research article emodin and aloe-emodin suppress breast...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 376123 12 pageshttpdxdoiorg1011552013376123

Research ArticleEmodin and Aloe-Emodin Suppress Breast Cancer CellProliferation through ER120572 Inhibition

Pao-Hsuan Huang1 Chih-Yang Huang23 Mei-Chih Chen14 Yueh-Tsung Lee15

Chia-Herng Yue16 Hsin-Yi Wang17 and Ho Lin1489

1 Department of Life Sciences National Chung Hsing University Taichung 40227 Taiwan2Department of Health and Nutrition Biotechnology Asia University Taichung 41354 Taiwan3Graduate Institute of Basic Medical Science China Medical University Taichung 40402 Taiwan4Department of Urology University of Texas Southwestern Medical Center Dallas TX 75390 USA5Department of Surgery Chang Bing Show Chwan Memorial Hospital Changhua 50544 Taiwan6Department of Surgery Tungsrsquo Taichung MetroHarbor Hospital Taichung 43304 Taiwan7Department of Nuclear Medicine Taichung Veterans General Hospital Taichung 40705 Taiwan8Department of Agricultural Biotechnology Center National Chung Hsing University Taichung 40227 Taiwan9Graduate Institute of Rehabilitation Science China Medical University Taichung 40402 Taiwan

Correspondence should be addressed to Ho Lin hlindragonnchuedutw

Received 25 April 2013 Accepted 6 June 2013

Academic Editor Gautam Sethi

Copyright copy 2013 Pao-Hsuan Huang 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

The anthraquinones emodin and aloe-emodin are abundant in rhubarb Several lines of evidence indicate that emodin and aloe-emodin have estrogenic activity as phytoestrogens However their effects on estrogen receptor 120572 (ER120572) activation and breastcancer cell growth remain controversial The goal of this study is to investigate the effects and molecular mechanisms of emodinand aloe-emodin on breast cancer cell proliferation Our results indicate that both emodin and aloe-emodin are capable ofinhibiting breast cancer cell proliferation by downregulating ER120572 protein levels thereby suppressing ER120572 transcriptional activationFurthermore aloe-emodin treatment led to the dissociation of heat shock protein 90 (HSP90) and ER120572 and increased ER120572ubiquitination Although emodin had similar effects to aloe-emodin it was not capable of promoting HSP90ER120572 dissociation andER120572 ubiquitination Protein fractionation results suggest that aloe-emodin tended to induce cytosolic ER120572 degradation Althoughemodin might induce cytosolic ER120572 degradation it primarily affected nuclear ER120572 distribution similar to the action of estrogenwhen protein degradation was blocked In conclusion our data demonstrate that emodin and aloe-emodin specifically suppressbreast cancer cell proliferation by targeting ER120572 protein stability through distinct mechanisms These findings suggest a possibleapplication of anthraquinones in preventing or treating breast cancer in the future

1 Introduction

Many phytochemicals derived from plants includinganthraquinone have been reported to have anticancerpotential The anthraquinone derivatives emodin (138-trihydroxy-6-methylanthraquinone) and aloe-emodin (18-dihydroxy-3-hydroxyl-methylanthraquinone) are the mainbioactive components of rhubarb (Rheum palmatum) whichhas been used in traditional Chinese medicine [1] Aloe-emodin is also abundant in the leaves of the common plant

Aloe vera [2] Emodin has been widely investigated for itsantibacterial [3] anti-inflammatory [4] and antiproliferativeeffects in several types of cancer [1] Notably emodin maydownregulate androgen receptor (AR) and lead to theinhibition of prostate cancer cell growth [5] suggestingthat anthraquinone derivatives might modulate steroidreceptor activity Several lines of evidence indicate thatemodin and aloe-emodin have estrogenic activity andmodulate breast cancer cell proliferation as phytoestrogencompounds [6 7] However the pharmacological effects

2 Evidence-Based Complementary and Alternative Medicine

and molecular mechanisms of emodin and aloe-emodin inestrogen receptor 120572 (ER120572) modulation and breast cancer cellgrowth remain elusive

Breast cancer is a commonmalignancywith high lethalityin women Because ER120572 activation plays an important role inthe initiation development and progression of breast cancerestrogen replacement therapy is the most common strategyto suppress breast cancer progression [8] By mimicking thestructure of estrogen synthetic estrogen-like compounds areused to compete for the binding of endogenous estrogenwith ER120572 and therefore inhibit ER120572-dependent growth ofbreast cancer cells [9 10] However synthetic estrogens haveside effects that increase the risk of cancer development dueto unselective estrogenic action [11] Although the potencyof natural phytoestrogens is generally lower than that ofsynthetic estrogens in terms of estrogenic action naturalphytoestrogens are relatively safer with fewer side effects[12] Therefore studies investigating the effects and molec-ular mechanisms of natural herbal medicines that containphytoestrogens as potential treatments to breast cancer are ofinterest

We found that the inhibition of proliferation by emodinand aloe-emodin was ER120572-dependent in breast cancer celllines Importantly aloe-emodin treatment promotes ER120572protein degradation by repressing the association of ER120572 andheat shock protein 90 (HSP90) Moreover the dissociatedER120572 is ubiquitinated and targeted for proteasome-dependentdegradation in the cytosol The findings for aloe-emodinare distinct from those for emodin Based on the aboveobservations these two anthraquinones could potentially beused as specific phytoestrogens to treat breast cancer

2 Materials and Methods

21 Cell Lines and Cell Culture The human breast cancercell lines MCF-7 and MDA-MB-453 were obtained from theBioresource Collection and Research Center (BCRC) FoodIndustry Research andDevelopment Institute TaiwanMCF-7 cells were grown in minimum essential medium (GibcoCarlsbad CA USA) containing 10 fetal bovine serum(Gibco) 15 gL NaHCO

3 01mM nonessential amino acids

(Gibco) 1mM sodium pyruvate (Gibco) and 1 penicillin-streptomycin (Sigma St Louis MO USA) MDA-MB-453cells weremaintained in Dulbeccorsquos modified Eaglersquos medium(Gibco) supplemented with 10 fetal bovine serum 15 gLNaHCO

3 and 1 penicillin-streptomycin All cells were

incubated at 37∘C in a humidified atmosphere with 5 CO2

22 Cell Viability Assay Cells were incubated for 24 hoursafter attachment Cell numbers were calculated by directcounting of cells excluding cells that stained positive for 02trypan blue stain (Sigma St Louis MO USA) [13] Cellswere treated with different concentrations of aloe-emodinor emodin (ChromaDex Irvine CA USA) for the indicatednumber of days and then the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT Sigma St Louis MOUSA) assay was used to quantify cell proliferation The MTTstock solution (5mgmL) was diluted to 05mgmL with

complete culturemedium and 01mLwas added to eachwellThe yellow MTT was converted to blue formazan by livingcells a reaction that is dependent on mitochondrial enzymeactivity After using DMSO to dissolve the blue formazan theabsorbance of converted MTT could be measured at 570 nm120582 [14]

