactions in aqueous solution Moreover it pos-sesses 4 nitrogen atoms which have 1s22s22p3 electronicarrangement Each nitrogen atom has a lone pair of electronswhich is able to pair with a proton from Asc Therefore we
AscTETAAscTETA
Catalase
[O2]
(120583m
olL
)
0
50
100
150
200
250
300
500 1000 1500 2000 2500 30000Time (s)
Figure 1 The effects of TETA on Asc oxidation Oxygen consump-tion rate (OCR) of Asc in aqueous solution was determined bythe use of an electrode oxygen monitor 1mM Asc alone 30 120583MTETA1mM Asc or 30120583M TETA alone was added to DMEM with10 FBS 600UmL catalase was then given into each reaction todetermine the return of O
2
hypothesized that TETA could enhance Asc autoxidation inbreast cancer cells and thus elevate intracellular H
2O2 which
will further boost Asc derived selective cytotoxicity to breastcancer cells (Figure 1) In the present study we investigatedthe effect TETA has on H
2O2production from Asc solution
We then used in vitro models to study its role in regulatingbreast cancer cell apoptosis as well as underlying molecularmechanisms by the use of an in vivo animal model
2 Materials and Methods
21 Cell Culture All the cell lines were purchased from TheCell Bank of Chinese Academy of Sciences (Shanghai)Cells were cultured in Dulbeccorsquos modified Eaglersquos medium(DMEM HyClone SH3002201B USA) supplemented with10 Fetal Bovine Serum (FBS) (Gibco USA) at 37∘C in ahumidified atmosphere with 5 CO
2
22 Oxygen Consumption Assay The rate of oxygen con-sumption (OCR 119889[O
2]119889119905) was determined as previously
described [7] Briefly a Clark electrode oxygen monitor (YSIInc) was connected to an ESA Biostat multielectrode systemin DMEM (10 FBS) The effect of TETA on the OCR of Ascwas thenmeasured and recorded Accumulation ofH
2O2was
determined by adding catalase (Sigma C9322-1G Germany)
23MTTAssay MCF-7 cells were seeded in 96-well plates (3times 103 cellswell) followed by 12 h or 24 h of treatments Themedia containing Asc was removed before being subjected toMTT assay because the oxidative products of Asc interferewith the MTT assay 100 120583L of serum-free DMEM medium
Oxidative Medicine and Cellular Longevity 3
was applied into each well and then 20 120583L of 3-(45)-dime-thylthiahiazo(-z-y1)-35-diphenytetrazoliumromide (MTT)(Sigma Germany) was added to each well Followed by 4 h ofincubation at 37∘C all media were removed and then 150 120583Lof dimethyl sulfoxide (DMSO) (Sigma Germany) was addedto each well after 10min of shaking the value of OD
490was
recorded by a Varioskan Flash (Thermo Fisher Finland)
24 ROS Measurement The intracellular ROS was detectedby using H
2DCF (Sigma Germany) as previously reported
[18] Briefly 10 120583MofH2DCFwas added onto cells for 30min
and then cells were harvested and analyzed by a fluorescencemicroscope (KEYENCE Corporation)
25 Western Blotting MCF-7 cells were harvested by theuse of ice-cold RIPA lysis buffer (Beyotime BiotechnologyShanghai China) and protein concentration was determinedusing the BCA protein quantification kit (Beyotime Biotech-nology Shanghai China) western blotting was performed aspreviously described [19] Primary antibodies of anti-Ac-H3anti-SOD1 anti-CAT anti-ERK anti-p-ERK anti-Cyt-C andanti-caspase 9 were purchased from Bioworld Technology(Nanjing China) anti-PARP rabbit anti-caspase 3 and anti-Ac-H3 were purchased from Cell Signaling TechnologyDanvers USA anti-RAS was purchased from BD USAand anti-GAPDH was purchased from Earthox MillbraeUSA The secondary antibodies were purchased from BiogotBiotechnology (Nanjing China) and ECL SuperSignal WestFemto Maximum Sensitivity Substrate was purchased fromThermo Fisher
26 Transient Transfection 1times105 cells were plated into eachwell of a 24-well plate 24 hours before transfection shRNA(600 ngwell) or Plasmids (800 ngwell) were transfectedwith Lipofectamine 3000 (Invitrogen USA) according to themanufacturerrsquos protocol
SOD1 NM 0004543-582s1c1 CCGGGCTGTAGA-AATGTATCCTGATCTCGAGATCAGGATACA-TTTCTACAGCTTTTTGCAT NM 0017522-1371s1c1 CCGGCGGAGATT-CAACACTGCCAATCTCGAGATTGGCAGTGT-TGAATCTCCGTTTTTG
27 Xenograft Study 6-week-old female nudemice (BALBc-nu) were purchased from Vital River Laboratories (BeijingChina) the animal experiments were approved by the Medi-cal Ethics Committee of Chongqing Medical University andall of the procedures were in accordance with the NationalInstitutes of Health guide for the care and use of Laboratoryanimals In short MCF-7 cells (5 times 106 in 200120583L) weresubcutaneously delivered into the hind leg of mice anda 072mg 90 daysrsquo release 17120573-estradiol pellet (InnovativeResearch of America USA) was implanted subcutaneouslyinto the front-back area to facilitate optimal tumor growthThe tumors were allowed to grow 14 days to reach the greatestdimension of about 3ndash5mm and treatments were initiatedon the 14th day Mice were randomly divided into 4 groups
including control (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) and Asc (3 gkg body weight)plus TETA (30mgkg body weight) 10 mice in each groupTreatments were given via intraperitoneal injection daily for25 consecutive days Tumor size was measured every 2 daysusing a vernier caliper while tumor volume was estimatedbased on the following formula tumor volume (mm3) = 119871 times11988222 where 119871 is the greatest dimension of the tumor and119882 means the dimension of the tumor in the perpendiculardirection Animals were sacrificed by CO
2euthanasia when
the tumor size reached 1000mm3
28 Statistical Analysis Data are expressed as mean plusmn SD Avariety of statistical tests using GraphPad Prism 5 softwarewere used on the basis of the design required for the specificquestion being asked This meant using 119905-tests and 2-wayANOVA A value of 119901 lt 005 was considered statisticallysignificant
3 Results
31 TETA Synergizes Ascorbic Acid Oxidation To investigatethe effect of TETA on promoting H
2O2generation from Asc
oxygen consumption of Asc in the presence and absence ofTETA has been measured respectively As shown in Figure 11mM Asc in DMEM with 10 FBS resulted in an OCRof 55 nmolLs and additional 30 120583M of TETA increasedOCR to 110 nmolLs while 30 120583M of TETA alone barelyconsumes O
2and generates H
2O2 However in the presence
of catalase (600UmL) H2O2accumulation in AscTETA
was dramatically suppressed compared toAsc or TETA aloneTaken together this evidence strongly suggested that TETAenhanced Asc-dependent H
2O2generation
32 AscTETA Combination Promotes Apoptosis in MCF-7Cells To further examine the cytotoxicity of AscTETAderived H
2O2to breast cancer cells MCF-7 cells were
treated with Asc (1mM) along with different doses of TETA(10 120583M 30 120583M and 50 120583M) for 12 hours MTT assay resultsdemonstrated that Asc alone resulted in a 40 reduction inMCF-7 proliferation while TETA alone did not show anyeffect on cell viability Intriguingly the combinations of Ascand TETA significantly decreased cell viability compared toAsc alone (Figure 2(a)) Moreover a prolonged incubationof AscTETA for 24 hours led to further reduction in cellviability indicating that the anticancer effect of AscTETAcombination is in a time-dose manner (Figure 2(b)) Furtherwestern blotting unveiled that AscTETA suppresses MCF-7 cell viability by elevating apoptotic signaling of caspase 9and caspase 3 (Figure 2(c)) Morphology and cell cloningexperiments confirmed that TETA synergizes Asc mediatedcytotoxicity on MCF-7 cells (Figures 2(d) and 2(e)) Takentogether these results strongly suggest that TETA synergizesthe anti-breast cancer effect of Asc in vitro
33 Asc and TETA Synergize to Enhance Cytotoxicity In VitroTo further validate whether the synergistic effects of Asc andTETA on cell death are specific for cancer cells in addition to
4 Oxidative Medicine and Cellular Longevity
1m
M A
sc+50120583
M T
ETA
1m
M A
sc+30120583
M T
ETA
1m
M A
sc+10120583
M T
ETA
50120583
M T
ETA
30120583
M T
ETA
10120583
M T
ETA
Con
trol
1m
M A
sc
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
lowast
(a)
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
6 12 18 240(Hrs)
(b)
GAPDH
Cleaved Caspase 9
Cleaved PARP
Procaspase-3
110 330 550
Cleaved Caspase 3
Control H2O2
AscTETA (mM120583M)
(c)
20x20x 20x 20x
Control AscTETATETAAsc
(d)
0
20
40
60
80
100
120
Clon
ogen
icity
()
Asc TETA AscTETAControl
lowast
(e)
