Breast Cancer Cell Apoptosis with Phytoestrogens Is Dependent on an Estrogen-Deprived State

Published OnlineFirst June 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0061

Cancer Prevention Research Article Research

Ifeyinwa E. Obiorah, Ping Fan, and V. Craig Jordan

Abstract Phytoestrogens have been investigated as natural alternatives to hormone replacement therapy and their potential as chemopreventive agents. We investigated the effects of equol, genistein, and coumestrol on cell growth in fully estrogenized MCF7 cells, simulating the perimenopausal state, and long-term estrogen- deprived MCF7:5C cells, which simulate the postmenopausal state of a woman after years of estrogen

deprivation, and compared the effects with that of steroidal estrogens: 17b estradiol (E2) and equilin present in conjugated equine estrogen. Steroidal and phytoestrogens induce proliferation of MCF7 cells at physiologic concentrations but inhibit the growth and induce apoptosis of MCF7:5C cells. Although steroidal and phytoestrogens induce estrogen-responsive genes, their antiproliferative and apoptotic effects are mediated through the estrogen receptor. Knockdown of ERa using siRNA blocks all estrogen-induced apoptosis and growth inhibition. Phytoestrogens induce endoplasmic reticulum stress and inflammatory response stress–related genes in a comparable manner as the steroidal estrogens. Inhibition of inflammation using dexamethasone blocked both steroidal- and phytoestrogen-induced apoptosis and growth inhibition as well as their ability to induce apoptotic genes. Together, this suggests that phytoestrogens can potentially be used as chemopreventive agents in older postmenopausal women but caution should be exercised when used in conjunction with steroidal anti-inflammatory agents due to their antiapoptotic effects. Cancer Prev Res; 7(9); 939–49. 2014 AACR.

Introduction breast cancer who had previous exhaustive anti-hormone Acquired resistance to antihormone therapy occurs therapy. Ellis and colleagues (7) showed that postmeno- despite the successful use of endocrine treatment to improve pausal women with aromatase inhibitors resistant meta- survival in patients with breast cancer. Early laboratory static breast cancer, had a 29% clinical benefit with low- models show that retransplantation of tamoxifen-resistant dose estrogen (6-mg daily) but the same clinical benefit but tumors into ovarectomized athymic mice led to tumor more side effects with high-dose estrogen (30-mg daily). Additional clinical evidence for the antitumor action of low- growth in response to tamoxifen and estradiol (E2; refs. 1, 2). Continued retransplantation of the tamoxifen-stimulat- dose estrogen comes from the Women Health Initiative ed tumors in nude mice for up to 5 years resulted to a rapid (WHI) trial, which compared conjugated equine estrogen (CEE) therapy with placebo in hysterectomized postmen- regression of the tumors in response to E2 (3). This corre- opausal women that show a persistent decrease in the lates with the finding that E2 induces apoptosis in long-term estrogen-deprived MCF7 breast cancer cells (4, 5). The use incidence and mortality of breast cancer in women who of estrogens has been beneficial in the treatment of meta- received estrogen alone therapy (8, 9). Studies in vitro show static breast cancer in postmenopausal women with that constituents of CEE cause apoptosis in long-term acquired resistance to endocrine therapy. A clinical study estrogen-deprived MCF7 cells (10). The clinical and labo- (6) found that high-dose diethylstilbestrol induced an ratory studies suggest that the ability of estrogen therapy to objective response in 30% of patients with postmenopausal treat or prevent tumors is most apparent in the postmenopausal state of a woman and how long they have been physiologically deprived of estrogen (10). Phytoestrogens are plant-derived polyphenolic com- Authors' Affiliation: Department of Oncology, Lombardi Comprehensive pounds that are structurally similar to E . Phytoestrogens Cancer Center, Georgetown University Medical Center, Washington, DC 2 consists of isoflavones (genistein and diadzein), coume- Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/). stans (coumestrol), the lignans (enterolactone and enter- odiol), and stilbenes (resveratrol). Isoflavones are princi- Corresponding Author: V. Craig Jordan, Georgetown University Medical Center, 3970 Reservoir Road North West, Research Building, Suite E501, pally found in soy-based products, which are staple foods Washington, DC 20057. Phone: 202-687-2897; Fax: 202-687-6402; E-mail: in many Asian countries and are becoming increasing [email protected] popular in Western countries. An inverse relationship found doi: 10.1158/1940-6207.CAPR-14-0061 between soy consumption in Asian countries and decreased 2014 American Association for Cancer Research. breast cancer risk has sparked a sustained interest in the use