23 Cell Fractionation and Western Blot Analysis Cells werecollected using a rubber scraper and homogenized withNa3VO4diluted in PBS (1 100) After centrifugation cells

were resuspended with lysis buffer as previously described[15ndash19] and protease inhibitor cocktail (Roche Applied Sci-ence Mannheim Germany) was added followed by incuba-tion on ice for 45 minutes The cell lysate was centrifugedand the supernatant was collected as total protein extractThe protein extract was mixed with sample buffer and boiledfor 10 minutes Then western blotting was performed aspreviously described [19 20] Briefly SDS-polyacrylamidegel electrophoresis (SDS-PAGE)was performed and proteinswere transferred from the SDS-PAGE gel onto a polyvinyli-dene fluoride (PVDF) membrane Primary antibodies wereincubated with the membrane overnight and horseradishperoxidase- (HRP-) conjugated secondary antibodies (Jack-son ImmunoResearch Laboratory West Grove PA USA)were applied The ECL (western lighting chemiluminescencereagent plus PerkinElmer Life Sciences Shelton CT USA)reaction was performed and the membranes were exposedto X-ray films to visualize protein staining (Fujifilm TokyoJapan) Antibodies directed against the following proteinswere used in this study poly(ADP-ribose) polymerase (PARP06-557 Upstate Biotechnology Lake Placid NY USA) 120572-tubulin (05-829 Upstate Biotechnology) ER120572 (sc-543 andsc-8005 Santa Cruz Biotechnology Santa Cruz CA USA)cyclin D1 (sc-20044 Santa Cruz Biotechnology) HSP 90120572120573(sc-59577 Santa Cruz Biotechnology) ubiquitin (sc-8017Santa Cruz Biotechnology) and 120573-actin (MAB1501 Milli-pore Temecula CA USA) The quantification software usedwas MCID Image Analysis Evaluation

24 Immunoprecipitation Cell lysate was extracted in lysisbuffer and immunoprecipitation was performed as previ-ously described [19] Briefly the beadsantibody precipitatedcomplex was prepared by mixing Protein G Mag SepharoseXtra beads (GEHealthcareWaukeshaWI USA) and specificantibody at 10 120583g 1120583g for 2 h at room temperature Celllysates were mixed with beadsantibody for 12 h at 4∘C andprotein was isolated by precipitation under magnetic attrac-tion with three rounds of PBST washes The precipitatedproteins were analyzed by western blotting after denaturationfollowing dilution in sample buffer and boiling for 10minutes

25 Quantitative Real-Time PCR Total RNA was extractedfrom cells using a Miniprep Purification Kit (GenemarkTaipei Taiwan) and reverse transcription-PCR was per-formed using a High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems Foster City CA USA) followingthe standard procedures recommended by the manufacturerFor reverse transcription 2120583g of total RNA was used as the

Evidence-Based Complementary and Alternative Medicine 3

first-strand cDNA template for the subsequent amplificationprocedure The following primers were used to amplifythe cDNAs ER120572 (51015840-TGGAGATCTTCGACATGCTG-31015840 and51015840-TCCAGAGACTTCAGGGTGCT-31015840) [21] and 120573-actin (51015840-TTGCCGACAGGATGCAGAA-31015840 and 51015840-GCCGATCCA-CACGGAGTACT-31015840) cDNA and primers weremixedwithinFastStart Universal SYBR Green Master (Roche AppliedScience) and measured using a real-time PCR instrument(Applied Biosystems) Data presented by Ct values wereanalyzed and adjusted relative to levels of the 120573-actin house-keeping gene

26 Transfection and Reporter Assays Cells were plated for atleast 24 h and had reached 80 confluency prior to transfec-tion Expression plasmidwas premixedwithin Lipofectamine2000 Reagent (Invitrogen Carlsbad CA USA) according tothe manufacturerrsquos instructions The liposomeplasmid com-plex was transfected into MCF-7 cells incubated with Opti-MEM (Gibco) for 6 h and then culture medium was addedfor exogenous protein expression The pCMV5-ER120572 expres-sion plasmid was kindly provided by Professor Chih-YangHuang Graduate Institute of Basic Medical Science ChinaMedical University Taichung Taiwan A reporter assay wasperformed to detected ER120572 transcriptional activity after cellswere transfectedwith 3timesERE-containing-promoter luciferasereporter gene (pGL2-TK-3 times ERE gift from Dr Chih-YangHuang China Medical University Taichung Taiwan) andinternal control pSV-120573-galactosidase expression plasmid (giftfrom Dr Jeremy J W Chen National Chuang Hsing Uni-versity Taichung Taiwan) Reporter gene luciferase produc-tion was performed using the Dual-Light System (AppliedBiosystems) andmeasurements were performed using a 1420Multilabel Counter VICTOR3 instrument (PerkinElmer LifeSciences) The raw data were normalized to 120573-galactosidaseactivity to control for varying transfection efficiencies [19]

27 Statistics All values are presented as themeanplusmn standarderror of themean (SEM) In all cases Studentrsquos 119905-test was usedto assess the results of cell proliferation and reporter assaysA difference between two means was considered statisticallysignificant when 119875 lt 005

3 Results

31 ER120572 Is Important for the Growth Inhibition Induced byEmodin and Aloe-Emodin The effects of different concen-trations (0 6 125 25 50 or 100120583M) of emodin and aloe-emodin on the growth of the ER120572-positive breast cancercell line MCF-7 were determined by cell number counting(0ndash6 days) and MTT assays (4 days) Emodin and aloe-emodin treatment led to dose-dependent suppression ofMCF-7 growth (Figures 1(a) and 1(b)) Notably 125 120583M orhigher concentrations of aloe-emodin had stronger effectson MCF-7 growth compared to the same dosage of emodinMTT assays showed significant inhibition of MCF-7 pro-liferation in a dose-dependent manner following emodintreatment at concentrations of 25 to 100120583M (Figure 1(c)) andof aloe-emodin treatment at concentrations of 6 to 100 120583M

(Figure 1(d)) Whereas emodin was more effective againstthe ER120572-negative breast cancer cell line MDA-MB-453 thanagainst MCF-7 (Figure 1(e)) the inhibitory effects of aloe-emodin on the growth of MDA-MB-453 was moderate com-pared to the effects on MCF-7 cells (Figure 1(f)) In which25 120583M of aloe-emodin was not able to affect MDA-MB-453cell proliferation (Figure 1(f)) while 25 120583M of emodin sig-nificantly reduced that cell proliferation (Figure 1(e)) impliesthat the effects of aloe-emodin might be associated with ER120572and distinct to emodin To further investigate whether ER120572was involved in the inhibitory effects of the emodin and aloe-emodin treatments the effects of emodin and aloe-emodinon the growth of MCF-7 with or without ER120572 overexpres-sion were investigated Following a 25120583M treatment withemodin (Figure 1(g)) or aloe-emodin (Figure 1(h)) the ER120572-overexpressing cells were more sensitive to drug treatmentscompared to control cells Similar results were also observedin another ER120572-positive cell line T47D (data not shown)These data indicate that ER120572 protein plays an importantrole in the emodin- and aloe-emodin-induced suppression ofbreast cancer cell proliferation although the potency of thesetwo compounds are slightly different

32 Emodin and Aloe-Emodin Decrease ER120572 Protein Levelsin a Time- and Dose-Dependent Manner ER120572 activationis triggered by estrogen and promotes the initiation andprogression of breast cancer [22 23] ER120572 protein levelswere detected following treatment with various dosages ofemodin or aloe-emodin (0ndash100 120583M Figures 2(a) and 2(c))for different time intervals (0ndash48 h Figures 2(b) and 2(d))The quantitative results were provided in the lower panels ofthe accompanying figures The data suggest that emodin andaloe-emodin triggered a decrease in ER120572 protein level in atime- and dose-dependent manner