Figure 2 AscTETA combination induces apoptosis in MCF-7 cells MCF-7 cells were treated with 1mM Asc along with 10120583M 30 120583Mor 50 120583M of TETA for 12 hours (a) MTT assay was performed to assess MCF-7 viability lowast119901 lt 0005 119901 lt 00001 119899 = 6 (b) viability ofMCF-7 cells was measured by MTT assay after 6 12 and 24 hours of 1mM Asc10 120583M TETA treatment 119899 = 6 (c) Effects of different dosageof AscTETA (1 100) on proapoptotic signaling were examined by western blotting (d) MCF-7 cells cloning formation experiments wereperformed after 12 hours of 1mM Asc10120583M TETA treatment (e) Statistic analysis of 3 independent experiments lowast119901 lt 005 versus control119901 lt 001 versus Asc 119899 = 3
various cancer cell lines such asMCF-7MDA-MB-157MDA-MB-231 U87 HCC-9204 and H1299 multiple normal celllines including Hs578Bst HUVEC and V79 were incubatedwith different concentrations of TETA (5120583M 10 120583M 30 120583Mand 50 120583M) and corresponding doses of Asc (05mM 1mM3mM and 5mM) in accordance with the 1 100 TETAAscratio MTT assay results demonstrated that Asc and TETAhave synergistic cytotoxicity on cancer cells but much mildereffects on the viability of normal cells (Figure 3)
34 H2O2 Is Critical for AscTETA Induced Cytotoxicity toMCF-7 Cell We then assessed the ROS level in MCF-7 cellsAfter 4 hours of incubation Asc alone moderately elevatedROS level inMCF-7 cells while TETA alone did not show anyeffect on ROS generation However AscTETA coincubationresulted in a dramatic increase of ROS compared to the othergroups while in the presence of N-acetyl-L-cysteine (NACSigma V900429 Germany) which degrade ROS exceptH2O2 AscTETA showed much less fluorescence staining
Oxidative Medicine and Cellular Longevity 5
Normal cell
Cancer cell0
20
40
60
80
100
Cel
l via
bilit
y (
cont
rol)
321 4 50Asc concentration (mM)
MCF-7MDA-MB-157MDA-MB-231
U87HCC-9204H1299
HS578BstHVVecsV79
Figure 3 Selective cytotoxicity of AscTETA on various cell lines MCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 H1299 Hs578BstHUVEC or V79 cells were incubated with 1mM 3mM or 5mMofAsc respectively alongwith 10120583M 30120583M or 50 120583Mof TETA tomaintain1 100 TETA-to-Asc ratio MTT assay was performed to assess the cytotoxicity of AscTETA on different cell lines after 12 hours of incubationexperiment was repeated three times
(Figure 4(a)) Taken together these data suggested TETApotentiated not only H
2O2but also other types of ROS
production from Asc in MCF-7 cellsNevertheless ROS include hydrogen peroxide (H
2O2)
superoxide anions (O2
∙minus) and hydroxyl radical to ascertainwhether the cytotoxicity of AscTETA specifically resultsfrom H
2O2 NAC was applied to MCF-7 cells along with
AscTETAWestern blotting showed that AscTETA combination
treatment resulted in inhibition of RAS expression whichwere not rescued by extraNAC treatment Inversely althoughAscTETA also lead to elevation of H3 acetylation in dose-dependent manner such effect was totally reversed by 5mMNAC (Figure 4(b)) Moreover the effects of AscTETA onRAS expression and H3 acetylation demonstrated a time-dependent manner (Figure 4(c)) The data suggests thatTETAAsc suppresses RAS expression in MCF-7 cells prin-cipally through H
2O2 while it upregulates H3 acetylation
mainly through the other types of ROS Most importantly5mM NAC failed to eliminate the cytotoxicity caused byAscTETA Taken together these results indicate that H
2O2
is the primary cytotoxic ROS induced by AscTETA inMCF-7 cells and possibly through the inhibition of RAS expression
35 The Selective Cytotoxicity of AscTETA Derived H2O2to MCF-7 Cells due to Compromised CAT Expression Wesubsequently investigated the mechanism underlying thediscrepancy of the cytotoxicity of AscTETA combinationin cancer cells and normal cells The effect of catalaseon AscTETA induced cell death was examined by cloneformation experiment the data (Figure 5(a)) shows thatAsc alone moderately reduced plating efficiency of MCF-7cells while neither TETA nor catalase shows similar effectHowever AscTETA combination significantly suppressedplating efficiency of MCF-7 cells but the reduction was
almost fully restored in the presence of catalase We thendetermined the expression levels of CAT in MCF-7 cellsand normal cells by immunoblotting It was shown thatCAT expression level is significantly compromised in MCF-7 cells compared to the normal cells (Figure 5(b)) Tofurther determine the importance of CAT in resistance toAscTETA induced cell death shCATwas transfected into thenormal breast epithelial HS578Bst cells it only dramaticallyrepressed CAT expression in HS578Bst cells (Figure 5(c))but also significantly impaired cell viability (Figure 5(d))This strongly suggests that the selective toxicity to cancercells could be attributed to CAT downregulation and thusthe cytotoxicity of combined TETA and Asc use is primarilymediated by H
2O2
36 AscTETA Induced Cytotoxicity to MCF-7 Cells Is NotMediated by O2
∙minus Although H2O2has been found critical
for TETAAsc mediated cytotoxicity the possibility of O2
∙minus
being involved in such anticancer effects has yet to beruled out To distinguish the roles of O
2
∙minus and H2O2in
TETAAsc combination derived cytotoxicity 100UmL SODwas applied onto MCF-7 cells with 1mMAsc and 10120583MTETA In contrast to CAT SOD failed to rescue cell survivalin the presence of Asc and TETA but exacerbated Asc andTETA combination induced cell death (Figure 6(a)) Thisindicates that O
2
∙minus does not contribute to TETA and Ascmediated cytotoxicity to cancer cells Not surprisingly SODprotein expression in MCF-7 cells was significantly higherthan normal cells (Figure 6(b)) To further confirm thisfinding SOD1 in MCF-7 cells was knocked down by shRNA(Figure 6(c)) In combined AscTETA treatment shSOD1 didnot result in enhanced viability of MCF-7 cell but led to morecell death (Figure 6(d)) This data suggests that the selectivecytotoxicity of AscTETA to MCF-7 cells is not mediated byO2
∙minus
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
RAS
Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
p = 0134
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
Asc
TETA
Asc
TET
A
Asc
TET
AN
AC
Con
trol
NAC
(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 3
was applied into each well and then 20 120583L of 3-(45)-dime-thylthiahiazo(-z-y1)-35-diphenytetrazoliumromide (MTT)(Sigma Germany) was added to each well Followed by 4 h ofincubation at 37∘C all media were removed and then 150 120583Lof dimethyl sulfoxide (DMSO) (Sigma Germany) was addedto each well after 10min of shaking the value of OD
490was
recorded by a Varioskan Flash (Thermo Fisher Finland)
24 ROS Measurement The intracellular ROS was detectedby using H
2DCF (Sigma Germany) as previously reported
[18] Briefly 10 120583MofH2DCFwas added onto cells for 30min
and then cells were harvested and analyzed by a fluorescencemicroscope (KEYENCE Corporation)
25 Western Blotting MCF-7 cells were harvested by theuse of ice-cold RIPA lysis buffer (Beyotime BiotechnologyShanghai China) and protein concentration was determinedusing the BCA protein quantification kit (Beyotime Biotech-nology Shanghai China) western blotting was performed aspreviously described [19] Primary antibodies of anti-Ac-H3anti-SOD1 anti-CAT anti-ERK anti-p-ERK anti-Cyt-C andanti-caspase 9 were purchased from Bioworld Technology(Nanjing China) anti-PARP rabbit anti-caspase 3 and anti-Ac-H3 were purchased from Cell Signaling TechnologyDanvers USA anti-RAS was purchased from BD USAand anti-GAPDH was purchased from Earthox MillbraeUSA The secondary antibodies were purchased from BiogotBiotechnology (Nanjing China) and ECL SuperSignal WestFemto Maximum Sensitivity Substrate was purchased fromThermo Fisher
26 Transient Transfection 1times105 cells were plated into eachwell of a 24-well plate 24 hours before transfection shRNA(600 ngwell) or Plasmids (800 ngwell) were transfectedwith Lipofectamine 3000 (Invitrogen USA) according to themanufacturerrsquos protocol
SOD1 NM 0004543-582s1c1 CCGGGCTGTAGA-AATGTATCCTGATCTCGAGATCAGGATACA-TTTCTACAGCTTTTTGCAT NM 0017522-1371s1c1 CCGGCGGAGATT-CAACACTGCCAATCTCGAGATTGGCAGTGT-TGAATCTCCGTTTTTG
