apoptosis induction via atm phosphorylation, cell cycle...

Research ArticleApoptosis Induction via ATM Phosphorylation Cell CycleArrest and ER Stress by Goniothalamin and ChemodrugsCombined Effects on Breast Cancer-Derived MDA-MB-231 Cells

Patompong Khaw-on 1 Wilart Pompimon2 and Ratana Banjerdpongchai 1

1Department of Biochemistry Faculty of Medicine Chiang Mai University Chiang Mai 50200ailand2Laboratory of Natural Products Faculty of Science Lampang Rajabhat University Lampang 52100 ailand

Correspondence should be addressed to Ratana Banjerdpongchai ratanabcmuacth

Received 20 April 2018 Revised 17 September 2018 Accepted 10 November 2018 Published 26 November 2018

Academic Editor Melchiorre Cervello

Copyright copy 2018 Patompong Khaw-on et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Goniothalamin (GTN) a styryl-lactone exhibits inhibitory effects onmany kinds of cancer cells in vitroThe objectives of this studywere to investigate the anticancer activities of GTN and molecular signaling pathways associated with cell death in human breastcancerMDA-MB-231 cell line GTN inhibited the growth of MDA-MB-231 cells Apoptosis was confirmed by annexin V-FITC andPI staining and apoptotic morphology was observed by microscopy Reduction of mitochondrial transmembrane potential andenhanced caspases activities were found in GTN-treated MDA-MB-231 cells GTN significantly altered apoptosis-related proteinexpressions including Noxa PUMA Bax Bim Bad Bcl-2 Bcl-xL and DIABLO which was related to the gene expression levelsMitochondrial calcium released to the cytosol and ER stress related proteins increased which correlatedwith increases in ER stressgene expression levels GTN induced hydrogen peroxide and superoxide anion radicals in MDA-MB-231 cells associated with cellcycle arrest in G2M phase which was induced by phosphorylation and ATM gene expression Moreover GTN had synergisticeffects when combined with cyclophosphamide 5-fluorouracil paclitaxel and vinblastine and additive effect with methotrexatethrough caspases enzyme-acceleration In conclusion goniothalamin-induced MDA-MB-231 cell apoptosis occurred via intrinsicand extrinsic pathways along with ER stress These pathways provide new targeted drug strategies for advancements in anticancermedicine

1 Introduction

Goniothalamin (GTN) is isolated and characterized fromGoniothalamus griffithii [1] It has been widely used as afolk medicine in south-east Asia Goniothalamin exhibits aninhibitory effect on growth and the proliferation on varioustypes of cancerous cell lines and noncancerousmurine fibrob-last (NIH3T3) cell line that were cultured in vitro [2] GTNinduces apoptosis in Jurkat T-cells via caspase-3-7 andPARP-cleavage [3] and intrinsic apoptosis in human promye-locytic leukemia HL-60 cells [4] Notably the apoptosisinduction pathway depends on the cancer cell type [1] GTNinduces cell cycle arrest at the G

2M phase in human breast

cancer MDA-MB-231 cells [5] In addition GTN causes DNAdamage which subsequently leads to apoptosis in many celllines [6ndash8]

Apoptosis a programmed cell death consists of deathreceptor- andor mitochondria-mediated pathways Chem-ical compounds drugs and ultraviolet (UV) light inducethe mitochondrial pathway by generating mitochondrialstress with reduced mitochondrial transmembrane potential(MTP) After the death receptors bind with its ligandsthe death receptor pathway initiates with Death-InducingSignaling Complex (DISC) formation and triggers initiatorcaspase-8 followed by effector caspase-3 activation to inducecell death Caspase-8 also cleaves proapoptosis Bid to becometruncated Bid (tBid) which induces mitochondrial poreformation by Bax-Bax Bax-Bak or Bak-Bak dimers for thechannel formation Notably pro- and antiapoptotic proteinshave important roles in apoptosis pathways [9]

Reactive oxygen species (ROS) play a crucial role in apop-tosis in cancer cells [10] since GTN causes oxidative damage

HindawiBioMed Research InternationalVolume 2018 Article ID 7049053 15 pageshttpsdoiorg10115520187049053

2 BioMed Research International

in many types of cancer cells [11 12] ROS induces cancercells to undergo apoptosis via interrupting the mitochondriaoxidative phosphorylation lipid peroxidation and a double-strand DNA break [8] The DNA break can induce cell cyclearrest by ATMATR activation which is induced by p53[13] Furthermore the p53-independent pathway has alsobeen shown to be influential in the activation of DNA dam-age sensing molecules and proapoptosis proteins PUMANoxa for apoptosis execution [14] ER stress related proteinsand heat shock 70kD protein 5GRP78HSPA5 equilibratecytosolic calcium which is released from cellular organelle-induced apoptosis [15] Ca2+ is released to the cytosol whilechaperone proteins and ER stress related proteins play crucialroles in programmed cell death induction in cancer cells [16]

Chemotherapeutic drugs have been developed and usedfor cancer treatments but are still associated with poor out-comes perhaps due to a lack of compliance and their com-plicated side effects The use of combination treatmentswith rationale on different mechanisms also synergizes theeffects of single targets and kills the cancer cells moreeffectively Providing selective synergism against multipletargets drug combinations are widely used and have becomethe leading choice for the treatment of cancer [17] Targeteddrug therapies are aimed directly at the cancerous cells orat the molecules that regulate or control the proliferation ofcancer cells These drugs are used in combination with othertherapies for the advantages of fewer or less severe side effects[18] Triple negative breast cancer MDA-MB-231 cells whichare characterized as negative for estrogen receptor (ER) pro-gesterone (PR) and epidermal growth factor receptor (EGFRor HER2) become indicated of invasive breast cancer asa consequence of poor prognosis and have a potential tobecome drug-resistant [19] In this study the human invasivebreast cancer MDA-MB-231 cell line was used as a modelfor an investigation whether GTN induced apoptosis andits cytotoxic effects were considered when the treatment iscombined with conventional chemodrugs in addition to itsrelated mechanism(s)

2 Materials and Methods

21 Chemicals The leaves and twigs of Goniothalamusgriffithii were collected in January 2011 from Chiang MaiProvince Thailand and identified by the Forest HerbariumDepartment of National Park Wildlife and Plant Conser-vation Ministry of Natural Resources and EnvironmentBangkok Thailand where a voucher specimen (BKF16447)has been deposited GTN was extracted purified and iden-tified by Professor Wilart Pompimon Lampang RajabpatUniversity Lampang Province Thailand The phytochemicalcompound goniothalamin was isolated from G griffithii ashas been previously reported [20]

GTN stock solution was prepared in DMSO at 20mMand GTN was diluted in Dulbeccorsquos Modified Eagle Medium(DMEM Gibco Carlsbad CA USA) Annexin V Fluos-staining kit and Protease Inhibitor Cocktail tablets wereobtained from Roche Diagnostics (Mannheim Germany)Ficoll-Hypaque reagent (HISTOPAQUE-1077) dihy-droethidium (DHE) and 2101584071015840ndashdichlorodihydrofluorescein

diacetate (DCFH-DA) 331015840-dihexyloxacarbocyanine iodide(DiOC

6) and 3-(45 dimethylthiazol-2yl)-25 diphenyl-

tetrazolium bromide (MTT) were obtained from Sigma-Aldrich MO USA Rhod-2 acetoxymethyl (AM) ester andFluo-3 AM were obtained from Thermo Fisher ScientificInc USA Caspases-3 -8 and -9 determination kits wereobtained from Invitrogen Thermo Fisher Scientific IncUSA Primary and secondary antibodies and horse-radishperoxidase-conjugated antibodies were obtained fromAbcam Cambridge UK RNA isolation kit was obtainedfrom GE Healthcare UK Total RNA was reversed tocomplementary DNA (cDNA) by using a cDNA SynthesisKit and quantitative real-time PCR assays were performedwith Reagents Kit (Bioline Reagents Ltd USA)

22 Cell Culture Triple negative breast cancer MDA-MB-231 cell line was cultured in DMEM supplemented with 10fetal bovine serum penicillin and streptomycin at 37∘C ina humidified atmosphere of 5 CO

2at 37∘C After the cells

grew to 80 confluence the cell lines were trypsinized by005 trypsin Peripheral blood mononuclear cells (PBMCs)were isolated from heparinized blood obtained from adultvolunteers as blood donors at Blood Bank Unit Maha RajNakorn Chiang Mai Hospital affiliated with the Faculty ofMedicine Chiang Mai University Each blood donor wasinformed of the objectives and signed the written consentform in order to comply as individual volunteers accordingto the Institutional Review Board Research Ethics Com-mittee Faculty of Medicine Chiang Mai University PBMCswere separated from the whole blood of healthy volunteersby using Ficoll-Hypaque reagent as has been previouslydescribed [21]

23 Cell Viability Assay The MTT (3-(45 dimethylthiazol-2yl)-25 diphenyltetrazolium bromide) assay [22] was per-formed by seeding MDA-MB-231 cells in 96-well cultureplates GTN treatment conditions were prepared in variousconcentrations and incubated for 24 hours The cell viableactivity in each well will be determined by MTT assay andcompared to the untreated cells [23]

24 Drug Combination Assay A process for analyzing theefficiency of combined drugs and the net effect was employedby the method of Chou TC and Talalay P [24] Briefly MDA-MB-231 cells were cotreated with various concentrations ofGTN and conventional therapeutic drugs with a noncon-stant ratio combination The chemotherapeutic drugs thatwere used in this study include vinblastine paclitaxel 5-fluorouracil methotrexate and cyclophosphamide After 24hours of treatment the cells were evaluated for viabilityusing MTT assay and a function of the effect level (Fa)valueswas calculatedTheCompuSyn Softwarewas employedfor determination of combination index (CI) values andvalidated as a combination effect

25 Fluorescence Microscopy MDA-MB-231 cells were seed-ed on culture slides and treated with GTN After 24 hours theslides were fixed in ice-cold absolute alcohol and stained with10120583gml propidium iodide (PI) The slides were investigated

BioMed Research International 3

Table 1 Primers for specific genes in the real-time RT-PCRmethod

Symbol Synonym Name Sequence of primers (51015840-gt31015840)

GAPDH glyceraldehyde-3-phosphatedehydrogenase

F TGCACCACCAACTGCTTAGCR GGCATGGACTGTGGTCATGAG

BAD BCL2 associated agonist of celldeath

F GCACAGCAACGCAGATGCR AAGTTCCGATCCCACCAGG

DIABLO smac diablo IAP-bindingmitochondrial protein

F GAAGCTGGAAACCACTTGGATGAR TGAATGTGATTCCTGGCGGTTA

BBC3 PUMA BCL2 binding component 3 F GCAGGCACCTAATTGGGCTR ATCATGGGACTCCTGCCCTTA

PMAIP1 NOXAphorbol-12-myristate-13-acetate-

induced protein1

F GCTGGAAGTCGAGTGTGCTAR CCTGAGCAGAAGAGTTTGGA

HSPA5 GRP78 heat shock protein family A(Hsp70) member 5

F GCCTGTATTTCTAGACCTGCCR TTCATCTTGCCAGCCAGTTG

CALR calreticulin F AAATGAGAAGAGCCCCGTTCTTCCTR AAGCCACAGGCCTGAGATTTCATCTG

ATM ATM serinethreonine kinase F TGCCAGACAGCCGTGACTTACR ACCTCCACCTGCTCATACACAAG

for apoptotic morphology using a fluorescence microscopeApoptosis positive cells were scored from 200 cells per trialsof three independent experiments

26 Apoptosis Assay After treatment with GTN for 24hours the cells were trypsinized and the cell pellets werewashed with phosphate-buffered saline (PBS) After that thecells were stained with the reagent annexin V-fluoresceinisothiocyanate (FITC) and PI for 15 minutes and were thenprocessed using a flow cytometer

27 Cell Cycle Analysis Cell cycle analysis was performedon the goniothalamin-treated cells by using FlowCellectCell Cycle Checkpoint ATM DNA Damage Kit (MerckMillipore Corporation Germany) and operated by GuavaEasyCyte 5HT Benchtop Flow Cytometer (Merck MilliporeCorporation Germany) The data were analyzed for p-ATMpositive cells and the percentages of each phase in the cellcycle were recorded

28 Assessment of Mitochondrial Transmembrane Potential Cytosolic vs Mitochondrial Calcium Ion Levels MDA-MB-231 cells were treated with GTN for 24 hours The cells werethen combined with 40 nM 331015840-dihexyloxacarbocyanineiodide (DiOC

6) 250 nM Rhod-2 AM and 10 120583M and Fluo-

3 AM solutions They were then incubated at 37∘C for 15minutes The cells were washed twice with PBS and analyzedusing the flow cytometry technique

29 Intracellular Reactive Oxygen Species (ROS) Genera-tion Assay Intracellular ROS including superoxide anionradicals and hydrogen peroxide species were detected byfluorescence probes viz dihydroethidium (DHE) and 2101584071015840-dichlorodihydrofluorescein diacetate (DCFH-DA) respec-tively and then measured using a fluorescence microplate

reader (BioTek Winooski VT USA) [25 26] Briefly MDA-MB-231 cells were preincubated with each probe for an hourand then treated with GTN at IC

20and IC

50concentrations

for four hours with or without N-acetylcysteine to countertreatment of the ROS inhibitory effect during each oxidantproduction cycle

210 Determination of Caspases-3 -8 and -9 ActivitiesDetermination of caspases activity was performed by col-orimetric protease assay Briefly the cell pellets were lysedwith lysis buffer on ice and an equal amount of protein wasprepared Caspase-8 (IETD-pNA) caspase-3 (DEVD-pNA)and caspase-9 (LEHD-pNA) chromogenic substrates wereadded for an hour at 37∘C The optical density was measuredat a wavelength of 405 nm using a microplate reader (BioTekWinooski VT USA)

211 Immunoblotting Proteins were extracted in RIPA con-taining supplements with Protease Inhibitor Cocktail tabletsThe immunoblotting was performed as has been previouslydescribed [27] Antibodies against Noxa PUMA Bax BimBad p112-Bad Bcl-2 Bcl-xL DIABLO GRP78 GADD153Calreticulin COX-4 and 120573-Actin were purchased fromAbcam UK

212 Quantitative Real-Time Reverse Transcription-Polymer-ase Chain Reaction (Real-Time RT-PCR) RNA was isolatedfrom the cell pellets by using the Illustra RNAspin Mini Kit(GE Healthcare UK) Total RNA was reversed to comple-mentary DNA (cDNA) by using Tetro cDNA Synthesis Kit(Bioline Reagents Ltd USA) Quantitative real-time PCRassays were performed with the SensiFAST SYBRLo-ROXKit (Bioline Reagents Ltd USA) and the QuantStudio 6Flex Real-Time PCR System (Thermo Fisher Scientific IncUSA) All the data were normalized by theGAPDH geneThedetails of all gene primers are listed in Table 1


213 Statistical Analysis Data are presented as the mean plusmnstandard deviation (SD) from triplicate trials of three inde-pendent experiments The data were analyzed by one-wayanalysis of variance (ANOVA) with a comparison betweengroups of data by Tukeyrsquos test All analyses were conductedusing free statistic PSPP Software Statistical significance wasconsidered when lowast p lt 005 or lowastlowast p lt 001

3 Results

31 Cytotoxic Effect and Cell Cycle Arrest on MDA-MB-231 Cells aer Treatment with Goniothalamin To determinethe cytotoxic effect and cell cycle distribution histogram ofgoniothalamin-treated MDA-MB-231 cells GTN was foundto be toxic against MDA-MB-231 cells at 24 hours oftreatment (Figure 1(a)) with an inhibitory concentration of50 percent (IC

50) at 37 120583M However GTN was found

to be less toxic to PBMCs as a normal cell control withIC50

of gt 80 120583M Cell cycle progression is also an eventresponding to DNA damage which is usually signaled via theATMATRp53p21 pathway [28] Cell cycle and phosphory-lated ATMwere investigated and analyzed by flow cytometryand are illustrated in Figure 1(b) Consequently GTN-treatedcells were arrested at G

2M phase (Figure 1(c)) Since p-

ATM functions in DNA injuries or genome instability it isa marker of cancer cell DNA damage [29] PhosphorylatedATM increased significantly in a dose-dependent manner(Figure 1(d)) Moreover the gene expression levels of ATMincreased in GTN-treated MDA-MB-231 cells in a dose-dependent manner (Figure 1(e))

32 Apoptosis Induction and the Pathways of GTN-treatedMDA-MB-231 Cell Death To investigate apoptosis in GTN-treated breast cancer MDA-MB-231 cells fluorescencemicro-graphs revealed the typical apoptosis morphology as con-densed nuclei and apoptotic bodies (see Figure 2(a)) Theapoptotic positive cells from fluorescence micrographs werescored which increased in a dose-dependent manner(see Figure 2(b)) and transmission electron micrographsalso exhibited apoptotic cell death (Figure 2(c)) GTN-treated cells also increased annexin-V-FITC signals dose-dependently as early apoptosis characters (Figure 2(d)) Tostudy the pathways of GTN-induced apoptosis cancer cellsexhibited the disruption of mitochondrial transmembranepotential (MTP) or loss of MTP (Figure 2(e)) Caspases-3-8 and-9 activities also increased significantly in a dose-dependent manner and this effect was attenuated by a pan-caspase inhibitor (z-VAD-fmk) (Figure 2(f))