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of phytoestrogens in breast cancer prevention. However, the In this study, we have evaluated the apoptotic and clear beneficial effects of these estrogens remain controver- potential chemopreventive effects of phytoestrogens sial. Several meta-analysis (11–13) that assessed soy expo- using a unique cell model that simulates a postmeno- sure and breast cancer risk revealed that studies conducted pausal cellular environment. Genistein, coumestrol, and in Asian countries showed a significant trend of a reduced equol, a gastrointestinal metabolite of diadzein, are used risk with increased soy intake in both pre- and postmeno- in comparison with E2 and equilin a constituent of CEE in pausal Asian women. On the other hand, no association hormone replacement therapy (HRT) to determine their was observed between soy consumption and breast cancer proliferative and apoptotic potential using fully estrogen- risk in low soy consuming Western populations (11, 13), ized and an estrogen-deprived breast cancer cells, respec- suggesting that consumption of soy products in amounts tively. Here, we test the hypothesis that the phytoestro- taken in the Asian population may have protective benefits. gens have biologic effects similar to that of E2 and CEE in Evaluation of the breast cancer–protective effects of isofla- breast cancer prevention and this may have clinical impli- vones stratified by menopausal status is still undefined. cations for the strategic use of phytoestrogens as alter- Trock and colleagues (14) reported in their meta-analysis, natives to HRT in postmenopausal populations. a stronger association between soy exposure and breast cancer risk in premenopausal women. However, the anal- Materials and Methods yses included studies with incomplete measurements, Cell culture and reagents potential confounders, and lack of a dose–response that Cell culture media were purchased from Invitrogen Inc. make the findings inconclusive. On the other hand, another and fetal calf serum (FCS) was obtained from HyClone study reported that adult or adolescent soy consumption Laboratories. Compounds E2, equilin, equol, genistein was associated with reduced risk of premenopausal breast and coumestrol (Supplementary Fig. S1), ICI 182,780, cancer (15) and no significant associations were reported and 4-hydroxytamoxifen (4OHT; were obtained from Sig- for the risk of postmenopausal breast cancer. Furthermore, ma). Dexamethasone was obtained from Tocris Bios- there is increased evidence that the chemoprotective effects ciences. MCF7:5C were derived from MCF7 cells obtained of isoflavones are dependent on early exposure. High soy from the Dr. Dean Edwards (University of Texas, San consumption during adolescence is associated with reduced Antonio, TX) as reported previously (35). MCF7:WS8 cells risk of adult breast cancer (15–17). This concurs with the were derived from MCF7 cells as previously described (35) findings in animal model experiments, in which prepuber- and maintained in RPMI media supplemented with 10% tal exposure to genistein causes mammary gland differen- FCS, 6 ng/mL bovine insulin and penicillin and strepto- tiation, thereby resulting in increased breast cancer preven- mycin. The MCF7:WS8 and the MCF7:5C cells have been tion (18, 19). The effect of phytoestrogens in breast cancer fully characterized and experiments were done as previ- cells has been extensively studied. At low pharmacologic ously reported (36). The expected growth/apoptotic concentrations, phytoestrogens stimulate the growth of responses to E2, biomarker statuses of ERa, progesterone estrogen receptor (ER)–positive breast cancer cells (20– receptor (PgR), and HER2, and ER-regulated transcription- > m 22). In contrast at high concentrations ( 5 mol/L), these al activity were confirmed in both cell lines. Protein levels plant-derived estrogens inhibit the growth of the cancer of ERa, PgR, and HER2 were characterized by semiquan- cells (21, 23, 24). Ingestion of soy isoflavones in healthy titative immunoblot analysis and ER transcriptional activ- premenopausal women resulted in increased breast tissue ity was evaluated using an estrogen-responsive element– proliferation (25), epithelial hyperplasia (26), and a weak regulated dual luciferase reporter gene system (36). The estrogenic response in inducing estrogen-regulated mar- last characterization was reported in (37) and the DNA kers (27). On the other hand in postmenopausal women, fingerprinting patterns of the cell lines were consistent soy supplementation resulted in either a protective effect with the report by the American Type Culture Collection. (28) or no effect (29–31) on breast cancer risk. Only MCF7 cells are cultured in phenol-red–free RPMI media one of the postmenopausal studies (31) consisted of containing 10% charcoal dextran–treated FCS, 6 ng/mL healthy subjects, the rest included patients with breast bovine insulin and penicillin and streptomycin for 3 days cancer. Fink and colleagues (32) reported a decreased all- before starting experiments. MCF7:5C cells were main- cause mortality in patients with pre- and postmenopausal tained in phenol-red–free RPMI media containing 10% breast cancer who had a high intake of isoflavones, dextran-coated charcoal-treated FCS, 6 ng/mL bovine insu- whereas a reduced breast cancer–related mortality was lin and penicillin and streptomycin. The cells were treated observed in postmenopausal women. However, the Diet- with indicated compounds (with media change every 48 CompLyf study (33), which investigated associations hours) for the specified time and were subsequently har- between phytoestrogens and breast cancer recurrence and vested for tissue culture experiments. survival, found no significant associations between pre- diagnosis phytoestrogen intake and reduced breast cancer Cell growth assay risk. Interestingly, Shu and colleagues (34) found that soy The cell growth was monitored by measuring the total food consumption was significantly associated with DNA content per well in 24-well plates. Fifteen thousand decreased risk of death and recurrence in patients with cells were plated per well and treatment with indicated breast cancer. concentrations of compounds was started after 24 hours,

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in triplicates. Media containing the specific treatments were reagent from Bio-Rad Laboratories. Of note, 25 mg of total changed every 48 hours. On day 7, the cells were harvested protein was separated on 10% sodium dodecyl sulfate– and total DNA was assessed using a fluorescent DNA quan- polyacrylamide gel and transferred to a nitrocellulose mem- tification kit (Cat # 170-2480; Bio-Rad) and was performed brane. The membrane was probed with primary antibodies as previously described (4). followed by incubation with secondary antibody conjugated with horseradish peroxidase and reaction with Western RNA isolation and real-time PCR Lighting plus-ECL enhanced chemiluminescent substrate Total RNA was isolated using TRizol reagent (Invitrogen) (PerkinElmer Inc.). ERa and b antibodies were from Santa and RNAeasy kit according to the manufacturer’s instruc- Cruz Biotechnology. Phosphorylated eIF2a, total eIF2a, tions. Real-time PCR (RT-PCR) was performed as previously IRE1a, and b-actin antibodies were from Cell Signaling described (38). The sequences for all primers are documen- Technology. Protein bands were visualized by exposing the ted in Supplementary Table S1. The change in expression membrane to X-ray film. of transcripts was determined as described previously and used the ribosomal protein 36B4 mRNA as the internal Small interfering RNA transfection control (38). For transient transfections, MCF7:5C cells were seeded at a density of 50% to 70% in 6-well plates in estrogen- Apoptosis assay free RPMI media containing 10% SFS. The following day, In brief, MCF7:5C cells were seeded in 100-mm dishes cells were transfected with 100 nmol/L small interfering and cultured overnight in estrogen-free RPMI-1640 medi- RNAs (siRNA) for ER a (Dharmacon; SMART pool: ON- um containing 10% stripped fetal calf serum (SFS). The next TARGETplus ESR1 siRNA product number L-003401-00- day, cells were treated with <0.1% ethanol (control), E2 0005) and ERb (Dharmacon; SMART pool: ON-TARGET- (1 nmol/L), equilin (1 nmol/L), equol (1 mmol/L), genis- plus ESR2 siRNA product number L-003402-00-0005) tein (1 mmol/L), and coumestrol (1 mmol/L) for 72 hours using DharmaFECT transfection reagent (Dharmacon; and the cells were detached using accutase (Life Technol- product number T-2001-03), according to the manufac- ogies), a marine-origin enzyme with proteolytic and col- turer’s recommended protocol. Nontarget siRNA was lagenolytic activity, in 1:3 dilution using PBS (Invitrogen) as purchased from Dharmacon and was used as a control the diluent. The cells were collected by centrifugation for (Silencer-negative control siRNA, product number D- 2 minutes at 500 Â g. Cells were then resuspended and 001810-01-20). The cells were harvested 72 hours post- stained simultaneously with either FITC-labeled Annexin V transfection and analyzed by Western blot analysis (as and propidium iodide (PI; Pharmingen) or DNA-binding described above). Transfected cells were also treated with dye, YO-PRO-1, and PI (Life Technologies). Apoptosis was vehicle, steroidal estrogens, or phytoestrogens for either verified on the basis of loss of plasma membrane integrity. an additional 72 hours, or 6 days and apoptotic cells and Viable cells excluded these dyes, whereas apoptotic cells DNA content were measured using Annexin V staining allowed moderate staining. Cells were analyzed using a and DNA quantification assays, respectively (as described fluorescence-activated cell sorter (FACS) flow cytometer above). (Becton Dickinson). The percentage of apoptosis was cal- culated by adding the percentage of cells stained with either Statistical analysis Annexin V alone (early apoptosis) in the right lower quad- All data are expressed as the mean of at least three rant and those stained with both PI and Annexin V (late determinations, unless otherwise stated. The differences apoptosis) in the right upper quadrant. Experiments are between the treatment groups and the control group were repeated three times with similar results. determined by one-factor analysis of variance [ANOVA, with Tukey posttest and two-way ANOVA with Bonferroni Cell cycles analysis posttest using GraphPad Prism, version 5.00 (GraphPad MCF7:5C cells were cultured in dishes and were treated Software Inc.)]. Results were considered statistically signif- < with <0.1% ethanol (control), E2 (1 nmol/L), equilin (1 icant if the P 0.05. nmol/L), equol (1 mmol/L), genistein (1 mmol/L), and coumestrol (1 mmol/L) for the indicated times. Cells were Results harvested and gradually fixed with 75% EtOH on ice. After Effect of phytoestrogens on breast cancer cells staining with PI, cells were analyzed using a FACS flow On the basis of the controversy surrounding breast cancer cytometer (Becton Dickinson), and the data were analyzed risk and the use of phytoestrogens, we decided to determine with Modfit software. the biologic properties of the genistein, equol, and coumestrol in comparison with E2 and equilin in two different Immunoblotting models of breast cancer cell models. Estrogens have been Proteins were extracted in cell lysis buffer (Cell Signaling shown to regulate the growth of ER-positive MCF7 breast Technology) supplemented with Protease Inhibitor Cock- cancer cells. First, we tested the ability of test compound to tail (Roche) and Phosphatase Inhibitor Cocktail Set I and induce proliferation in MCF7:WS8 cells, which are estro- Set II (Calbiochem). Total protein content of the lysate was gen-responsive breast cancer cells grown in fully estrogen- determined by a standard bicinchoninic acid assay using the ized medium. MCF7:WS8 cells were grown in estrogen-free

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media for 3 days and treated with various concentrations (14.89%), and coumestrol (17.83%) all show increased of genistein, coumestrol, and equol and their effects were apoptotic staining compared with the control-treated cells compared with E2 and equilin (Fig. 1A). The phytoestro- (Fig. 2A). A similar effect was noted using a DNA-binding À9 gens, equol (EC50, 1.72 Â 10 ), genistein (EC50, 1.08 Â stain, YO-PRO-1 (Supplementary Fig. S2). E2, equilin, and À8 À9 10 ), and coumestrol (EC50, 3.07 Â 10 ), all stimulated all phytoestrogens induced apoptotic genes; BCL2L11/ cell growth in a concentration-related manner with maxi- BIM, TNF, FAS, and FADD (Fig. 2B and C) after 48 hours mum stimulation occurring at 0.1 mmol/L, whereas E2 of treatment. Induction of these genes is consistent with À12 À11 (EC50, 3.11 Â 10 ) and equilin (EC50, 1.01 Â 10 ) the apoptotic status determined using the flow cytometry. maximally induced cell growth at 10 pmol/L and 0.1 Although evidence of apoptosis occurs with the phytoes- nmol/L, respectively. trogens by 48 hours, a consistent increase in the S phase Growth inhibition was observed with the phytoestrogens when compared with the control was observed with all À À at 10 mmol/L with genistein (10 5 mol/L vs. 10 7 mol/L; P < estrogens (Supplementary Fig. S3). In contrast with other 0.05). Next, we investigated the growth properties of the reports (23, 39), which indicate that genistein causes a genistein, equol, and coumestrol in long-term estrogen- G2–M arrest, no checkpoint blockade was noted after deprived MCF7:5C cells in comparison with E2 and equilin treatment with all compounds, indicating that the initial À8 (Fig. 1B). Genistein (IC50, 2.77 Â 10 ), equol (IC50, 4.67 Â response of the cells to estrogens is growth, then apopto- À8 À8 10 ), and coumestrol (IC50, 2.34 Â 10 ) drastically sis in MCF7:5C cells. inhibited the growth of the MCF7:5C cells at higher concentrations compared with E2. Maximum growth inhibition Phytoestrogens possess estrogenic properties was observed with all phytoestrogens at 0.1 mmol/L. E2 mediated through the ER in the MCF7:5C cells À11 (IC50, 2.06 Â 10 ) achieved maximum growth inhibition We explored the ability of phytoestrogens to regulate Â À10 at 0.1 nmol/L, whereas equilin (IC50, 2.32 10 ) reached estrogen response genes in comparison with E2 and maximum growth inhibition at 1 nmol/L after 7 days of equilin. Genistein, equol, and coumestrol were all able treatment. to induce TFF1/PS2 and GREB1 (Fig.3A).Phytoestrogens have been shown to induce apoptosis through an ER- Phytoestrogens induce apoptosis in a long-term independent mechanism (21, 40). To evaluate the estrogen-deprived breast cancer cell line involvement of ER in the effects of the phytoestrogens, On the basis of the fact that the decrease in cell growth we investigated their antiproliferative effects in the pres- observed with the steroidal estrogens is due to apoptosis ence of 4OHT (Fig. 3B) and ICI 182 780 (Supplementary (10), we investigated whether the antiproliferative effects Fig. S4). The combination of various concentrations of of the phytoestrogens were also due to an increase in 4OHT or ICI 182 780 with E2, equilin and each phytoes- apoptosis. MCF7:5C cells were treated with E2 (1 nmol/L), trogens blocked estrogen-induced apoptosis, suggesting equilin (1 nmol/L), genistein (1 mmol/L), equol (1 mmol/L), that the phytoestrogens mediate apoptosis via the ER. We and coumestrol (1 mmol/L) for 72 hours and stained with sought to examine the effects of genistein, equol, and Annexin V–FITC and PI fluorescence and cells were ana- coumestrol on the ER. Following treatment of MCF7:5C lyzed using the flow cytometry. In the control-treated cells with E2, equilin, and the phytoestrogens for 24 group, only 6.8% of cells stained for apoptosis, whereas hours, ERa levels were determined by Western blotting. E2 (24.56%), equilin (17.49%), genistein (14.79%), equol All phytoestrogens caused a decrease in the ERa protein

E2 14 12 E A Equilin B 2 Equilin Equol Equol Figure 1. Growth characteristics of Genistein 12 Genistein 10 17b-estradiol, equilin, and Coumestrol Coumestrol phytoestrogens in breast cancer 10 8 cells. A, MCF7:WS8 cells were seeded in 24-well plate and treated 8 with steroidal and phytoestrogens 6 over a range of doses for 7 days. 6 * Cell growth was assessed as DNA 4 content in each well. B, inhibition of DNA ( m g/well) DNA ( m g/well) 4 cell growth in MCF7:5C cells by genistein, equol, and coumestrol 2 2 was assessed in comparison with E2 and equilin. Each data point, 0 0 average Æ SD of three replicates; Ã, P < 0.05. -log [M] -log [M]

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A Control E2 Equilin a102813_217.fcs a102813_205.fcs a102813_206.fcs 104 104 104 2.79% 4.67% 5.06% 16.81% 5.05% 13.96% 103 103 103

2 2 PI PI 2 10 10 PI 10

101 101 101 90.37% 2.17% 70.37% 7.75% 77.46% 3.53% 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Annexin V–FITC Annexin V–FITC Annexin V–FITC

Equol Genistein Coumestrol

a102813_207.fcs a102813_208.fcs a102813_209.fcs 4 4 4 10 5.23% 10 5.33% 10 11.52% 12.49% 7.95% 15.49% 103 103 103

PI 2 PI 2 10 10 PI 10

101 101 101 79.92% 3.33% 79.88% 2.30% 74.23% 2.34% 100 100 100 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Annexin V–FITC Annexin V–FITC Annexin V–FITC B C 4.0 7 FAS BIM * 6 3.5 * TNF * FADD * 3.0 * * 5 ** * * * * 2.5 * 4 * * * 2.0 * * * 3 1.5

2 1.0 1 0.5 Fold difference vs. control Fold difference vs. control 0 0.0 Control E Equilin Equol Genistein Coumestrol Control E2 Equilin Equol Genistein Coumestrol 2

Figure 2. Induction of apoptosis by phytoestrogens and steroidal estrogens. A, MCF7:5C cells were treated with 0.1% ethanol vehicle (control), or 1 nmol/L E2, 1 nmol/L equilin, or phytoestrogens (1 mmol/L) for 72 hours and then stained with Annexin V–FITC and PI and analyzed by ﬂow cytometry. Increased apoptotic effect is observed in the right upper and lower quadrants. E2, equilin, and phytoestrogens increase BIM and TNF (B), FAS and FADD (C) mRNA levels. PCR data values are presented as fold difference versus vehicle-treated cells Æ SEM; Ã, P < 0.05.

levels in a comparable manner as E2 and equilin (Fig. 3C). did not prevent the ability of the steroidal or phytoestro- Similarly, the same effect was noted with all estrogens on gens to either induce apoptosis (Fig. 4E) or inhibit the the ERa mRNA levels (Fig. 3D). Interestingly, E2, equilin, growth (Fig. 4F) of the MCF7:5C cells. Taken together, genistein, equol, and coumestrol, all have no effect on the this indicates that ERa is the initial site for the indicated ERb protein and mRNA levels, suggesting different regu- estrogens to cause growth inhibition and apoptosis in the latory effects the phytoestrogens may have on the ERa MCF7:5C cells. and ERb. A relative ratio of ERa to ERb in MCF7:5C cells is shown in (Supplementary Fig. S5). Phytoestrogens induce endoplasmic reticulum stress and inflammatory stress response genes ERa is important for steroidal- and phytoestrogen- Microarray analysis indicates that endoplasmic reticulum induced apoptosis and growth inhibition stress (ERS) and inflammatory response genes are top To determine whether ER a or b is required for the scoring pathways associated with E2-induced apoptosis antiproliferative and apoptotic effects of the estrogens, (36). To investigate whether phytoestrogens induce ERS MCF7:5C cells were transfected with either ERa or ERb genes, we used RT-PCR to quantitate mRNA levels. After 48 siRNA or nontarget siRNA (control) for 72 hours. Knock- hours of treatment, genistein, equol, coumestrol, and equi- down of ERa and ERb protein levels were determined by lin and E2 all induce DDIT3 (also known as CHOP), a Western blot analysis (Fig. 4A and D). RNA interference– marker of ERS associated with cell death, and inositol mediated inhibition of ERa abolished both steroidal- and requiring protein 1 alpha (IRE1a), an unfolded-protein- phytoestrogen-induced apoptosis (Fig. 4B) and growth response (UPR) sensor, which is activated to relieve stress inhibition (Fig. 4C) compared with cells transfected with (Fig. 5A). Significant induction of IRE1a and phospho- the control siRNA. Interestingly, loss of ERb using siRNA eukaryotic translation initiation factor-2a (p-eIF2a),

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A B

40 PS2 7 35 * GREB1 E2 30 * 6 Equilin * Equol 25 * * 5 Genistein 20 * * * * 4 Coumestrol 15 * 10 3

5 2 DNA ( m g/well) 0 * ** 1 * * * ** * * * ** * **** * * 0 * mRNA (fold difference vs. vehicle) Control- 10 9 8 7 6 6

4OHT – log [M]

C D Coumestrol Genistein Control Equilin 2.5 Equol ERa

E 2.0 ERb 2

ERa 1.5

1.0 ERb ** 0.5 * ** b-Actin Fold difference vs. control 0.0

Figure 3. Steroidal and phytoestrogens act as agonists via an ER-dependent mechanism. A, MCF7:5C cells were treated with 0.1% ethanol vehicle (control), 1 nmol/L E2, 1 nmol/L equilin, or phytoestrogens (1 mmol/L). Total RNA was isolated after 24 hours and reverse transcribed, and PS2, GREB1, PgR mRNA levels were obtained using RT-PCR. B, various concentrations of 4OHT block steroidal estrogen- or phytoestrogen-mediated growth inhibition. C, MCF7:5C cells were treated with vehicle (control) and steroidal and phytoestrogens for 24 hours. ERa and ERb proteins were detected by immunoblotting. D, ERa and ERb mRNA was quantiﬁed with RT-PCR; Ã, P < 0.05, compared with control.

another UPR sensor, protein levels occur by 24 hours (Fig. inflammatory properties was used to inhibit inflammation 5B). Next, we determined whether genistein, coumestrol, in the MCF7:5C cells. Cells were treated with 1 nmol/L E2 and equol induce proinflammatory response genes using or equilin or 1 mmol/L phytoestrogens and various con- RT-PCR. At 48 hours, E2, equilin, and all phytoestrogens centrations of dexamethasone were added to block the activate caspase-4, an inflammatory caspase; CEBPb, which biologic effects of the compounds. Although dexameth- is known to bind to IL1 response element in IL6 and a asone has an inhibitory effect in the MCF7:5C cells, it was downstream target of ERS; IL6, a proinflammatory cytokine; able to reverse the steroidal estrogen– or phytoestrogen- lymphotoxin beta (LTB), an inducer of inflammation inhibited growth (Fig. 6A). Similarly, flow cytometry response (Fig. 5C and D). This indicates that the phytoes- studies revealed that 1 mmol/L dexamethasone reversed trogens activate similar genes involved in the apoptotic the apoptotic effects mediated by E2, equilin, genistein, pathway of E2. equol, and coumestrol (Fig. 6B). To determine that inflammatory stress response was inhibited by dexameth- Inflammation is required for phytoestrogen-mediated asone, MCF7:5C cells were treated with the indicated apoptosis estrogens for 48 hours and total RNA was extracted and Next, we investigated the importance of inflammatory reverse transcribed. Dexamethasone inhibited the ability response in phytoestrogen-mediated apoptosis. Dexa- of all estrogens to induce caspase-4, CEBPb, BIM,andTNF methasone, a synthetic glucocorticoid with potent anti- (Fig. 6C and D). Together, this suggests that inflammation

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AB45 C 35 40 Consi ERasiRNA 30 35 30 25 ERa 25 20

20 15 b-Actin 15 Apoptosis (%) * 10 10 * *** DNA ( m g/well) * * * * 5 5 * 0 0 2 E Geni Consi Equol Coum ER a si Equilin +ER a si 2 E Geni+ER a si Equol+ER a si Coum+ER a si Equilin+ER a si D E F 40 Consi ERbsiRNA 50 35 45 30 ERb 40 35 25 30 20 b-Actin 25

20 15 DNA ( m g/well) Apoptosis (%) 15 10 10 5 5 0 0 2 E Geni ER b si Consi Equol Coum +Er b si Equilin 2 E Geni+Er b si Equol+Er b si Coum+Er b si Equilin+Er b si

Figure 4. ERa is required for estrogen-induced growth inhibition and apoptosis. MCF7:5C cells were transfected with either nontarget RNA (consi) or siRNA of ERa for 72 hours. A, ERa was detected by immunoblotting. Then, cells were treated with either control (0.1% EtOH), 1 nmol/L steroidal estrogens, or 1 mmol/L phytoestrogens for 72 hours (B) and apoptosis was determined using Annexin V–binding assay. C, growth inhibition in the transfected cells was assessed after 6 days of treatment with indicated compounds using DNA quantiﬁcation assay; Ã, P < 0.05. ERb is not required for estrogen-induced growth inhibition and apoptosis. Cells were transfected with either non-target RNA (consi) or siRNA of ERb for 72 hours. D, immunoblots. E, apoptosis. F, growth inhibition as per ERa. is important for both steroidal- and phytoestrogen-medi- phytoestrogens will induce apoptosis in MCF7:5C cells, ated apoptosis. which simulate a postmenopausal state that is dependent on the duration of estrogen deprivation following menopause. Genistein, equol, and coumestrol all increase cell Discussion growth in MCF7:WS8 (which simulate the premenopausal Phytoestrogen consumption is associated with a decrease or perimenopausal state) after 3 days of estrogen depri- in the incidence of breast cancer in the Asian population vation at low concentrations. These cells have adapted to probably due to early exposure to a high soy diet. This an estrogen-rich environment and will grow with a nat- correlates with animal studies that suggest that it is due to ural resupply of estrogens provided with exogenous phy- mammary cell differentiation and a decrease in terminal toestrogens treatment. This correlates with the results of end buds which are sites of early tumor proliferation (41, Andrade and colleagues (43), who show that long-term 42). Phytoestrogens increase cell growth of ER-positive consumption of low genistein doses (500 ppm) pro- breast cancer cells but induce apoptosis at high concen- motes MCF7 tumor growth in vivo.However,atlow trations in these cells. Although studies (11, 28) may concentrations <1 mmol/L, all phytoestrogens inhibit support use of phytoestrogens in postmenopausal wom- cell growth. In contrast, the phytoestrogens, although less en, their full chemopreventive properties are yet to be potent than E2 and equilin, induce apoptosis in MCF7 clearly deﬁned. E2 and CEE induce apoptosis in long-term cells that have undergone long-term estrogen depriva- estrogen-deprived breast cancer cells. Therefore, we tion. Therefore, a potential use of phytoestrogens at phys- addressed the question of whether low concentrations of iologic concentrations will be in an estrogen-deprived

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A B Coumestrol 3.5 Genistein Control IRE1a * Equilin 3.0 * Equol DDIT3 * E 2 2.5 * * * 2.0 * * peIF2a * * 1.5 eIF2a 1.0

0.5 IRE1 Fold difference vs. control 0.0 b-Actin Control E2 Equilin Equol Genistein Coumestrol

C D

4.0 3.5 IL6 CEBPb * 3.5 * 3.0 LTB * Caspase-4 * * 3.0 * 2.5 * * 2.5 ** ** * * * * 2.0 2.0 * * * 1.5 1.5 1.0 1.0

0.5 0.5 Fold difference vs. control

0.0 Fold difference vs. control 0.0 Control E2 Equilin Equol Genistein Coumestrol Control E2 Equilin Equol Genistein Coumestrol

Figure 5. Endoplasmic reticulum stress and inﬂammatory stress response are involved in phytoestrogen-induced apoptosis. A, the indicated estrogens induce endoplasmic reticulum stress–related genes, DDTT3 and IRE1a. B, MCF7:5C were treated with E2 (1 nmol/L), equilin (1 nmol/L), or phytoestrogen (1 mmol/L) for 24 hours. IRE1a and phosphorylated eIF2a were used as indicators of UPR activation and their protein expression were examined by immunoblotting. Total eIF2a and b-actin were determined for loading controls. Indicators of inﬂammatory stress response caspase-4, CEBPb (C), IL6 and LTB Ã (D) were activated by E2, equilin, and phytoestrogens; , P < 0.05.

environment, which is induced either by natural with- ptosis, suggesting that ERa signaling is required for their drawal of estrogens caused by menopause or by treatment biologic actions. with exhaustive anti-estrogen therapy for breast cancer Genistein, equol, and coumestrol induce ERS and inflam- with aromatase inhibitors or tamoxifen. matory stress response, intrinsic and extrinsic apoptosis– Studies (44–46) suggest that phytoestrogens possess related genes, which correlates with results of differential anti-estrogenic properties that may be responsible for gene expression in response to E2 interrogated using agilent- their chemopreventive effects. Here, we show that the based microarray analysis (36). Activated ERS genes indi- phytoestrogens do in fact induce estrogen-responsive cate that E2 prevents protein folding leading to accumula- genes just like steroidal estrogens in the estrogen-deprived tion of unfolded proteins and widespread inhibition of MCF7:5C cells and that their growth inhibition and protein translation and cross-talk with inflammatory apoptosis are mediated through the ER. In contrast, it response genes and subsequent induction of cell death. has been reported that genistein mediates apoptosis Inhibition of PERK/EIF2AK3, a key ERS sensor of UPR and throughanER-independentmechanismintheMCF7 inducer of pEIF2a (48), prevents E2-mediated apoptosis cells (40, 44) and the ability of phytoestrogens to induce (49). PERK is also known to induce apoptosis by sustaining apoptosis is observed maximally in the presence of E2.It levels of DDIT3 (50), another major ERS gene involved in is important to note, however, that apoptosis was medi- apoptosis, which is known to dimerize with CEBPb under ated by the phytoestrogens only at high concentrations in stress conditions (51, 52). Ablation of CEBPb using siRNA these studies (40, 44). As another potential mechanism of decreases expression of DDIT3 (52), suggesting a cross-talk apoptosis, phytoestrogens show increased binding affin- between ERS and inflammatory stress response. Similarly, ity to ERb (47), which is thought to be responsible for its inhibition of caspase-4, an inflammatory response gene and growth inhibitory properties. In our study, loss of ERb did a downstream target of ERS, using caspase-4 inhibitor not affect the antiproliferative and apoptotic properties of z-LEVD-fmk also blocks E2-induced apoptosis. To show the steroidal and phytoestrogens. However, we deter- that inflammation is important in phytoestrogen-induced mined that knockdown of ERa prevents both steroidal- apoptosis, dexamethasone was used to block inflammation and phytoestrogen-mediated growth inhibition and apo- globally, resulting in inhibition of all estrogen-induced

946 Cancer Prev Res; 7(9) September 2014 Cancer Prevention Research

Phytoestrogen-Induced Apoptosis

A B 14 E2 30 12 Equilin Equol 25 10 Genistein Coumestrol 8 20

6 15

4 10 DNA ( m g/well) Apoptosis (%) 2 5

0 0 Control - 10 9 8 7 6 6

Dexamethasone -log [M]

C D 4.0 BIM b CEBP 3.5 4.5 TNF Caspase-4 3.0 4.0 3.5 2.5 3.0 2.0 2.5 2.0 1.5 1.5 1.0 1.0 0.5 0.5 Fold difference vs. control 0.0

Fold difference vs. control 0.0

Figure 6. Inflammation is important for phytoestrogen-mediated apoptosis. A, cells were treated with the indicated estrogens in presence of increasing concentration of dexamethasone (dexa). B, dexamethasone completely reverses E2, equilin, and all phytoestrogen-induced apoptosis. Apoptosis was assessed using the flow cytometry. Dexamethasone blocked the induction of CEBPb and caspase-4 (C), BIM and TNF (D) by E2, equilin, and phytoestrogens; Ã, P < 0.05. apoptosis and their ability to induce inflammatory response women more than 5 years postmenopausal (35%) when and apoptosis-related genes. Therefore, the clinical impli- compared with women who were less than 5 years post- cation is that caution should be exercised in the use of menopausal (9%). In more recent clinical studies, about steroidal anti-inflammatory agents in conjunction with 30% of patients with advanced breast cancer who have been these phytoestrogens, which could prevent the full chemo- exposed to exhaustive anti-hormone therapy show an preventive benefits. objective clinical response with estrogen therapy (6, 7). Successful use of estrogens to treat or prevent tumors is CEEs reduced the incidence and mortality from breast dependent on the timing of estrogen withdrawal. Estrogen cancer but this is probably because the majority of these therapy was the first chemical used in the treatment of women were over 65 years (9). Furthermore, 10 years advanced breast cancer in postmenopausal women and this adjuvant tamoxifen therapy produced a further reduction therapy resulted in the regression of 30% of tumors in the in recurrence and mortality from breast cancer when com- first reported clinical trial (53). It was noted that "the pared with 5 years of tamoxifen therapy (56), suggesting beneficial responses were three times more frequent in that it was the woman’s own estrogen that destroys the women over the age of 60 years than in those under that appropriately sensitive tamoxifen-resistant micrometastasis age; that estrogens may, on the contrary, accelerate the once long-term tamoxifen is stopped (57). course of mammary cancer in younger women, and that In conclusion, it is important to note that to obtain the their therapeutic use should be restricted to cases 5 years full breast cancer chemopreventive benefits of phytoestro- beyond the menopause" (54). Stoll and colleagues (55) gens, it is necessary to begin up to 5 years following noted that objective remission rate from estrogen treatment menopause. Commencing soy consumption during peri- in 407 patients with advanced breast cancer was higher in menopause may cause growth of nascent ER-positive breast

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Obiorah et al.

tumors, which may increase breast cancer risk, whereas Writing, review, and/or revision of the manuscript: I.E. Obiorah, V.C. Jordan phytoestrogen therapy 5 years after menopause will most Administrative, technical, or material support (i.e., reporting likely induce apoptotic cell death and enhanced patient or organizing data, constructing databases): I.E. Obiorah, P. Fan, survival. V.C. Jordan

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support This work was supported by the Department of Defense Breast Pro- gram under Award number W81XWH-06-1-0590 Center of Excellence (to Disclaimer V.C. Jordan); the Susan G Komen for the Cure Foundation under Award The views and opinions of the author(s) do not reflect those of the U.S. number SAC100009, the Lombardi Comprehensive Cancer 1095 Center Army or the Department of Defense. Support Grant (CCSG) Core Grant NIH P30 CA051008. The grant recipientsinclude:V.C.Jordan,P.Fan,andI.E.Obiorah. Authors' Contributions The costs of publication of this article were defrayed in part by the Conception and design: I.E. Obiorah, V.C. Jordan payment of page charges. This article must therefore be hereby marked Development of methodology: I.E. Obiorah, P. Fan, V.C. Jordan advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acquisition of data (provided animals, acquired and managed patients, this fact. provided facilities, etc.): I.E. Obiorah, V.C. Jordan Analysis and interpretation of data (e.g., statistical analysis, Received February 20, 2014; revised April 25, 2014; accepted May 21, biostatistics, computational analysis): I.E. Obiorah, P. Fan, V.C. Jordan 2014; published OnlineFirst June 3, 2014.

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www.aacrjournals.org Cancer Prev Res; 7(9) September 2014 949

Breast Cancer Cell Apoptosis with Phytoestrogens Is Dependent on an Estrogen-Deprived State

Ifeyinwa E. Obiorah, Ping Fan and V. Craig Jordan

Cancer Prev Res 2014;7:939-949. Published OnlineFirst June 3, 2014.

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