27 Xenograft Study 6-week-old female nudemice (BALBc-nu) were purchased from Vital River Laboratories (BeijingChina) the animal experiments were approved by the Medi-cal Ethics Committee of Chongqing Medical University andall of the procedures were in accordance with the NationalInstitutes of Health guide for the care and use of Laboratoryanimals In short MCF-7 cells (5 times 106 in 200120583L) weresubcutaneously delivered into the hind leg of mice anda 072mg 90 daysrsquo release 17120573-estradiol pellet (InnovativeResearch of America USA) was implanted subcutaneouslyinto the front-back area to facilitate optimal tumor growthThe tumors were allowed to grow 14 days to reach the greatestdimension of about 3ndash5mm and treatments were initiatedon the 14th day Mice were randomly divided into 4 groups
including control (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) and Asc (3 gkg body weight)plus TETA (30mgkg body weight) 10 mice in each groupTreatments were given via intraperitoneal injection daily for25 consecutive days Tumor size was measured every 2 daysusing a vernier caliper while tumor volume was estimatedbased on the following formula tumor volume (mm3) = 119871 times11988222 where 119871 is the greatest dimension of the tumor and119882 means the dimension of the tumor in the perpendiculardirection Animals were sacrificed by CO
2euthanasia when
the tumor size reached 1000mm3
28 Statistical Analysis Data are expressed as mean plusmn SD Avariety of statistical tests using GraphPad Prism 5 softwarewere used on the basis of the design required for the specificquestion being asked This meant using 119905-tests and 2-wayANOVA A value of 119901 lt 005 was considered statisticallysignificant
3 Results
31 TETA Synergizes Ascorbic Acid Oxidation To investigatethe effect of TETA on promoting H
2O2generation from Asc
oxygen consumption of Asc in the presence and absence ofTETA has been measured respectively As shown in Figure 11mM Asc in DMEM with 10 FBS resulted in an OCRof 55 nmolLs and additional 30 120583M of TETA increasedOCR to 110 nmolLs while 30 120583M of TETA alone barelyconsumes O
2and generates H
2O2 However in the presence
of catalase (600UmL) H2O2accumulation in AscTETA
was dramatically suppressed compared toAsc or TETA aloneTaken together this evidence strongly suggested that TETAenhanced Asc-dependent H
2O2generation
32 AscTETA Combination Promotes Apoptosis in MCF-7Cells To further examine the cytotoxicity of AscTETAderived H
2O2to breast cancer cells MCF-7 cells were
treated with Asc (1mM) along with different doses of TETA(10 120583M 30 120583M and 50 120583M) for 12 hours MTT assay resultsdemonstrated that Asc alone resulted in a 40 reduction inMCF-7 proliferation while TETA alone did not show anyeffect on cell viability Intriguingly the combinations of Ascand TETA significantly decreased cell viability compared toAsc alone (Figure 2(a)) Moreover a prolonged incubationof AscTETA for 24 hours led to further reduction in cellviability indicating that the anticancer effect of AscTETAcombination is in a time-dose manner (Figure 2(b)) Furtherwestern blotting unveiled that AscTETA suppresses MCF-7 cell viability by elevating apoptotic signaling of caspase 9and caspase 3 (Figure 2(c)) Morphology and cell cloningexperiments confirmed that TETA synergizes Asc mediatedcytotoxicity on MCF-7 cells (Figures 2(d) and 2(e)) Takentogether these results strongly suggest that TETA synergizesthe anti-breast cancer effect of Asc in vitro
33 Asc and TETA Synergize to Enhance Cytotoxicity In VitroTo further validate whether the synergistic effects of Asc andTETA on cell death are specific for cancer cells in addition to
4 Oxidative Medicine and Cellular Longevity
1m
M A
sc+50120583
M T
ETA
1m
M A
sc+30120583
M T
ETA
1m
M A
sc+10120583
M T
ETA
50120583
M T
ETA
30120583
M T
ETA
10120583
M T
ETA
Con
trol
1m
M A
sc
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
lowast
(a)
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
6 12 18 240(Hrs)
(b)
GAPDH
Cleaved Caspase 9
Cleaved PARP
Procaspase-3
110 330 550
Cleaved Caspase 3
Control H2O2
AscTETA (mM120583M)
(c)
20x20x 20x 20x
Control AscTETATETAAsc
(d)
0
20
40
60
80
100
120
Clon
ogen
icity
()
Asc TETA AscTETAControl
lowast
(e)
Figure 2 AscTETA combination induces apoptosis in MCF-7 cells MCF-7 cells were treated with 1mM Asc along with 10120583M 30 120583Mor 50 120583M of TETA for 12 hours (a) MTT assay was performed to assess MCF-7 viability lowast119901 lt 0005 119901 lt 00001 119899 = 6 (b) viability ofMCF-7 cells was measured by MTT assay after 6 12 and 24 hours of 1mM Asc10 120583M TETA treatment 119899 = 6 (c) Effects of different dosageof AscTETA (1 100) on proapoptotic signaling were examined by western blotting (d) MCF-7 cells cloning formation experiments wereperformed after 12 hours of 1mM Asc10120583M TETA treatment (e) Statistic analysis of 3 independent experiments lowast119901 lt 005 versus control119901 lt 001 versus Asc 119899 = 3
various cancer cell lines such asMCF-7MDA-MB-157MDA-MB-231 U87 HCC-9204 and H1299 multiple normal celllines including Hs578Bst HUVEC and V79 were incubatedwith different concentrations of TETA (5120583M 10 120583M 30 120583Mand 50 120583M) and corresponding doses of Asc (05mM 1mM3mM and 5mM) in accordance with the 1 100 TETAAscratio MTT assay results demonstrated that Asc and TETAhave synergistic cytotoxicity on cancer cells but much mildereffects on the viability of normal cells (Figure 3)
34 H2O2 Is Critical for AscTETA Induced Cytotoxicity toMCF-7 Cell We then assessed the ROS level in MCF-7 cellsAfter 4 hours of incubation Asc alone moderately elevatedROS level inMCF-7 cells while TETA alone did not show anyeffect on ROS generation However AscTETA coincubationresulted in a dramatic increase of ROS compared to the othergroups while in the presence of N-acetyl-L-cysteine (NACSigma V900429 Germany) which degrade ROS exceptH2O2 AscTETA showed much less fluorescence staining
Oxidative Medicine and Cellular Longevity 5
Normal cell
Cancer cell0
20
40
60
80
100
Cel
l via
bilit
y (
cont
rol)
321 4 50Asc concentration (mM)
MCF-7MDA-MB-157MDA-MB-231
U87HCC-9204H1299
HS578BstHVVecsV79
Figure 3 Selective cytotoxicity of AscTETA on various cell lines MCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 H1299 Hs578BstHUVEC or V79 cells were incubated with 1mM 3mM or 5mMofAsc respectively alongwith 10120583M 30120583M or 50 120583Mof TETA tomaintain1 100 TETA-to-Asc ratio MTT assay was performed to assess the cytotoxicity of AscTETA on different cell lines after 12 hours of incubationexperiment was repeated three times
(Figure 4(a)) Taken together these data suggested TETApotentiated not only H
2O2but also other types of ROS
production from Asc in MCF-7 cellsNevertheless ROS include hydrogen peroxide (H
2O2)
superoxide anions (O2
∙minus) and hydroxyl radical to ascertainwhether the cytotoxicity of AscTETA specifically resultsfrom H
2O2 NAC was applied to MCF-7 cells along with
AscTETAWestern blotting showed that AscTETA combination
treatment resulted in inhibition of RAS expression whichwere not rescued by extraNAC treatment Inversely althoughAscTETA also lead to elevation of H3 acetylation in dose-dependent manner such effect was totally reversed by 5mMNAC (Figure 4(b)) Moreover the effects of AscTETA onRAS expression and H3 acetylation demonstrated a time-dependent manner (Figure 4(c)) The data suggests thatTETAAsc suppresses RAS expression in MCF-7 cells prin-cipally through H
2O2 while it upregulates H3 acetylation
mainly through the other types of ROS Most importantly5mM NAC failed to eliminate the cytotoxicity caused byAscTETA Taken together these results indicate that H
2O2
is the primary cytotoxic ROS induced by AscTETA inMCF-7 cells and possibly through the inhibition of RAS expression
35 The Selective Cytotoxicity of AscTETA Derived H2O2to MCF-7 Cells due to Compromised CAT Expression Wesubsequently investigated the mechanism underlying thediscrepancy of the cytotoxicity of AscTETA combinationin cancer cells and normal cells The effect of catalaseon AscTETA induced cell death was examined by cloneformation experiment the data (Figure 5(a)) shows thatAsc alone moderately reduced plating efficiency of MCF-7cells while neither TETA nor catalase shows similar effectHowever AscTETA combination significantly suppressedplating efficiency of MCF-7 cells but the reduction was
