research article green tea catechin, egcg, suppresses pcb...

Research ArticleGreen Tea Catechin EGCG Suppresses PCB 102-InducedProliferation in Estrogen-Sensitive Breast Cancer Cells

Kimberly Mantzke Baker1 and Angela C Bauer2

1Department of Biology University of Indianapolis Lilly Science Hall 30 1400 East Hanna Avenue Indianapolis IN 46227 USA2Department of Biology High Point University Congdon 217 833 Montlieu Avenue High Point NC 27268 USA

Correspondence should be addressed to Kimberly Mantzke Baker bakerkmuindyedu

Received 11 August 2015 Revised 19 November 2015 Accepted 23 November 2015

Academic Editor Vladimir F Semiglazov

Copyright copy 2015 K M Baker and A C BauerThis 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 persistence of polychlorinated biphenyls (PCBs) in the environment is of considerable concern since they accumulate in humanbreast tissue andmay stimulate the growth of estrogen-sensitive tumors Studies have shown that EGCG from green tea canmodifyestrogenic activity and thus may act as a cancer chemopreventive agent In the present study we evaluated the individual andcombined effects of PCB 102 andEGCGon cell proliferation using an estrogen-sensitive breast cancer cell lineMCF-7BOS PCB 102(1ndash10 120583M) increased cell proliferation in a dose-dependent manner Furthermore the proliferative effects of PCB 102 weremediatedby ER120572 and could be abrogated by the selective ER120572 antagonistMPP EGCG (10ndash50 120583M) caused a dose-dependent inhibition of PCB102-induced cell proliferation with nearly complete inhibition at 25 120583MEGCGThe antiproliferative action of EGCGwasmediatedby ER120573 and could be blocked by the ER120573-specific inhibitor PHTPP In conclusion EGCG suppressed the proliferation-stimulatingactivity of the environmental estrogen PCB 102 which may be helpful in the chemoprevention of breast cancer

1 Introduction

Endocrine disrupting chemicals (EDCs) are environmentalcontaminants that have the ability to interfere with hormonesignaling in the body EDCs can disrupt endocrine functionin a variety of ways through enhancement or inhibition ofhormone synthesis [1 2] through activation or antagonismof hormone receptors [3 4] andor through blockade ofhormone intracellular signaling [5] A variety of substancesmdashboth natural and man-mademdashhave been identified as EDCsincluding (but not limited to) pesticides [1 6] plasticizers(such as bisphenol A [5 7]) pharmaceuticals [8] sunscreens[9] and triclosan (found in antibacterial soaps [10 11])Exposure to some EDCs is associated with negative healtheffects in both humans and wildlife These negative healtheffects include infertility [12] intersexed conditions [12 13]increased risk of endometriosis [14] and increased risk ofcertain cancers (especially hormone-sensitive cancers likebreast and prostate cancers [12])

Xenoestrogens are a specific group of EDCs that disrupthormone signaling by mimicking the actions of estrogen

within the body Several of the polychlorinated biphenyls(PCBs)mdashpersistent organic pollutants found in soil airwater and foodmdashare categorized as xenoestrogens given thefact that they exert estrogenic effects through direct bindingof estrogen receptors particularly ER120572 [15ndash17] A varietyof human tissuesmdashblood adipose tissue and milkmdashexhibitsignificant accumulation of PCBs due to the low degradationrate and fat solubility of these estrogenic pollutants [1819] The PCB concentrations detected in these tissuesmdashinparticular in breast fat and milkmdashfall within the range ofthose found in laboratory studies to exert physiologic effectsvia estrogen receptors [19 20] As a result many scientistshave postulated a potential role for PCBs in the increasedincidence of estrogen-sensitive cancers including breast can-cer Indeed some studies suggest that levels of specific PCBspresent in the breast fat of women are positively correlatedwith the incidence of malignant tumors [21 22] and certainPCBs have been shown to enhance the proliferation ofestrogen-sensitive breast cancer cells in vitro [3 16 17 20]

Recent studies indicate that certain polyphenolic com-pounds found in foods (green tea red wine chocolate and

Hindawi Publishing CorporationInternational Journal of Breast CancerVolume 2015 Article ID 163591 7 pageshttpdxdoiorg1011552015163591

2 International Journal of Breast Cancer

fruits) can also act like xenoestrogens and exert biologiceffects through the activation of estrogen receptors [23 24]However unlike the proliferative effects of certain PCBsexerted via estrogen receptors within cancer cells thesepolyphenolic compounds exert chemopreventive actions viaestrogen receptors in cancer cells Epigallocatechin gallate(EGCG) the major catechin found in green tea is one ofthese chemopreventive compounds in vitro EGCG has beenshown to bind to both ER120572 and ER120573 [23] and to inhibitproliferation of the estrogen-sensitive MCF-7 breast cancercell line [24] In addition to antiproliferative effects exertedvia estrogen receptors EGCG also exerts ER-independentactions that result in inhibition of aryl hydrocarbon- (AhR-)regulated genes and induction of apoptosis [25ndash27]

Several epidemiologic and experimental studies havedemonstrated a positive correlation between the consump-tion of estrogenic polyphenolic compounds and cancer pre-vention [28 29] Furthermore some experiments havedemonstrated that the proliferative effects of environmentalEDCs on cancer cells can be partially or fully inhibitedby cotreatment with polyphenolic compounds [30] Suchfindings suggest that the detrimental health effects of EDCslike the PCBs could potentially be counteracted by a dietthat is rich in polyphenolic chemopreventive compoundslike EGCG (found in green tea) In light of this possibilitythe present study was conducted in order to determinewhether EGCG can inhibit the proliferative effects of anestrogenic PCB (specifically PCB 102) on the proliferation ofthe estrogen-sensitive breast cancer cell line MCF-7BOS

2 Materials and Methods

21 Chemicals and Reagents Epigallocatechin-3-gallate(EGCG) 17120573-estradiol (E2) and dimethylsulfoxide (DMSO)were purchased from Sigma (St Louis MO) 2210158404561015840-Pentachlorobiphenyl (PCB 102) was obtained from Accu-Standard (New Haven CT) The selective ER120572 antagonist13-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth-oxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydro-chloride) and the selective ER120573 antagonist 4-[2-phenyl-57-bis(trifluoromethyl)pyrazolo[15-a]pyrimidin-3-yl]phenol(PHTPP) were purchased from RampD Systems Inc (Minnea-polis MN)