33 Apoptosis-Related Proteins and Gene Expression Alter-ations in GTN-treated MDA-MB-231 Cells To investigate theroles of Bcl-2 family proteins in signaling pathways GTN-treated MDA-MB-231 cells were investigated by Westernblotting and band density analyses as shown in Figures 3(a)and 3(b) The proapoptosis proteins levels Noxa PUMABax Bim and Bad increased whereas the antiapoptoticproteins Bcl-2 and Bcl-xL decreased Bad protein was alsoactivated by dephosphorylating at serine 112 Mitochondrial

intermembranous space protein DIABLO was also releasedfrom mitochondria to the cytosol after GTN treatment in adose-dependent manner which is illustrated in Figures 3(a)3(b) and 3(c) The expressions of apoptotic related-genesincluding PMAIP1Noxa BBC3PUMA BAD and DIABLOalso increased afterMDA-MB231 cellswere treatedwithGTN(Figure 3(d))

34 GTN-ActivatedReactiveOxygen Species (ROS)Generationand Cellular Calcium Ion Levels Alteration-induced-ER Stressin MDA-MB-231 Cells In the apoptosis of cancer cells theROS generation and ER stress occur as pathways of apoptoticcell death signaling [13] Thus to investigate whether ornot GTN induced ROS production in MDA-MB-231 cellsintracellular ROS were determined by using DCFH-DA andDHE assays

The ROS levels in GTN-induced MDA-MB-231 cells weremeasured in time variations using GTN at 50 120583M It wasexhibited that the DCF fluorescence intensity levels signifi-cantly increased after an hour when compared to the control(Supplement Figure 1) However the DCF intensity slightlyincreased at 1 to 12 hours of treatment and then displayed atrend to decrease to a normal level after 6 hours of treatmentbut not significantly (p gt 005) (as shown in Supplement Fig-ure 1) Hence we chose to investigate this result by employingconcentration variations instead and used a constant GTN-incubation time at an hour After GTN treatment dichlo-rofluorescein (DCF) and ethidium (E) mean fluorescenceintensities significantly increased in GTN-treated MDA-MB231 cells for which N-acetylcysteine (NAC) an antioxidantattenuated the effect of GTN-mediated-intracellular ROSgenerations both as hydrogenperoxide and superoxide anionradicals (Figures 4(a) and 4(b)) respectively

To determine whether or not the cellular calcium inducedER stress Fluo-3 AM fluorescence dye as a cytosolic calciumion level marker was used and its intensity also increasedin the GTN-treated MDA-MB-231 cells However Rhod-2 AM fluorescence dye as a mitochondrial calcium ionmarker revealed that its fluorescence intensity decreasedsignificantly in a dose-dependent manner but the calciumion concentrations were inversely related to each compart-ment ie high in the cytosol and low in mitochondria(Figure 4(c)) In addition ER stress HSPA5GRP78 mRNAand HSPA5GRP78 protein levels and GADD153 proteinlevels increased after GTN treatment in MDA-MB 231 cellsAnother ER stress geneCALR increased significantly only inmRNA level but the expression of Calreticulin protein leveldid not change (Figures 4(d) 4(e) and 4(f))

35 Goniothalamin and Conventional ChemotherapeuticDrugs Combination Effect on MDA-MB-231 Cells A recentstudy has shown that the combination of a natural productand conventional chemodrugs is widely used for cancertreatment which has been found to reduce side effects andincrease therapeutic outcomes [30] To conduct drug combi-nation effects of GTNwith chemotherapeutic drugs inMDA-MB-231 cells the cytotoxic effect of the drug combinationswas assessed in nonconstant ratio combinations by varying


C

ell v

iabi

lity

GTN Concentration (M)0 20 40 60 80 100

0

20

40

60

80

100

120

MDA-MB-231 PBMC

lowastlowastlowastlowastlowastlowastlowastlowast

(a)

Control

PI

Cel

l cou

nt

PI

Ant

i-pAT

M-A

lexa

488

Plot P05 gated on P01R1singlecell Plot P05 gated on P01R1singlecell Plot P05 gated on P01R1singlecell

S

G1 G2M

S

G1 G2M

S

G1 G2M


Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

100

101

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103

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101

102

103

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100

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Ant

i-pAT

M A

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(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

）20 ）50

(b)

Gat

ed p

opul

atio

n (

)Cell cycle phase

G1 SG2M

0

20

40

60

80

0 ）20 ）50

lowastlowastlowastlowast

(c)

GTN Concentration

pATM

pos

itive

(ga

ted)

Control

00

25

50

75

100

IC 20 IC 50

lowast

lowastlowast

(d)

ATM

Goniothalamin

Rela

tive Q

uant

ifica

tion

(fold

of c

ontr

ol)

control00

05

10

15

20

IC20 IC50

lowastlowast

lowastlowast

(e)

Figure 1 Cytotoxicity of goniothalamin (GTN) against human breast cancer MDA-MB-231 cells Percent cell viability (a) of adherent MDA-MB-231 compared with PBMCs as mean plusmn SD Cell cycle analysis of MDA-MB-231 cells after treatment with GTN at IC

20and IC

50((b) upper

panel) Dot plot of cells positive for phospho-ATM was accomplished by using specific Guava Cell Cycle reagent ((b) lower panel) Bargraphs of percent cells in each phase of the cell cycle are shown as mean plusmn SD analyzed by using Guava Flow Cytometry easyCyte Systems(c) Bar graphs of cells that were positive for phospho-ATM as mean plusmn SD (d) Bar graphs are presented of ATM gene expression in GTN-treatedMDA-MB-231 cells (e)The significance of statistical values compared to control (without treatment) was marked with lowast plt005 lowastlowastplt001


0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)

Control GTN TreatmentI50

(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4

(d)GTN Concentration

0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20

GTN ConcentrationCON IC20 IC50



lowastlowast

lowast

lowastlowastlowast lowastlowast lowastlowast lowastlowast

lowastlowast

lowastlowast

(f)

Figure 2 Apoptosis induction by GTN inMDA-MB-231 cells Cell morphology of apoptotic cells was condensed nuclei and apoptotic bodies(arrows) after staining with propidium iodide (PI) (a) Bar graphs of percent apoptotic cells were obtained from counting positive apoptoticcells stained with PI of total 200 cells in a sample from three independent experiments as mean plusmn SD (b) A transmission electronmicrographof the representative apoptotic cell that was compared to the normal cell after GTN treatment for 24 h (c) Dot plots of early apoptosis wereconfirmed by staining with annexin V-FITC and PI employing flow cytometry (d) Bar graphs of early apoptotic cells as evidenced by positivecells for annexin V-FITC as mean plusmn SD (e) Caspases-3 -8 and -9 activities increased dose dependently after being incubated with GTN atIC20and IC

50for 24 hours and they were then compared to controlThe three caspase activities were attenuated by pre-treatment for an hour

with z-VAD-fmk at the concentration of 10 120583M (f)The significance of statistical values compared to control (without treatment) was markedwith lowast plt005 lowastlowast plt001


GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

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evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

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cont

rol)

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GTN concentration0I20

I50

lowast

lowast

lowast

lowast

lowastlowast


lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast


lowastlowast

lowastlowast


Noxa PUMA Bax Bim Bad Bad(p112)

Bcl-2 Bcl-xL DIABLO DIABLO(Mito) (Cyto)

(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50

cytosolicmitochondria

(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)

PMAIP1 BBC3 BAD DIABLO0

1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)

Figure 3 Alteration of apoptosis-related proteins and gene expressions in Bcl-2 family pro-apoptotic such as Bax and BH3-only proteinseg Noxa PUMA Bim Bad phospho112-Bad and anti-apoptotic proteins in Bcl-2 family such as Bcl-2 Bcl-xL were determined byWesternblotting (a) The relative levels of protein expressions of MDA-MB-231 cells treated with GTN were obtained using densitometry from threeindependent experiments of Western blotting as mean plusmn SD (b) DIABLO a protein in the intermembranous space of the mitochondriawas released into the cytosol in apoptotic cells The amount of DIABLO protein increased in the cytoplasm and decreased in mitochondriadetected by using digitonin-induced-cell fractionation followed by Western blotting (c) Some related corresponding gene expressions weremeasured by real-time RT-PCR and calculated using the Ct method for relative quantifications The data are shown as mean plusmn SD fromthree independent experiments (d) The significance of statistical values compared to the control (without treatment) was marked with lowastplt005 lowastlowast plt001


00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)

GTN ConcentrationCON IC 20 IC 50

lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast

lowastlowast lowastlowast

lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50


GRP78 CalreticulinGADD153

(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)

Figure 4 Reactive oxygen species (ROS) production and alteration of endoplasmic reticulum (ER) Calcium ion levels and ER proteinexpressions ROS generation is exhibited as mean plusmn SD of fluorescence intensity in folds compared to that of the control (without treatment)from three independent experiments by using fluorescence probes DCFH-DA (a) and DHE (b) measured by a fluorescence microplatereader N-acetylcysteine (NAC) was used to confirm the oxidative stress involvement in the signaling pathway ER stress was determinedby Ca2+ ion level determination probes in the cytoplasm and mitochondria ER stress was exhibited as evidenced by the increased levels ofCa2+ in the cytosol and decreased in the mitochondria The data were shown as mean plusmn SD of fluorescence intensity as folds compared tothe control (without treatment) from three independent experiments (c) The ER chaperone protein expression levels GRP78 GADD153and Calreticulin were illustrated by Western blotting after MDA-MB-231 cells were treated with GTN at IC

20and IC

50for 24 hours (d) The

densitometry of protein bands was calculated by Image J Software and is shown as mean plusmn SD of three independent experiments (e) Bargraphs of mRNA relative levels of HSPA5GRP78 and CALR by real-time RT-PCR and results were calculated by using the Ct method forrelative quantifications The data are presented as mean plusmn SD from three independent experiments (f) The significance of statistical valuescompared to the control (without treatment) was marked with lowast plt005 lowastlowast plt001

GTN concentrations and fixing the chemodrug concentra-tion values to minimize the chemodrug adverse effects (seeFigures 5(a)ndash5(e)) The cell viability of drug combinationswas illustrated by their statistical significancewhen comparedwith single GTN or chemodrug treatments alone (see Figures5(a) 5(c) 5(d) 5(e) and 6(a)) Combination index (CI) valuewas calculated by CompuSyn Software The combination of625 120583M GTN with 0125 120583M paclitaxel (PTX) 125 120583Mvinblastine (VBT) 10 120583M 5-fluorouracil (5-FU) or 100 120583Mcyclophosphamide (CPP) revealed a synergistic effect forwhich CI was less than 10 and the additive effect in 20120583M methotrexate (MTX) combination with CI value wasestimated to equal 10 (see Table 2 and Figures 5(f)ndash5(j))Thesynergistic effect of GTNwas also exhibited in caspases-3 and

-9 activities which markedly increased when compared withGTN alone or in single chemodrug treatments but caspase-8activity significantly increased in GTN-combined treatmentswith VBT and 5-FU (see Figures 6(b)ndash6(d))

4 Discussion

GTN was previously reported to possess anticancer proper-ties against various cancer cells via intrinsic and extrinsicapoptotic pathways [31] In this study the cytotoxicity alsoexhibited the fifty-percent inhibition growth concentrationat 37 120583M in the MDA-MB-231 cell line for 24 hoursof incubation (Figure 1(a)) The general janitor genes areDNA repair and mitotic checkpoint genes DNA damage


Combination index for GTN+MTX

00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000

Fractional inhibition (Fa)

Cytotoxicity of GTN+MTX

GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity

Combination index for GTN+PTX

00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000


Cytotoxicity of GTN+PTX

GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity

Combination index for GTN+VBT

00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000


Cytotoxicity of GTN+VBT

GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued


Combination index for GTN+CPP

00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000


Cytotoxicity of GTN+CPP

GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity

Combination index for GTN+5FU

00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000


Cytotoxicity of GTN+5FU

GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)

Figure 5 Combined effects of GTN and 5 chemodrugs on MDA-MB-231 cells The non-constant ratio combination of cytotoxicity of 625125 25 50 and 75 120583M GTN and fixed concentration chemodrugs 125 120583M vinblastine (VBT) (a) 0125 120583M paclitaxel (PTX) (b) 20 120583Mmethotrexate (MTX) (c) 10 120583M 5-fluorouracil (5-FU) (d) and 20 120583M cyclophosphamide (CPP) (e) The cell viability was measured by MTTassay Combination index (CI) plot corresponds to the cell viabilities of each drug vinblastine (VBT) (f) paclitaxel (PTX) (g) methotrexate(MTX) (h) 5-fluorouracil (5-FU) (i) and cyclophosphamide (CPP) (j) which were interpreted from CompuSyn Software analysis

Table 2 GTN and chemodrug concentrations in combination treatments and the combination index (CI) used to determine the synergisticcombination effects calculated using CompuSyn Software

Goniothalamin (GTN) (120583M) CombinationIndex (CI)

625 625 625 625 625Paclitaxel (PTX) (120583M) 0125 00709Vinblastine (VBT) (120583M) 125 0182715-Fluorouracil (5FU) (120583M) 10 080856Cyclophosphamide (CPP)(120583M) 100 063287

Methotrexate (MTX) (120583M) 20 093235


25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)

Figure 6 Combined effects of GTN and 5 chemodrugs on MDA-MB-231 cells The cytotoxicity of 625 120583M GTN and chemodrugs 0125120583M paclitaxel (PTX) 125 120583M vinblastine (VBT) 20 120583M methotrexate (MTX) 10 120583M 5-fluorouracil (5-FU) and 20 120583M cyclophosphamide(CPP) in non-constant ratio combination by fixed chemodrug concentrations and cell viability was measured by MTT assay (a) Caspase-3(b) caspase-9 (c) and caspase-8 (d) activities were measured in GTN and chemodrug combinations were performed by using their specificsubstratesThe significance of statistical values compared to single treatments was marked with plt005 and plt001

checkpoint genes ataxia telangiectasia mutated (ATM) andthe tumor suppressor p53 induce apoptosis in response toDNA damage [32] Activation of ATM by autophosphoryla-tion on Ser1981 occurs in response to exposed DNA double-stranded breaks Several downstream targets have been iden-tifiedThese substrates includemany tumor suppressors suchas p53 which respond to the S-phase checkpoint and BRCA1to facilitate the G

2checkpoint respectively [33] The triple

negative breast cancer MDA-MB-231 cell line has a mutanttumor suppressor p53 but it has a wild type of BRCA1 thatis responsible for DNA double-strand break and G

2cell cycle

arrest [34] GTN has been reported to induce DNA double-strand breaks in oral cancer cells which induced apoptosis[12]

The mode of MDA-MB-231 cell death was apoptosisconfirmed by the morphologies under fluorescence andtransmission electron micrographs (Figures 2(a) and 2(c))The externalization of phosphatidylserine (PS) to the outerlayer of the cell membrane also increased suggesting earlyapoptosis in cell death mode Percentage of cells in earlyapoptosis increased in a dose-dependent manner whichinvolved the cells in the right lower quadrant (annexin V-FITC-positive cells PI-negative cells) (Figure 2(d)) Percent-age of cells with a loss of mitochondrial transmembranepotential increased dose-dependently (Figure 2(e)) indicat-ing the influence of the apoptosis and regulated necroptosispathways The caspases-3 -8 and -9 activities were activatedwhen compared to the control (untreated cells) However


the pan-caspase inhibitor z-VAD-fmk attenuated all threecaspases activities when compared to the GTN-treated cellsindividually (Figure 2(f)) Cumulatively at this point theapoptosis pathways of GTN-induced MDA-MB-231 cellswere completed via both mitochondrial and death receptor(caspase-8) pathways

Furthermore Bcl-2 family proteins and genes play signif-icant roles in apoptotic cell deaths [35] Hence GTN-inducedapoptotic cells were investigated for the expressions of someof the Bcl-2 family proteins and corresponding mRNAs Theproapoptotic Bax BH3-only Noxa PUMA Bim Bad andp112-Bad and antiapoptotic Bcl-2 and Bcl-xL proteins weremeasured quantitatively by Western blotting and densito-metry It exhibited that Bax and BH3-only proteins wereupregulated and antiapoptotic proteins were downregulatedwhen compared to the control This study also demonstratedthe activation of Bad as dephosphorylated-Bad and the highexpression of Bim (Figures 3(a) and 3(b)) which are respon-sible for DNA damage cell cycle arrest and ATM activationvia ERK activation by GTN [11 36] The mitochondrialpathway was also established by the DIABLO release frommitochondrial intermembranous space DIABLO which islocated in the mitochondrial intermembranous space func-tions to inhibit a family of proteins called ldquoinhibitors ofapoptosis protein (IAP)rdquo Therefore DIABLO plays a role inapoptosis induction We illustrated that DIABLO levels inmitochondria were abrogated and in cytosol were enhancedin a concentration-dependent manner (Figures 3(b) and3(c)) However the levels of mRNA expression of someapoptotic genes such as PMAIP1noxa BBC3PUMA badand DIABLO were determined by real-time RT-PCR andcompared with the protein levels by immunoblotting anddensitometer The mRNA expression levels were found tocorrespond to the protein expression levels

Oxidative stress is reported to induce cancer cell deathvia ROSRNS production in signaling via the mitochondrialpathway [21] The roles of oxidative stress in GTN-inducedapoptosis were investigated and reported as the major causesof apoptosis in many cancer cell types namely Jurkat T-cells melanoma cells and Ca9-22 oral cancer cells [8 11 1237] This study focused on which types of ROS that GTNwere induced by DCFH-DA and DHE relative fluorescenceintensity and this was confirmed by the reduction of flu-orescence intensity when the cancer cells were pretreatedwith N-acetylcysteine (NAC) (Figures 4(a) and 4(b)) anantioxidant molecule The reactive oxygen species includinghydrogen peroxide was similarly reported in the previousinvestigation using DCFH-DA as a probe [31] and superoxideanion radicals which was first shown in this study byemploying DHE as an indicator [38] The increase of ROSdemonstrated by both DCF and E fluorescence intensitycontained an increased trend of dose response mannersHowever there are many factors that can influence thelevels of ROS generation in the cells such as the levelsof reduced glutathione NADPH reducing agent (a coen-zyme) and the status of mitochondrial electron transportchain [39] These might affect the levels of ROS produc-tion that are induced by GTN-treatment which caused theresponse to not be determined in a concentration manner

remarkedly of both hydrogen peroxide and superoxide anionradicals

Calcium ion levels also alter apoptosis and other kindsof cell death such as necroptosis [21] Hence the calciumion levels were measured using fluorescence dye Rhod-2AM specific for mitochondrial compartment whereas Fluo-3 AM dye was used specifically for Ca2+ levels in the cytoso-lic compartment Calcium ion levels in the cytoplasmiccompartment were higher while those in the mitochondriawere lower in GTN-treated breast cancer MDA-MB-231 cellswhen compared to the control (untreated cells) (Figure 4(c))Intracellular ROS and calcium ions levels were involved inoxidative and endoplasmic reticulum (ER) stresses-inducedapoptosis [40] ER stress is also considered as a mechanismof cancer cell apoptosis [41] Consequently the expressionsof some ER stress proteins and mRNAs were investigatedsuch as GRP78 (glucose-regulated protein 78) GADD153(growth arrest- and DNA damage-inducible gene 153) andCalreticulin proteins and HSPA5GRP78 and CALR mRNAlevels The ER stress proteins HSPA5GRP78 and GADD153were upregulated (as shown in Figures 4(d) 4(e) and 4(f))except for Calreticulin which did not changeThismight havebeen due to the transient mRNA expression of CALRmRNAwhereas the protein half-life is short and is degraded quickly[42 43] Calreticulin has many biological functions underboth physiological and pathological conditions Calreticulinis located at the cell membrane and functions as an ldquoeat merdquosignal and brings the cancer cells to their microenvironmentsuch as by the extracellular matrix (ECM) in an inside-out or outside-in signaling manner through integrins or thecytoskeletal protein network It also plays a role in angiogene-sismetastasis cell adhesion and tumorigenesis [44]HSPA5GRP78 functions as a chaperone in protein proper foldingin ER [45] Another ER stress-mediated apoptosis proteinis the CEBP homologous protein (CHOP) which is alsoknown as growth arrest- and DNA damage-inducible gene153 (GADD153) which downregulates Bcl-2 and perturbscancer cell oxidationreduction or the redox state [46]

Combination chemotherapy has been widely used inbreast cancer treatments Drug combinations can improveprognosis by overcoming drug resistance in invasive breastcancer patients [47] Currently natural products have becomea potential strategy for drug combinations in cancer thera-pies Phytochemical compounds are less toxic to normal cellsand have a different target when compared to conventionalchemotherapeutic drugs [48] A synergistic effect is anenhanced effectwhen twoormore compounds are combinedwhich improves toxicitywhileminimizing the adverse effectsMostly a nonconstant combination ratio is used to reducethe adverse effects of chemodrugs [49] The cytotoxicity ofcommonly used chemodrugs on human breast cancer MDA-MB-231 cells has been previously reported [50] the sameindividual concentration which was used in this study rangedfrom twenty to fifty percent inhibitory concentrations anddisplayed less toxicity than twenty percent in the normal cells(data not shown) The additive effect of GTN and MTX aslightly synergistic effect for GTN and 5-FU or CPP wasgrouped by drug actions as antimetabolites or alkylatingagents which were less sensitive to triple negative breast


DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+

Figure 7 Illustration representing the mechanism of goniothalamin (GTN)-induced human breast-derived MDA-MB-231 cancer cellapoptosis GTN induced apoptosis by DNA damage from oxidative stress and an increase of ROS production GTN directly inducedmitochondria-mediated stress leading to ROS production mitochondrial intermembranous protein release and activation with sequentialcaspases GTN induced a cellular stress response which led to the upregulation of pro-apoptotic molecules GTN also induced ER-stress andan increase in the levels of the ER-stress proteins leading to apoptosis

MDA-MB-231 cancer cells [51] However mitotic spindletargeted chemodrugs PTX and VBT had a synergistic effectwhen combined with GTN (Figure 5(a) and Table 2) becauselow concentrations of PTX and VBT increased arrest inmitosis [52]The synergistic effect when combined with GTNis a target via mitotic arrest (Figure 1(c)) All combinationsof GTN with chemodrugs enhanced the apoptotic caspases-3and -9 activities whereas those of caspase-8 increased inVBTand 5-FU plus with GTN (Figures 6(b)ndash6(d)) The mecha-nisms of drug combinations with GTN treatments showedsynergistic effects via the mechanisms that are required tobe illustrated while the molecular signaling pathways requirefurther study to clarify the apoptotic andor mitotic arrestsignaling pathways

5 Conclusion

Goniothalamin induced adherent human breast cancerMDA-MB-231 cells apoptosis via the intrinsic and extrin-sic pathways initiated by intracellular oxidative and ERstresses which altered Bcl-2 family proteinsmRNAs levelsand ER chaperone proteinsmRNA expression levels G2Mcell cycle arrest was illustrated in GTN-treated MDA-MB-231 cells Notably levels of p-ATM increased indicating DNAinstability in breast cancer cells after GTN treatment Thenovel findings of this study included the illustration of ERand oxidative stress pathways phosphorylated-ATM DNAdamage or instability and combined five chemotherapeuticdrugs with GTN to demonstrate the relevant synergistic andadditive effects The synergistic combination effect of GTNwas exhibited in cell viability reduction when combined withchemodrugs which also enhanced some caspase activitiesas evidenced by the intrinsic andor extrinsic apoptosispathways The signaling pathway of GTN-induced cell death

is illustrated in Figure 7 GTN has a high potential as a breastcancer treatment agent and as a single natural compound oras one that could be used in combination with conventionalchemotherapeutic drugs to reduce the required dosage ofeffective chemotherapeutic drugs and to cause less severeadverse side effects

Data Availability

The data collected in the present study are properly analyzedand summarized inMethods and Results and all are availablefrom the corresponding author upon reasonable request Allmaterials used in this study are properly included inMethods

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This work was supported by Endowment Research Fund ofFaculty of Medicine Chiang Mai University (0642016) forRatana Banjerdpongchai Graduate Student Supportive FundFaculty ofMedicine ChiangMaiUniversity (year 2014ndash2016)for Patompong Khaw-on National Research Council ofThai-land (NRCT) of the year 2013 for Ratana Banjerdpongchaiand Financial Support in Publishing Research Work fromGraduate School Chiang Mai University

Supplementary Materials

Supplement Figure 1 A Bar graph demonstrating DCFintensity of 50 120583MGTN treatment in MDA-MB-231 cells for1 to 24 hours It exhibited significant alteration at an h our


when compared to without treatment (p lt 005) but therewas no significant change among groups at 1 to 12 h ours ( pgt 005) (Supplementary Materials)

References

[1] R Banjerdpongchai P Khaw-On and W Pompimon ldquoPhyto-chemicals from Goniothalamus griffithii induce human cancercell apoptosisrdquo Asian Pacific Journal of Cancer Prevention vol17 no 7 pp 3281ndash3287 2016

[2] A M Ali M M Mackeen M Hamid et al ldquoCytotoxicity andelectron microscopy of cell death induced by goniothalaminrdquoPlanta Medica vol 63 no 1 pp 81ndash83 1997

[3] S H Inayat-Hussain A B Osman L B Din et al ldquoCaspases-3 and -7 are activated in goniothalamin-induced apoptosis inhuman Jurkat T-cellsrdquo FEBS Letters vol 456 no 3 pp 379ndash3831999

[4] S H Inayat-Hussain B O Annuar L B Din A M Ali andD Ross ldquoLoss of mitochondrial transmembrane potential andcaspase-9 activation during apoptosis induced by the novelstyryl-lactone goniothalamin in HL-60 leukemia cellsrdquoToxicol-ogy in Vitro vol 17 no 4 pp 433ndash439 2003

[5] W Chen CWu Y Lan F Chang C Teng and Y Wu ldquoGonio-thalamin induces cell cycle-specific apoptosis by modulatingthe redox status in MDA-MB-231 cellsrdquo European Journal ofPharmacology vol 522 no 1-3 pp 20ndash29 2005

[6] K M Chan N F Rajab M H A Ishak et al ldquoGoniothalamininduces apoptosis in vascular smooth muscle cellsrdquo Chemico-Biological Interactions vol 159 no 2 pp 129ndash140 2006

[7] K M Chan N F Rajab D Siegel L B Din D Ross andS H Inayat-Hussain ldquoGoniothalamin induces coronary arterysmooth muscle cells apoptosis the p53-dependent caspase-2activation pathwayrdquo Toxicological Sciences vol 116 no 2 pp533ndash548 2010

[8] S H Inayat-Hussain K M Chan N F Rajab et al ldquoGoniot-halamin-induced oxidative stress DNA damage and apoptosisvia caspase-2 independent and Bcl-2 independent pathways inJurkat T-cellsrdquo Toxicology Letters vol 193 no 1 pp 108ndash1142010

[9] D J McConkey ldquoTherapy-induced apoptosis in primarytumorsrdquo Advances in Experimental Medicine and Biology vol608 pp 31ndash51 2007

[10] M Redza-Dutordoir and D A Averill-Bates ldquoActivation ofapoptosis signalling pathways by reactive oxygen speciesrdquoBiochimica et Biophysica Acta (BBA) - Molecular Cell Researchvol 1863 no 12 pp 2977ndash2992 2016

[11] S Tangchirakhaphan S Innajak S Nilwarangkoon N Tanjap-atkul W Mahabusrakum and R Watanapokasin ldquoMechanismof apoptosis induction associatedwith ERK12 upregulation viagoniothalamin inmelanoma cellsrdquo Experimental anderapeu-tic Medicine vol 15 no 3 pp 3052ndash3058 2018

[12] C Yen C Chiu R Haung et al ldquoAntiproliferative effects ofgoniothalamin on Ca9-22 oral cancer cells through apoptosisDNA damage and ROS inductionrdquoMutation Research - GeneticToxicology and Environmental Mutagenesis vol 747 no 2 pp253ndash258 2012

[13] AMarechal andL Zou ldquoDNAdamage sensing by theATMandATR kinasesrdquo Cold Spring Harbor Perspectives in Biology vol 5no 9 2013

[14] J Yang Z-P Xu Y Huang H E Hamrick P J Duerksen-Hughes and Y-N Yu ldquoATM and ATR sensing DNA damagerdquo

World Journal of Gastroenterology vol 10 no 2 pp 155ndash1602004

[15] Z Yu H Luo W Fu and M P Mattson ldquoThe endoplasmicreticulum stress-responsive protein GRP78 protects neuronsagainst excitotoxicity and apoptosis Suppression of oxidativestress and stabilization of calcium homeostasisrdquo ExperimentalNeurology vol 155 no 2 pp 302ndash314 1999

[16] J Boelens S Lust F OffnerM E Bracke and BW VanhoeckeldquoThe endoplasmic reticulum a target for new anticancer drugsrdquoIn Vivo vol 21 no 2 pp 215ndash226 2007

[17] T C Chou ldquoTheoretical basis experimental design and com-puterized simulation of synergism and antagonism in drugcombination studiesrdquo Pharmacological Reviews vol 58 no 3pp 621ndash681 2006

[18] G Acs N N Esposito Z Rakosy C Laronga and P J ZhangldquoInvasive ductal carcinomas of the breast showing partialreversed cell polarity are associated with lymphatic tumorspread and may represent part of a spectrum of invasivemicropapillary carcinomardquo e American Journal of SurgicalPathology vol 34 no 11 pp 1637ndash1646 2010

[19] D L Holliday and V Speirs ldquoChoosing the right cell line forbreast cancer researchrdquo Breast Cancer Research vol 13 no 4article 215 2011

[20] P Petsophonsakul W Pompimon and R BanjerdpongchaildquoApoptosis induction in human leukemic promyelocytic HL-60andmonocytic U937 cell lines by GoniothalaminrdquoAsian PacificJournal of Cancer Prevention vol 14 no 5 pp 2885ndash2889 2013

[21] R Banjerdpongchai P Kongtawelert O Khantamat et alldquoMitochondrial and endoplasmic reticulum stress pathwayscooperate in zearalenone-induced apoptosis of humanleukemic cellsrdquo Journal of Hematology amp Oncology vol 3article no 50 2010

[22] J C Stockert A Blazquez-Castro M Canete R W Horobinand A Villanueva ldquoMTT assay for cell viability intracellularlocalization of the formazan product is in lipid dropletsrdquo ActaHistochemica vol 114 no 8 pp 785ndash796 2012

[23] R Banjerdpongchai Y Chanwikruy V Rattanapanone andB Sripanidkulchai ldquoInduction of apoptosis in the humanleukemic u937 cell line by kaempferia parviflora wallexbakerextract and effects of paclitaxel and camptothecinrdquoAsian PacificJournal of Cancer Prevention vol 10 no 6 pp 1137ndash1140 2009

[24] T Chou and P Talalay ldquoQuantitative analysis of dose-effectrelationships the combined effects of multiple drugs or enzymeinhibitorsrdquo Advances in Enzyme Regulation vol 22 pp 27ndash551984

[25] D C Joshi and J C Bakowska ldquoDetermination of mitochon-drial membrane potential and reactive oxygen species in live ratcortical neuronsrdquo Journal of Visualized Experiments no 51 2011

[26] W Mao X Chen T Yang et al ldquoA rapid fluorescent screen-ing method for cellular sensitivity to anti-cancer compoundrdquoCytotechnology vol 64 no 4 pp 451ndash457 2012

[27] P Khaw-on andR Banjerdpongchai ldquoInduction of intrinsic andextrinsic apoptosis pathways in the human leukemic MOLT-4 cell line by terpinen-4-olrdquo Asian Pacific Journal of CancerPrevention vol 13 no 7 pp 3073ndash3076 2012

[28] S-J Li X-Y Liang H-J Li et al ldquoLow-dose irradiationpromotes proliferation of the human breast cancer MDA-MB-231 cells through accumulation of mutant p53rdquo InternationalJournal of Oncology vol 50 no 1 pp 290ndash296 2017

[29] S Banin L Moyal S-Y Shieh et al ldquoEnhanced phosphoryla-tion of p53 by ATM in response to DNA damagerdquo Science vol281 no 5383 pp 1674ndash1677 1998


[30] S Chen Z Wang Y Huang et al ldquoGinseng and anticancerdrug combination to improve cancer chemotherapy a crit-ical reviewrdquo Evidence-Based Complementary and AlternativeMedicine vol 2014 Article ID 168940 13 pages 2014

[31] M A Seyed I Jantan and S N Bukhari ldquoEmerging anticancerpotentials of goniothalamin and its molecular mechanismsrdquoBioMed Research International vol 2014 Article ID 536508 10pages 2014

[32] S Negrini V G Gorgoulis and T D Halazonetis ldquoGenomicinstabilitymdashan evolving hallmark of cancerrdquo Nature ReviewsMolecular Cell Biology vol 11 no 3 pp 220ndash228 2010

[33] X Tang Z-G Hui X-L Cui R Garg M B Kastan andB Xu ldquoA novel ATM-dependent pathway regulates proteinphosphatase 1 in response to DNA damagerdquo Molecular andCellular Biology vol 28 no 8 pp 2559ndash2566 2008

[34] Y GuMHelenius K Vaananen et al ldquoBRCA1-deficient breastcancer cell lines are resistant to MEK inhibitors and showdistinct sensitivities to 6-thioguaninerdquo Scientific Reports vol 6Article ID 28217 2016

[35] R Banjerdpongchai and P Khaw-On ldquoTerpinen-4-ol inducesautophagic and apoptotic cell death in human leukemic HL-60cellsrdquo Asian Pacific Journal of Cancer Prevention vol 14 no 12pp 7537ndash7542 2013

[36] D Tang D Wu A Hirao et al ldquoERK activation mediates cellcycle arrest and apoptosis after DNA damage independently ofp53rdquo e Journal of Biological Chemistry vol 277 no 15 pp12710ndash12717 2002

[37] C Chiu P Liu K Huang et al ldquoGoniothalamin inhibits growthof human lung cancer cells through DNA damage apoptosisand reduced migration abilityrdquo Journal of Agricultural and FoodChemistry vol 59 no 8 pp 4288ndash4293 2011

[38] E Owusu-Ansah A Yavari and U Banerjee ldquoA protocol forin vivo detection of reactive oxygen speciesrdquo Protocol Exchange2008

[39] D L Nelson and M M Cox Lehninger Principles of Biochem-istry Freeman and Company NY USA 2008

[40] G Hajnoczky E Davies and M Madesh ldquoCalcium signalingand apoptosisrdquo Biochemical and Biophysical Research Commu-nications vol 304 no 3 pp 445ndash454 2003