almost fully restored in the presence of catalase We thendetermined the expression levels of CAT in MCF-7 cellsand normal cells by immunoblotting It was shown thatCAT expression level is significantly compromised in MCF-7 cells compared to the normal cells (Figure 5(b)) Tofurther determine the importance of CAT in resistance toAscTETA induced cell death shCATwas transfected into thenormal breast epithelial HS578Bst cells it only dramaticallyrepressed CAT expression in HS578Bst cells (Figure 5(c))but also significantly impaired cell viability (Figure 5(d))This strongly suggests that the selective toxicity to cancercells could be attributed to CAT downregulation and thusthe cytotoxicity of combined TETA and Asc use is primarilymediated by H
2O2
36 AscTETA Induced Cytotoxicity to MCF-7 Cells Is NotMediated by O2
∙minus Although H2O2has been found critical
for TETAAsc mediated cytotoxicity the possibility of O2
∙minus
being involved in such anticancer effects has yet to beruled out To distinguish the roles of O
2
∙minus and H2O2in
TETAAsc combination derived cytotoxicity 100UmL SODwas applied onto MCF-7 cells with 1mMAsc and 10120583MTETA In contrast to CAT SOD failed to rescue cell survivalin the presence of Asc and TETA but exacerbated Asc andTETA combination induced cell death (Figure 6(a)) Thisindicates that O
2
∙minus does not contribute to TETA and Ascmediated cytotoxicity to cancer cells Not surprisingly SODprotein expression in MCF-7 cells was significantly higherthan normal cells (Figure 6(b)) To further confirm thisfinding SOD1 in MCF-7 cells was knocked down by shRNA(Figure 6(c)) In combined AscTETA treatment shSOD1 didnot result in enhanced viability of MCF-7 cell but led to morecell death (Figure 6(d)) This data suggests that the selectivecytotoxicity of AscTETA to MCF-7 cells is not mediated byO2
∙minus
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
RAS
Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
p = 0134
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
Asc
TETA
Asc
TET
A
Asc
TET
AN
AC
Con
trol
NAC
(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
1m
M A
sc+50120583
M T
ETA
1m
M A
sc+30120583
M T
ETA
1m
M A
sc+10120583
M T
ETA
50120583
M T
ETA
30120583
M T
ETA
10120583
M T
ETA
Con
trol
1m
M A
sc
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
lowast
(a)
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
6 12 18 240(Hrs)
(b)
GAPDH
Cleaved Caspase 9
Cleaved PARP
Procaspase-3
110 330 550
Cleaved Caspase 3
Control H2O2
AscTETA (mM120583M)
(c)
20x20x 20x 20x
Control AscTETATETAAsc
(d)
0
20
40
60
80
100
120
Clon
ogen
icity
()
Asc TETA AscTETAControl
lowast
(e)
Figure 2 AscTETA combination induces apoptosis in MCF-7 cells MCF-7 cells were treated with 1mM Asc along with 10120583M 30 120583Mor 50 120583M of TETA for 12 hours (a) MTT assay was performed to assess MCF-7 viability lowast119901 lt 0005 119901 lt 00001 119899 = 6 (b) viability ofMCF-7 cells was measured by MTT assay after 6 12 and 24 hours of 1mM Asc10 120583M TETA treatment 119899 = 6 (c) Effects of different dosageof AscTETA (1 100) on proapoptotic signaling were examined by western blotting (d) MCF-7 cells cloning formation experiments wereperformed after 12 hours of 1mM Asc10120583M TETA treatment (e) Statistic analysis of 3 independent experiments lowast119901 lt 005 versus control119901 lt 001 versus Asc 119899 = 3
various cancer cell lines such asMCF-7MDA-MB-157MDA-MB-231 U87 HCC-9204 and H1299 multiple normal celllines including Hs578Bst HUVEC and V79 were incubatedwith different concentrations of TETA (5120583M 10 120583M 30 120583Mand 50 120583M) and corresponding doses of Asc (05mM 1mM3mM and 5mM) in accordance with the 1 100 TETAAscratio MTT assay results demonstrated that Asc and TETAhave synergistic cytotoxicity on cancer cells but much mildereffects on the viability of normal cells (Figure 3)
34 H2O2 Is Critical for AscTETA Induced Cytotoxicity toMCF-7 Cell We then assessed the ROS level in MCF-7 cellsAfter 4 hours of incubation Asc alone moderately elevatedROS level inMCF-7 cells while TETA alone did not show anyeffect on ROS generation However AscTETA coincubationresulted in a dramatic increase of ROS compared to the othergroups while in the presence of N-acetyl-L-cysteine (NACSigma V900429 Germany) which degrade ROS exceptH2O2 AscTETA showed much less fluorescence staining
Oxidative Medicine and Cellular Longevity 5
Normal cell
Cancer cell0
20
40
60
80
100
Cel
l via
bilit
y (
cont
rol)
321 4 50Asc concentration (mM)
MCF-7MDA-MB-157MDA-MB-231
U87HCC-9204H1299
HS578BstHVVecsV79
Figure 3 Selective cytotoxicity of AscTETA on various cell lines MCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 H1299 Hs578BstHUVEC or V79 cells were incubated with 1mM 3mM or 5mMofAsc respectively alongwith 10120583M 30120583M or 50 120583Mof TETA tomaintain1 100 TETA-to-Asc ratio MTT assay was performed to assess the cytotoxicity of AscTETA on different cell lines after 12 hours of incubationexperiment was repeated three times
(Figure 4(a)) Taken together these data suggested TETApotentiated not only H
2O2but also other types of ROS
production from Asc in MCF-7 cellsNevertheless ROS include hydrogen peroxide (H
2O2)
superoxide anions (O2
∙minus) and hydroxyl radical to ascertainwhether the cytotoxicity of AscTETA specifically resultsfrom H
2O2 NAC was applied to MCF-7 cells along with
AscTETAWestern blotting showed that AscTETA combination
treatment resulted in inhibition of RAS expression whichwere not rescued by extraNAC treatment Inversely althoughAscTETA also lead to elevation of H3 acetylation in dose-dependent manner such effect was totally reversed by 5mMNAC (Figure 4(b)) Moreover the effects of AscTETA onRAS expression and H3 acetylation demonstrated a time-dependent manner (Figure 4(c)) The data suggests thatTETAAsc suppresses RAS expression in MCF-7 cells prin-cipally through H
2O2 while it upregulates H3 acetylation
mainly through the other types of ROS Most importantly5mM NAC failed to eliminate the cytotoxicity caused byAscTETA Taken together these results indicate that H
2O2
is the primary cytotoxic ROS induced by AscTETA inMCF-7 cells and possibly through the inhibition of RAS expression
35 The Selective Cytotoxicity of AscTETA Derived H2O2to MCF-7 Cells due to Compromised CAT Expression Wesubsequently investigated the mechanism underlying thediscrepancy of the cytotoxicity of AscTETA combinationin cancer cells and normal cells The effect of catalaseon AscTETA induced cell death was examined by cloneformation experiment the data (Figure 5(a)) shows thatAsc alone moderately reduced plating efficiency of MCF-7cells while neither TETA nor catalase shows similar effectHowever AscTETA combination significantly suppressedplating efficiency of MCF-7 cells but the reduction was
almost fully restored in the presence of catalase We thendetermined the expression levels of CAT in MCF-7 cellsand normal cells by immunoblotting It was shown thatCAT expression level is significantly compromised in MCF-7 cells compared to the normal cells (Figure 5(b)) Tofurther determine the importance of CAT in resistance toAscTETA induced cell death shCATwas transfected into thenormal breast epithelial HS578Bst cells it only dramaticallyrepressed CAT expression in HS578Bst cells (Figure 5(c))but also significantly impaired cell viability (Figure 5(d))This strongly suggests that the selective toxicity to cancercells could be attributed to CAT downregulation and thusthe cytotoxicity of combined TETA and Asc use is primarilymediated by H
2O2
36 AscTETA Induced Cytotoxicity to MCF-7 Cells Is NotMediated by O2
∙minus Although H2O2has been found critical
for TETAAsc mediated cytotoxicity the possibility of O2
∙minus
being involved in such anticancer effects has yet to beruled out To distinguish the roles of O
2
∙minus and H2O2in
TETAAsc combination derived cytotoxicity 100UmL SODwas applied onto MCF-7 cells with 1mMAsc and 10120583MTETA In contrast to CAT SOD failed to rescue cell survivalin the presence of Asc and TETA but exacerbated Asc andTETA combination induced cell death (Figure 6(a)) Thisindicates that O
2
∙minus does not contribute to TETA and Ascmediated cytotoxicity to cancer cells Not surprisingly SODprotein expression in MCF-7 cells was significantly higherthan normal cells (Figure 6(b)) To further confirm thisfinding SOD1 in MCF-7 cells was knocked down by shRNA(Figure 6(c)) In combined AscTETA treatment shSOD1 didnot result in enhanced viability of MCF-7 cell but led to morecell death (Figure 6(d)) This data suggests that the selectivecytotoxicity of AscTETA to MCF-7 cells is not mediated byO2