22 Cell Culture MCF-7BOS human breast cancer cellswere kindly provided by Dr Ana Soto (Tufts UniversityBoston MA) The cells were grown in Dulbeccorsquos modi-fied Eaglersquos medium (DMEM) (Hyclone Logan UT) sup-plemented with 5 fetal bovine serum (FBS) (MediatechInc Manassas VA) 100UmL penicillin 100 120583gmL strep-tomycin and 025 120583gmL amphotericin B (Hyclone LoganUT) Cells were cultured as monolayers and maintained at37∘C in a 5 CO

2humidified environment

23 Cell Proliferation Assay MCF-7BOS cells were seededin 12-well plates (Corning Inc Corning NY) at 8 times 104 cellsper well for 24 hours to allow the cells to attach The culturemedium was then changed to phenol red-free DMEM F12

supplemented with 5 CharcoalDextran treated FBS (CD-FBS) (Hyclone Logan UT) containing the test compoundsPCB 102 (1ndash10 120583M) EGCG (10ndash50 120583M) 1 nM E2 1 120583M MPPand 1 120583M PHTPP dissolved in DMSO (final concentration01ndash02) In the assays with PCB 102 plusmn MPP PHTPPor EGCG the cells were cotreated for 72 hours and thenharvested by trypsinization For the EGCG plusmn ER antagonistassays cells were pretreated with 1120583M MPP or PHTPP for24 hours and then treated with EGCG for an additional 72hours before harvesting Cell proliferation was assessed bycounting with a hemocytometer and results were expressedas a percentage of the control cells (media with DMSO) on aplate-by-plate basis In selective experiments the trypan blue(Hyclone Logan UT) dye exclusion assay was used to assesscell viability

24 Statistical Analysis SAS (v 9) for Windows was usedfor statistical analysis Data were expressed as the mean plusmnstandard error (SE) of 3 independent experiments performedin duplicate Statistical differences among the groups wereanalyzed using one-way analysis of variance (ANOVA) fol-lowedbyDunnettrsquos or Tukeyrsquosmultiple comparison testwhereappropriate A statistically significant difference was set at119875 lt 005

3 Results

31 Effect of PCB 102 on MCF-7BOS Breast Cancer CellProliferation The growth of MCF-7BOS cells was increasedby PCB 102 in a dose-dependent manner (Figure 1) MCF-7BOS cells were incubated with 1 25 5 and 10 120583M of PCB102 for 72 hours Cell proliferation was determined by hemo-cytometer 1 120583M of PCB 102 elicited approximately a 40increase in cell proliferation in comparison to the DMSO-treated control cells whereas 5 120583M of PCB 102 induced thehighest stimulation of cell proliferation (250 of control)

32 Effect of ER Antagonists on PCB 102-Mediated Cell Pro-liferation Since some PCBs are known to exhibit estrogenicactivity (reviewed in Discussion) we performed experimentsto determine whether PCB 102-induced cell proliferation wasestrogen receptor- (ER-) mediated To address this questionwe utilized two types of antiestrogens MPP an ER120572-selectiveantagonist and PHTPP an ER120573-selective antagonist Asshown in Figure 2 in the presence of PCB 102 (5120583M) or E2(1 nM) MCF-7BOS cell proliferation increased to approx-imately 250 of control This increase in cell proliferationwas completely blocked with the addition of the ER120572-specificinhibitor MPP (1120583M) whereas the ER120573-specific inhibitorPHTPP (1 120583M) did not inhibit PCB- or E2-induced cellproliferation thus confirming the role of ER120572 rather thanER120573 in mediating the stimulatory effects of PCB 102 on cellproliferation

33 Effect of EGCGAlone and inCombinationwith PCB 102 onMCF-7BOS Breast Cancer Cell Proliferation To determinewhether EGCG can modulate MCF-7BOS cell proliferationcells were incubated with 10 25 and 50120583M EGCG alone for

International Journal of Breast Cancer 3

0

50

100

150

200

250

300

0 1 25 5 10

Cell

pro

lifer

atio

n (

of c

ontro

l)

PCB 102 (120583M)

lowast

lowastlowast

lowastlowast

lowastlowast

Figure 1 PCB 102 increases MCF-7BOS breast cancer cell prolif-eration MCF-7BOS cells were treated with varying concentrationsof PCB 102 (1 25 5 and 10 120583M) for 72 hours Cell numbers weredetermined by hemocytometer and expressed as a percentage of theDMSO control (set at 100) Values are expressed as mean plusmn SE(119899 = 3) Significance of differences between means lowast119875 lt 005lowastlowast119875 lt 0001 compared to DMSO control

0

50

100

150

200

250

300

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOE2PCB 102

lowast lowast

lowast lowast

⧧

Figure 2 PCB 102-induced cell proliferation is mediated by ER120572MCF-7BOS cells were treated with 1 nM E2 or 5 120583M PCB 102 withand without the ER120572-specific inhibitor MPP (1120583M) or the ER120573-specific inhibitor PHTPP (1 120583M) for 72 hours Cell numbers weredetermined by hemocytometer and expressed as a percentage of theDMSO control (set at 100) Values are expressed as mean plusmn SE(119899 = 3) lowastSignificantly different compared to control 119875 lt 005significantly different compared to E2 treatment 119875 lt 005 andsignificantly different compared to PCB treatment 119875 lt 005

72 hours Neither 10 120583M EGCG nor 25 120583M EGCG alone hadan effect on cell proliferation (Figure 3) In contrast 50120583MEGCG alone decreased cell growth approximately 65 with-out a reduction in overall cell viability To determine if EGCGcould suppress PCB 102-induced cell proliferation cells wereincubated with 5120583MPCB 102 alone and in combination with10 25 or 50 120583MEGCGThe addition of EGCG caused a dose-dependent inhibition of PCB 102-induced cell proliferationwith nearly complete inhibition at 25120583M