33 Emodin and Aloe-Emodin Decrease Both Nuclear andCytosolic ER120572 and Inhibit ER120572 Activation Because ER120572protein levels were affected by emodin and aloe-emodin theactivation status of ER120572 was investigated Fractionation ofcellular protein was performed and indicated that emodinand aloe-emodin repressed both the nuclear and cytosolicdistribution of ER120572 protein (upper panels in Figures 3(a)and 3(b)) The quantitative results are presented in thelower panels of Figures 3(a) and 3(b) Additionally cytosolicER120572 was more sensitive to treatment than nuclear ER120572An ER120572 reporter assay was performed in MCF-7 cellsThe data showed that aloe-emodin significantly inhibitedER120572-targeted promoter activity in a dose-dependent manner(Figure 3(d)) Compared to aloe-emodin emodin in highdosages (25ndash100120583M) moderately inhibited ER120572 activationwhereas low dosages of emodin (6 and 125 120583M) tended toincrease ER120572 activation Furthermore the expressions of ER120572downstream genes might be affected by those two com-pounds Figures 3(e) and 3(f) were the evidence indicatingthat the protein expression of cyclin D1 which is one ofER120572-regulated protein was indeed decreased by treatmentsof 25120583M emodin or 25 120583M aloe-emodin for 48 h


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Figure 1 Emodin and aloe-emodin inhibit breast cancer cell growth MCF-7 cells were treated with various concentrations of emodin (a)or aloe-emodin (b) for 0 to 6 days Cell numbers were calculated by trypan blue staining (cndashf) MCF-7 and MDA-MB-453 cells were treatedwith various concentrations of emodin or aloe-emodin for 4 days Cell proliferation was determined by an MTT assay The control cellproliferation rate was set at 1 MCF-7 cells were transiently transfected with empty vector or pCMV5-ER120572 and treated with 25120583M emodin(g) or aloe-emodin (h) for 4 days The transfected efficiencies of ER120572 overexpression and cell numbers were detected by western blotting andtrypan blue staining respectively The results were presented as the mean plusmn SEM lowast and lowastlowast correspond to 119875 lt 005 and 119875 lt 001 respectivelyversus the control group or empty vector group

34 Emodin and Aloe-Emodin Treatment Leads to DecreasedER120572 Protein through Proteasomal Degradation Because ER120572protein levels were affected by both emodin and aloe-emodinin the previous experiments the gene expression and proteinstability of ER120572 were investigated First ER120572 messengerRNA expression was detected by quantitative real-time PCRfollowing treatment We found that treatments of 25120583Memodin and 25120583M aloe-emodin for 24 h did not have asignificant effect compared to the control group (Figure 4(a))Second because ubiquitin-proteasome-dependent degrada-tion is the main process involved in ER120572 proteolysis [23] theproteasome inhibitorMG132was used to prevent ER120572proteindegradation and drug effects can be observed The resultsof this experiment indicated that MG132 could rescue ER120572degradation after emodin or aloe-emodin treatment (Figures4(b) and 4(c))These findings suggest that the decreased ER120572protein levels observed following emodin or aloe-emodintreatment resulted from proteasome degradation

35 Aloe-Emodin Promotes the Dissociation of ER and HeatShock Protein 90 and Causes ER120572 Ubiquitination ER120572 isprocessed by ubiquitination after disassociating from heatshock protein 90 (HSP90) [24] Using immunoprecipitationin the presence of MG132 to prevent protein degradation wefound that aloe-emodin apparently blocked the protein inter-action between ER120572 and HSP90 (Figure 5(a)) however theeffect induced by emodin was not as significant (Figure 5(b))The quantitative graphs were provided in the lower panels ofaccompanying figures Subsequently ER120572 ubiquitination fol-lowing drug treatmentwas investigated by detecting the levelsof ubiquitinated ER120572 in drug-treated cell extracts followingMG132 administration The data indicated that aloe-emodintreatment enhanced ubiquitin-conjugated ER120572 levels andprotein degradation was prevented by MG132 treatment

regardless of whether ER120572 or ubiquitin was immunoprecipi-tated first (Figure 5(c)) Although emodin slightly promotedER120572HSP90 dissociation (Figure 5(b)) no increase in ER120572ubiquitination was observed following emodin treatment(Figure 5(d)) The related quantitative results were shownin the lower panels It suggests that ubiquitination was notrequired for emodin-induced ER120572 degradation by protea-some These results indicate that aloe-emodin specificallyreduces ER120572 protein levels by promoting ER120572 ubiquitinationwhich results in proteasome-dependent degradation

36 Comparison of EmodinAloe-Emodin and Estrogen onER120572 Behaviors Ligand binding by estrogen or estrogen-like molecules promotes ER120572 transactivation during whichER120572 first dissociates from HSP90 in the cytoplasm andthen translocates into the nucleus [8] Because the effects ofaloe-emodin and emodin on the ER120572 ubiquitination processare distinct (Figure 5) it is of interest to understand howthe effects of these two compounds compare to those ofestrogen to mediate ER120572 behavior Immunoprecipitationexperiment results indicate that aloe-emodin had similareffects to synthetic estrogen (estradiol benzoate (EB)) onER120572HSP90 dissociation (Figure 6(a)) whereas emodin didnot (Figure 5(b)) However in contrast to EB treatmentaloe-emodin treatment led to a decrease in the nuclearand cytoplasmic levels of ER120572 protein Furthermore thedecrease in ER120572 protein levels following aloe-emodin treat-ment was blocked by MG132 suggesting that aloe-emodinpromotes ER120572 degradation (Figure 6(b)) Comparing thedata in Figure 3(b) we suggest that aloe-emodin preferen-tially induces ER120572 degradation in the cytoplasm of breastcancer cells Interestingly nuclear ER120572 was increased byemodin treatment and protein degradation was preventedby MG132 (Figure 6(c)) suggesting that although emodin


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Figure 2 Emodin and aloe-emodin reduce ER120572 protein levels MCF-7 cells were treated with emodin or aloe-emodin in a dose-dependentmanner for 24 h (a c) and via a time course using 25 120583M of the drug (b d) ER120572 protein levels were detected by western blotting 120573-Actinwas used as an internal control The quantification of ER120572 levels is presented as fold increase or decrease compared to control levels and wasevaluated in three independent experiments The results were presented as the mean plusmn SEM lowast and lowastlowast correspond to 119875 lt 005 and 119875 lt 001respectively versus the control group

induces ER120572 degradation in both the nucleus and cyto-plasm (compared to Figure 3(a)) it seems that emodin isable to promote ER120572 translocation into the nucleus whichis in contrast to the actions of aloe-emodin This mightexplain why low concentrations of emodin showed slightstimulation in the ER120572 reporter assay (Figure 3(c)) Takentogether these results demonstrate that aloe-emodin inducesthe dissociation of ER120572HSP90 and subsequently promotesER120572 ubiquitin-proteasome-dependent degradation in thecytoplasm thereby inhibiting ER120572 translocation into nucleus

where ER120572 would otherwise be activated as a positivemodulator

4 Discussion

Breast cancer is a commonmalignancy in women and estro-gen plays an important role in early cancer development [25]Estrogen which usually denotes 17120573-estradiol (E2) binds toits primary receptor ER120572 and stimulates ER120572 transcriptionalactivity to regulate downstream gene expression and cell


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Figure 3 Emodin and aloe-emodin diminish both nuclear and cytoplasmic ER120572 levels and transcriptional activity MCF-7 cells were treatedwith 25120583M emodin (a) or aloe-emodin (b) for 24 h prior to cell lysis and protein fractionation as described in Section 2 ER120572 proteinwas detected by western blotting and PARP and 120572-tubulin served as markers for the nuclear and cytoplasmic fractions respectively Thequantification of ER120572 levels was presented as fold increase or decrease relative to controls andwas evaluated in three independent experimentsThe effects of emodin (c) and aloe-emodin (d) on ER120572 activation were evaluated using a 3 timesERE- (estrogen-responsive element-) containingluciferase reporter assay in MCF-7 cells for 48 h 120573-galactosidase expression served as the internal control The data were presented as thefold change compared to control levels (0120583M)The results were presented as the mean plusmn SEM lowast and lowastlowast correspond to 119875 lt 005 and 119875 lt 001respectively versus control group MCF-7 cells were treated with emodin (e) or aloe-emodin (f) in a dose-dependent manner for 48 h andthe expressions of ER120572-regulated protein cyclin D1 were detected by western blotting


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Figure 4 Emodin and aloe-emodin decrease ER120572 protein levels by promoting protein degradation (a) MCF-7 cells were treated with 25 120583Mof emodin or aloe-emodin for 24 h prior to quantitative real-time PCR to assess ER120572 mRNA expression Data were presented as the foldchange compared to control levels The experiments were performed in triplicate and each condition was replicated four times within eachexperiment The results are presented as the mean plusmn SEM (b c) MCF-7 cells were treated with various concentrations of emodin or aloe-emodin in the presence of MG132 (proteasome inhibitor 5 120583M) for 24 h ER120572 protein levels were detected by western blotting 120573-Actin wasused as an internal control

growth [26 27] Tamoxifen is designed to interfere with E2binding and thus block ER120572 transcriptional activity to treatER120572-dependent diseases such as breast cancer [28] Howeveralternative compounds that are safer and associated withfewer side effects than Tamoxifen are desired Numerousstudies suggest that phytoestrogens possess organ-specificestrogenic and antiestrogenic effects [29] Phytoestrogensmainly consist of isoflavones such as genistein and daidzeinand are used in the treatment of menopausal symptomsas well as breast cancer [30 31] In this study we provideevidence indicating that two phytoestrogens emodin andaloe-emodin might significantly inhibit the proliferation ofER120572-positive breast cancer cells through ER120572 degradationAlthough both drugs had dose-responsive effects on inhibit-ing proliferation of breast cancer cells low doses (25120583M orless) of aloe-emodin could not affect proliferation of ER120572-negative cells (Figure 1(f)) Therefore 25 120583M of drugs wereutilized in other experiments (Figure 3 to Figure 6) Inter-estingly both compounds had inhibitory effects on ER120572 butthey utilized different inhibitory mechanisms Aloe-emodininhibited ER120572 activation through HSP90ER120572 dissociationand ubiquitin-dependent degradation whereas emodin did

not share the same molecular pathway Therefore thesefindings illustrate that emodin and aloe-emodin might serveas estrogen receptor modulators with different molecularmechanisms The therapeutic application of these two com-pounds should be investigated further in the future

Anthraquinones are phytoestrogens that have beendemonstrated to possess anticancer properties through theinhibition of cell proliferation the induction of apoptosisand the prevention of metastasis [1] The effects of theanthraquinone derivatives emodin and aloe-emodin havebeen extensively investigated in several cancer types throughstudies on their signaling targets Emodin has been reportedto sensitize Her2neu-overexpressing lung cancer cells tochemotherapeutic treatments and to suppress Her2neu-overexpressing breast cancer growth by inhibiting tyrosinekinase activity [32ndash34] Notably emodin has been shown todownregulate androgen receptor (AR) and inhibit prostatecancer growth [5] suggesting that there is a connectionbetween anthraquinone derivatives and steroid receptor inhormone-related cancers Although the estrogenic abilityof emodin is higher than that of aloe-emodin as deter-mined by ER120572 binding studies the antiproliferation effect


HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572

poly-Ub-ER120572 120573-Actin

ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)

Figure 5 Only aloe-emodin promotes the dissociation of HSP90ER120572 and subsequent ER120572 ubiquitinationThe interaction between ER120572 andHSP90 in MCF-7 cells following 25120583M treatment of aloe-emodin (a) or emodin (b) in the presence of MG132 (5 120583M) for 24 h was evaluatedby ER120572 immunoprecipitation followed by western blotting for HSP90with specific antibodiesThe ubiquitin-conjugated ER120572 levels inMCF-7cells following 25 120583M treatment of aloe-emodin (c) and emodin (d) in the presence ofMG132 (5 120583M)were examined by immunoprecipitationwith anti-ER120572 or antiubiquitin antibodies followed by western blottingThe lysates were used in the western blotting experiments to indicateprotein levels Immunoprecipitation by IgG served as a negative control 120573-Actin served as an internal control for western blotting Thequantitative data were obtained from the results of three times immunoprecipitation experiments and presented as fold change of controlThe quantitative graphs were presented as the mean plusmn SEM lowast and lowastlowast correspond to 119875 lt 005 and 119875 lt 001 respectively versus the controlgroup


C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input

C E EB C E EBER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)

Figure 6 Effects of aloe-emodin and emodin on ER120572 subcellular distribution in comparison to EB (a b) MCF-7 cells were treated withaloe-emodin (25 120583M) or estradiol benzoate ((EB) 10 nM) in the presence of MG132 (5 120583M) for 24 hThe interaction between HSP90 and ER120572was evaluated by ER120572 immunoprecipitation followed by western blotting of HSP90 with specific antibodies Immunoprecipitation by IgGserved as a negative control The fractionation of cellular proteins was performed and ER120572 protein was detected by western blotting (c)MCF-7 cells were treated with emodin (25120583M) or EB (10 nM) in the presence of MG132 (5120583M) for 24 hThe fractionation of cellular proteinswas performed and ER120572 protein was detected by western blotting PARP and 120572-tubulin served as markers for the nuclear and cytoplasmicfractions respectively The lysates were used in the western blotting experiments to indicate protein levels 120573-Actin served as an internalcontrol for western blotting

of aloe-emodin is more efficient than that of emodin inbreast cancer cells [7] Kang et al also showed that thecytotoxicity of aloe-emodin is higher in ER120572-positive cellsthan in ER120572-negative cells [7] These observations suggestthat aloe-emodin might be a stronger inhibitor of ER120572-positive cancer cell growth than emodin

The goal of this study was to investigate the differencesbetween emodin and aloe-emodin in their efficiency andmechanism of blocking breast cancer cell growth Our datashowed that aloe-emodin is a more potent growth inhibitorthan emodin and has a unique mechanism for the growthinhibition of breast cancer cells MCF-7 cell growth wasabolished following treatment with 125 120583M aloe-emodin for6 days and only 50 of cells survived at 25120583M aloe-emodintreatment for 4 days (Figure 1(b)) Although a previous studysuggested that the IC

50of aloe-emodin is 126 plusmn 083 120583gmL

(approximately 46 120583M) on MCF-7 cells [7] the IC50

ofaloe-emodin in our system was only approximately 25 120583M(Figure 1(d)) With regard to side effects on normal cells theprevious report indicated that 25 120583M aloe-emodin is a moresignificant proliferation inhibitor in human skin epidermoidcarcinoma cells than in noncancerous cells [35] Howeveraloe-emodin did not significantly affect the proliferation ofMDA-MB-453 ER120572-negative cells (Figure 1(f)) The overex-pression of ER120572 in MCF-7 cells increased the sensitivityto aloe-emodin treatment (Figure 1(h)) These results areconsistent with previous findings that aloe-emodin has a

higher cytotoxic potential to MCF-7 (ER120572-positive) cellsthan to MDA-MB-231 (ER120572-negative) cells [7] A low dosage(10 120583M) of aloe-emodin [6] showed that both cell growth(Figures 1(b) and 1(d)) and ER120572 activation (Figure 3(d)) weresignificantly repressed by aloe-emodin in a dose-dependentmanner even at low dosages (6 120583M) Additionally aloe-emodin treatment reduced ER120572 protein levels not only intotal lysates (Figures 2(c) and 2(d)) but also in nuclear andcytoplasmic fractions (Figure 3(b)) In the nuclear and cyto-plasmic fractions the impact of aloe-emodin treatment oncytoplasmic ER120572 levels seems stronger than that on nuclearER120572 levels (Figure 3(b)) As shown in Figures 2 and 4 we con-clude that the decrease in ER120572 levels induced by aloe-emodinwas due to protein degradation This correlates with ER120572proteasome-dependent degradation because of the observedelevation in ubiquitin-conjugated levels (Figure 5(c)) Inter-estingly the dissociation of ER120572 and HSP90 was increasedby aloe-emodin treatment (Figure 5(a)) and ER120572 releasedfrom HSP90 protection was subjected to ubiquitination fordegradation rather than translocation to the nucleus foractivation (Figure 6(b)) These phenomena are similar tothe actions of antiestrogens such as ICI 164384 ICI 182780and RU 58668 which may induce ER120572 degradation andresult in rapid turnover [24] As a ligand with an inhibitoryeffect on ER120572 we suggest that aloe-emodin might qualify asan estrogen receptor modulator to negatively regulate ER120572activity and inhibit breast cancer cell growth