∙minus
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
RAS
Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
p = 0134
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
Asc
TETA
Asc
TET
A
Asc
TET
AN
AC
Con
trol
NAC
(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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EndocrinologyInternational Journal of
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Disease Markers
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BioMed Research International
OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 5
Normal cell
Cancer cell0
20
40
60
80
100
Cel
l via
bilit
y (
cont
rol)
321 4 50Asc concentration (mM)
MCF-7MDA-MB-157MDA-MB-231
U87HCC-9204H1299
HS578BstHVVecsV79
Figure 3 Selective cytotoxicity of AscTETA on various cell lines MCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 H1299 Hs578BstHUVEC or V79 cells were incubated with 1mM 3mM or 5mMofAsc respectively alongwith 10120583M 30120583M or 50 120583Mof TETA tomaintain1 100 TETA-to-Asc ratio MTT assay was performed to assess the cytotoxicity of AscTETA on different cell lines after 12 hours of incubationexperiment was repeated three times
(Figure 4(a)) Taken together these data suggested TETApotentiated not only H
2O2but also other types of ROS
production from Asc in MCF-7 cellsNevertheless ROS include hydrogen peroxide (H
2O2)
superoxide anions (O2
∙minus) and hydroxyl radical to ascertainwhether the cytotoxicity of AscTETA specifically resultsfrom H
2O2 NAC was applied to MCF-7 cells along with
AscTETAWestern blotting showed that AscTETA combination
treatment resulted in inhibition of RAS expression whichwere not rescued by extraNAC treatment Inversely althoughAscTETA also lead to elevation of H3 acetylation in dose-dependent manner such effect was totally reversed by 5mMNAC (Figure 4(b)) Moreover the effects of AscTETA onRAS expression and H3 acetylation demonstrated a time-dependent manner (Figure 4(c)) The data suggests thatTETAAsc suppresses RAS expression in MCF-7 cells prin-cipally through H
2O2 while it upregulates H3 acetylation
mainly through the other types of ROS Most importantly5mM NAC failed to eliminate the cytotoxicity caused byAscTETA Taken together these results indicate that H
2O2
is the primary cytotoxic ROS induced by AscTETA inMCF-7 cells and possibly through the inhibition of RAS expression
35 The Selective Cytotoxicity of AscTETA Derived H2O2to MCF-7 Cells due to Compromised CAT Expression Wesubsequently investigated the mechanism underlying thediscrepancy of the cytotoxicity of AscTETA combinationin cancer cells and normal cells The effect of catalaseon AscTETA induced cell death was examined by cloneformation experiment the data (Figure 5(a)) shows thatAsc alone moderately reduced plating efficiency of MCF-7cells while neither TETA nor catalase shows similar effectHowever AscTETA combination significantly suppressedplating efficiency of MCF-7 cells but the reduction was
almost fully restored in the presence of catalase We thendetermined the expression levels of CAT in MCF-7 cellsand normal cells by immunoblotting It was shown thatCAT expression level is significantly compromised in MCF-7 cells compared to the normal cells (Figure 5(b)) Tofurther determine the importance of CAT in resistance toAscTETA induced cell death shCATwas transfected into thenormal breast epithelial HS578Bst cells it only dramaticallyrepressed CAT expression in HS578Bst cells (Figure 5(c))but also significantly impaired cell viability (Figure 5(d))This strongly suggests that the selective toxicity to cancercells could be attributed to CAT downregulation and thusthe cytotoxicity of combined TETA and Asc use is primarilymediated by H
2O2
36 AscTETA Induced Cytotoxicity to MCF-7 Cells Is NotMediated by O2
∙minus Although H2O2has been found critical
for TETAAsc mediated cytotoxicity the possibility of O2
∙minus
being involved in such anticancer effects has yet to beruled out To distinguish the roles of O
2
∙minus and H2O2in
TETAAsc combination derived cytotoxicity 100UmL SODwas applied onto MCF-7 cells with 1mMAsc and 10120583MTETA In contrast to CAT SOD failed to rescue cell survivalin the presence of Asc and TETA but exacerbated Asc andTETA combination induced cell death (Figure 6(a)) Thisindicates that O
2
∙minus does not contribute to TETA and Ascmediated cytotoxicity to cancer cells Not surprisingly SODprotein expression in MCF-7 cells was significantly higherthan normal cells (Figure 6(b)) To further confirm thisfinding SOD1 in MCF-7 cells was knocked down by shRNA(Figure 6(c)) In combined AscTETA treatment shSOD1 didnot result in enhanced viability of MCF-7 cell but led to morecell death (Figure 6(d)) This data suggests that the selectivecytotoxicity of AscTETA to MCF-7 cells is not mediated byO2
∙minus
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
RAS
Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
p = 0134
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
Asc
TETA
Asc
TET
A
Asc
TET
AN
AC
Con
trol
NAC
(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
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Oxidative Medicine and Cellular Longevity
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Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
AscTETA
Control Asc TETA
AscTETANAC NAC
100120583M 100120583M 100120583M
100120583M100120583M100120583M
(a)
ControlAscTETANAC
RAS
Acetyl-H3
H3
GAPDH
minusminus +
3mM 3mM1mM10120583M 30120583M 30120583M
(b)
RAS
Acetyl-H3
H3
GAPDH
Control AscTETA6hrs 12hrs 24hrs
(c)lowast
p = 0134
0
30
60
90
120
Cel
l via
bilit
y (
cont
rol)
Asc
TETA
Asc
TET
A
Asc
TET
AN
AC
Con
trol
NAC
(d)
Figure 4 AscTETA induces H2O2-dependent RAS downregulation and apoptosis in MCF-7 cells MCF-7 cells were treated with 1mMAsc
10120583M TETA 1mM Asc10 120583M TETA 5mM NAC or 1mM Asc10 120583M TETA and 5mM NAC respectively for 4 hours (a) ROS generationin MCF-7 cells was examined by H2DCF staining (b) MCF-7 cells were treated with 1mM Asc10120583M TETA 3mM Asc30 120583M TETA or3mMAsc30 120583MTETA with 5mMNAC for 12 hours RAS expression and H3 acetylation were assessed by western blotting (c) MCF-7 cellswere treated with 1mM Asc10120583M TETA for 6 12 and 24 hours respectively RAS expression and H3 acetylation were assessed by westernblotting (d) MCF-7 cells were treated with 1mM Asc 10120583M TETA 5mM NAC 1mM Asc10 120583M TETA or 1mM Asc10 120583M TETA plusNAC for 12 hours cell viability was measured with MTT assay lowast119901 lt 0005 119899 = 4 Experiment was repeated three times
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
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OncologyJournal of
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7
lowast
Asc
TET
A
Asc
TET
Ac
atal
ase
Asc
Cata
lase
Con
trol
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
CAT
HS578Bst MCF-7
(b)
CAT
GAPDH
Vector CAT shRNA
(c)Vector CAT shRNA
0
20
40
60
Cel
l via
bilit
y (
cont
rol) lowast
(d)
Figure 5 CAT expression levels in breast cancer cells and normal cells (a) MCF-7 cells were treated with 1mMAsc 10120583MTETA 300UmLcatalase 1mM Asc10 120583MTETA or 1mM Asc10120583MTETA plus catalase for 12 hours cloning formation assay was performed lowast119901 lt 00001119901 lt 00001 119899 = 4 (b) CAT expression levels in HS578Bst and MCF-7 cells were determined by western blotting (c) CAT expression inHS578Bst was suppressed by CAT shRNA (d) HS578Bst cell viability after CAT shRNA treatment was measured by MTT assay 119901 lt 0005119899 = 4
37 The Signaling Associated with AscTETA Treatment inCancer Cells We then investigated which signaling pathwaysare involved in AscTETA induced cell death As shown inFigure 7 western blots illustrated that AscTETA treatmentsuppressed ERK12 and SOD1 expression in MCF-7 cells Tofurther determine the type of cell death caused by Asc andTETA CytochromeC (Cyt-C) the key regulator of apoptosishas been measured AscTETA induced Cyt-C release wastime dependent which is consistent with previous MTTresults These data strongly suggest that AscTETA inducedcancer cell apoptosis is probably mediated by RAS-ERKpathway
38 TETA Enhances Asc-Induced Cytotoxicity In VivoIn order to examine whether combined AscTETA hascytotoxicity to tumor cells in vivo the combination was
