Breast Cancer Cell Apoptosis with Phytoestrogens Is Dependent on An

Home , Phytoestrogen

Author Manuscript Published OnlineFirst on June 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0061 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Title

Breast Cancer Cell Apoptosis with Phytoestrogens is dependent on an

estrogen deprived state

Authors and affiliations

Ifeyinwa E. Obiorah1, Ping Fan1 and V. Craig Jordan1

1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University

Medical Center, Washington, DC 20057

Running title: Phytoestrogen induced apoptosis

Keywords: Phytoestrogen, estradiol, apoptosis, inflammation, endosplasmic reticulum

stress(ERS).

Financial Support for all authors

This work (VCJ) was supported by the Department of Defense Breast Program under

Award number W81XWH-06-1-0590 Center of Excellence; the Susan G Komen for the Cure

Foundation under Award number SAC100009, the Lombardi Comprehensive Cancer 1095

Center Support Grant (CCSG) Core Grant NIH P30 CA051008. The grant recipients include:

V.C. Jordan, P. Fan and I. Obiorah. The views and opinions of the author(s) do not reflect those

of the US Army or the Department of Defense.

*Corresponding Author

V. Craig Jordan OBE, PhD, DSc, FMedSci

Vincent T. Lombardi Chair of Translational Cancer Research

Downloaded from cancerpreventionresearch.aacrjournals.org on September 25, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0061 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Professor of Oncology and Pharmacology

Georgetown University Medical Center

3970 Reservoir Rd NW. Research Building, Suite E501, Washington, DC 20057

Tel: 202.687.2897

Fax: 202.687.6402

E-mail: [email protected]

Potential Conflicts of Interests

None

Number of words in abstract: 206

Number of text words: 4963

Number of figures: 6

Number of tables: 0

Number of references: 56

ABSTRACT

Phytoestrogens have been investigated as natural alternatives to hormone replacement therapy

and their potential as chemopreventive agents. We investigated the effects equol, genistein and

coumestrol on cell growth in fully estrogenized MCF-7 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 to that of steroidal

estrogens: 17β estradiol (E2) and equilin present in conjugated equine estrogen. Steroidal and

phytoestrogens induce proliferation of MCF-7 cells at physiologic concentrations but inhibit the

growth and induce apoptosis of MCF7:5C cells. Although steroidal and phytoestrogens induce estrogen responsive genes, their anti-proliferative and apoptotic effects are mediated through the estrogen receptor. Knockdown of ERα 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 anti-apoptotic effects.

INTRODUCTION

Acquired resistance to anti-hormone therapy occurs despite the successful use of endocrine

treatment to improve survival in breast cancer patients. Early laboratory models show that re-

transplantation of tamoxifen resistant tumors into ovarectomized athymic mice led to tumor

growth in response to tamoxifen and estradiol(E2)(1, 2). Continued retransplantation of the tamoxifen stimulated tumors in nude mice for up to 5years resulted to a rapid regression of the tumors in response to E2 (3). This correlates with the finding that E2 induces apoptosis in long

term estrogen deprived MCF-7 breast cancer cells (4, 5). The use of estrogens has been

beneficial in the treatment of metastatic breast cancer in postmenopausal women with acquired

resistance to endocrine therapy. A clinical study (6) found that high dose diethylstilbestrol

induced an objective response in 30 percent of postmenopausal breast cancer patients who had

previous exhaustive antihormone therapy. Ellis and colleagues (7) showed that postmenopausal

women with aromatase inhibitors resistant metastatic breast cancer, had a 29% clinical benefit

with low dose estrogen (6mg daily) but the same clinical benefit but more side effects with high

dose estrogen (30mg daily). Additional clinical evidence for the antitumor action of low dose

estrogen comes from the Women Health Initiative (WHI) trial which compared conjugated

equine estrogen (CEE) therapy with placebo in hysterectomised postmenopausal women which

show a persistent decrease in the incidence and mortality of breast cancer in women who

received estrogen alone therapy(8, 9). Studies in vitro show that constituents of CEE cause

apoptosis in long term estrogen deprived MCF7 cells (10). The clinical and laboratory studies

suggest that the ability of estrogen therapy to 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 compounds that are structurally similar to