0

50

100

150

200

250

300

350

0 10 25 50

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOPCB 102

EGCG (120583M)

lowast

lowast

lowast

Figure 3 EGCG suppresses PCB 102-induced cell proliferationMCF-7BOS cells were treated with 5 120583M PCB 102 alone and inthe presence of EGCG (10ndash50 120583M) for 72 hours Cell numbers weredetermined by hemocytometer and expressed as a percentage of theDMSO control (set at 100) Values are expressed as mean plusmn SE(119899 = 3) lowastSignificantly different compared to control 119875 lt 005significantly different compared to PCB treatment 119875 lt 005

34 Effect of EGCG and ER Antagonists on MCF-7BOSBreast Cancer Cell Proliferation Since PCB 102-induced cellproliferation was antagonized by EGCG and blocked by theER120572-specific inhibitor MPP we evaluated the effect of EGCGin combination with the ER antagonists on MCF-7BOScell proliferation We questioned whether the antiestrogenswould block the decrease in cell proliferation mediated by50 120583M EGCG To address this issue MCF-7BOS cells werepretreated with 1 120583MMPP or 1 120583M PHTPP for 24 hours andthen treated with 50 120583M EGCG for an additional 72 hoursCells treated with 50120583M EGCG alone were pretreated withDMSO for 24 hours and then treated with 50 120583M EGCG foran additional 72 hours

As a result of the pretreatments the cells grew for 48 hoursprior to EGCG exposure and thus 50 120583M EGCG alone inthis set of experiments decreased cell growth approximately34 (Figure 4) Also as a result of the pretreatments the cellswere exposed to the ER120572-specific inhibitor MPP (1120583M) andthe ER120573-specific inhibitor PHTPP (1120583M) for a total of 96hours in this set of experiments A time-dependent inhibitionof cell proliferation was observed for cells exposed to ER120572-specific inhibitor MPP alone the 96-hour exposure resultedin a 44 decrease in cell proliferation compared to the con-trol In contrast cells exposed to the ER120573-specific inhibitorPHTPP alone for 96 hours resulted in a 20 increase incell proliferation compared to the control Importantly theantiproliferative effects of 50120583M EGCG on MCF-7BOScells were effectively abrogated by the ER120573-specific inhibitorPHTPP but not by the ER120572-specific inhibitor MPP

4 Discussion

In this study we evaluated the effects of PCB 102 and the greentea catechin EGCG individually and in combination on cell


0

20

40

60

80

100

120

140

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOEGCG

lowast

lowast

lowast

lowastsect

sect

Figure 4 ER120573-specific inhibitor PHTPP blocks inhibition of cellproliferation by EGCG MCF-7BOS cells were pretreated with theER120572-specific inhibitor MPP (1120583M) or the ER120573-specific inhibitorPHTPP (1 120583M) for 24 hours and then treated with 50120583M EGCGfor an additional 72 hours Cell numbers were determined byhemocytometer and expressed as a percentage of the DMSO control(set at 100) Values are expressed as mean plusmn SE (119899 = 3)lowastSignificantly different compared to control 119875 lt 005 sectsignificantlydifferent compared to treatment with EGCG alone 119875 lt 005

proliferation in estrogen-sensitive MCF-7BOS breast cancercells PCB 102 found in the commercially used PCB mixtureAroclor 1242 was selected for our study because Aroclor 1242is a major source of local contamination in the Fox RiverWisconsin due to use by the paper industry in this region[31] We found that PCB 102 stimulated cell proliferation inMCF-7BOS breast cancer cells in a dose-dependent mannerThe proliferative effects of PCB 102 were mediated by ER120572 afinding consistent with what has been observed with otherPCBs exhibiting estrogenic activity [15 32] and could beblockedwith the ER120572-specific inhibitorMPP but not with theER120573-specific inhibitor PHTPP To our knowledge this is thefirst report to show that PCB 102 exhibits estrogenic activityAdditionally the proliferative effects of E2 in MCF-7BOScells (used as positive control) were completely abrogated bythe selective ER120572 antagonist MPP in MCF-7BOS cells asobserved previously in normalmammary epithelial cells PC3prostate cancer cells and MCF-7 breast cancer cells [33ndash35]

Interestingly the proliferative effects of PCB 102 via ER120572could also be antagonized by the green tea catechin EGCGWe found that EGCG caused a dose-dependent inhibitionof PCB 102-induced cell proliferation with nearly completeinhibition at 25120583M EGCG Green tea extract and EGCGthe major catechin in green tea both suppress the activityof estrogen via ER120572 and block ER120572-dependent transcription[36 37] Our results suggest that EGCG also has the abilityto suppress the activity of environmental estrogens Similarfindings have been reported for the phytoestrogens genisteinand luteolin which suppress the estrogenic activity of theindustrial environmental estrogens alkylphenols and bisphe-nol A (BPA) respectively [38]

Previous studies have shown that EGCG inhibits breastcancer cell proliferation by downregulating cyclin and CDK

expression as well as inducing expression of the CDKinhibitors p21 and p27 thereby triggering G1 cell cycle arrest[39 40] Sensitivity to EGCG treatment is highly dependenton cell type [24 41] and in some cancer cell lines highconcentrations of EGCG (ge85 120583M) induce apoptosis [4142] In our studies with MCF-7BOS cells treatment withEGCG alone did not inhibit cell growth until the highestconcentration of 50120583M was reached Our results are similarto other studies usingMCF-7 cells in which 40ndash50120583MEGCGtreatment for 72 hours resulted in inhibition of proliferationwithout a significant decrease in cell viability [42ndash44] Inaddition we found that the susceptibility to EGCG treatmentalone in MCF-7BOS cells was affected by the timing ofEGCG exposure after plating Adding 50120583M EGCG aloneto cells 24 hours after plating (as used for the PCB plusmnEGCG cotreatment experiments) resulted in a 65 decreasein proliferation whereas adding the same concentrationto MCF-7BOS cells 48 hours after plating (as used forthe EGCG plusmn ER antagonists experiment due to 24-hourpretreatment with antagonists) resulted in a 34 decreasein proliferation A possible explanation for this difference inEGCG susceptibility after platingmay be the fact that the cellsexposed at a later time are physiologically more stable

While the concentrations of EGCG (10ndash50 120583M) used inour experiments are within the same range as many othershort-term cell culture studies the concentrations exceed theaverage maximum plasma concentration of 8 120583M detected inhumans after oral administration of EGCG [45] Howeverlong-term culture of breast cancer cells with 8120583MEGCG alsoresulted in growth inhibition [46] Furthermore sera frombreast cancer patients given EGCG orally for several weeksin combination with radiotherapy inhibited in vitro culturesof metastatic breast cancer cells [47] Together these findingssupport the chemoprotective potential of EGCG in vivo

Although EGCG is able to bind to both ER isoformssince the 3-gallate group mimics the 7120572-position of E2 [48]it has a greater binding affinity for ER120573 than ER120572 [23] MCF-7BOS cells express both ER120572 and ER120573 however ER120572 isthe predominant ER type expressed [49] In cells expressingboth receptor types ER120573 acts as a dominant repressor ofER120572 function thus ER120573 negatively modulates ER120572-mediatedtranscriptional activity [50ndash52] and inhibits ER120572-mediatedbreast cancer cell proliferation and tumor formation [51 5354] Given the protective effect of ER120573 due to its ability tocounteract ER120572 activity we wanted to investigate whetherER120573 plays an important role in mediating inhibition of cellproliferation by 50120583M EGCG alone The antiproliferativeaction of EGCGalone inMCF-7BOS cells was blocked by theER120573-specific inhibitor PHTPP but not by the ER120572-specificinhibitor MPP thus our results indicate that EGCG is mostlikely acting as an ER120573 agonist to inhibit cell proliferationEGCG has been previously shown to act as an ER120573 agonistin MCF-7 cells at the same concentrations used in ourstudy (10ndash50120583M) [23] Other phytoestrogens have also beenshown to preferentially bind [55] and utilize ER120573 over ER120572to modulate transcriptional activity and cell proliferation[56] The antiproliferative action of the flavonoid apigenin inprostate and breast cancer cells is mediated by ER120573 [57] as arethe antiproliferative effects of soy isoflavones in colon cancer


cells [58] and the phytoestrogen farrerol in vascular smoothmuscle cells [59] In all cases inhibition of cell proliferationwas reversed by using the ER120573-specific antagonist PHTPPor siRNA knockdown of ER120573 but not by the ER120572-specificantagonist MPP or siRNA knockdown of ER120572 [57 59]

We also found that inhibition of ER120573 using the ER120573-specific antagonist PHTPP resulted in a significant increasein MCF-7BOS cell proliferation even in the absence of E2A similar effect has been observed in normal mammaryepithelial cells and T47D and MCF-7 breast cancer cells[33 60 61] In contrast inhibition of ER120572 using the ER120572-specific inhibitor MPP for an extended amount of time(96 hours) resulted in a significant decrease in MCF-7BOScell proliferation These results were in accordance to thoseobtained previously with normal mammary epithelial cells[33] Together these findings suggest that cell proliferation inMCF-7BOS cells is influenced by the ER120572ER120573 ratio Thusthe ability of EGCG to mediate its antiproliferative actionvia ER120573 and modify the estrogenic activity of industrialenvironmental estrogens via ER120572 may contribute to its roleas a dietary chemopreventive agent

While the focus of this study is on the ability of EGCG tosuppress the proliferative effects of PCBs in estrogen respon-sive cells EGCG may also be able to counteract the effects ofPCBs in estrogen receptor negative (ERminus) breast cancer cellsPCBs induce overexpression of vascular endothelial growthfactor (VEGF) [62] enhance cell migration and promotemetastasis in ERminus breast cancer cells [63] In contrast EGCGinhibits VEGF expression migration and invasion of ERminusbreast cancer cells [64] In addition breast cancer patientsgiven EGCG orally plus radiotherapy showed significantlylower serum levels of VEGF and reduced activity of invasionpromotingmetalloproteinases [47] compared to patients whoreceived radiotherapy alone Thus the ability of EGCG tocounteract the detrimental effects of PCBs in ERminus breastcancer cells warrants further investigation

5 Conclusion

Our results demonstrate that PCB 102 exhibits estrogenicactivity and induces cell proliferation in estrogen-sensitiveMCF-7BOSbreast cancer cells via ER120572 in a dose-dependentmanner Furthermore this study demonstrates that EGCGcan suppress the estrogenic activity of polychlorinatedbiphenyls specifically PCB 102 in breast cancer cells invitro The antiproliferative action of EGCG was blocked bythe ER120573-specific inhibitor PHTPP thus the results suggestthat EGCG can act as an ER120573 agonist to induce growthinhibition These findings are significant since they indicatethat EGCG has the ability to mitigate the detrimental effectsof industrial environmental estrogens in human breast cancercells Moreover they further illustrate how consumptionof green tea may play an important role in breast cancerchemoprevention

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was funded in part by a grant from the Universityof Wisconsin-Green Bay Institute for Research (to KimberlyMantzke Baker)The authors thankMrM Damie andMs AGruen for technical assistance with this project The authorsalso thank Dr D Dolan for assistance and advice regardingthe statistical analysis of their data

References

[1] J T SandersonW Seinen J P Giesy andM Van Den Berg ldquo2-Chloro-s-triazine herbicides induce aromatase (CYP19) activityin H295R human adrenocortical carcinoma cells a novelmechanism for estrogenicityrdquo Toxicological Sciences vol 54no 1 pp 121ndash127 2000

[2] D M Kristensen M L Skalkam K Audouze et al ldquoManyputative endocrine disruptors inhibit prostaglandin synthesisrdquoEnvironmental Health Perspectives vol 119 no 4 pp 534ndash5412011

[3] A M Soto C Sonnenschein K L Chung M F Fernandez NOlea and F Olea Serrano ldquoThe E-SCREEN assay as a tool toidentify estrogens an update on estrogenic environmental pol-lutantsrdquoEnvironmentalHealth Perspectives vol 103 supplement7 pp 113ndash122 1995

[4] M R Fielden I Chen B Chittim S H Safe and T RZacharewski ldquoExamination of the estrogenicity of 2462101584061015840-pentachlorobiphenyl (PCB 104) its hydroxylated metabolite2462101584061015840-pentachloro-4-biphenylol (HO-PCB 104) and a fur-ther chlorinated derivative 246210158404101584061015840-hexachlorobiphenyl(PCB 155)rdquo Environmental Health Perspectives vol 105 no 11pp 1238ndash1248 1997

[5] P Alonso-Magdalena O Laribi A B Ropero et al ldquoLow dosesof bisphenol A and diethylstilbestrol impair Ca2+ signals inpancreatic 120572-cells through a nonclassical membrane estrogenreceptor within intact islets of Langerhansrdquo EnvironmentalHealth Perspectives vol 113 no 8 pp 969ndash977 2005

[6] T B Hayes V Khoury A Narayan et al ldquoAtrazine inducescomplete feminization and chemical castration in male Africanclawed frogs (Xenopus laevis)rdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 107 no10 pp 4612ndash4617 2010

[7] S Flint T Markle S Thompson and E Wallace ldquoBisphenol Aexposure effects and policy a wildlife perspectiverdquo Journal ofEnvironmental Management vol 104 pp 19ndash34 2012

[8] M F Rahman E K Yanful and S Y Jasim ldquoEndocrine disrupt-ing compounds (EDCs) and pharmaceuticals and personal careproducts (PPCPs) in the aquatic environment implications forthe drinking water industry and global environmental healthrdquoJournal of Water and Health vol 7 no 2 pp 224ndash243 2009

[9] M Krause A Klit M B Jensen et al ldquoSunscreens are theybeneficial for health An overview of endocrine disruptingproperties of UV-filtersrdquo International Journal of Andrology vol35 no 3 pp 424ndash436 2012

[10] T E Stoker E K Gibson and L M Zorrilla ldquoTriclosan expo-sure modulates estrogen-dependent responses in the femalewistar ratrdquo Toxicological Sciences vol 117 no 1 pp 45ndash53 2010

[11] G W Louis D R Hallinger and T E Stoker ldquoThe effect oftriclosan on the uterotrophic response to extended doses ofethinyl estradiol in the weanling ratrdquo Reproductive Toxicologyvol 36 pp 71ndash77 2013


[12] S De Coster and N van Larebeke ldquoEndocrine-disruptingchemicals associated disorders and mechanisms of actionrdquoJournal of Environmental and Public Health vol 2012 ArticleID 713696 52 pages 2012

[13] L Gaspari F Paris C Jandel et al ldquoPrenatal environmentalrisk factors for genital malformations in a population of 1442French male newborns a nested casecontrol studyrdquo HumanReproduction vol 26 no 11 pp 3155ndash3162 2011

[14] D Balabanic M Rupnik and A K Klemencic ldquoNegativeimpact of endocrine-disrupting compounds on human repro-ductive healthrdquoReproduction Fertility andDevelopment vol 23no 3 pp 403ndash416 2011

[15] S Tavolari L Bucci V Tomasi and T Guarnieri ldquoSelectedpolychlorobiphenyls congeners bind to estrogen receptor alphain human umbilical vascular endothelial (HUVE) cells modu-lating angiogenesisrdquo Toxicology vol 218 no 1 pp 67ndash74 2006

[16] B R DeCastro S A Korrick J D Spengler and A M SotoldquoEstrogenic activity of polychlorinated biphenyls present inhuman tissue and the environmentrdquo Environmental Science ampTechnology vol 40 no 8 pp 2819ndash2825 2006

[17] S Kitamura N Jinno T Suzuki et al ldquoThyroid hormone-likeand estrogenic activity of hydroxylated PCBs in cell culturerdquoToxicology vol 208 no 3 pp 377ndash387 2005

[18] S Safe ldquoToxicology structure-function relationship andhuman and environmental health impacts of polychlorinatedbiphenyls progress and problemsrdquo Environmental HealthPerspectives vol 100 pp 259ndash268 1993

[19] M S Wolff P G Toniolo E W Lee M Rivera and N DubinldquoBlood levels of organochlorine residues and risk of breastcancerrdquo Journal of the National Cancer Institute vol 85 no 8pp 648ndash652 1993

[20] K Nesaretnam D Corcoran R R Dils and P Darbreldquo343101584041015840-Tetrachlorobiphenyl acts as an estrogen in vitro andin vivordquo Molecular Endocrinology vol 10 no 8 pp 923ndash9361996

[21] K J Aronson A BMiller C GWooleott et al ldquoBreast adiposetissue concentrations of polychlorinated biphenyls and otherorganochlorines and breast cancer riskrdquo Cancer EpidemiologyBiomarkers and Prevention vol 9 no 1 pp 55ndash63 2000

[22] A Demers P Ayotte J Brisson S Dodin J Robert and EDewailly ldquoPlasma concentrations of polychlorinated biphenylsand the risk of breast cancer a congener-specific analysisrdquoAmerican Journal of Epidemiology vol 155 no 7 pp 629ndash6352002

[23] M G Goodin K C Fertuck T R Zacharewski and R JRosengren ldquoEstrogen receptor-mediated actions of polypheno-lic catechins in vivo and in vitrordquo Toxicological Sciences vol 69no 2 pp 354ndash361 2002

[24] PWang SMHenning andDHeber ldquoLimitations ofMTTandMTS-based assays for measurement of antiproliferative activityof green tea polyphenolsrdquo PLoS ONE vol 5 no 4 Article IDe10202 2010

[25] S G Han S-S Han M Toborek and B Hennig ldquoEGCG pro-tects endothelial cells against PCB 126-induced inflammationthrough inhibition of AhR and induction of Nrf2-regulatedgenesrdquo Toxicology and Applied Pharmacology vol 261 no 2 pp181ndash188 2012

[26] A M Roy M S Baliga and S K Katiyar ldquoEpigallocatechin-3-gallate induces apoptosis in estrogen receptor-negative humanbreast carcinoma cells via modulation in protein expressionof p53 and Bax and caspase-3 activationrdquo Molecular CancerTherapeutics vol 4 no 1 pp 81ndash90 2005