administrated into nude mice with transplanted tumorsAfter 25 days of treatment there was no significant differ-ence in viscera index between the 4 groups as has beenobserved (Figure 8(a)) indicating AscTETA has low tox-icity for these organs However there was difference inthe day 25 tumor volume between the groups 9711 plusmn2420mm3 in control group 8980 plusmn 1603mm3 in TETAgroup and 7465 plusmn 1444mm3 in Asc group but it signif-icantly reduced to 2780 plusmn 1642mm3 in AscTETA groupIn congruence the tumor weights were 1276 plusmn 0097 g0969 plusmn 0095 g 1226 plusmn 0087 g in control Asc and TETAgroup respectively but dramatically declined to 0478 plusmn0094 g by AscTETA treatment (Figures 8(b) 8(c) and 8(d))These results indicate that TETA and Asc have synergisticantitumor effects in vivo without notable toxicity to internalorgans
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
lowast
Asc
TET
A
SOD
Asc
TET
AS
OD
Con
trol
Asc
TETA
0
30
60
90
120
Plat
ing
effici
ency
()
(a)
GAPDH
SOD1
HS578Bst MCF-7
(b)
GAPDH
SOD1
Vector SOD1 shRNA
(c)
Vector SOD1 shRNA
lowast
0
10
20
30
40
Cel
l via
bilit
y (
cont
rol)
(d)
Figure 6The effects of SOD on AscTETA induces apoptosis (a) 1mMAsc 10 120583MTETA 100UmL SOD1 1mMAsc10 120583MTETA or 1mMAsc10 120583M TETA plus SOD was applied onto MCF-7 cells for 12 hours Cloning formation assay was performed to determine the platingefficiency lowast119901 lt 00001 119901 lt 005 119899 = 4 (b) SOD1 expression levels in HS578Bst and MCF-7 cells were assessed by western blotting (c)downregulation of SOD1 expression inMCF-7 cells by SOD1 shRNAwas determined by western blotting (d) MCF-7 cell viability after SOD1shRNA treatment was measured by MTT assay lowast119901 lt 005 119899 = 4
AscTETA
GAPDH
Cyt-C
SOD1
ERK
Con
trol
30120583
M T
ETA
3m
M A
sc
6hrs 12hrs 24hrs
Figure 7 The effects of AscTETA on ERK and SOD expressionMCF-7 cells were incubated with 3mM Asc 30 120583M TETA or3mMAsc30 120583MTETA for various duration ERK RAS and Cyt-Cprotein levels were assessed by western blotting
4 Discussion
Given that one of the typical characteristics of breast can-cer is SOD overexpression along with compromised CATexpression the intracellular oxidative stress is higher in
the cancer cells compared to normal cells [20] Wlassoff etal have shown that hydroxyl radicals derived from H
2O2
promote breast cancer cell apoptosis in the presence ofa tamoxifenndashferrocene conjugate [21] Other groups havedemonstrated that the excessive H
2O2production and accu-
mulation in cancer cells could trigger cancer cell cycle arrestand apoptosis [6] suggesting the enhancement of intracel-lular H
2O2concentration could be a promising therapy for
breast cancerTherefore a natural compound which is able toselectively induce H
2O2generation in cancer cells could be
an ideal therapy for breast cancerAsc is a potent natural antioxidant that has long been
assumed to be beneficial for cancer treatment Intriguinglyit has been reported that in cancer cells Asc is oxidized bydonating an electron which had an oxidation reaction withthe metal ion (copper iron and manganese) producing theanticancer H
2O2[12] Another group has also shown that
4 h of 1mM Asc treatment on C6 cells triggers intracellularCu release [22] which can further promote Asc oxidationindicating H
2O2generated by Asc autoxidation might be
beneficial for cancer treatment However the effectivenessof Asc derived H
2O2on treating cancer remains debatable
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 9
Heart Liver Spleen Lung Kidney0
2
4
6
8
Visc
era i
ndex
()
ControlAsc
TETAAscTETA
(a)
Control Asc TETA Asc+TETA
(b)
10 20 300Days
ControlAsc
TETAAscTETA
Tum
or v
olum
e (m
m3)
0
200
400
600
800
1000
(c)
Control Asc TETA AscTETA00
05
10
15
20
Tum
or w
eigh
t (g)
lowast
(d)
Figure 8 AscTETA suppresses tumorigenesis inmouse breast cancermodel 5times106MCF-7 cells were subcutaneously delivered into the hindleg of female nude mice 14 days later mice were randomly divided into 4 groups and given vehicle (001M PBS) Asc (3 gkg body weight)TETA (30mgkg body weight) Asc (3 gkg body weight) plus TETA (30mgkg body weight) respectively via intraperitoneal injection once aday On the 25th day of treatment (a) viscera index (b) representative pictures of tumor from different groups (c) tumor volume 119901 lt 00001119899 = 6 (d) tumor weight lowast119901 lt 005 119901 lt 0005 119899 = 6
Firstly there is no strong association between plasma Ascconcentration and breast cancer risk [23] secondly H
2O2
has been found to enhance growth of breast cancer [5] Bothof them indicate that not only is a high dose of Asc neededto induce a dramatic elevation of intracellular H
2O2flux
but also a reagent that could potentiate or promote H2O2
production from Asc autoxidation might be critical for Ascclinically utilized in breast cancer therapy
Considering human circulating Asc concentration isrelative high ranging from 20 to 80120583molL [24 25] and quitestable in various countries [26] it is reasonable to pursuethe effects of pharmacologic Asc on breast cancer Indeedcumulative evidence indicates that high doses of Asc couldbe beneficial for breast cancer treatment Yun et al recentlyreported that high dose Asc selectively kills KRAS and BRAFmutant colorectal cancer cells [27] Similarly another group
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Oxidative Medicine and Cellular Longevity
has also shown that pharmacologic concentrations of Ascinhibit proliferation and induce apoptosis in various colorec-tal cancer lines probably by promoting intracellular oxidativestress [28] Asc has also exhibited anticancer properties inpancreatic cancer cells [29] providing further evidence tosuggest that high dose of Asc induces H
2O2flux in the
presence of catalytic metal ions resulting in oxidative stressin cancer cells which ultimately leads to apoptosis Theseresults suggest that pharmacologic Asc may be needed tocause breast cancer cell death Our data confirmed that a highdose of Asc is essential for inducing MCF-7 cell apoptosis invitro and inhibiting tumorigenesis in vivo Although manystudies have shown Asc can be used as an adjuvant for breastcancer chemotherapy [30] therapy that primarily dependson Asc-induced oxidative stress in cancer cells has yet to beinvestigated
TETA is a charge-deficient isosteric analogue of spermi-dine and a Cu (II) chelating compound It is usually used fortreating Wilsonrsquos disease Recently its potential in treatingcancer has been unveiled growing evidence demonstratesthat TETAplays a role against cancer by inhibiting telomerase[31] in antiangiogenesis [32] and in suppressing cancercell proliferation by modulating metabolism [33] as wellas in induction of cell withered death [34] However noneof these studies focused on the effect of TETArsquos chemicalproperties on cancer specifically the two amino groups andtheir role as a potential catalyst for Asc autoxidation Ourwork is the first to report that in aqueous solution TETAenhances H
2O2production by increasing Asc oxidation
Thereafter we examined whether AscTETA combinationcould sufficiently lead to breast cancer cell death In vitro andin vivo experiments consistently demonstrated that high doseAsc alone only resulted in mild toxicity to cancer cells whileTETA alone barely shows any effects However AscTETAcombination significantly inhibited MCF-7 cell viabilitystrongly suggesting that AscTETA combination could bea promising neoadjuvant therapy for breast cancer Never-theless the underlying molecular mechanism remains to bediscovered
To examine the cytotoxicity of AscTETA to breast cancercells fluorescent staining of ROS has been performed by theuse of H2DCF The results suggested that AscTETA signif-icantly promoted ROS generation in cancer cells We thendetermined which type of ROS is responsible for AscTETAinduced cancer cell death Adding NAC does not inhibit thecytotoxicity of AscTETA strongly indicating that H
2O2is
the major type of ROS derived fromAscTETA and the causeof the selective cytotoxicity to breast cancer cell Howevermany studies have shown that chronic high extracellularH2O2 over several months promotes breast cancer cell pro-
liferation and results in an aggressive phenotype [5 35 36]Nevertheless our results are consistent with Wlassoff et alwho reported that H
2O2is the hydroxyl radical which stim-
ulates apoptosis in tamoxifenndashferrocene conjugate treatedMCF-7 cells [21]This indicates that the best therapeutic win-dowofH