The effect of emodin was also investigated in this studyAlthough the inhibitory effects of emodin on breast cancergrowth and ER120572 activation were similar to those of aloe-emodin the molecular mechanism of emodin inhibition wasunexpectedly distinct In Figures 1(a) and 1(c) the inhibitorypotency of emodin onMCF-7 cell growth was lower than thatof aloe-emodin (Figures 1(b) and 1(d)) Interestingly emodinsignificantly suppressed cell proliferation of the ER120572-negativecell line MDA-MB-453 (Figure 1(e)) Because emodin mayact as a tyrosine kinase inhibitor by inhibiting the binding ofHer2neu andHSP90 thereby depletingHer2neu via protea-somal degradation [36] it is possible that emodinmight differin its effect on the growth of MDA-MB-453 cells (which arecharacterized by Her2 overexpression) in ER120572-independentregulation in comparison to aloe-emodin Like aloe-emodinemodin targeted ER120572 by proteasomal degradation (Figures 2and 4) however the degradation pathway was independentof HSP90ER120572 dissociation and ubiquitination (Figures 5(b)and 5(d)) Although the ubiquitin-proteasome degradationis an important process for modulation of many proteinsrecent studies demonstrate that the proteasome-dependentprotein degradation possibly goes through ubiquitination-independent pathway [37 38] Previous reports indicatethat the estrogenic activity of emodin is higher than thatof aloe-emodin [6 7] Our ERE reporter assay resultsshowed that emodin induced the inhibition of ER120572 activationonly at high dosages (over 25 120583M Figure 3(c)) whereasthe aloe-emodin data showed inhibitory effects starting atlow dosages (6 120583M Figure 3(d)) Notably emodin treat-ment led to a slight increase in ER120572 activation at 6 and125 120583M which differed from the aloe-emodin treatmentresults (Figure 3(c)) Unlike aloe-emodin emodin treatmentelevated nuclear ER120572 protein levels in the presence of MG132similar to the effects of estradiol benzoate (EB) treatment(Figure 6(c)) Taken together with the data in Figure 3(a)we suggest that emodin using a distinct mechanism fromaloe-emodin might cause ER120572 shuttling into the nucleusand subsequently promote nuclear ER120572 degradation throughubiquitination-independent pathway whereas cytoplasmicER120572 is less affected This hypothesis is similar to previousstudies indicating that the chemical structure of the liganddirectly affects the nuclear fate and protein turnover rate ofER120572 independently of transcriptional regulation [39] Theseobservations suggest that the essential difference betweenemodin and aloe-emodin lies in the mechanism of ER120572regulation and Her2 inhibition

In conclusion we provide evidence showing that the anti-cancer mechanisms of the anthraquinone derivatives emodinand aloe-emodin are mediated via an ER120572-dependent path-way in breast cancer cells Although emodin and aloe-emodindisplay distinct differences in efficiency and ER120572 activationmechanism on breast cancer cell growth both compoundsare potential selectively therapeutic treatments for breastcancer

Abbreviations

ER120572 Estrogen receptor 120572HSP90 Heat shock protein 90

AE Aloe-emodinE Emodin

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors thank Dr H C Chen Dr T H Lee Dr Y MLiou Dr J W Chen and Dr H C Cheng (National ChungHsing University Taiwan) for technical support This workwas supported by the following Grants NSC100-2320-B-005-002 and NSC101-2320-B-005-004-MY3 from the TaiwanNational Science Council (to H Lin) Taichung VeteransGeneral HospitalNational Chung Hsing University JointResearch Program (TCVGH-NCHU1017607 and TCVGH-NCHU1027606) Chang Bing Show Chwan Memorial Hos-pital Research Grant (CBBC001 to Y-T Lee) and theTaiwan Ministry of Education under the Aiming for the TopUniversity plan

References

[1] Q Huang G Lu H-M Shen M C M Chung and NO Choon ldquoAnti-cancer properties of anthraquinones fromrhubarbrdquo Medicinal Research Reviews vol 27 no 5 pp 609ndash630 2007

[2] A Dutta S Bandyopadhyay C Mandal and M ChatterjeeldquoAloe vera leaf exudate induces a caspase-independent celldeath in Leishmania donovani promastigotesrdquo Journal of Medi-cal Microbiology vol 56 no 5 pp 629ndash636 2007

[3] H-H Wang and J-G Chung ldquoEmodin-induced inhibition ofgrowth and DNA damage in the Helicobacter pylorirdquo CurrentMicrobiology vol 35 no 5 pp 262ndash266 1997

[4] C-H Chang C-C Lin J-J Yang T Namba and M HattorildquoAnti-inflammatory effects of emodin fromVentilago leiocarpardquoAmerican Journal of ChineseMedicine vol 24 no 2 pp 139ndash1421996

[5] T-L Cha L Qiu C-T Chen Y Wen and M-C HungldquoEmodin down-regulates androgen receptor and inhibitsprostate cancer cell growthrdquo Cancer Research vol 65 no 6 pp2287ndash2295 2005

[6] H Matsuda H Shimoda T Morikawa and M Yoshi-kawa ldquoPhytoestrogens from the roots of Polygonum cuspi-datum (Polygonaceae) structure-requirement of hydroxyan-thraquinones for estrogenic activityrdquo Bioorganic and MedicinalChemistry Letters vol 11 no 14 pp 1839ndash1842 2001

[7] S C Kang C M Lee E S Choung et al ldquoAnti-proliferativeeffects of estrogen receptor-modulating compounds isolatedfrom Rheum palmatumrdquo Archives of Pharmacal Research vol31 no 6 pp 722ndash726 2008

[8] F Acconcia andR Kumar ldquoSignaling regulation of genomic andnongenomic functions of estrogen receptorsrdquo Cancer Lettersvol 238 no 1 pp 1ndash14 2006

[9] S Ali and R C Coombes ldquoEndocrine-responsive breast cancerand strategies for combating resistancerdquoNature Reviews Cancervol 2 no 2 pp 101ndash112 2002

[10] C K Osborne ldquoTamoxifen in the treatment of breast cancerrdquoTheNew England Journal of Medicine vol 339 no 22 pp 1609ndash1618 1998


[11] G A Colditz S E Hankinson D J Hunter et al ldquoThe useof estrogens and progestins and the risk of breast cancer inpostmenopausal womenrdquoTheNewEngland Journal ofMedicinevol 332 no 24 pp 1589ndash1593 1995

[12] H Adlercreutz and W Mazur ldquoPhyto-oestrogens and Westerndiseasesrdquo Annals of Medicine vol 29 no 2 pp 95ndash120 1997