[27] Y-C Hsu and Y-M Liou ldquoThe anti-cancer effects of (-)-Epigalocathine-3-gallate on the signaling pathways associatedwith membrane receptors in MCF-7 cellsrdquo Journal of CellularPhysiology vol 226 no 10 pp 2721ndash2730 2011

[28] C S Yang P Maliakal and XMeng ldquoInhibition of carcinogen-esis by teardquoAnnual Review of Pharmacology and Toxicology vol42 pp 25ndash54 2002

[29] S Ulrich F Wolter and J M Stein ldquoMolecular mechanismsof the chemopreventive effects of resveratrol and its analogs incarcinogenesisrdquoMolecular Nutrition and Food Research vol 49no 5 pp 452ndash461 2005

[30] S-H Tu C-Y Ku C-T Ho et al ldquoTea polyphenol (-)-epi-gallocatechin-3-gallate inhibits nicotine- and estrogen-induced1205729-nicotinic acetylcholine receptor upregulation in humanbreast cancer cellsrdquo Molecular Nutrition amp Food Research vol55 no 3 pp 455ndash466 2011

[31] I Imamoglu K Li E R Christensen and J K McMullinldquoSources and dechlorination of polychlorinated biphenyl con-geners in the sediments of Fox River Wisconsinrdquo Environmen-tal Science amp Technology vol 38 no 9 pp 2574ndash2583 2004

[32] P Ruiz E Myshkin P Quigley et al ldquoAssessment of hydrox-ylated metabolites of polychlorinated biphenyls as potentialxenoestrogens a QSAR comparative analysisrdquo SAR and QSARin Environmental Research vol 24 no 5 pp 393ndash416 2013

[33] L A Helguero M H Faulds J A Gustafsson and L-AHaldosen ldquoEstrogen receptors alfa (ER120572) and beta (ER120573)differentially regulate proliferation and apoptosis of the normalmurine mammary epithelial cell line HC11rdquo Oncogene vol 24no 44 pp 6605ndash6616 2005

[34] E Powell E Shanle A Brinkman et al ldquoIdentification of estro-gen receptor dimer selective ligands reveals growth-inhibitoryeffects on cells that co-express ER120572 and ER120573rdquo PLoS ONE vol 7no 2 Article ID e30993 2012

[35] H-J Li L-Y Wang H-N Qu et al ldquoP2Y2 receptor-mediatedmodulation of estrogen-induced proliferation of breast cancercellsrdquoMolecular andCellular Endocrinology vol 338 no 1-2 pp28ndash37 2011

[36] R Kuruto-Niwa S Inoue S Ogawa M Muramatsu andR Nozawa ldquoEffects of tea catechins on the ERE-regulatedestrogenic activityrdquo Journal of Agricultural and Food Chemistryvol 48 no 12 pp 6355ndash6361 2000

[37] M R Sartippour R Pietras D C Marquez-Garban et al ldquoThecombination of green tea and tamoxifen is effective againstbreast cancerrdquo Carcinogenesis vol 27 no 12 pp 2424ndash24332006

[38] D-H Han M S Denison H Tachibana and K YamadaldquoRelationship between estrogen receptor-binding and estro-genic activities of environmental estrogens and suppression byflavonoidsrdquo Bioscience Biotechnology and Biochemistry vol 66no 7 pp 1479ndash1487 2002

[39] Y-C Liang S-Y Lin-Shiau C-F Chen and J-K Lin ldquoInhi-bition of cyclin-dependent kinases 2 and 4 activities as well asinduction of Cdk inhibitors p21 and p27 during growth arrest ofhuman breast carcinoma cells by (-)-epigallocatechin-3-gallaterdquoJournal of Cellular Biochemistry vol 75 no 1 pp 1ndash12 1999

[40] K T Kavanagh L J Hafer D W Kim et al ldquoGreen tea extractsdecrease carcinogen-induced mammary tumor burden in ratsand rate of breast cancer cell proliferation in culturerdquo Journal ofCellular Biochemistry vol 82 no 3 pp 387ndash398 2001

[41] F Farabegoli C Barbi E Lambertini and R Piva ldquo(minus)-Epigallocatechin-3-gallate downregulates estrogen receptor


alpha function inMCF-7 breast carcinoma cellsrdquo Cancer Detec-tion and Prevention vol 31 no 6 pp 499ndash504 2007

[42] A Mittal M S Pate R C Wylie T O Tollefsbol and S KKatiyar ldquoEGCG down-regulates telomerase in human breastcarcinoma MCF-7 cells leading to suppression of cell viabilityand induction of apoptosisrdquo International Journal of Oncologyvol 24 no 3 pp 703ndash710 2004

[43] T-C Hsieh and J MWu ldquoSuppression of cell proliferation andgene expression by combinatorial synergy of EGCG resveratroland 120574-tocotrienol in estrogen receptor-positive MCF-7 breastcancer cellsrdquo International Journal of Oncology vol 33 no 4pp 851ndash859 2008

[44] G-J Du Z Zhang X-D Wen et al ldquoEpigallocatechin gallate(EGCG) is the most effective cancer chemopreventive polyphe-nol in green teardquo Nutrients vol 4 no 11 pp 1679ndash1691 2012

[45] H-H S Chow I AHakimD RVining et al ldquoEffects of dosingcondition on the oral bioavailability of green tea catechinsafter single-dose administration of Polyphenon E in healthyindividualsrdquo Clinical Cancer Research vol 11 no 12 pp 4627ndash4633 2005

[46] E P Moiseeva G M Almeida G D D Jones and M MManson ldquoExtended treatment with physiologic concentrationsof dietary phytochemicals results in altered gene expressionreduced growth and apoptosis of cancer cellsrdquo MolecularCancer Therapeutics vol 6 no 11 pp 3071ndash3079 2007

[47] G Zhang Y Wang Y Zhang et al ldquoAnti-cancer activities oftea epigallocatechin-3-gallate in breast cancer patients underradiotherapyrdquo Current Molecular Medicine vol 12 no 2 pp163ndash176 2012

[48] H Fang W Tong L M Shi et al ldquoStructuremdashactivity rela-tionships for a large diverse set of natural synthetic andenvironmental estrogensrdquo Chemical Research in Toxicology vol14 no 3 pp 280ndash294 2001