E2. Phytoestrogens consists of isoflavones (genistein, diadzein), coumestans (coumestrol), the

lignans (enterolactone, enterodiol) and stilbenes (resveratrol).Isoflavones are principally found in

soy based products which are staple foods in many Asian countries and are becoming increasing popular in western countries. An inverse relationship found between soy consumption in Asian countries and decreased breast cancer risk has sparked a sustained interest in the use of

phytoestrogens in breast cancer prevention. However, the clear beneficial effects of these

estrogens remain controversial. Several meta-analysis(11-13) that assessed soy exposure and

breast cancer risk revealed that studies conducted in Asian countries showed a significant trend

of a reduced risk with increased soy intake in both pre- and postmenopausal Asian women. On

the other hand no association was observed between soy consumption and breast cancer risk in

low soy consuming western populations(11, 13), suggesting that consumption of soy products in

amounts taken in the Asian population may have protective benefits. Evaluation of the breast

cancer protective effects of isoflavones stratified by menopausal status is still undefined. Trock

and colleagues(14) reported in their meta-analysis, a stronger association between soy exposure

and breast cancer risk in premenopausal women. However, the analyses included studies with

incomplete measurements, potential confounders and lack of a dose response that make the

findings inconclusive. On the other hand, another study reported that adult or adolescent soy

consumption was associated with reduced risk of premenopausal breast cancer(15) and no

significant associations were reported for the risk of postmenopausal breast cancer. Furthermore,

there is increased evidence that the chemo-protective effects of isoflavones are dependent on

early exposure. High soy consumption during adolescence is associated with reduced risk of

adult breast cancer (15-17). This concurs with the findings in animal model experiments, where

prepubertal exposure to genistein causes mammary gland differentiation, thereby resulting in

increased breast cancer prevention(18, 19). The effect of phytoestrogens in breast cancer cells

have been extensively studied. At low pharmacological concentrations, phytoestrogens stimulate the growth of estrogen receptor positive breast cancer cells (20-22). In contrast at high concentrations (>5µM), these plant derived estrogens inhibit the growth of the cancer cells (21,

23, 24). Ingestion of soy isoflavones in healthy premenopausal women resulted in increased breast tissue proliferation (25),epithelial hyperplasia (26) and a weak estrogenic response in inducing estrogen regulated markers (27). On the other hand in postmenopausal women, soy supplementation resulted in either a protective effect(28) or no effect (29-31) on breast cancer risk. Only one of the postmenopausal studies(31) consisted of healthy subjects, the rest included breast cancer patients. Fink et al (32) reported a decreased all-cause mortality in pre- and postmenopausal breast cancer patients who had a high intake of isoflavones, whereas a reduced breast cancer mortality was observed in postmenopausal women. However the DietCompLyf study (33) which investigated associations between phytoestrogens and breast cancer recurrence and survival found no significant associations between pre-diagnosis phytoestrogen intake and reduced breast cancer risk. Interestingly Shu and colleagues (34) found that soy food consumption was significantly associated with decreased risk of death and recurrence in breast cancer patients.

In this study we have evaluated the apoptotic and potential chemopreventive effects of phytoestrogens using a unique cell model that simulates a postmenopausal cellular environment.

Genistein, coumestrol and equol, a gastrointestinal metabolite of diadzein are used in comparison to E2 and equilin a constituent of conjugated equine estrogen (CEE) in hormone replacement

therapy (HRT) to determine their proliferative and apoptotic potential using fully estrogenised

and an estrogen deprived breast cancer cells respectively. Here, we test the hypothesis that the

phytoestrogens have biologic effects similar to that of E2 and CEE in breast cancer prevention

and this may have clinical implications for the strategic use of phytoestrogens as alternatives to

HRT in postmenopausal populations.