[13] M C Chen C Y Huang S L Hsu et al ldquoRetinoic acidInduces apoptosis of prostate cancer DU145 cells through Cdk5overactivationrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 580736 11 pages 2012

[14] H Lin and P S Wang ldquoInhibitory effects of digoxin ontestosterone production in rat Leydig cellsrdquo Adaptive Medicinevol 4 pp 165ndash172 2012

[15] H Lin T-Y Lin and J-L Juang ldquoAbl deregulates Cdk5 kinaseactivity and subcellular localization in Drosophila neurodegen-erationrdquo Cell Death and Differentiation vol 14 no 3 pp 607ndash615 2007

[16] H Lin M-C Chen C-Y Chiu Y-M Song and S-Y LinldquoCdk5 regulates STAT3 activation and cell proliferation inmedullary thyroid carcinoma cellsrdquo Journal of Biological Chem-istry vol 282 no 5 pp 2776ndash2784 2007

[17] H Lin M-C Chen and C-T Ku ldquoCyclin-dependent kinase 5regulates steroidogenic acute regulatory protein and androgenproduction in mouse Leydig cellsrdquo Endocrinology vol 150 no1 pp 396ndash403 2009

[18] H Lin J-L Juang and P S Wang ldquoInvolvement of Cdk5p25in digoxin-triggered prostate-cancer cell apoptosisrdquo Journal ofBiological Chemistry vol 279 no 28 pp 29302ndash29307 2004

[19] F-N Hsu M-C Chen M-C Chiang et al ldquoRegulationof androgen receptor and prostate cancer growth by cyclin-dependent kinase 5rdquo Journal of Biological Chemistry vol 286no 38 pp 33141ndash33149 2011

[20] M-C Chen S-W Wang S-F Kan S-C Tsai Y-C Wu andP S Wang ldquoStimulatory effects of propylthiouracil on preg-nenolone production through upregulation of steroidogenicacute regulatory protein expression in rat granulosa cellsrdquoToxicological Sciences vol 118 no 2 Article ID kfq302 pp 667ndash674 2010

[21] M Dalvai and K Bystricky ldquoCell cycle and anti-estrogeneffects synergize to regulate cell proliferation and er target geneexpressionrdquo PLoS One vol 5 no 6 Article ID e11011 2010

[22] N Heldring A Pike S Andersson et al ldquoEstrogen receptorshow do they signal and what are their targetsrdquo PhysiologicalReviews vol 87 no 3 pp 905ndash931 2007

[23] Y Miyoshi K Murase M Saito M Imamura and K OhldquoMechanisms of estrogen receptor-120572 upregulation in breastcancersrdquoMedical Molecular Morphology vol 43 no 4 pp 193ndash196 2010

[24] M Fan A Park and K P Nephew ldquoCHIP (carboxyl ter-minus of Hsc70-interacting protein) promotes basal andgeldanamycin-induced degradation of estrogen receptor-120572rdquoMolecular Endocrinology vol 19 no 12 pp 2901ndash2914 2005

[25] R AWalker ldquoOestrogen receptor and its potential role in breastcancer developmentrdquo The Journal of Pathology vol 188 no 3pp 229ndash230 1999

[26] D J Mangelsdorf C Thummel M Beato et al ldquoThe nuclearreceptor super-family the second decaderdquo Cell vol 83 no 6pp 835ndash839 1995

[27] J M Hall J F Couse and K S Korach ldquoThe multifacetedmechanisms of estradiol and estrogen receptor signalingrdquoJournal of Biological Chemistry vol 276 no 40 pp 36869ndash36872 2001

[28] V C Jordan ldquoTamoxifen (ICI46474) as a targeted therapy totreat and prevent breast cancerrdquo British Journal of Pharmacol-ogy vol 147 no 1 pp S269ndashS276 2006

[29] A L Murkies G Wilcox and S R Davis ldquoPhytoestrogensrdquoJournal of Clinical Endocrinology and Metabolism vol 83 no2 pp 297ndash303 1998

[30] ENikander A KilkkinenMMetsa-Heikkila et al ldquoA random-ized placebo-controlled crossover trial with phytoestrogens intreatment of menopause in breast cancer patientsrdquo Obstetricsand Gynecology vol 101 no 6 pp 1213ndash1220 2003

[31] Y Imai S Tsukahara S Asada and Y SugimotoldquoPhytoestrogensflavonoids reverse breast cancer resistanceproteinABCG2-mediated multidrug resistancerdquo CancerResearch vol 64 no 12 pp 4346ndash4352 2004

[32] H Jayasuriya NMKoonchanok R L Geahlen J LMcLaugh-lin and C-J Chang ldquoEmodin a protein tyrosine kinaseinhibitor from Polygonum cuspidatumrdquo Journal of NaturalProducts vol 55 no 5 pp 696ndash698 1992

[33] L Zhang C-J Chang S S Bacus and M-C Hung ldquoSup-pressed transformation and induced differentiation of HER-2neu-overexpressing breast cancer cells by emodinrdquo CancerResearch vol 55 no 17 pp 3890ndash3896 1995

[34] L Zhang and M-C Hung ldquoSensitization of HER-2neu-overexpressing non-small cell lung cancer cells to chemother-apeutic drugs by tyrosine kinase inhibitor emodinrdquo Oncogenevol 12 no 3 pp 571ndash576 1996

[35] T-H Chou and C-H Liang ldquoThe molecular effects of Aloe-Emodin (AE)liposome-AE on human nonmelanoma skin can-cer cells and skin permeationrdquoChemical Research in Toxicologyvol 22 no 12 pp 2017ndash2028 2009

[36] Y-Y Yan L-S Zheng X Zhang et al ldquoBlockade of Her2neubinding to Hsp90 by emodin azide methyl anthraquinonederivative induces proteasomal degradation of Her2neurdquoMolecular Pharmaceutics vol 8 no 5 pp 1687ndash1697 2011

[37] Y Murakami S Matsufuji T Kameji et al ldquoOrnithine decar-boxylase is degraded by the 26S proteasome without ubiquiti-nationrdquo Nature vol 360 no 6404 pp 597ndash599 1992

[38] G Asher and Y Shaul ldquop53 proteasomal degradation poly-ubiquitination is not the whole storyrdquo Cell Cycle vol 4 no 8pp 1015ndash1018 2005

[39] S Kocanova M Mazaheri S Caze-Subra and K BystrickyldquoLigands specify estrogen receptor alpha nuclear localizationand degradationrdquo BMC Cell Biology vol 11 article 98 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


and molecular mechanisms of emodin and aloe-emodin inestrogen receptor 120572 (ER120572) modulation and breast cancer cellgrowth remain elusive

Breast cancer is a commonmalignancywith high lethalityin women Because ER120572 activation plays an important role inthe initiation development and progression of breast cancerestrogen replacement therapy is the most common strategyto suppress breast cancer progression [8] By mimicking thestructure of estrogen synthetic estrogen-like compounds areused to compete for the binding of endogenous estrogenwith ER120572 and therefore inhibit ER120572-dependent growth ofbreast cancer cells [9 10] However synthetic estrogens haveside effects that increase the risk of cancer development dueto unselective estrogenic action [11] Although the potencyof natural phytoestrogens is generally lower than that ofsynthetic estrogens in terms of estrogenic action naturalphytoestrogens are relatively safer with fewer side effects[12] Therefore studies investigating the effects and molec-ular mechanisms of natural herbal medicines that containphytoestrogens as potential treatments to breast cancer are ofinterest