2O2-dependent breast cancer treatmentwould be no
longer than 6 months However in future studies it would bepertinent to perform a real timemeasurement of intracellular
H2O2flux under AscTETA treatment as previously reported
[37]It is quite interesting to understand whether TETAAsc
is selectively toxic to cancer cells It has previously beendemonstrated that Asc arrested growth of some cancer celllines like HeLa SK-BR-3 SK-BR-3-Dox L929 and Mel B16but did not influence the growth of others Hef OVCARHEp2 HEp2VA3 and V79 [38] Our results have shown thatAscTETA is selectively toxic to cancer cell lines includingMCF-7 MDA-MB-157 MDA-MB-231 U87 HCC-9204 andH1299 but has muchmilder toxicity to normal cell lines suchas Hs578Bst HUVEC and V79 Taken together these datasuggest AscTETA selectively kill certain type of cancer cellsincluding the ER-positive MCF-7 breast cancer cells
To investigate the mechanism of the selective cytotox-icity of AscTETA in breast cancer cell the expression ofSOD1 and CAT was modulated using shRNA Interestinglydownregulation of SOD1 failed to ameliorate the cytotoxicityof AscTETA to cancer cells in contrast overexpression ofcatalase effectively halted the cancer cell death induced byAscTETA This confirmed that H
2O2rather than O
2
∙minus isthe causative factor for AscTETA induced cell death Inaddition to further study whether the disturbed expressionpattern of SOD1 and CAT in breast cancer cell is relatedwith cell viability SOD1 and CAT were knocked down byshRNA respectively SOD1 is overexpressed in the MCF-7cell compared to normal cells and shSOD1RNA treatmentresulted in significant reduction in viability of the MCF-7cell indicating endogenous SOD1 is critical for maintainingbreast cancer cell physiological function Probably a productof the SOD1 H
2O2 is an important signaling molecule for
cancer cells It is known that certain levels of H2O2promote
cancer cell proliferation and growth [5] Furthermore due tothe low levels of expression of CAT in MCF-7 cells we thendownregulated CAT in HS578Bst cells which demonstratedaugmented cell viability This suggests low level of CAT maybe beneficial for cells possibly by blocking the removal ofH2O2and thus maintaining a beneficial intracellular H
2O2
concentrationNumerous studies have shown that RAS-ERK pathway
is involved in breast cancer cell growth and metastasis [3940] Our data also suggested that AscTETA suppressed RASand ERK expression in a time- and dose-dependent mannerIt also suppressed subsequent cancer cell apoptosis as evi-denced by release of Cyt-C and caspases cleavage while nei-ther TETA nor Asc alone showed similar effects In this workwe also observed AscTETA treatment result in global H3acetylation inMCF-7 cells which is not associatedwithH
2O2
production and the viability of MCF-7 cells but evidence hasshown that elevating histone acetylation by SIRT1 inhibitioninduces breast cancer cell apoptosis [41] possibly throughthe upregulation of p21 [42] Therefore it will be interestingto further elucidate whether histone modification in thepromoter region of specific genes is involved in AscTETAinduced breast cancer cell apoptosisMoreover we found thatAscTETA treatment also leads to a downregulation of SOD1inMCF-7 cells which is in accordance with the SOD1 shRNAexperiment result indicating the anti-breast cancer effectsof AscTETA combination are partially mediated by the
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 11
inhibition of SOD1 Despite other studies also suggesting Ascmay be involved in regulating breast cancer cell proliferationand migration through Akt and RhoA respectively [43 44]it is unclear whether AscTETA is effective through the samepathways and warrants further investigation
Xenograft experiments validated our findings fromthe cell model Administration of AscTETA dramaticallyreduced tumor size and weight while visceral organs inthe nude mice remained unaffected This result confirmedthat TETA synergizes Ascrsquos anti-breast cancer effects in vivoHowever the dose used in this experiment is 3 grams per kgbody weight which is a far higher dose of Asc compared towhat has been used previously Further pharmacodynamicsand pharmacokinetics studies for AscTETA are needed toexplore the optimal administrative approach and dosagefor breast cancer treatment Likewise a treatment of Ascin combination with another natural mixture includinglysine proline and green tea extract significantly reducedtumor weight and inhibited metastasis to lung spleenliver kidney and heart in a breast cancer murine model[45] Similarly a natural antioxidant mixture containingAsc has also been shown to effectively repress radiation-induced carcinogenesis in animal experiments [46] It isalso reported that oral administration of high dose of Ascrepresents strong anticarcinogenic effect and prolongs sur-vival time of a rat cancer model induced by benzo[a]pyrene[47]
In addition to the H2O2mediated apoptotic effects of
AscTETA on cancer cells the synergistic antioxidant prop-erties hold potential in preventing normal cell damage fromhigh intercellularintracellular ROS Findings from othergroups suggest these additional antitumorigenic effects maybe mediated by inhibiting HIF1 [48] However evidencesuggests that pharmacological Asc might have side effectsAsc induced procoagulant and prothrombotic activation ofRBCs and increased thrombosis has been reported in vivoRBCs from cancer patients exhibited increased sensitivity tothe prothrombotic effects of Asc reflecting that intravenousgram-dose vitamin C therapy needs to be carefully revisited[49] An Asc derivative 6-deoxy-6-chloro-ascorbic acidexhibited more potent effects in suppressing cancer cellproliferation and is more likely to be used clinically to treatbreast cancer [38]
In conclusion the present work demonstrates that TETAsynergizes with pharmacologic Asc in breast cancer toenhance hydrogen peroxide production thus inhibitingtumorigenic RASERK signaling pathways and inducingapoptosis
Competing Interests
No potential competing interests were disclosed
Acknowledgments
This study was supported by grants from the Natural Sci-ence Foundation of China (81100399) Yuzhong DistrictNatural Science Foundation (20150117) Chongqing Medical
University (CYYQ201507) and Chongqing Education Com-mittee (KJ1500223)
References
[1] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995
[2] S L Payne B Fogelgren A R Hess et al ldquoLysyl oxidase reg-ulates breast cancer cell migration and adhesion through ahydrogen peroxide-mediated mechanismrdquo Cancer Researchvol 65 no 24 pp 11429ndash11436 2005
[3] C J Weydert T A Waugh J M Ritchie et al ldquoOverexpressionof manganese or copper-zinc superoxide dismutase inhibitsbreast cancer growthrdquo Free Radical Biology and Medicine vol41 no 2 pp 226ndash237 2006
[4] J Ferlay H-R Shin F Bray D Forman C Mathers and DM Parkin ldquoEstimates of worldwide burden of cancer in 2008GLOBOCAN2008rdquo International Journal of Cancer vol 127 no12 pp 2893ndash2917 2010
[5] S Sen B Kawahara andG Chaudhuri ldquoMaintenance of higherH2O2 levels and its mechanism of action to induce growth inbreast cancer cells important roles of bioactive catalase andPP2Ardquo Free Radical Biology and Medicine vol 53 no 8 pp1541ndash1551 2012
[6] P-J Chua G W-C Yip and B-H Bay ldquoCell cycle arrestinduced by hydrogen peroxide is associated with modulation ofoxidative stress related genes in breast cancer cellsrdquo Experimen-tal Biology and Medicine vol 234 no 9 pp 1086ndash1094 2009
[7] M Rawal S R Schroeder B A Wagner et al ldquoManganopor-phyrins increase ascorbate-induced cytotoxicity by enhancingH2O2generationrdquo Cancer Research vol 73 no 16 pp 5232ndash
5241 2013[8] Y Kuang K Balakrishnan V Gandhi and X Peng ldquoHydrogen
peroxide inducible DNA cross-linking agents targeted anti-cancer prodrugsrdquo Journal of the American Chemical Society vol133 no 48 pp 19278ndash19281 2011
[9] G R Buettner ldquoThe pecking order of free radicals and antioxi-dants lipid peroxidation120572-tocopherol and ascorbaterdquoArchivesof Biochemistry and Biophysics vol 300 no 2 pp 535ndash543 1993
[10] C Jacobs B Hutton T Ng R Shorr andM Clemons ldquoIs therea role for oral or intravenous ascorbate (Vitamin C) in treatingpatients with cancer A systematic reviewrdquo Oncologist vol 20no 2 pp 210ndash223 2015