[49] S Wang J M M J G Aarts N M Evers A A C M Peij-nenburg I M C M Rietjens and T F H Bovee ldquoProliferationassays for estrogenicity testing with high predictive value for thein vivo uterotrophic effectrdquoThe Journal of Steroid Biochemistryand Molecular Biology vol 128 no 3ndash5 pp 98ndash106 2012

[50] T F H Bovee R J R Helsdingen I M C M Rietjens J Keijerand R L A P Hoogenboom ldquoRapid yeast estrogen bioassaysstably expressing human estrogen receptors 120572 and 120573 and greenfluorescent protein a comparison of different compounds withboth receptor typesrdquo The Journal of Steroid Biochemistry andMolecular Biology vol 91 no 3 pp 99ndash109 2004

[51] E C Chang J Frasor B Komm and B S KatzenellenbogenldquoImpact of estrogen receptor 120573 on gene networks regulated byestrogen receptor 120572 in breast cancer cellsrdquo Endocrinology vol147 no 10 pp 4831ndash4842 2006

[52] J Matthews B Wihlen M Tujague J Wan A Strom andJ-A Gustafsson ldquoEstrogen receptor (ER) 120573 modulates ER120572-mediated transcriptional activation by altering the recruitmentof c-Fos and c-Jun to estrogen-responsive promotersrdquo Molecu-lar Endocrinology vol 20 no 3 pp 534ndash543 2006

[53] A Strom J Hartman J S Foster S Kietz J Wimalasena andJ-A Gustafsson ldquoEstrogen receptor 120573 inhibits 17120573-estradiol-stimulated proliferation of the breast cancer cell line T47DrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 6 pp 1566ndash1571 2004

[54] S Paruthiyil H Parmar V Kerekatte G R Cunha G LFirestone and D C Leitmant ldquoEstrogen receptor 120573 inhibitshuman breast cancer cell proliferation and tumor formation by

causing a G2 cell cycle arrestrdquoCancer Research vol 64 no 1 pp423ndash428 2004

[55] G G J M Kuiper J G Lemmen B Carlsson et al ldquoInteractionof estrogenic chemicals and phytoestrogens with estrogenreceptor 120573rdquo Endocrinology vol 139 no 10 pp 4252ndash4263 1998

[56] J An C Tzagarakis-Foster T C Scharschmidt N Lomri andD C Leitman ldquoEstrogen receptor 120573-selective transcriptionalactivity and recruitment of coregulators by phytoestrogensrdquoTheJournal of Biological Chemistry vol 276 no 21 pp 17808ndash178142001

[57] P Mak Y-K Leung W-Y Tang C Harwood and S-MHo ldquoApigenin suppresses cancer cell growth through ER120573rdquoNeoplasia vol 8 no 11 pp 896ndash904 2006

[58] A Bielecki J Roberts RMehta and J Raju ldquoEstrogen receptor-120573 mediates the inhibition of DLD-1 human colon adenocarci-noma cells by soy isoflavonesrdquo Nutrition and Cancer vol 63no 1 pp 139ndash150 2011

[59] Q-Y Li L Chen Y-H Zhu M Zhang Y-P Wang andM-W Wang ldquoInvolvement of estrogen receptor-120573 in farrerolinhibition of rat thoracic aorta vascular smooth muscle cellproliferationrdquo Acta Pharmacologica Sinica vol 32 no 4 pp433ndash440 2011

[60] O Treeck C Lattrich A Springwald and O OrtmannldquoEstrogen receptor beta exerts growth-inhibitory effects onhuman mammary epithelial cellsrdquo Breast Cancer Research andTreatment vol 120 no 3 pp 557ndash565 2010

[61] A M Sotoca D Ratman P van der Saag et al ldquoPhytoestrogen-mediated inhibition of proliferation of the human T47D breastcancer cells depends on the ER120572ER120573 ratiordquo The Journal ofSteroid Biochemistry andMolecular Biology vol 112 no 4-5 pp171ndash178 2008

[62] S Y Eum Y W Lee B Hennig and M Toborek ldquoVEGFregulates PCB 104-mediated stimulation of permeability andtransmigration of breast cancer cells in human microvascularendothelial cellsrdquo Experimental Cell Research vol 296 no 2 pp231ndash244 2004

[63] S Liu S Li and Y Du ldquoPolychlorinated biphenyls (PCBs)enhance metastatic properties of breast cancer cells by activat-ing rho-associated kinase (ROCK)rdquo PLoS ONE vol 5 no 6Article ID e11272 2010

[64] C Braicu C D Gherman A Irimie and I Berindan-NeagoeldquoEpigallocatechin-3-gallate (EGCG) inhibits cell proliferationand migratory behaviour of triple negative breast cancer cellsrdquoJournal of Nanoscience and Nanotechnology vol 13 no 1 pp632ndash637 2013

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


fruits) can also act like xenoestrogens and exert biologiceffects through the activation of estrogen receptors [23 24]However unlike the proliferative effects of certain PCBsexerted via estrogen receptors within cancer cells thesepolyphenolic compounds exert chemopreventive actions viaestrogen receptors in cancer cells Epigallocatechin gallate(EGCG) the major catechin found in green tea is one ofthese chemopreventive compounds in vitro EGCG has beenshown to bind to both ER120572 and ER120573 [23] and to inhibitproliferation of the estrogen-sensitive MCF-7 breast cancercell line [24] In addition to antiproliferative effects exertedvia estrogen receptors EGCG also exerts ER-independentactions that result in inhibition of aryl hydrocarbon- (AhR-)regulated genes and induction of apoptosis [25ndash27]

Several epidemiologic and experimental studies havedemonstrated a positive correlation between the consump-tion of estrogenic polyphenolic compounds and cancer pre-vention [28 29] Furthermore some experiments havedemonstrated that the proliferative effects of environmentalEDCs on cancer cells can be partially or fully inhibitedby cotreatment with polyphenolic compounds [30] Suchfindings suggest that the detrimental health effects of EDCslike the PCBs could potentially be counteracted by a dietthat is rich in polyphenolic chemopreventive compoundslike EGCG (found in green tea) In light of this possibilitythe present study was conducted in order to determinewhether EGCG can inhibit the proliferative effects of anestrogenic PCB (specifically PCB 102) on the proliferation ofthe estrogen-sensitive breast cancer cell line MCF-7BOS