We found that the inhibition of proliferation by emodinand aloe-emodin was ER120572-dependent in breast cancer celllines Importantly aloe-emodin treatment promotes ER120572protein degradation by repressing the association of ER120572 andheat shock protein 90 (HSP90) Moreover the dissociatedER120572 is ubiquitinated and targeted for proteasome-dependentdegradation in the cytosol The findings for aloe-emodinare distinct from those for emodin Based on the aboveobservations these two anthraquinones could potentially beused as specific phytoestrogens to treat breast cancer

2 Materials and Methods

21 Cell Lines and Cell Culture The human breast cancercell lines MCF-7 and MDA-MB-453 were obtained from theBioresource Collection and Research Center (BCRC) FoodIndustry Research andDevelopment Institute TaiwanMCF-7 cells were grown in minimum essential medium (GibcoCarlsbad CA USA) containing 10 fetal bovine serum(Gibco) 15 gL NaHCO

3 01mM nonessential amino acids

(Gibco) 1mM sodium pyruvate (Gibco) and 1 penicillin-streptomycin (Sigma St Louis MO USA) MDA-MB-453cells weremaintained in Dulbeccorsquos modified Eaglersquos medium(Gibco) supplemented with 10 fetal bovine serum 15 gLNaHCO

3 and 1 penicillin-streptomycin All cells were

incubated at 37∘C in a humidified atmosphere with 5 CO2

22 Cell Viability Assay Cells were incubated for 24 hoursafter attachment Cell numbers were calculated by directcounting of cells excluding cells that stained positive for 02trypan blue stain (Sigma St Louis MO USA) [13] Cellswere treated with different concentrations of aloe-emodinor emodin (ChromaDex Irvine CA USA) for the indicatednumber of days and then the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide (MTT Sigma St Louis MOUSA) assay was used to quantify cell proliferation The MTTstock solution (5mgmL) was diluted to 05mgmL with

complete culturemedium and 01mLwas added to eachwellThe yellow MTT was converted to blue formazan by livingcells a reaction that is dependent on mitochondrial enzymeactivity After using DMSO to dissolve the blue formazan theabsorbance of converted MTT could be measured at 570 nm120582 [14]

23 Cell Fractionation and Western Blot Analysis Cells werecollected using a rubber scraper and homogenized withNa3VO4diluted in PBS (1 100) After centrifugation cells

were resuspended with lysis buffer as previously described[15ndash19] and protease inhibitor cocktail (Roche Applied Sci-ence Mannheim Germany) was added followed by incuba-tion on ice for 45 minutes The cell lysate was centrifugedand the supernatant was collected as total protein extractThe protein extract was mixed with sample buffer and boiledfor 10 minutes Then western blotting was performed aspreviously described [19 20] Briefly SDS-polyacrylamidegel electrophoresis (SDS-PAGE)was performed and proteinswere transferred from the SDS-PAGE gel onto a polyvinyli-dene fluoride (PVDF) membrane Primary antibodies wereincubated with the membrane overnight and horseradishperoxidase- (HRP-) conjugated secondary antibodies (Jack-son ImmunoResearch Laboratory West Grove PA USA)were applied The ECL (western lighting chemiluminescencereagent plus PerkinElmer Life Sciences Shelton CT USA)reaction was performed and the membranes were exposedto X-ray films to visualize protein staining (Fujifilm TokyoJapan) Antibodies directed against the following proteinswere used in this study poly(ADP-ribose) polymerase (PARP06-557 Upstate Biotechnology Lake Placid NY USA) 120572-tubulin (05-829 Upstate Biotechnology) ER120572 (sc-543 andsc-8005 Santa Cruz Biotechnology Santa Cruz CA USA)cyclin D1 (sc-20044 Santa Cruz Biotechnology) HSP 90120572120573(sc-59577 Santa Cruz Biotechnology) ubiquitin (sc-8017Santa Cruz Biotechnology) and 120573-actin (MAB1501 Milli-pore Temecula CA USA) The quantification software usedwas MCID Image Analysis Evaluation

24 Immunoprecipitation Cell lysate was extracted in lysisbuffer and immunoprecipitation was performed as previ-ously described [19] Briefly the beadsantibody precipitatedcomplex was prepared by mixing Protein G Mag SepharoseXtra beads (GEHealthcareWaukeshaWI USA) and specificantibody at 10 120583g 1120583g for 2 h at room temperature Celllysates were mixed with beadsantibody for 12 h at 4∘C andprotein was isolated by precipitation under magnetic attrac-tion with three rounds of PBST washes The precipitatedproteins were analyzed by western blotting after denaturationfollowing dilution in sample buffer and boiling for 10minutes

25 Quantitative Real-Time PCR Total RNA was extractedfrom cells using a Miniprep Purification Kit (GenemarkTaipei Taiwan) and reverse transcription-PCR was per-formed using a High-Capacity cDNA Reverse TranscriptionKit (Applied Biosystems Foster City CA USA) followingthe standard procedures recommended by the manufacturerFor reverse transcription 2120583g of total RNA was used as the





3 Results






0

10

20

30

40

50

60

0 2 4 6Day

Cel

l num

ber (times10

4ce

lls)

06125

2550100

Emodin (120583M)

(a)

0

10

20

30

40

50

60

Cel

l num

ber (times10

4ce

lls)

06125

2550100

0 2 4 6

AE (120583M)

Day

(b)

0

02

04

06

08

1

12

0 6 125 25 50 100

MCF-7

Cel

l pro

lifer

atio

n

Emodin(120583M)

lowastlowast

lowastlowast

lowast

(c)

0 6 125 25 50 100

MCF-7

AE(120583M)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio


lowastlowast


(d)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

0 6 125 25 50 100Emodin(120583M)

lowastlowast

lowastlowast


MDA-MB-453

(e)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

lowastlowastlowast

0 6 125 25 50 100AE(120583M)

MDA-MB-453

(f)

lowastlowast

lowastlowast

0

10

20

30

C

E

Emodin

ER120572

ER120572

ER120572

ER120572

Empty vector

EV ER120572EVC

Darker exp

120573-ActinCel

l num

ber (times10

4ce

lls)

(g)

Figure 1 Continued


lowastlowast

lowastlowast


Darker exp


C

ER120572

ER120572

120573-Actin0

10

20

30

C AE

Cel

l num

ber (times10

4ce

lls)

(h)







0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100Emodin

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (24 h)

ER120572

120573-Actin

lowastlowastlowast


lowastlowast

(120583M)

(120583M)

(a)

0 3 6 12 24 48

(h)

(h)

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (25 120583M)

Emodin (25 120583M)

ER120572

120573-Actin

lowastlowast


lowastlowast

(b)

0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100AE (24 h)

AE (24 h)

ER120572

120573-Actin

(120583M)

(120583M)

ER120572

leve

ls (fo

ld o

f con

trol)

lowastlowast



(c)

0 3 6 12 24 48

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

AE (25 120583M)

AE (25 120583M)(h)

(h)

ER120572

120573-Actin

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)




4 Discussion



EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results






0

10

20

30

40

50

60

0 2 4 6Day

Cel

l num

ber (times10

4ce

lls)

06125

2550100

Emodin (120583M)

(a)

0

10

20

30

40

50

60

Cel

l num

ber (times10

4ce

lls)

06125

2550100

0 2 4 6

AE (120583M)

Day

(b)

0

02

04

06

08

1

12

0 6 125 25 50 100

MCF-7

Cel

l pro

lifer

atio

n

Emodin(120583M)

lowastlowast

lowastlowast

lowast

(c)

0 6 125 25 50 100

MCF-7

AE(120583M)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio


lowastlowast


(d)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

0 6 125 25 50 100Emodin(120583M)

lowastlowast

lowastlowast


MDA-MB-453

(e)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

lowastlowastlowast

0 6 125 25 50 100AE(120583M)