[11] J Du J J Cullen and G R Buettner ldquoAscorbic acid chemistrybiology and the treatment of cancerrdquo Biochimica et BiophysicaActamdashReviews on Cancer vol 1826 no 2 pp 443ndash457 2012
[12] G R Buettner and B A Jurkiewicz ldquoCatalyticmetals ascorbateand free radicals combinations to avoidrdquo Radiation Researchvol 145 no 5 pp 532ndash541 1996
[13] N H Williams and J K Yandell ldquoReduction of oxidizedcytochrome c by ascorbate ionrdquo Biochimica et Biophysica Acta(BBA)mdashBioenergetics vol 810 no 2 pp 274ndash277 1985
[14] A Chakravarty K Bhowmik A Mukherjee and G DeldquoCu(2)O nanoparticles anchored on amine-functionalizedgraphite nanosheet a potential reusable catalystrdquoLangmuir vol31 no 18 pp 5210ndash5219 2015
[15] J Lu ldquoTriethylenetetramine pharmacology and its clinicalapplicationsrdquo Molecular Cancer Therapeutics vol 9 no 9 pp2458ndash2467 2010
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
12 Oxidative Medicine and Cellular Longevity
[16] V K Yellepeddi K K Vangara A Kumar and S PalakurthildquoComparative evaluation of small-molecule chemosensitizersin reversal of cisplatin resistance in ovarian cancer cellsrdquoAnticancer Research vol 32 no 9 pp 3651ndash3658 2012
[17] D P G Brown H Chin-Sinex B Nie M S Mendonca andMWang ldquoTargeting superoxide dismutase 1 to overcome cisplatinresistance in human ovarian cancerrdquo Cancer Chemotherapy andPharmacology vol 63 no 4 pp 723ndash730 2009
[18] Y Cui I Parra M Zhang et al ldquoElevated expression of mito-gen-activated protein kinase phosphatase 3 in breast tumors amechanism of tamoxifen resistancerdquo Cancer Research vol 66no 11 pp 5950ndash5959 2006
[19] C Tong A Morrison S Mattison et al ldquoImpaired SIRT1 nuc-leocytoplasmic shuttling in the senescent heart during ischemicstressrdquo FASEB Journal vol 27 no 11 pp 4332ndash4342 2013
[20] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006
[21] W A Wlassoff C D Albright M S Sivashinski A IvanovaJ G Appelbaum and R I Salganik ldquoHydrogen peroxideoverproduced in breast cancer cells can serve as an anticancerprodrug generating apoptosis-stimulating hydroxyl radicalsunder the effect of tamoxifen-ferrocene conjugaterdquo Journal ofPharmacy and Pharmacology vol 59 no 11 pp 1549ndash1553 2007
[22] D W Domaille L Zeng and C J Chang ldquoVisualizingascorbate-triggered release of labile copper within living cellsusing a ratiometric fluorescent sensorrdquo Journal of the AmericanChemical Society vol 132 no 4 pp 1194ndash1195 2010
[23] F Hu Z Wu G Li et al ldquoThe plasma level of retinol vitaminsA C and 120572-tocopherol could reduce breast cancer risk Ameta-analysis and meta-regressionrdquo Journal of Cancer Research andClinical Oncology vol 141 no 4 pp 601ndash614 2015
[24] L Cahill P N Corey and A El-Sohemy ldquoVitamin C deficiencyin a population of young canadian adultsrdquo American Journal ofEpidemiology vol 170 no 4 pp 464ndash471 2009
[25] A Jungert and M Neuhauser-Berthold ldquoThe lower vitamin Cplasma concentrations in elderly men compared with elderlywomen can partly be attributed to a volumetric dilution effectdue to differences in fat-free massrdquo British Journal of Nutritionvol 113 no 5 pp 859ndash864 2015
[26] B Olmedilla F Granado S Southon et al ldquoSerum concentra-tions of carotenoids and vitamins A E andC in control subjectsfrom five European countriesrdquo British Journal of Nutrition vol85 no 2 pp 227ndash238 2001
[27] J Yun E Mullarky C Lu et al ldquoVitamin C selectively killsKRAS and BRAF mutant colorectal cancer cells by targetingGAPDHrdquo Science vol 350 no 6266 pp 1391ndash1396 2015
[28] A S Pires C R Marques J C Encarnacao et al ldquoAscorbic acidand colon cancer an oxidative stimulus to cell death dependingon cell profilerdquo European Journal of Cell Biology vol 95 no 6-7pp 208ndash218 2016
[29] J A Cieslak and J J Cullen ldquoTreatment of pancreatic can-cer with pharmacological ascorbaterdquo Current PharmaceuticalBiotechnology vol 16 no 9 pp 759ndash770 2015
[30] E Guerriero A Sorice F Capone et al ldquoVitamin C effect onmitoxantrone-induced cytotoxicity in human breast cancer celllinesrdquo PLoS ONE vol 9 no 12 Article ID e115287 2014
[31] G Lixia Y Fei J Jiajia and L Jianhui ldquoTriethylene tetraminea novel ligand of G-quadruplex induces senescence of MCF-7cellsrdquo Biotechnology Letters vol 30 no 1 pp 47ndash53 2008
[32] E D Harris ldquoA requirement for copper in angiogenesisrdquoNutrition Reviews vol 62 no 2 pp 60ndash64 2004
[33] M T Hyvonen S Ucal M Pasanen et al ldquoTriethylenete-tramine modulates polyamine and energy metabolism andinhibits cancer cell proliferationrdquo Biochemical Journal vol 473no 10 pp 1433ndash1441 2016
[34] S Kadowaki D Endoh T Okui and M Hayashi ldquoTrientine aCopper-chelating agent induced apoptosis in murine fibrosar-coma cells by activation of the p38 MAPK pathwayrdquo Journal ofVeterinary Medical Science vol 71 no 11 pp 1541ndash1544 2009
[35] P K S Mahalingaiah L Ponnusamy and K P Singh ldquoChronicoxidative stress causes estrogen-independent aggressive phe-notype and epigenetic inactivation of estrogen receptor alphain MCF-7 breast cancer cellsrdquo Breast Cancer Research andTreatment vol 153 no 1 pp 41ndash56 2015
[36] P K Mahalingaiah and K P Singh ldquoChronic oxidative stressincreases growth and tumorigenic potential of MCF-7 breastcancer cellsrdquo PLoS ONE vol 9 no 1 Article ID e87371 2014
[37] F Liu S Ge J YuM Yan andX Song ldquoElectrochemical devicebased on a Pt nanosphere-paper working electrode for in situand real-time determination of the flux of H2O2 releasing fromSK-BR-3 cancer cellsrdquo Chemical Communications vol 50 no71 pp 10315ndash10318 2014
[38] MOsmak I Kovacek I Ljubenkov R Spaventi andM Eckert-Maksic ldquoAscorbic acid and 6-deoxy-6-chloro-ascorbic acidpotential anticancer drugsrdquo Neoplasma vol 44 no 2 pp 101ndash107 1997
[39] J Gao X Liu F Yang T Liu Q Yan and X Yang ldquoByinhibiting RasRafERK and MMP-9 knockdown of EpCAMinhibits breast cancer cell growth and metastasisrdquo Oncotargetvol 6 no 29 pp 27187ndash27198 2015
[40] N R Treff D Pouchnik G A Dement R L Britt and RReeves ldquoHigh-mobility group A1a protein regulates RasERKsignaling in MCF-7 human breast cancer cellsrdquo Oncogene vol23 no 3 pp 777ndash785 2004
[41] A M Kalle A Mallika J Badiger Alinakhi P Talukdar andSachchidanand ldquoInhibition of SIRT1 by a small moleculeinduces apoptosis in breast cancer cellsrdquo Biochemical and Bio-physical Research Communications vol 401 no 1 pp 13ndash192010
[42] Y Al Dhaheri S Attoub K Arafat et al ldquoSalinomycin inducesapoptosis and senescence in breast cancer upregulation of p21downregulation of survivin and histoneH3 andH4 hyperacety-lationrdquo Biochimica et Biophysica Acta vol 1830 no 4 pp 3121ndash3135 2013
[43] F Ourique M R Kviecinski K B Felipe et al ldquoDNA damageand inhibition of akt pathway inMCF-7 cells and ehrlich tumorin mice treated with 14-naphthoquinones in combination withascorbaterdquoOxidativeMedicine and Cellular Longevity vol 2015Article ID 495305 10 pages 2015
[44] L Ma W-Z Zhu T-T Liu et al ldquoH2O2inhibits proliferation
and mediates suppression of migration via DLC1RhoA signal-ing in cancer cellsrdquo Asian Pacific Journal of Cancer Preventionvol 16 no 4 pp 1637ndash1642 2015
[45] M W Roomi T Kalinovsky N M Roomi J Cha M Rathand A Niedzwiecki ldquoIn vitro and in vivo effects of a nutrientmixture on breast cancer progressionrdquo International Journal ofOncology vol 44 no 6 pp 1933ndash1944 2014
[46] A R Kennedy J HWareW Carlton and J G Davis ldquoSuppres-sion of the later stages of radiation-induced carcinogenesis byantioxidant dietary formulationsrdquo Radiation Research vol 176no 1 pp 62ndash70 2011
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 13
[47] KCharalabopoulos S Karkabounas PDimicco et al ldquoThe roleof ascorbic acid selenium and glutathione on benzo[a]pyrene-induced carcinogenesis in wistar ratsrdquo Journal of BUON vol 9pp 187ndash192 2004
[48] P Gao H Zhang R Dinavahi et al ldquoHIF-dependent antitu-morigenic effect of antioxidants in vivordquoCancer Cell vol 12 no3 pp 230ndash238 2007
[49] K Kim O-N Bae S-H Koh et al ldquoHigh-dose vitamin Cinjection to cancer patients may promote thrombosis throughprocoagulant activation of erythrocytesrdquo Toxicological Sciencesvol 147 no 2 pp 350ndash359 2015
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpswwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom