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Carcinogenesls vol.17 no.2 pp.271-275, 1996

Molecular effects of genistein on estrogen receptor mediatedpathways

Thomas T.Y.Wang, Neeraja Sathyamoorthy andJames M.PhangLaboratory of Nutritional and Molecular Regulation, NCIFrederick CancerResearch and Development Center, NIH, Frederick, MD 21702-1201, USAGenistein, a component of soy products, may play a rolein the prevention of breast and prostate cancer. However,little is known about the molecular mechanisms involved.In the present study, we examined the effects of genisteinon the estrogen receptor positive human breast cancercell line MCF-7. We observed that genistein stimulatedestrogen-responsive pS2 mRNA expression at concentra-tions as low as 10~ 8 M and these effects can be inhibitedby tamoxifen. We also showed that genistein competed with[3H]estradiol binding to the estrogen receptor with 50inhibition at 5 X 10~ 7 M. Thus, the estrogenic effect ofgenistein would ap pear to be a result of an interaction w iththe estrogen receptor. The effect of genistein on growth ofMCF-7 cells was also examined. Genistein produced aconcentration-dependent effect on the growth of MCF-7cells. At lower concentrations (10~8 - 1 0 ~ 6 M) genisteinstimulated growth, but at higher concentrations (> 10 ~ 5M )genistein inhibited growth. The effects of genistein ongrowth at lower concentrations appeared to be via theestrogen receptor pathway, while the effects at higherconcentrations were independent of the estrogen receptor.We also found that genistein, though estrogenic, can inter-fere with the effects of estradiol. In addition, prolongedexposure to genistein resulted in a decrease in estrogenreceptor mRNA level as well as a decreased response tostimulation by estradiol.

IntroductionIn epidemiological studies, an increased intake of soy productsis associated with a decreased risk of hormone-dependentcancers (1,2). In rodents, ingestion of soy products was foundto inhibit mammary tumorigenesis (3). It has been suggestedthat genistein, an isoflavone abundant in soy products, maycontribute to the chemopreventive effects of soy products forhormone-dependent cancers (2,4). However, the molecularmechanisms by which genistein exerts its effects remain poorlyunderstood. This is not due to a lack of proposed and identifiedeffects. On the one hand, genistein has been shown to stimulateproliferation, presumably through estrogen-like effects (5).On the other hand, it is considered to be antiproliferative,presumably due to inhibition of tyrosine kinases (6-8) andtopoisomerase (9). Until its mechanisms of action are betterunderstood, the chemopreventive potential of genistein isunlikely to be fully realized.

In the present study, we focused on the effect of genisteinA bb rev iat ion s: ER, estrogen receptor; CD S, charcoal dextran-treated fetalbovine serum; DIG, digoxigenin; G3PDH, glyceraldehyde 3-phosphatedehydrogenase.

on the estrogen receptor (ER*) pathway. Using the humanbreast cancer MCF-7 cells, we examined the effects of RNAon: (i) the expression of pS2 mRNA; (ii) the displacement ofestrogen from binding to the ER; (iii) cellular proliferation;and (iv) regulation of the ER. The results from these studieswere used to address the following questions, (i) Is genisteinestrogenic? (ii) Could genistein be anti-estrogenic? (iii) Whatcontributes to the paradoxical ob servation that genistein stimu-lates both proliferative and anti-proliferative responses?Materials and methodsChemicalsGenistein was purchased from Sigma Chemical Co. (St Louis, MO). Quercetinwas purchased from Indofine Chemical Co. (Somerville, NJ). Tamoxifen waspurchased from Aldrich Chemical Co. (Milwaukee, WI). These compoundswere >95% pure. All other chemicals were from the best sources available.Cells and cell cultureMCF-7 cells were maintained in Medium A [RPMI 1640 with 2 mM L-glutamine, hydrocortisone (3.5 ng/ml), insulin (1.5 ng/ml), penicillin (100 U/ml) and streptomycin (100 mg/ml)] with 5% fetal bovine serum in 75 cm 2flasks (Falcon) and grown in the presence of 5% CO2 in air at 37C. Oneweek before initiation of the experiment, cells were switched to Medium Asupplemented with 5% charcoal dextran-treated fetal bovine serum (CDS).Cells were grown to confluence and passaged using trypsinEDTA. Viablecells were counted by Trypan blue exclusion. A day prior to treatment thecells were switched to Medium B [phenol-red free RPMI 1640 containing2 mM glutamine, penicillin (100 U/ml), streptomycin (100 mg/ml) and5% CDS].ro e for pS2 and ERDigoxigenin (DIGHabeled pS2 cRNA probe (10) was synthesized using anucleic acid labeling kit following the manu facturer's instructions (BoehringerMannheim Biochemicals, Indianapolis, IN). For production of the DIG-ERcRNA probe, plasmid pOR3 carrying the ER cDNA (11) was obtained fromthe American Type Culture Collection. The pOR3 was digested with EcoRIand a 1.35 kb cDNA fragment coding for the ER was inserted into the EcoRIsite of plasmid pGEM-4Z. The resulting plasmid was designated pGEM/ER.DIG-labeled ER cRNA probe was generated from Nari linearized pGEM/ER using the SP6 RNA polymerase following the procedure describedpreviously (10).Total RNA isolation. Northern blot hybridization and detectionFor isolation of total RNA, MCF-7 cells were grown in six-well Costar plates(1 X 1 0 * cells/well in 3 ml of Medium B ) and isolated as described previously(10). RNA was resuspended in 70-100 ul DEPC-treated water, and 10 ul ofRNA was routinely used for Northern blot analysis. For detection of mRNA,10 fj.1 of total RNA was separated on a 1% agarose formaldehyde gel andtransferred to nylon membrane (Boehringer Mannheim) using the PossiBlotPressure Blotter (Stratagene, La Jolla, CA). The RNA was fixed onto themembrane by UV crosslinking in a Stratalinker (Stratagene) for 3 min at thehighest power. The membrane was then hybridized with DIG-labeled RNAprobe and detected by a chemiluminescence nucleic acid detection methodaccording to the manufacturer's protocol (Boehringer Mannheim). All RNAsamples were also probed for either glyceraldehyde 3-phosphate dehydrogenase(G3PDH) or pVactin mRNA, both of which were used to normalize specificexpression stimulated by various treatments. After exposure to X-ray film(Kodak, Rochester, NY), the intensity of the pS2, ER, G3PDH or fj-actinmRNA bands were quantitated by densitometry (Analtech Uniscan, Newark,DE). Densitometer readings were normalized for either G3PDH or p*-actinRNA content and expressed as relative expression level.Growth assayCell growth was determined by the sulforhodamine colorimetric assay asdescribed by Skehan et al. (12) except that the cells were seeded in 24-well

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T.T.Y.Wang et al.plates instead of 96-microtiter plates. Routinely, 1 X 10 4 cells in 1 ml ofMedium B were seeded to each well of a 24-well plate (Costar, Cambridge,MA). Twenty-four hours after plating, either genistein or vehicle was added.The medium was changed every 24 h and the test compounds were replenishedwith each medium change. After color development, aliquots were pipettedinto a 96-well microtiter plate and the intensity of the developed color wasmeasured at 570 nm using an ELISA microplate reader (Molecular Devices,Menlo Park, CA).ER binding studyMCF-7 cells were grown to confluence in 150 cm 2 flasks (Becton DickinsonLabware, Lincoln Park, NJ) and cultured in Medium A supplemented with5% fetal bovine serum at 37C in the presence of 5% CO2 in air. The cellswere rinsed twice with PBS, scraped off in 5 ml of PBS and pelleted at1500 r.p.m. for 10 min. After aspiration of the supernatant, the cell pelletswere suspended in lysis buffer consisting of 10 raM Tris-HCl, pH 7.4, 1.5 mMEDTA and 0.5 mM dithiothreitol. The cell suspension was homogenized usinga Tekmar Tissumizer (Tekmar Co., Cincinnati, OH) for 45 s. The homogenatewas then centrifuged at 40 000 r.p.m. for 50 min and the supernatant saved.After determination of the protein concentration, the supernatant was used forER binding assays. ER binding assay was performed essentially as describedby McGuire (13). Briefly, the ER binding studies were carried out as follows:200 (il of the cytosol fraction (2.0 mg/ml protein) was incubated with 10~ 9M[3H]estradiol (New England Nuclear, Boston, MA) for 18 h at 4C. Followingincubation, 0.5 ml of dextran-coated charcoal was added to the assay mixture,and incubated at 4C for 30 min with vortexing at 5 min intervals. The assaymixture was then centrifuged at 2000 g for 10 min and 0.5 ml of thesupernatant was removed for measurement of the bound [ 3H]estradiol byliquid scintillation spectrometry. For competition experiments, binding assayswere run as described above in the presence of increasing amounts ofcompetitor (0-10~ 6M) while the concentration of estradiol was held constantat 10 9 M .

ResultsEffect of genistein on pS2 mRNA expression and estradiolbinding to ERThe structure of the compounds used in the study is illustratedin Figure 1. To characterize the estrogen-like property ofgenistein we monitored the expression of estrogen-dependentpS2 mRNA using a chemiluminescence detection methodreported previously (10). As illustrated in Figure 2, stimulationof pS2 mRNA expression occurred in a concentration-depend-ent fashion. The stimulatory effect of genistein on pS2 mRNAexpression was observed at concentrations as low as 10~ 8M .As illustrated in Figure 3(A), the stimulation ofpS2expressionby genistein (10~ 7 M) could be blocked by the additionof tamoxifen (10~ 5 M). In addition, actinomycin D at aconcentration of 3 ^g/ml inhibited the genistein-stimulatedpS2 expression (Figure 3B). These results support the hypo-

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thesis that genistein acts through an ER-mediated transcrip-tional event.To characterize further the interaction between genistein andthe ER pathway, we examined the ability of genistein tocompete with [3H]estradiol (10~ 9 M) for binding to the ER.As shown in Figure 4, genistein can compete with estradiolfor binding to the ER. In contrast, quercetin, which does notstimulate pS2 expression (10), did not compete with estradiolfor binding to ER. The concentration required for genistein toproduce a 50% inhibition of binding was 5 X 10~ 7M. Theseresults, together with the results from the pS2 expression assay,supported the interpretation that genistein induces an estrogenicresponse by interacting with the ER.Anti-estrogenic effects of genisteinIt is known that anti-estrogens such as tamoxifen can also actas weak estrogens (14). Hence, like tamoxifen, genistein may

Genistein109 10 s1 0 ' 1 06 1 0 5 Cone. (M)

0 - 8 . , , - 7Genistein (M) 10

Figs. 2. Concentration-dependent stimulation of pS2 expression bygenistein. MCF-7 cells were cultured in Media B in the presence of variousconcentrations of genistein (10~ 9 - 1 0 ~ 5 M). Total RNA was isolated after48 h. Northern blot and detection for pS2 and fi-actin mRNA wasperformed as described in Materials and methods. The results are expressedas relative pS2 expression.

A BTamo xifen Actinomycin D

Fig. 1. Structure of estradiol, genistein and quercetin.27 2

Fig. 3. Effects of tamoxifen and actinomycin D on genistein-stimulated pS2mRNA level. (A) Effects of tamoxifen on genistein-stimulated pS2 mRNAexpression. Cells were incubated in the presence of genistein (10~ 7 M) andwith or without tamoxifen (10~ 5 M) for 48 h. Total RNA was isolated after48 h and pS2 mRNA level determined as described in Materials andmethods. (B) Effects of actinomycin D on genistein-stimulated pS2 mRNAexpression. Cells were incubated in the presence of genistein (10~ 7 M) withor without actinomycin D (3 Jig/ml) for 48 h. Total RNA was isolated after48 h and pS2 mRNA level determined as described in Materials andmethods.

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Molecular effects of genlstein on estrogen receptor mediated pathways

Enndiol

Qucrcctlii

O-M I O- ' M[Compe t i to r ]

Fig. 4. Effects of genistein and quercetin on the binding of [3H]estradiol tothe ER. ER binding assays were performed as described in Materials andmethods. Competitors (estradiol, genistein and quercetin) were added at theindicated concentrations with 10~ 9 M [3H]estradiol. The results (mean SE,n = 3) are expressed as a percentage of control.

Control+Estradiol

0.00 0- 7 0 - 6

Fig. 5. Effects of genistein on estradiol-stimulated pS2 mRNA expression.MCF-7 cells were incubated with genistein (10~ 7 or 1O~6M) alone,estradiol ( 1 0 M) alone, or media containing both genistein and estradiol.Total RNA was isolated after 48 h and pS2 mRNA expression wasdetermined as described in Materials and methods. The results (meanSE,n = 3) are expressed as relative level of pS2 mRNA expression. Data wereanalyzed using ANOVA and post hoc comparison using Fisher's PLSD test.Bars with different letters are significantly different from each other P

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MCF-7MDA-MB-231

0 1 0 8 1 0 * 1 0 7 10- 1CTS 1 0 4

Genistcin(M)Fig. 8. Effects of varied concentrations of genistein on growth of ER-positive (MCF-7) and ER-negative (MDA-MB-231) cells. Cells werecultured in the presence of various concentrations of genistein( 1 0 ~ 9 - 1 0 ~ 4 M) for 5 d ays. Cell growth was determined as described inMaterials and methods. The results (meanSE, n = 4) are expressedrelative to cells grown without genistein.

work in this area. In cultured cells, both proliferative and anti-proliferative effects have been ascribed to genistein (5-9). Wefound that in the ER-positive human breast cancer cell lineMCF-7, the concentration-dependent effect of genistein ongrowth was in fact biphasic. At low (1 0 ~ 8 - 1 0 5M) concentra-tions genistein stimulated cell growth, while at high(2.5 X 10~ 5 - 1 0 ~ 4 M) concentrations genistein inhibited cellgrowth. Since genistein failed to stimulate the proliferation ofER-ne gative c ells, we concluded that the proliferative effectof genistein was mediated through ER. The effects of genisteinon pS2 mRNA expression and binding of estradiol to ERprovide additional support for this conclusion because: (i)genistein stimulated both proliferation and pS2 expression ina concentration-depend ent m anner over the concentration rangeof 10~ 8 - 1 0 ~ 5 M; (ii) the affinity of genistein towards ERK, = 10~7 M) is in the same concentration range thatwe observed increased pS2 mRNA expression and cellularproliferation; (iii) stimulation of pS2 mRNA expression bygenistein was inhibited by the anti-estrogen tamoxifen.

By contrast, the anti-proliferative effect on MCF-7 cellsobserved with high concentrations of genistein (2.5 X1 0 5 - 1 0 ~ 4 M) appeared independent of ER. This growthinhibition was also observed in ER-negative cells in which nogrowth stimulatory effect was observed at low genisteinconcentrations. Since the growth inhibitory concentration isin the range for inhibition of tyrosine kinases (6-8) andtopoisomerases (9), it is likely that growth inhibition seen intissue culture at genistein concentrations >2.5 X 10~5M mayresult from perturbation of post-receptor signaling pathwaysand/or interference with DNA synthesis, and is, in fact,independe nt of ER . Initially, the aforementioned effects mightappear to be involved in the mechanism by which genisteincontributes to the decreased risk in hormone-dependent cancersassociated with ingestion of soy products. However, this isunlikely since in humans the circulating level of genisteindoes not exceed 2.5 X 10~ 5 M (15,16). Even with increasedintake of soy products, circulating levels of genistein normallydo not exceed 10~ 6M (15,16). It is more likely that the effectsof dietary genistein on tumorigenesis are mediated through dieER, for which it has a relatively high affinity K{ = 10 ~7M ).

Results from the present study provide evidence for theanti-estrogenic effects of genistein. We observed that not onlydoes genistein compete with estradiol for binding to the ER,but it also interferes w ith the effects of estrogen . When genisteinand estradiol were added together in the pS2 expression assay,the expression of pS2 mRNA was lower than when thesecompounds were added individually. In addition, the durationof exposure to genistein also appeared to be important. MCF-7 cells treated with genistein showed a decrease in the responseto estrogen as well as a decrease in ER mRNA levels. This issimilar to cells treated with estradiol, as has been describedby others (17). Taken together, the results support the ideathat genistein at physiologically achievable concentrationsmay, in fact, interfere with the action of estrogen throughdirect competition for binding to ER and by reducing ERexpression. These effects could be considered anti-estrogenic,consistent with the proposed cancer-preventive effects ofsoy diet.In summary, our findings provide support for a model inwhich phyto-estrogens such as genistein, at physiologicalconcentrations, may exert their effect by modulating estrogenicpathways. This interaction may occur directly through com-petition with estrogen for binding to ER. In addition, pro-longed exposure may result in the reduction of ER expressionand hence lead to decreased responsiveness to endogenousestrogens.