MATERIALS AND METHODS

Cell Culture and Reagents

Cell culture media were purchased from Invitrogen Inc. (Grand Island, NY) and fetal calf serum

(FCS) was obtained from HyClone Laboratories (Logan, UT). Compounds E2 , equilin,

equol,genistein and coumestrol (Supplementary Fig. S1), ICI 182,780 and 4-hydroxytamoxifen

(4OHT) (were obtained from Sigma, St. Louis, MO). Dexamethasone was obtained from Tocris

Biosciences, Bristol, UK. MCF7:5C were derived from MCF7 cells obtained from the Dr. Dean

Edwards, San Antonio, Texas as reported previously (35). MCF7:WS8 cells were derived from

MCF-7 cells as previously described(35) and maintained in RPMI media supplemented with

10% FCS, 6 ng/ml bovine insulin and penicillin and streptomycin. The MCF7:WS8 and the

MCF7:5C cells have been fully characterized and experiments were done as previously reported

(36). The expected growth/apoptotic responses to E2, biomarker statuses of estrogen receptor-α

(ERα), PgR, and HER2, and ER-regulated transcriptional activity were confirmed in both cell lines. Protein levels of ERα, PgR, and HER2 were characterized by semiquantitative immunoblot analysis and ER transcriptional activity was evaluated using an estrogen responsive element

(ERE) -regulated dual luciferase reporter gene system(36). The last characterization was reported in (37) and the DNA fingerprinting patterns of the cell lines were consistent with the report by

the American Type Culture Collection. MCF7 cells are cultured in phenol-red free RPMI media

containing 10% charcoal dextran treated FCS, 6ng/ml bovine insulin and penicillin and

streptomycin for three days prior to starting experiments. MCF7:5C cells were maintained in

phenol-red free RPMI media containing 10% dextran coated charcoal-treated FCS, 6ng/ml

bovine insulin and penicillin and streptomycin. The cells were treated with indicated compounds

(with media changes every 48 h) for the specified time and were subsequently harvested for

tissue culture experiments.

Cell growth assay

The cell growth was monitored by measuring the total DNA content per well in 24 well plates.

Fifteen thousand cells were plated per well and treatment with indicated concentrations of

compounds was started after 24h, in triplicates. Media containing the specific treatments

were changed every 48h. On day 7 the cells were harvested and total DNA was assessed using a fluorescent DNA quantification kit (Cat # 170-2480; Bio-Rad,Hercules, CA, USA) and was performed as previously described (38).

RNA isolation and real time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) and RNAeasy kit

according to the manufacturer’s instructions. Real-time PCR was performed as previously

described (39). The sequences for all primers are documented in Supplementary Table S1. The

change in expression of transcripts was determined as described previously and used the

ribosomal protein 36B4 mRNA as the internal control (39).

Apoptosis assay

In brief, MCF-7:5C cells were seeded in 100-mm dishes and cultured overnight in estrogen-free

RPMI 1640 medium containing 10% SFS. The next day, cells were treated with <0.1% ethanol

(control), E2 (1 nM ) equilin (1 nM ), equol (1 µM ), genistein(1 µM ) and coumestrol(1 µM )

for 72h and the cells were detached using accutase (Life Technologies, Carlsbad, California), a

marine-origin enzyme with proteolytic and collagenolytic activity, in 1:3 dilution using PBS

(Invitrogen, Grand Island, NY) as the diluent. The cells were collected by centrifugation for 2 min at 500 × g. Cells were then resuspended and stained simultaneously with either FITC- labeled annexin V and propidium iodide (PI) (Pharmingen, San Diego, CA) or DNA binding dye, YO-PRO-1 and PI (Life technologies, (Grand Island, NY). Apoptosis was verified based on loss of plasma membrane integrity. Viable cells excluded these dyes, whereas apoptotic cells allowed moderate staining. Cells were analyzed using a fluorescence- activated cell sorter

(FACS) flow cytometer (Becton Dickinson, San Jose, CA). The percentage of apoptosis was calculated by adding the percentage of cells stained with either annexin V alone (early apoptosis) in the right lower quadrant and those stained with both PI and annexin V (late apoptosis) in the right upper quadrant. Experiments are repeated three times with similar results.

Cell cycles analysis

MCF7:5C cells were cultured in dishes and were treated with with <0.1% ethanol (control), E2 (1 nM ) equilin (1 nM ), equol (1 µM ), genistein(1 µM ) and coumestrol(1 µM ) for the indicated times. Cells were harvested and gradually fixed with 75% EtOH on ice. After staining with PI, cells were analyzed using a fluorescence-activated cell sorter (FACS) flow cytometer (Becton

Dickinson, San Jose, CA), and the data were analysed with Modfit software (Topsham, ME).