[41] R Banjerdpongchai P Punyati A Nakrob W Pompimonand P Kongtawelert ldquo4rsquo-hydroxycinnamaldehyde fromAlpiniagalanga (Linn) induces human leukemic cell apoptosis viamitochondrial and endoplasmic reticulum stress pathwaysrdquoAsian Pacific Journal of Cancer Prevention vol 12 no 3 pp 593ndash598 2011

[42] C Vogel and E M Marcotte ldquoInsights into the regulation ofprotein abundance from proteomic and transcriptomic analy-sesrdquo Nature Reviews Genetics vol 13 no 4 pp 227ndash232 2012

[43] V E Goitea and M E Hallak ldquoCalreticulin and arginylatedcalreticulin have different susceptibilities to proteasomal degra-dationrdquoe Journal of Biological Chemistry vol 290 no 26 pp16403ndash16414 2015

[44] Yi-Chien Lu Wen-Chin Weng and Hsinyu Lee ldquoFunctionalRoles of Calreticulin in Cancer Biologyrdquo BioMed ResearchInternational vol 2015 Article ID 526524 9 pages 2015

[45] J Behnke M J Feige and L M Hendershot ldquoBiP and ItsNucleotide Exchange Factors Grp170 and Sil1 Mechanisms ofAction and Biological Functionsrdquo Journal of Molecular Biologyvol 427 no 7 pp 1589ndash1608 2015

[46] S Oyadomari and M Mori ldquoRoles of CHOPGADD153 inendoplasmic reticulum stressrdquoCell DeathampDifferentiation vol11 no 4 pp 381ndash389 2004

[47] D A Yardley ldquoDrug Resistance and the Role of CombinationChemotherapy in Improving Patient Outcomesrdquo InternationalJournal of Breast Cancer vol 2013 Article ID 137414 15 pages2013

[48] S Hemalswarya and M Doble ldquoPotential synergism of naturalproducts in the treatment of cancerrdquoPhytotherapy Research vol20 no 4 pp 239ndash249 2006

[49] T Chou ldquoDrug combination studies and their synergy quantifi-cation using the chou-talalaymethodrdquoCancer Research vol 70no 2 pp 440ndash446 2010

[50] OMoneo F Garcia CMGalmarini NGuillenMAviles andN Santamaria Combinationerapy with an Antitumor Antibi-otic PharmaMar Sa European Patent Office Ed PharmaMarSa 2014

[51] S J Isakoff ldquoTriple-negative breast cancer Role of specificchemotherapy agentsrdquo Cancer Journal vol 16 no 1 pp 53ndash612010

[52] A L Blajeski V A Phan T J Kottke and S H KaufmannldquoG1and G

2cell-cycle arrest following microtubule depolymer-

ization in human breast cancer cellsrdquo e Journal of ClinicalInvestigation vol 110 no 1 pp 91ndash99 2002

Stem Cells International

Hindawiwwwhindawicom Volume 2018


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Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

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Volume 2018

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Computational and Mathematical Methods in Medicine


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Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

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in many types of cancer cells [11 12] ROS induces cancercells to undergo apoptosis via interrupting the mitochondriaoxidative phosphorylation lipid peroxidation and a double-strand DNA break [8] The DNA break can induce cell cyclearrest by ATMATR activation which is induced by p53[13] Furthermore the p53-independent pathway has alsobeen shown to be influential in the activation of DNA dam-age sensing molecules and proapoptosis proteins PUMANoxa for apoptosis execution [14] ER stress related proteinsand heat shock 70kD protein 5GRP78HSPA5 equilibratecytosolic calcium which is released from cellular organelle-induced apoptosis [15] Ca2+ is released to the cytosol whilechaperone proteins and ER stress related proteins play crucialroles in programmed cell death induction in cancer cells [16]

Chemotherapeutic drugs have been developed and usedfor cancer treatments but are still associated with poor out-comes perhaps due to a lack of compliance and their com-plicated side effects The use of combination treatmentswith rationale on different mechanisms also synergizes theeffects of single targets and kills the cancer cells moreeffectively Providing selective synergism against multipletargets drug combinations are widely used and have becomethe leading choice for the treatment of cancer [17] Targeteddrug therapies are aimed directly at the cancerous cells orat the molecules that regulate or control the proliferation ofcancer cells These drugs are used in combination with othertherapies for the advantages of fewer or less severe side effects[18] Triple negative breast cancer MDA-MB-231 cells whichare characterized as negative for estrogen receptor (ER) pro-gesterone (PR) and epidermal growth factor receptor (EGFRor HER2) become indicated of invasive breast cancer asa consequence of poor prognosis and have a potential tobecome drug-resistant [19] In this study the human invasivebreast cancer MDA-MB-231 cell line was used as a modelfor an investigation whether GTN induced apoptosis andits cytotoxic effects were considered when the treatment iscombined with conventional chemodrugs in addition to itsrelated mechanism(s)

2 Materials and Methods

21 Chemicals The leaves and twigs of Goniothalamusgriffithii were collected in January 2011 from Chiang MaiProvince Thailand and identified by the Forest HerbariumDepartment of National Park Wildlife and Plant Conser-vation Ministry of Natural Resources and EnvironmentBangkok Thailand where a voucher specimen (BKF16447)has been deposited GTN was extracted purified and iden-tified by Professor Wilart Pompimon Lampang RajabpatUniversity Lampang Province Thailand The phytochemicalcompound goniothalamin was isolated from G griffithii ashas been previously reported [20]

GTN stock solution was prepared in DMSO at 20mMand GTN was diluted in Dulbeccorsquos Modified Eagle Medium(DMEM Gibco Carlsbad CA USA) Annexin V Fluos-staining kit and Protease Inhibitor Cocktail tablets wereobtained from Roche Diagnostics (Mannheim Germany)Ficoll-Hypaque reagent (HISTOPAQUE-1077) dihy-droethidium (DHE) and 2101584071015840ndashdichlorodihydrofluorescein

diacetate (DCFH-DA) 331015840-dihexyloxacarbocyanine iodide(DiOC

6) and 3-(45 dimethylthiazol-2yl)-25 diphenyl-

tetrazolium bromide (MTT) were obtained from Sigma-Aldrich MO USA Rhod-2 acetoxymethyl (AM) ester andFluo-3 AM were obtained from Thermo Fisher ScientificInc USA Caspases-3 -8 and -9 determination kits wereobtained from Invitrogen Thermo Fisher Scientific IncUSA Primary and secondary antibodies and horse-radishperoxidase-conjugated antibodies were obtained fromAbcam Cambridge UK RNA isolation kit was obtainedfrom GE Healthcare UK Total RNA was reversed tocomplementary DNA (cDNA) by using a cDNA SynthesisKit and quantitative real-time PCR assays were performedwith Reagents Kit (Bioline Reagents Ltd USA)

22 Cell Culture Triple negative breast cancer MDA-MB-231 cell line was cultured in DMEM supplemented with 10fetal bovine serum penicillin and streptomycin at 37∘C ina humidified atmosphere of 5 CO

2at 37∘C After the cells

grew to 80 confluence the cell lines were trypsinized by005 trypsin Peripheral blood mononuclear cells (PBMCs)were isolated from heparinized blood obtained from adultvolunteers as blood donors at Blood Bank Unit Maha RajNakorn Chiang Mai Hospital affiliated with the Faculty ofMedicine Chiang Mai University Each blood donor wasinformed of the objectives and signed the written consentform in order to comply as individual volunteers accordingto the Institutional Review Board Research Ethics Com-mittee Faculty of Medicine Chiang Mai University PBMCswere separated from the whole blood of healthy volunteersby using Ficoll-Hypaque reagent as has been previouslydescribed [21]

23 Cell Viability Assay The MTT (3-(45 dimethylthiazol-2yl)-25 diphenyltetrazolium bromide) assay [22] was per-formed by seeding MDA-MB-231 cells in 96-well cultureplates GTN treatment conditions were prepared in variousconcentrations and incubated for 24 hours The cell viableactivity in each well will be determined by MTT assay andcompared to the untreated cells [23]

24 Drug Combination Assay A process for analyzing theefficiency of combined drugs and the net effect was employedby the method of Chou TC and Talalay P [24] Briefly MDA-MB-231 cells were cotreated with various concentrations ofGTN and conventional therapeutic drugs with a noncon-stant ratio combination The chemotherapeutic drugs thatwere used in this study include vinblastine paclitaxel 5-fluorouracil methotrexate and cyclophosphamide After 24hours of treatment the cells were evaluated for viabilityusing MTT assay and a function of the effect level (Fa)valueswas calculatedTheCompuSyn Softwarewas employedfor determination of combination index (CI) values andvalidated as a combination effect

25 Fluorescence Microscopy MDA-MB-231 cells were seed-ed on culture slides and treated with GTN After 24 hours theslides were fixed in ice-cold absolute alcohol and stained with10120583gml propidium iodide (PI) The slides were investigated












induced protein1














20and IC

50concentrations







3 Results















C

ell v

iabi

lity


0

20

40

60

80

100

120

MDA-MB-231 PBMC


(a)

