Breast Cancer Cell Apoptosis with Phytoestrogens is dependent on

Transcription

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
1
Department 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
1
Downloaded from cancerpreventionresearch.aacrjournals.org on October 20, 2016. © 2014 American Association for
Cancer Research.
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
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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 antiinflammatory agents due to their anti-apoptotic effects.
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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 retransplantation 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).
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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
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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
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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
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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
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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 FITClabeled 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
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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 D001810-01-20). 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).
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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
phytoestrogens, equol [EC50: 1.72 x 10-9], genistein [EC50: 1.08 x 10-8] and coumestrol[EC50:
3.07 x 10-9], all stimulated cell growth in a concentration related manner with maximum
stimulation occurring at 0.1µM, whereas E2 [EC50: 3.11x 10-12] and equilin [EC50: 1.01 x 10-11]
maximally induced cell growth at 10 pM and 0.1 nM respectively.
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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.
1B). Genistein[IC50: 2.77 x 10-8], equol[IC50: 4.67 x 10-8] and coumestrol[IC50: 2.34 x 10-8]
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 1011
] achieved maximum growth inhibition at 0.1nM, while equilin [IC50: 2.32 x 10-10] 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
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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 4hydroxytamoxifen (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
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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
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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
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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
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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,
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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 antiinflammatory 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).
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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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Cancer Research.
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23
Cancer Research.
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
24
Cancer Research.
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]
25
Cancer Research.
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]
26
Cancer Research.
Equol
2
2
0
0
4
-log [M]
-log[M]
8
6
4
DN
NA (ug/well))
12
Genistein
10
14
E2
Equilin
12
*
DNA ((ug/well)
6
E2
Equilin
Equol
q
Genistein
Coumesterol
10
8
B
A
Coumesterol
Cancer Research.
Figure 1
Figure 2
Control
10
10
2.79%
3
10
2
10
1
10
90.37%
10
10
4.67%
0
10
1
10
2
Annexin V FITC
10
3
5.06%
10
2
10
4
10
10
0
10
10
5.23%
10
11.52%
3
10
2
1
10
10
79.92%
79
92%
10
0
4
10
1
Annexin V FITC
2
1
3
77.46%
77
46%
10
10
4
10
2
Annexin V FITC
10
3
10
10
0
10
12.49%
10
10
2
1
10
10
0
4
10
1
10
2
10
Annexin V FITC
3
10
4
10
2
Annexin V FITC
10
3
7.95%
15.49%
3
2
1
2.30%
2
30%
74.23%
0
10
1
a102813_209.fcs
5.33%
79.88%
79
88%
10
4
3.53%
3
53%
0
Coumestrol
B
10
10
4
2.34%
0
10
0
10
1
10
2
Annexin V FITC
10
3
10
C
4.0
BIM
6
*
TNF
5
4
*
*
* *
*
*
3
*
*
2
1
fold diffference vs. control
7
fold diffference vs. control
10
3
3.33%
3
33%
0
10
2
P
I
10
10
13.96%
3
a102813_208.fcs
P
I
P
I
10
1
5.05%
Genistein
a102813_207.fcs
4
4
7.75%
0
Equol
10
10
1
70.37%
10
10
16.81%
3
2.17%
0
10
4
a102813_206.fcs
P
I
P
I
10
4
a102813_205.fcs
P
I
10
Equilin
E2
a102813_217.fcs
3.5
3.0
FAS
FADD
*
*
2.5
*
*
*
2.0
*
*
*
*
*
1.5
1.0
0.5
0.0
0
Control
E2
Equilin
Equol
Genistein Coumestrol
Control
E2
Equilin
Equol
4
Cancer Research.
A
Figure 3
B
mRNA (fold difference vs. veh)
40
PS2
35
*
GREB1
30
*
25
20
*
*
15
7
*
*
*
*
*
E2
Equilin
Equol
Genistein
Coumestrol
6
DNA (ug/well)
A
*
10
5
0
5
4
3
2
*
*
***** ** ** * **** *
1
0
Control
-
10
9
***
*
8
7
6
4OHT – log [M]
C
D
Coumestrol
ER α
ER β
β actin
fold difference vs. control
Genistein
Equol
Equilin
E2
control
2.5
2.0
ERα
ERβ
1.5
1.0
0.5
*
*
0.0
Cancer Research.
*
*
*
6
ERβsi
Coum+Erβsi
Coum
Geni+Erβsi
Geni
Equol+Erβsi
Equol
Equilin+Erβsi
25
30
ERαsi
Cou
um+ERαsi
Coum
Ge
eni+ERαsi
Geni
Equ
uol+ERαsi
Equol
Equiilin+ERαsi
Equilin
E2+ERαsi
0
Equilin
0
E2
Consi
5
E2+Erβsi
35
*
5
E2
30
*
*
*
DNA (ug/well)
D
20
Consi
DNA (ug/welll)
25
10
0
0
5
5
10
15
Apoptosis (%))
Apoptosis (%)
40
Cancer Research.
45
15
20
35
50
20
25
β actin
40
ER β
25
*
10
*
*
*
10
*
*
15
β actin
40
ERβsiRNA
Consi
15
20
F
E
D
30
ER α
30
35
ERαsiRNA
Consi
Figure 4
35
C
45
B
A
B
*
*
*
*
2.0
*
*
*
*
1.5
E2
2.5
Equol
*
DDIT3
Genistein
IRE1α
Equilin
3.0
Figure 5
Coumestrol
3.5
Control
A
peIF2α
*
eIF2α
1.0
IRE1
0.5
0.0
Control
E2
Equilin
Equol
β actin
D
C
3.5
IL6
3.0
LTB
2.5
* *
CEBPβ
3.5
*
Caspase 4
3.0
* *
2.5
* *
*
* *
*
*
2.0
1.5
1.0
0.5
0.0
Control
E2
Equilin
Equol
4.0
*
*
*
2.0
*
*
*
*
1.5
1.0
0.5
0.0
Control
E2
Equilin
Cancer Research.
Equol
14
A
E2
Equilin
Equol
Genistein
Coumestrol
12
8
Figure 6
30
25
20
Apoptosis (%)
DNA (ug/well)
10
B
6
4
2
15
10
5
0
0
Control
-
10
9
8
7
6
6
Dexamethasone –log [M]
C
D
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
3.5
Caspase 4
3.0
4.0
CEBPβ
BIM
TNF
2.5
2.0
1.5
1.0
0.5
0.0
Cancer Research.
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.
Updated version
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Cancer Research.

Breast Cancer Cell Apoptosis with Phytoestrogens is dependent on

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