Immunoblotting

Proteins were extracted in cell lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase

Inhibitor Cocktail Set I and Set II (Calbiochem, San Diego, CA). Total protein content of the lysate was determined by a standard bicinchoninic acid assay using the reagent from Bio-Rad

Laboratories (Hercules, CA). Twenty five micrograms of total protein was separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a nitrocellulose membrane.

The membrane was probed with primary antibodies followed by incubation with secondary antibody conjugated with HRP and reaction with Western Lighting™ plus-ECL enhanced chemiluminescent substrate (PerkinElmer Inc., Waltham MA). ERα and β antibody was from

Santa Cruz Biotechnology (Dallas, TX). Phosphorylated eIF2α, total eIF2α, IRE1α and β actin antibodies were from Cell Signaling Technology (Danvers,MA). Protein bands were visualised by exposing the membrane to X-ray film.

Small Interfering RNA Transfection

For transient transfections, MCF7:5C cells were seeded at a density of 50-70% in 6 well plates in estrogen free RPMI media containing 10% SFS. The following day, cells were transfected with

100 nM small interfering RNAs (siRNAs) for ER α (Dharmacon Pittsburgh PA, SMART pool:

ON-TARGETplus ESR1 siRNA product number L-003401-00-0005) and ERβ (Dharmacon,

SMART pool: ON-TARGETplus ESR2 siRNA product number L-003402-00-0005) using

DharmaFECT transfection reagent (Dharmacon Pittsburgh PA, product number T-2001-03), according to the manufacturer’s recommended protocol. Non target siRNA was purchased from

Dharmacon and was used as a control (Silencer negative control siRNA, product number D-

001810-01-20). The cells were harvested 72h posttransfection and analyzed by western blot (as described above).Transfected cells were also treated with vehicle, steroidal estrogens or phytoestrogens for either an additional 72h, or 6 days and apoptotic cells and DNA content were measured using annexinV staining and DNA quantification assays respectively (as described above).

Statistical analysis

All data are expressed as the mean of at least three determinations, unless otherwise stated. The

differences between the treatment groups and the control group were determined by one-factor analysis of variance (ANOVA with Tukey’s post test and two-way ANOVA with Bonferroni post test using GraphPad Prism, version 5.00 (GraphPad Software Inc., La Jolla, CA). Results were considered statistically significant if the P < 0.05.

RESULTS

Effect of phytoestrogens on breast cancer cells

Based on the controversy surrounding breast cancer risk and the use of phytoestrogens, we decided to determine the biological properties of the genistein, equol and coumestrol in comparison to E2 and equilin in two different models of breast cancer cell models. Estrogens

have been shown to regulate the growth of ER positive MCF-7 breast cancer cells. First, we

tested the ability of test compound to induce proliferation in MCF7:WS8 cells which are

estrogen responsive breast cancer cells grown in fully estrogenised medium. MCF-7:WS8 cells

were grown in estrogen free media for 3 days and treated with various concentrations of

genistein, coumestrol and equol and their effects were compared to E2 and equilin (Fig. 1A). The

-9 -8 phytoestrogens, equol [EC50: 1.72 x 10 ], genistein [EC50: 1.08 x 10 ] and coumestrol[EC50:

3.07 x 10-9], all stimulated cell growth in a concentration related manner with maximum

-12 -11 stimulation occurring at 0.1µM, whereas E2 [EC50: 3.11x 10 ] and equilin [EC50: 1.01 x 10 ]

maximally induced cell growth at 10 pM and 0.1 nM respectively.

Growth inhibition was observed with the phytoestrogens at 10µM with genistein (10-5M vs 10-7M; P<0.05). Next we investigated the growth properties of the genistein, equol and

coumestrol in long term estrogen deprived MCF7:5C cells in comparison to E2 and equilin (Fig.

-8 -8 -8 1B). Genistein[IC50: 2.77 x 10 ], equol[IC50: 4.67 x 10 ] and coumestrol[IC50: 2.34 x 10 ] drastically inhibited the growth of the MCF7:5C cells at higher concentrations compared to E2.