Referencesl.DunnJ.E., Jr (1975) Cancer epidemiology in populations of the UnitedStatesemphasis on Hawaii and Californiaand Japan.Cancer Res.,3 5 ,3240-3245.2. MessinaJvlJ., Persky.V., Setchell,K.D.R. and Bames.S. (1994) Soy intakeand cancer risk: a review of the in-vitro and in-vivo data. Nutr. Cancer,21 , 415-131.3.Barnes,S., Grubbs,C. Setchell.K.D.R. and arlsonJ. (1990) Soybeansinhibit mammary tumors in models of breast cancer. In Pariza,M.,Felton J.S., A eschbacher,H.-U. and Sa to.S. (eds),Mutagens and Carcino-gens in the Diet. Wiley-Liss, New York, pp. 239-253.4.Lamartiniere,C.A., MooreJ., Holland.M. and Barnes.S. (1995) Neonatalgenistein chemoprevents mammary cancer. Proc. Soc. Exp. Biol. Med.,208, 120-123.5. Miksicek,RJ. (1993) Commonly occurring plant flavonoids have estrogenicactivity.Mol. Pharmacol., 44 , 37-43.6. Akiyama.T., IshidaJ., Naka gawa .S., Ogawara,H ., Watanabe,S.-I., Itoh.N.,Shibuya,M and Fukami.Y. (1987) Gen istein, a specific inhibitor oftyrosine-specific protein kinase. /. Biol. Chem., 262, 5592-5595.7.Hill,T.D., Dean,N.M., Mordan,LJ., Lau.A.F., Kanemitsu,M.Y. andBoynton,A.L. (1990) PDGF-induced activation of phospholipase C is notrequired for induction of DNA synthesis.Science,248, 1660-1663.8.Fotsis,T., Pepper,M., Adlercreutz,H., Fleischmann.G., Hase.T., Montesano,R. and Schweigerer.L. (1993) Genistein, a dietary derived inhibitor ofin vitro angiogenesis.Proc. NatlAcad. Sci. USA,90, 2690-2694.9.Okura,A., Arakawa.H., Oka,H., Yoshinari.T. and Monden,Y. (1988) Effectof genistein on topoisomerase activity on the growth of [Val 12]Ha-ros-transformed NIH3T3cells.Biochem. Biophys.Res.Commun.,157,183-189.lO.Sathyamoorthy.N., Wang.T.T.Y. and PhangJ.M. (1994) Stimulation of pS2expression by diet-derived compounds.CancerRes.,54,957-961.11.Walter,P., Green.S., Greene.G., Krust^A., BomerU-M., JeetschJ.-M.,StaubA, Jensen.E., Scrace.G., Waterfield,M. and Chambon.P. (1985)Cloning ofthe human estrogen receptor cDNA.Proc. NatlAcad. Sci. USA,82 ,7889-7893.12.Skehan,P, Storeng.R., Scudiero.D., M on ks A, McM ahonJ., Vistica,D.,Warren J.T., B okesch.H., Kenney .S. and Boyd Jtf.R. (1990) Newcolorimetric cytotoxicity assay for anticancer-drug screening. J.Nail CancerInst.,82, 1107-1112.13.McGuire,W. (1975) Quantitation of estrogen receptor in mammarycarcinoma. In O'Malley,B.W. and HardmanJ.G. (eds). Methods ofEnzymology.A cademic Press, New York, Vol. 36, pp. 248-25 4.

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Molecular effects of genistein on estrogen receptor mediated pathwaysW.WakelingAE. (1992) Steroid antagonists as nuclear receptor blockers. InParker,M.G. (ed.), Growth Regulation by Nuclear Hormone Receptors.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 71-S5.15.Adlercreutz,H., Markkanen,R and Watanabe.S. (1993) Plasma concen-tration of phytoestrogens in Japanese men. Lancet, 342, 1209-1210.16.Xu,X., W ang,H.-J., Murphy.P.A., Cook.L. and He ndrich.S. (1994) D aidzeinis a more bioavailable soymilk isoflavone than is genistein in adult wom en./ . Nutr., 124, 825-832.17.Read,L.D., Greene.G.L. and Katzenellenbogen.B.S. (1989) Regulation ofestrogen receptor messenger ribonucleic acid and protein levels in humanbreast cancer cell lines by sex steroid hormones, their antagonist, and

growth factors. Mol Endocrinol, 3, 295-304.Received on May 18, 1995; revised on October27 1995; accepted on October30, 1995

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