Control

PI

Cel

l cou

nt

PI

Ant

i-pAT

M-A

lexa

488


S

G1 G2M

S

G1 G2M

S

G1 G2M


Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

）20 ）50

(b)

Gat

ed p

opul

atio

n (

)Cell cycle phase

G1 SG2M

0

20

40

60

80

0 ）20 ）50


(c)

GTN Concentration

pATM

pos

itive

(ga

ted)

Control

00

25

50

75

100

IC 20 IC 50

lowast

lowastlowast

(d)

ATM

Goniothalamin

Rela

tive Q

uant

ifica

tion

(fold

of c

ontr

ol)

control00

05

10

15

20

IC20 IC50

lowastlowast

lowastlowast

(e)


20and IC

50((b) upper



0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)


(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4


0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20




lowastlowast

lowast


lowastlowast

lowastlowast

(f)





GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






























induced protein1














20and IC

50concentrations







3 Results















C

ell v

iabi

lity


0

20

40

60

80

100

120

MDA-MB-231 PBMC


(a)

Control

PI

Cel

l cou

nt

PI

Ant

i-pAT

M-A

lexa

488


S

G1 G2M

S

G1 G2M

S

G1 G2M


Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

）20 ）50

(b)

Gat

ed p

opul

atio

n (

)Cell cycle phase

G1 SG2M

0

20

40

60

80

0 ）20 ）50


(c)

GTN Concentration

pATM

pos

itive

(ga

ted)

Control

00

25

50

75

100

IC 20 IC 50

lowast

lowastlowast

(d)

ATM

Goniothalamin

Rela

tive Q

uant

ifica

tion

(fold

of c

ontr

ol)

control00

05

10

15

20

IC20 IC50

lowastlowast

lowastlowast

(e)


20and IC

50((b) upper



0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)


(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4


0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20




lowastlowast

lowast


lowastlowast

lowastlowast

(f)





GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















3 Results















C

ell v

iabi

lity


0

20

40

60

80

100

120

MDA-MB-231 PBMC


(a)

Control

PI

Cel

l cou

nt

PI

Ant

i-pAT

M-A

lexa

488


S

G1 G2M

S

G1 G2M

S

G1 G2M


Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

）20 ）50

(b)

Gat

ed p

opul

atio

n (

)Cell cycle phase

G1 SG2M

0

20

40

60

80

0 ）20 ）50


(c)

GTN Concentration

pATM

pos

itive

(ga

ted)

Control

00

25

50

75

100

IC 20 IC 50

lowast

lowastlowast

(d)

ATM

Goniothalamin

Rela

tive Q

uant

ifica

tion

(fold

of c

ontr

ol)

control00

05

10

15

20

IC20 IC50

lowastlowast

lowastlowast

(e)


20and IC

50((b) upper



0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)


(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4


0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20




lowastlowast

lowast


lowastlowast

lowastlowast

(f)





GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















C

ell v

iabi

lity


0

20

40

60

80

100

120

MDA-MB-231 PBMC


(a)

Control

PI

Cel

l cou

nt

PI

Ant

i-pAT

M-A

lexa

488


S

G1 G2M

S

G1 G2M

S

G1 G2M


Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0

Cou

nt0

5015

025

035

045

0PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000PI (RED-HLin)

1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

PI (RED-HLin)1000 3000 5000 7000 90000

100

101

102

103

104

100

101

102

103

104

100

101

102

103

104

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

Ant

i-pAT

M A

lexa

488

(GRN

-HLo

g)

）20 ）50

(b)

Gat

ed p

opul

atio

n (

)Cell cycle phase

G1 SG2M

0

20

40

60

80

0 ）20 ）50


(c)

GTN Concentration

pATM

pos

itive

(ga

ted)

Control

00

25

50

75

100

IC 20 IC 50

lowast

lowastlowast

(d)

ATM

Goniothalamin

Rela

tive Q

uant

ifica

tion

(fold

of c

ontr

ol)

control00

05

10

15

20

IC20 IC50

lowastlowast

lowastlowast

(e)


20and IC

50((b) upper



0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)


(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4


0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20




lowastlowast

lowast


lowastlowast

lowastlowast

(f)





GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















0

GTN Concentration

I20 I50

(a)

GTN Concentration

Perc

ent o

f apo

ptos

is p

ositi

ve ce

lls

00

20

40

60

80

I20 I50

lowastlowast

lowastlowast

(b)


(c)

GTN Concentration

AnnexinV-FITC

Prop

idiu

m Io

dide

0 I20 I50

102 103 104 105102 103 104 105102 103 104 105

102

103

104

105

102

103

104

105

102

103

104

105

Q1 Q2

Q3 Q4

Q1 Q2Q1 Q2

Q3 Q4Q3 Q4


0

Perc

ent o

f cel

ls w

ith lo

ss of

mito

chon

dria

l mem

bran

e pot

entia

l

0

20

40

60

80

100

IC20 IC50

lowast

lowastlowast

(e)

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

GTNGTN+z-VAD-fmk

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

00

05

10

15

20

00

05

10

15

20

00

05

10

15

20




lowastlowast

lowast


lowastlowast

lowastlowast

(f)





GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















GTN concentration

Noxa

PUMA

Bax

Bim

Bad

Bcl-2

Bcl-xL

-Actin

p112-Bad

0 I20 I50

(a)

Rela

tive l

evel

of a

popt

osis-

rela

ted

prot

eins

(Fol

d of

cont

rol)

0

1

2

3

5

10

15


I50

lowast

lowast

lowast

lowast

lowastlowast



lowastlowast

lowastlowast


lowastlowast

lowastlowast




(b)

GTN concentration

ndashActinCOX-4

DIABLO

0 I20 I500 I20 I50


(c)

Relat

ive g

ene q

uant

ifica

tion

(fold

of c

ontro

l)


1

2

3

4

5

6


I50

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)



00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















00

05

10

15

20

GTNGTN+NAC

DCF

fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


lowast


(a)

GTNGTN+NAC

00

05

10

15

DH

E flu

ores

cent

inte

nsity

mea

n(fo

ld o

f con

trol

)


lowast


lowastlowast

(b)

00

05

10

15

Fluo

resc

ent i

nten

sity

mea

n(fo

ld o

f con

trol

)


Cytosolic Ca2+

Mitochondria Ca2+

lowast lowast


(c)

GTN Concentration

Calreticulin

GADD153

GRP78

ndashActin

0 I20 I50

(d)

0

1

2

34

6

8

10

GTN concentration

Rela

tive l

evel

of E

R st

ress

pro

tein

s(F

old

of co

ntro

l)

0I20

I50



(e)

HSPA5GRP78 CALR00

05

10

15

20

25

Rela

tive g

ene q

uant

ifica

tion

(Fol

d of

cont

rol)


I50

lowastlowastlowast

(f)


20and IC





4 Discussion




00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+MTX20MTX20

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTN

GTN+PTX0125

PTX0125

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+VBT125VBT125

0

20

40

60

80

100

Cel

l via

bilit

y

20 40 60 800

Goniothalamin (M)

(h)

(g)

(f)

(c)

(b)

(a)

Figure 5 Continued



00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















00

05

10

15

20C

ombi

natio

n In

dex

(CI)

02 04 06 08 1000



GTNGTN+CPP100CPP100

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

C

ell v

iabi

lity


00

05

10

15

20

Com

bina

tion

Inde

x (C

I)

02 04 06 08 1000



GTNGTN+5FU105FU10

20 40 60 800

Goniothalamin (M)

0

20

40

60

80

100

Cel

l via

bilit

y

(j)

(i)

(e)

(d)







25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















25

0

50

75

100

125

CONGTNChemoCombined

C

ell v

iabi

lity

VBTPTX

5FUCPP

MTX

(a)

1

0

2

3

4

CONGTNChemoCombined

Cas

pase

-3 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(b)

00

05

10

15

20

CONGTNChemoCombined

Cas

pase

-9 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(c)

05

00

10

15

20

CONGTNChemoCombined

Cas

pase

-8 a

ctiv

ity(F

old

of C

ontr

ol)

VBTPTX

5FUCPP

MTX

(d)





2cell cycle











DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























DNA damage

Caspase-3Caspase-8

ROS

Bcl-2

Bcl-xL

Caspase-9IAP

Bax

DIABLO

Bax

NoxaBad

PumaCytochrome c

GTN

GRP78

APOPTOSIS

GADD153

Calreticulin

Cell Cycle Arrest

pATM

Bim GTN

GTN

GTN

ROS

ROS

ROS

Bax ΔΨm

2+



5 Conclusion



Data Availability




Acknowledgments






References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















References





























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















apoptosis induction via atm phosphorylation, cell cycle...

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