- Maximum growth inhibition was observed with all phytoestrogens at 0.1µM. E2 [IC50: 2.06 x 10

11 -10 ] achieved maximum growth inhibition at 0.1nM, while equilin [IC50: 2.32 x 10 ] reached

maximum growth inhibition at 1nM after 7 days of treatment.

Phytoestrogens induce apoptosis in a long term estrogen deprived breast cancer cell line

Based on the fact that the decrease in cell growth observed with the steroidal estrogens is due to

apoptosis(10), we investigated whether the anti-proliferative effects of the phytoestrogens was

also due to an increase in apoptosis. MCF7:5C cells were treated with E2 (1nM), equilin (1nM),

genistein(1µM), equol(1µM) and coumestrol(1µM) for 72h and stained with annexinV-FITC and

PI fluorescence and cells were analyzed using the flow cytometry. In the control treated group,

only 6.8% of cells stained for apoptosis, whereas E2(24.56%), equilin(17.49%), genistein

(14.79%), equol (14.89%) and coumestrol (17.83%) all show increased apoptotic staining

compared to the control treated cells (Fig. 2A). A similar effect was noted using a DNA binding

stain, YO-PRO-1 (Supplementary Fig. S2). E2, equilin and all phytoestrogens induced apoptotic

genes; BCL2L11/BIM, TNF, FAS and FADD (Fig. 2B-C) after 48h of treatment. Induction of

these genes is consistent with the apoptotic status determined using the flow cytometry.

Although evidence of apoptosis occurs with the phytoestrogens by 48h, a consistent increase in

the S phase when compared to the control was observed with all estrogens(Supplementary Fig.

S3). In contrast to other reports(23, 40) which indicate that genistein causes a G2/M arrest, no

checkpoint blockade was noted after treatment with all compounds, indicating that the initial

response of the cells to estrogens is growth, then apoptosis in MCF7:5C cells.

Phytoestrogens possess estrogenic properties mediated through the estrogen receptor in the

MCF7:5C cells

We explored the ability of phytoestrogens to regulate estrogen response genes in comparison to

E2 and equilin. Genistein, equol and coumestrol were all able to induce TFF1/PS2 and GREB1

(Fig. 3A). Phytoestrogens have been shown to induce apoptosis through an estrogen receptor

(ER) independent mechanism(21, 41). To evaluate the involvement of ER in the effects of the

phytoestrogens, we investigated their anti-proliferative effects in the presence of 4-

hydroxytamoxifen (4OHT) (Fig. 3B) and ICI 182 780 (Supplementary Fig. S4). The combination

of various concentrations of 4OHT or ICI 182 780 with E2, equilin and each phytoestrogens

blocked estrogen induced apoptosis suggesting that the phytoestrogens mediate apoptosis via the

ER. We sought to examine the effects of genistein, equol and coumestrol on the ER. Following

treatment of MCF7:5C cells with E2, equilin and the phytoestrogens for 24h, ERα levels were

determined by western blotting. All phytoestrogens caused a decrease in the ERα protein levels in a comparable manner as E2 and equilin (Fig. 3C). Similarly the same effect was noted with all

estrogens on the ERα mRNA levels (Fig. 3D). Interestingly, E2, equilin, genistein, equol and

coumestrol, all have no effect on the ERβ protein and mRNA levels suggesting different

regulatory effects the phytoestrogens may have on the ERα and ERβ . A relative ratio of ERα to

ERβ in MCF7:5C cells is shown in (Supplementary Fig. S5).

ERα is important for steroidal and phytoestrogen induced apoptosis and growth inhibition

To determine whether ER α or β is required for the antiproliferative and apoptotic effects of the

estrogens, MCF7:5C cells were transfected with either ERα or ERβ siRNA or nontarget

siRNA(control) for 72h. Knockdown of ERα and ER β protein level was determined by western

blot (Fig. 4A, D). RNA- interference mediated inhibition of ER α abolished both steroidal and

phytoestrogen induced apoptosis (Fig. 4B) and growth inhibition (Fig. 4C) compared with cells

transfected with the control siRNA. Interestingly, loss of ERβ using siRNA did not prevent the

ability of the steroidal or phytoestrogens to either induce apoptosis (Fig.4E) or inhibit the growth

(Fig.4F) of the MCF7:5C cells. Taken together, this indicates that ERα is the initial site for the

indicated estrogens to cause growth inhibition and apoptosis in the MCF7:5C cells.

Phytoestrogens induce endoplasmic reticulum stress and inflammatory stress response

genes

Microarray analysis indicates that endoplasmic reticulum stress (ERS) and inflammatory

response genes are top scoring pathways associated with E2 induced apoptosis(36). To investigate whether phytoestrogens induce ERS genes, we used RT-PCR to quantitate mRNA levels. After 48h of treatment, genistein, equol, coumestrol and equilin and E2 all induce

DDIT3(also known as CHOP), a marker of ERS associated with cell death, and inositol requiring protein 1 alpha (IRE1α) , an unfolded protein response sensor which is activated to relieve stress(Fig. 5A). Significant induction of IRE1α and phospho-eukaryotic translation initiation factor-2α (p-eIF2α), another UPR sensor, protein levels occur by 24h (Fig. 5B). Next we determined whether genistein, coumestrol and equol induce proinflammatory response genes using RT-PCR. At 48h, E2, equilin and all phytoestrogens activate caspase 4, an inflammatory

caspase; CEBP β which is known to bind to IL-1 response element in IL6 and a downstream

target of ERS; IL6, a proinflammatory cytokine; lymphotoxin beta (LTB),an inducer of

inflammation response, (Fig. 5C-D). This indicates that the phytoestrogens activate similar

genes involved in the apoptotic pathway of E2.

Inflammation is required for phytoestrogen mediated apoptosis

Next we investigated the importance of inflammatory response in phytoestrogen mediated

apoptosis. Dexamethasone, a synthetic glucocorticoid with potent anti-inflammatory properties

was used to inhibit inflammation in the MCF7:5C cells. Cells were treated with 1nM E2 or equilin or 1µM phytoestrogens and various concentrations of dexamethasone were added to block the biological effects of the compounds. Although dexamethasone has an inhibitory effect in the MCF7:5C cells, it was able to reverse the steroidal estrogen or phytoestrogen inhibited growth(Fig. 6A). Similarly, flow cytometry studies revealed that 1µM dexamethasone reversed the apoptotic effects mediated by E2, equilin, genistein, equol and coumestrol (Fig. 6B). To

determine that inflammatory stress response was inhibited by dexamethasone, MCF7:5C cells

were treated with the indicated estrogens for 48h and total RNA was extracted and reverse

transcribed. Dexamethasone inhibited the ability of all estrogens to induce caspase 4, CEBP β,

BIM and TNF (Fig. 6C-D). Together, this suggests that inflammation is important for both

steroidal and phytoestrogen mediated apoptosis.

DISCUSSION

Phytoestrogen consumption is associated with a decrease in the incidence of breast cancer in the

Asian population probably due to early exposure to a high soy diet. This correlates to animal

studies which suggest that it is due to mammary cell differentiation and a decrease in terminal

end buds which are sites of early tumor proliferation(42, 43). Phytoestrogens increase cell

growth of ER positive breast cancer cells but induce apoptosis at high concentrations in these

cells. Although studies (11, 28) may support use of phytoestrogens in postmenopausal women, their full chemopreventive properties is yet to be clearly defined. E2 and CEE induce apoptosis in

long term estrogen deprived breast cancer cells. Therefore we addressed the question of whether

low concentrations of phytoestrogens will induce apoptosis in MCF7:5C cells which simulate a

postmenopausal state that is dependent on the duration of estrogen deprivation following

menopause. Genistein, equol and coumestrol all increase cell growth in MCF7:WS8,(which

simulate the premenopausal or perimenopausal state) after 3 days of estrogen deprivation at low

concentrations. These cells have adapted to an estrogen rich environment and will grow with a

natural resupply of estrogens provided with exogenous phytoestrogens treatment. This correlates

with the results of Andrade and colleagues (44)who show that long-term consumption of low

GEN doses (≤500 ppm) promotes MCF-7 tumor growth in vivo. However at low concentrations

<1µM, all phytoestrogens inhibit cell growth. In contrast the phytoestrogens, although less potent

than E2 and equilin, induce apoptosis in MCF-7 cells that have undergone long term estrogen deprivation. Therefore a potential use of phytoestrogens at physiologic concentrations will be in

an estrogen deprived environment which is induced either by natural withdrawal of estrogens

caused by menopause or by treatment with exhaustive anti-estrogen therapy for breast cancer

with aromatase inhibitors or tamoxifen.

Studies (45-47)suggest that phytoestrogens possess anti-estrogenic properties which may

be responsible for their chemopreventive effects. Here we show that the phytoestrogens do in

fact induce estrogen responsive genes just like steroidal estrogens in the estrogen deprived

MCF7:5C cells and that their growth inhibition and apoptosis are mediated through the ER. In

contrast, it has been reported that genistein mediates apoptosis through an ER independent

mechanism in the MCF-7 cells (41, 45) and the ability of phytoestrogens to induce apoptosis is

observed maximally in the presence of E2. It is important to note however that apoptosis was

mediated by the phytoestrogens only at high concentrations in these studies (41, 45). As another

potential mechanism of apoptosis, phytoestrogens show increased binding affinity to ERβ(48)

which is thought to be responsible for its growth inhibitory properties. In our study, loss of ERβ

did not affect the anti-proliferative and apoptotic properties of the steroidal and phytoestrogens.

However, we determined that knockdown of ERα prevents both steroidal and phytoestrogen

mediated growth inhibition and apoptosis suggesting that ER α signaling is required for their

biological actions.

Genistein, equol and coumestrol induce ERS and inflammatory stress response, intrinsic

and extrinsic apoptosis related genes which correlates with results of differential gene expression

in response to E2 interrogated using agilent based microarray analysis(36). Activated ERS genes indicate that E2 prevents protein folding leading to accumulation of unfolded proteins and

widespread inhibition of protein translation and crosstalk with inflammatory response genes and

subsequent induction of cell death. Inhibition of PERK/EIF2AK3, a key ERS sensor of UPR and

inducer of pEIF2α (49) prevents E2 mediated apoptosis(50). PERK is also known to induce

apoptosis by sustaining levels of DDIT3(51),another major ERS gene involved in apoptosis,

which is known to dimerize with CEBPβ under stress conditions(52, 53). Ablation of CEBPβ

using siRNA decreases expression of DDIT3 (53) suggesting a crosstalk between ERS and

inflammatory stress response. Similarly, inhibition of caspase 4, an inflammatory response gene

and a downstream target of ERS, using caspase 4 inhibitor-z-LEVD-fmk also blocks E2 induced apoptosis. To show that inflammation is important in phytoestrogen induced apoptosis,

dexamethasone was used to block inflammation globally, resulting in inhibition of all estrogen

induced apoptosis and their ability to induce inflammatory response and apoptosis related genes.

Therefore the clinical implication is that caution should be exercised in the use of steroidal anti-

inflammatory agents in conjunction with these phytoestrogens, which could prevent the full

chemopreventive benefits.

Successful use of estrogens to treat or prevent tumors is dependent on the timing of

estrogen withdrawal. Estrogen therapy was the first chemical used in the treatment of advanced

breast cancer in postmenopausal women and this therapy resulted in the regression of 30% of

tumors in the first reported clinical trial(54). It was noted that “the beneficial responses were

three times more frequent in women over the age of 60 years than in those under that age; that estrogens may, on the contrary, accelerate the course of mammary cancer in younger women, and that their therapeutic use should be restricted to cases 5 years beyond the menopause”(55).

Stoll and colleagues(56) noted that objective remission rate from estrogen treatment in 407

patients with advanced breast cancer was higher in women more than 5 years postmenopausal(35%) when compared to women who were less than 5 years postmenopausal(9%). In more recent clinical studies, about thirty percent of patients with advanced breast cancer who have been exposed to exhaustive antihormone therapy show an objective clinical response with estrogen therapy(6, 7). CEEs reduced the incidence and mortality from breast cancer but this is probably because the majority of these women were over

65 years(9). Furthermore, 10 years adjuvant tamoxifen therapy produced a further reduction in recurrence and mortality from breast cancer when compared to 5 years of tamoxifen therapy(57) suggesting that it was the woman’s own estrogen that destroys the appropriately sensitive tamoxifen resistant micrometastasis once long term tamoxifen is stopped(58).