MDA-MB-453

(f)

lowastlowast

lowastlowast

0

10

20

30

C

E

Emodin

ER120572

ER120572

ER120572

ER120572

Empty vector

EV ER120572EVC

Darker exp

120573-ActinCel

l num

ber (times10

4ce

lls)

(g)

Figure 1 Continued


lowastlowast

lowastlowast


Darker exp


C

ER120572

ER120572

120573-Actin0

10

20

30

C AE

Cel

l num

ber (times10

4ce

lls)

(h)







0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100Emodin

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (24 h)

ER120572

120573-Actin

lowastlowastlowast


lowastlowast

(120583M)

(120583M)

(a)

0 3 6 12 24 48

(h)

(h)

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (25 120583M)

Emodin (25 120583M)

ER120572

120573-Actin

lowastlowast


lowastlowast

(b)

0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100AE (24 h)

AE (24 h)

ER120572

120573-Actin

(120583M)

(120583M)

ER120572

leve

ls (fo

ld o

f con

trol)

lowastlowast



(c)

0 3 6 12 24 48

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

AE (25 120583M)

AE (25 120583M)(h)

(h)

ER120572

120573-Actin

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)




4 Discussion



EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

0 2 4 6Day

Cel

l num

ber (times10

4ce

lls)

06125

2550100

Emodin (120583M)

(a)

0

10

20

30

40

50

60

Cel

l num

ber (times10

4ce

lls)

06125

2550100

0 2 4 6

AE (120583M)

Day

(b)

0

02

04

06

08

1

12

0 6 125 25 50 100

MCF-7

Cel

l pro

lifer

atio

n

Emodin(120583M)

lowastlowast

lowastlowast

lowast

(c)

0 6 125 25 50 100

MCF-7

AE(120583M)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio


lowastlowast


(d)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

0 6 125 25 50 100Emodin(120583M)

lowastlowast

lowastlowast


MDA-MB-453

(e)

0

02

04

06

08

1

12

Cel

l pro

lifer

atio

n

lowastlowastlowast

0 6 125 25 50 100AE(120583M)

MDA-MB-453

(f)

lowastlowast

lowastlowast

0

10

20

30

C

E

Emodin

ER120572

ER120572

ER120572

ER120572

Empty vector

EV ER120572EVC

Darker exp

120573-ActinCel

l num

ber (times10

4ce

lls)

(g)

Figure 1 Continued


lowastlowast

lowastlowast


Darker exp


C

ER120572

ER120572

120573-Actin0

10

20

30

C AE

Cel

l num

ber (times10

4ce

lls)

(h)







0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100Emodin

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (24 h)

ER120572

120573-Actin

lowastlowastlowast


lowastlowast

(120583M)

(120583M)

(a)

0 3 6 12 24 48

(h)

(h)

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (25 120583M)

Emodin (25 120583M)

ER120572

120573-Actin

lowastlowast


lowastlowast

(b)

0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100AE (24 h)

AE (24 h)

ER120572

120573-Actin

(120583M)

(120583M)

ER120572

leve

ls (fo

ld o

f con

trol)

lowastlowast



(c)

0 3 6 12 24 48

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

AE (25 120583M)

AE (25 120583M)(h)

(h)

ER120572

120573-Actin

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)




4 Discussion



EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowastlowast

lowastlowast


Darker exp


C

ER120572

ER120572

120573-Actin0

10

20

30

C AE

Cel

l num

ber (times10

4ce

lls)

(h)







0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100Emodin

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (24 h)

ER120572

120573-Actin

lowastlowastlowast


lowastlowast

(120583M)

(120583M)

(a)

0 3 6 12 24 48

(h)

(h)

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (25 120583M)

Emodin (25 120583M)

ER120572

120573-Actin

lowastlowast


lowastlowast

(b)

0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100AE (24 h)

AE (24 h)

ER120572

120573-Actin

(120583M)

(120583M)

ER120572

leve

ls (fo

ld o

f con

trol)

lowastlowast



(c)

0 3 6 12 24 48

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

AE (25 120583M)

AE (25 120583M)(h)

(h)

ER120572

120573-Actin

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)




4 Discussion



EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100Emodin

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (24 h)

ER120572

120573-Actin

lowastlowastlowast


lowastlowast

(120583M)

(120583M)

(a)

0 3 6 12 24 48

(h)

(h)

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

Emodin (25 120583M)

Emodin (25 120583M)

ER120572

120573-Actin

lowastlowast


lowastlowast

(b)

0 6 125 25 50 100

0

02

04

06

08

1

12

0 6 125 25 50 100AE (24 h)

AE (24 h)

ER120572

120573-Actin

(120583M)

(120583M)

ER120572

leve

ls (fo

ld o

f con

trol)

lowastlowast



(c)

0 3 6 12 24 48

0

02

04

06

08

1

12

0 3 6 12 24 48

ER120572

leve

ls (fo

ld o

f con

trol)

AE (25 120583M)

AE (25 120583M)(h)

(h)

ER120572

120573-Actin

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(d)




4 Discussion



EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















EC EC

PARP

0

02

04

06

08

1

12

Nucleus Cytoplasm

ControlEmodin

CytoplasmNucleus

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(a)

C AE C AE

0

02

04

06

08

1

12

Nucleus Cytoplasm

Nucleus Cytoplasm

PARP

ControlAE

120572-Tubulin

ER120572

leve

ls (fo

ld o

f con

trol)

ER120572


(b)

002040608

1121416

0 6 125 25 50 100Emodin

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast

lowastlowast

lowast

(120583M)

(c)

0

02

04

06

08

1

12

14

0 6 125 25 50 100AE

Fold

chan

ge in

3times

ERE

luci

fera

se ac

tivity

lowastlowast


lowast

(120583M)

(d)

Cyclin D1

Emodin 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(e)

0 6 125 25 50 100

Cyclin D1

AE

ER120572

120573-Actin

(120583M)

(f)



0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

C AE EFo

ld ch

ange

in E

R120572m

RNA

expr

essio

n(a)

Emodin

Control MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(b)

AEControl MG132

0 6 125 25 50 100 0 6 125 25 50 100

ER120572

120573-Actin

(120583M)

(c)






HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HSP90HSP90

Input

C AEWB IgG C AE

MG132

0

02

04

06

08

1

12

C AE

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)ER120572 ER120572

120573-Actin

IP ER120572

lowastlowast

(a)

0

02

04

06

08

1

12

C E

HSP

90E

R120572in

tera

ctio

n le

vels

(fold

of c

ontro

l)

HSP90HSP90

Input

C AEWB IgG C AE

MG132

ER120572 ER120572

120573-Actin

IP ER120572

(b)

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

0

05

1

15

2

C AE

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

lowast

(c)

0

02

04

06

08

1

12

C E

(fold

of c

ontro

l)

Ub

ER120572

inte

ract

ion

leve

ls

C AEC AE

WB

IP UbWB ER120572

minus70

minus100

minus130minus170

minus70

minus100

minus130minus170

Input

MG132

IP ER120572IgG

ER120572

ER120572


ER120572

120573-Actin

(d)



C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















C AE EBC AE EB

Input

HSP90

ER120572

HSP90

ER120572

120573-Actin

IP ER120572

IgG

MG132

(a)

C AE EB

Input

C AE EB C AE EB

ER120572

120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(b)

C AE EB

Input


120573-Actin

MG132

Nucleus Cytoplasm

PARP

120572-Tubulin

ER120572

(c)











Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Abbreviations





Acknowledgments


References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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