2 Materials and Methods

21 Chemicals and Reagents Epigallocatechin-3-gallate(EGCG) 17120573-estradiol (E2) and dimethylsulfoxide (DMSO)were purchased from Sigma (St Louis MO) 2210158404561015840-Pentachlorobiphenyl (PCB 102) was obtained from Accu-Standard (New Haven CT) The selective ER120572 antagonist13-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth-oxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydro-chloride) and the selective ER120573 antagonist 4-[2-phenyl-57-bis(trifluoromethyl)pyrazolo[15-a]pyrimidin-3-yl]phenol(PHTPP) were purchased from RampD Systems Inc (Minnea-polis MN)

22 Cell Culture MCF-7BOS human breast cancer cellswere kindly provided by Dr Ana Soto (Tufts UniversityBoston MA) The cells were grown in Dulbeccorsquos modi-fied Eaglersquos medium (DMEM) (Hyclone Logan UT) sup-plemented with 5 fetal bovine serum (FBS) (MediatechInc Manassas VA) 100UmL penicillin 100 120583gmL strep-tomycin and 025 120583gmL amphotericin B (Hyclone LoganUT) Cells were cultured as monolayers and maintained at37∘C in a 5 CO

2humidified environment

23 Cell Proliferation Assay MCF-7BOS cells were seededin 12-well plates (Corning Inc Corning NY) at 8 times 104 cellsper well for 24 hours to allow the cells to attach The culturemedium was then changed to phenol red-free DMEM F12

supplemented with 5 CharcoalDextran treated FBS (CD-FBS) (Hyclone Logan UT) containing the test compoundsPCB 102 (1ndash10 120583M) EGCG (10ndash50 120583M) 1 nM E2 1 120583M MPPand 1 120583M PHTPP dissolved in DMSO (final concentration01ndash02) In the assays with PCB 102 plusmn MPP PHTPPor EGCG the cells were cotreated for 72 hours and thenharvested by trypsinization For the EGCG plusmn ER antagonistassays cells were pretreated with 1120583M MPP or PHTPP for24 hours and then treated with EGCG for an additional 72hours before harvesting Cell proliferation was assessed bycounting with a hemocytometer and results were expressedas a percentage of the control cells (media with DMSO) on aplate-by-plate basis In selective experiments the trypan blue(Hyclone Logan UT) dye exclusion assay was used to assesscell viability

24 Statistical Analysis SAS (v 9) for Windows was usedfor statistical analysis Data were expressed as the mean plusmnstandard error (SE) of 3 independent experiments performedin duplicate Statistical differences among the groups wereanalyzed using one-way analysis of variance (ANOVA) fol-lowedbyDunnettrsquos or Tukeyrsquosmultiple comparison testwhereappropriate A statistically significant difference was set at119875 lt 005

3 Results

31 Effect of PCB 102 on MCF-7BOS Breast Cancer CellProliferation The growth of MCF-7BOS cells was increasedby PCB 102 in a dose-dependent manner (Figure 1) MCF-7BOS cells were incubated with 1 25 5 and 10 120583M of PCB102 for 72 hours Cell proliferation was determined by hemo-cytometer 1 120583M of PCB 102 elicited approximately a 40increase in cell proliferation in comparison to the DMSO-treated control cells whereas 5 120583M of PCB 102 induced thehighest stimulation of cell proliferation (250 of control)

32 Effect of ER Antagonists on PCB 102-Mediated Cell Pro-liferation Since some PCBs are known to exhibit estrogenicactivity (reviewed in Discussion) we performed experimentsto determine whether PCB 102-induced cell proliferation wasestrogen receptor- (ER-) mediated To address this questionwe utilized two types of antiestrogens MPP an ER120572-selectiveantagonist and PHTPP an ER120573-selective antagonist Asshown in Figure 2 in the presence of PCB 102 (5120583M) or E2(1 nM) MCF-7BOS cell proliferation increased to approx-imately 250 of control This increase in cell proliferationwas completely blocked with the addition of the ER120572-specificinhibitor MPP (1120583M) whereas the ER120573-specific inhibitorPHTPP (1 120583M) did not inhibit PCB- or E2-induced cellproliferation thus confirming the role of ER120572 rather thanER120573 in mediating the stimulatory effects of PCB 102 on cellproliferation

33 Effect of EGCGAlone and inCombinationwith PCB 102 onMCF-7BOS Breast Cancer Cell Proliferation To determinewhether EGCG can modulate MCF-7BOS cell proliferationcells were incubated with 10 25 and 50120583M EGCG alone for


0

50

100

150

200

250

300

0 1 25 5 10

Cell

pro

lifer

atio

n (

of c

ontro

l)

PCB 102 (120583M)

lowast

lowastlowast

lowastlowast

lowastlowast


0

50

100

150

200

250

300

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOE2PCB 102

lowast lowast

lowast lowast

⧧



0

50

100

150

200

250

300

350

0 10 25 50

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOPCB 102

EGCG (120583M)

lowast

lowast

lowast




4 Discussion



0

20

40

60

80

100

120

140

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOEGCG

lowast

lowast

lowast

lowastsect

sect












5 Conclusion




Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

300

0 1 25 5 10

Cell

pro

lifer

atio

n (

of c

ontro

l)

PCB 102 (120583M)

lowast

lowastlowast

lowastlowast

lowastlowast


0

50

100

150

200

250

300

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOE2PCB 102

lowast lowast

lowast lowast

⧧



0

50

100

150

200

250

300

350

0 10 25 50

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOPCB 102

EGCG (120583M)

lowast

lowast

lowast




4 Discussion



0

20

40

60

80

100

120

140

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOEGCG

lowast

lowast

lowast

lowastsect

sect












5 Conclusion




Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

120

140

+MPP +PHTPP

Cell

pro

lifer

atio

n (

of c

ontro

l)

DMSOEGCG

lowast

lowast

lowast

lowastsect

sect












5 Conclusion




Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion




Acknowledgments


References










































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article green tea catechin, egcg, suppresses pcb...

Documents