In conclusion, it is important to note that in order to obtain the full breast cancer chemopreventive benefits of phytoestrogens, it is necessary to begin up to five years following menopause. Commencing soy consumption during perimenopause may cause growth of nascent

ER positive breast tumors which may increase breast cancer risk, whereas phytoestrogen therapy

5 years after menopause will most likely induce apoptotic cell death and enhanced patient survival.

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FIGURE LEGENDS

Figure 1.

Growth characteristics of 17β-estradiol, equilin and phytoestrogens in breast cancer cells

(A) MCF7:WS8 cells were seeded in 24-well plate and treated with steroidal and phytoestrogens

over a range of doses for seven days. Cell growth was assessed as DNA content in each well. (B)

Inhibition of cell growth in MCF7:5C cells by genistein, equol and coumestrol was assessed in

comparison to E2 and equilin. Each data point is average +/- SD of three replicates. [* P< 0.05].

Figure 2.

Induction of apoptosis by phytoestrogens and steroidal estrogens

(A) MCF7:5C cells were treated with 0.1% ethanol vehicle (control), or 1nM E2, 1nM equilin or phytoestrogens (1µM) for 72h and then stained with annexin v-FITC and propidium iodide and analysed by flow cytometry. Increased apoptotic effect is observed in the right upper and lower quadrant. E2, equilin and phytoestrogens increase (B) BIM, TNF (C) FAS and FADD

mRNA levels. PCR data values are presented as fold difference versus vehicle treated cells ±

SEM. [* P< 0.05]

Figure 3.

Steroidal and phytoestrogens act as agonists via an estrogen receptor dependent

mechanism

(A) MCF7:5C cells were treated with 0.1% ethanol vehicle (control), 1nM E2, 1nM equilin or phytoestrogens (1µM). Total RNA was isolated after 24h and reverse transcribed and PS2,

GREB1, progesterone receptor (PgR) mRNA levels was obtained using RT-PCR. (B) Various

concentrations of 4-hydroxytamoxifen (4OHT) block steroidal estrogen- or phytoestrogen mediated growth inhibition (C) MCF-7:5C cells were treated with vehicle (control) and steroidal and phyto-estrogens for 24 hours. ERα and ERβ protein was detected by immunoblotting. (D)

ERα and ERβ mRNA was quantified with real time PCR (RT-PCR). *, P < 0.05, compared with control.

Figure 4

ERα is required for estrogen induced growth inhibition and apoptosis

MCF7:5C cells were transfected with either non target RNA (consi) or siRNA of ERα for 72 h.

(A) ERα was detected by immunoblotting. Then, cells were treated with either control (0.1%

EtOH), 1nM steroidal estrogens or 1µM phytoestrogens for (B) 72h 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. [*

P< 0.05]

Figure 5

Endoplasmic reticulum stress and inflammatory stress response are involved phytoestrogen induced apoptosis

(A) The indicated estrogens induce endoplasmic reticulum stress related genes, DDTT3 and

IRE1α. (B) MCF-7:5C were treated with E2 (1nM), equilin (1nM) or phytoestrogen (1µM) for

24h. IRE1α and phosphorylated eIF2α were used as indicators of UPR activation and their protein expression were examined by immunoblotting. Total eIF2α and β-actin were determined for loading controls. Indicators of inflammatory stress response (C) caspase4, CEBP β, (D) IL6 and LTB were activated by E2, equilin and phytoestrogens. [* P< 0.05]

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 (C) CEBP β,caspase 4 (D) BIM, and TNF by E2, equilin and phytoestrogens. [*

P< 0.05]

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fold difference vs. control vs. difference fold 0.0

Breast cancer cell apoptosis with phytoestrogens is dependent on an estrogen deprived state

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

Cancer Prev Res Published OnlineFirst June 3, 2014.

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