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Experimental Biology and Medicine 230:558-568 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Phytoestrogens Induce Differential Estrogen Receptor Alpha- or Beta-Mediated Responses in Transfected Breast Cancer Cells

D. M. Harris*,1, E. Besselink*, S. M. Henning*, V. L. W. Go{dagger} and D. Heber*

* The UCLA Center for Human Nutrition and {dagger} The David Geffen School of Medicine at UCLA, Los Angeles, California 90095

1To whom requests for reprints should be addressed at The UCLA Center for Human Nutrition, 13–145 Warren Hall, 900 Veteran Avenue, Los Angeles, CA 90095-1742. E-mail: dmharris{at}ucla.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Increased intake of phytoestrogens may be associated with a lower risk of cancer in the breast and several other sites, although there is controversy surrounding this activity. One of the mechanisms proposed to explain the activity of phytoestrogens is their ability to bind and activate human estrogen receptor {alpha} (ER{alpha}) and human estrogen receptor ß (ERß). Nine phytoestrogens were tested for their ability to transactivate ER{alpha} or ERß at a range of doses. Mammary adenocarcinoma (MCF-7) cells were co-transfected with either ER{alpha} or ERß, and an estrogen-response element was linked to a luciferase reporter gene. Dose-dependent responses were compared with the endogenous ligand 17ß-estradiol. Purified genistein, daidzein, apigenin, and coumestrol showed differential and robust transactivation of ER{alpha}- and ERß-induced transcription, with an up to 100-fold stronger activation of ERß. Equol, naringenin, and kaempferol were weaker agonists. When activity was evaluated against a background of 0.5 nM 17ß-estradiol, the addition of genistein, daidzein, and resveratrol superstimulated the system, while kaempferol and quercetin were antagonists at the highest doses. This transfection assay provides an excellent model to evaluate the activation of ER{alpha} and ERß by different phytoestrogens in a breast cancer context and can be used as a screening bioassay tool to evaluate the estrogenic activity of extracts of herbs and foods.

Key Words: estrogen receptor ß • phytoestrogens • isoflavonoids • MCF-7 • bioassay


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Phytoestrogens are secondary metabolites produced in a wide variety of plants that induce biologic responses in vertebrates and can mimic or modulate the actions of endogenous estrogens, usually by binding to estrogen receptors (ERs; Ref. 1). The broad classes of phytoestrogenic compounds include isoflavonoids, coumestans, lignans, and stilbenes (2, 3). Specific classes of isoflavonoids include the flavones (e.g., apigenin), flavonols (e.g., quercetin, kaempferol), flavanones (e.g., naringenin), isoflavones (e.g., genistein, daidzein, equol), and anthocyanidins. Coumestrol is a coumestan and resveratrol, a stilbene (3–5). All are biphenolic compounds with structures resembling natural and synthetic estrogens. Soybeans and clover, alfalfa sprouts, and oilseeds (e.g., flaxseed) are the most significant dietary sources of isoflavones, coumestans, and lignans, respectively, but phytoestrogens are found in a wide number of foodstuffs such as soy beans, black beans, onions, and citrus fruits, as illustrated in Table 1Go (3).


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  		Table 1. Phytoestrogens Tested and Food Sources
 
Epidemiologic evidence supports a protective effect of high phytoestrogen diets to reduce the incidence of certain hormone-responsive cancers, such as breast and prostate cancer (6). Relative to Asian countries, the incidence and mortality from breast cancer is much higher in the Western world. The contributing factors to this differential effect are various; however, several studies have focused on the environmental, and specifically dietary, differences among these two parts of the world. Immigration studies have shown that rates of breast cancer in first-generation immigrants are low, but they increase in the second and subsequent generations, presumably due to intake of an increasingly Western diet (7, 8). Much of the epidemiologic studies have focused on isoflavones and soy, but interest is increasing in the protective effects of lignans in breast cancer (9). Average daily dietary intake of soy and isoflavones in Asian countries is estimated to be 50 g daily and 30 mg daily, respectively, while in the West the intake is closer to 1 g daily and 1 mg daily of each (10). The lower breast cancer incidence in Asian countries has also been correlated to early exposure to phytoestrogens through breast milk (11).

The mechanisms for the protective effect of phytoestrogens are unclear. One theory is that the presence of these low-affinity, low-potency ligands for ERs can reduce the effect of potent endogenous estrogens such as estradiol when they are present in sufficient quantities, with the net effect of antagonizing the estrogen-responsive system (12). The abundance of isoflavones relative to endogenous estrogens could explain their bioactivity despite their weaker binding. Setchell and Cassidy (13) showed that consumption of 50 mg/day of isoflavones in an adult can give rise to plasma concentrations ranging from 200–3200 nM, whereas endogenous plasma estradiol levels range from 0.14–0.28 pmol/ml (14). In addition, phytoestrogens have a weaker affinity for serum proteins such as albumin and sex hormone–binding globulin, which renders a relatively greater proportion of unbound phytoestrogens (14). A number of the pleiotropic molecular effects of phytoestrogens, including modulation of cell-signaling pathways, regulation of the cell cycle, stimulation of apoptosis, and antioxidant activities, may occur independently of ER binding (15, 16).

However, the health consequences of phytoestrogen exposure is perhaps not universally beneficial and, in certain situations, could increase disease risk, particularly with use of dietary supplements that could provide exceedingly high levels of phytoestrogens relative to those provided in whole foods (12). The potential adverse effects could include procarcinogenic effects in peri- and postmenopausal women taking supplements to ameliorate menopausal symptoms, potential effects of natural antiestrogens on reproduction, and developmental effects on children prenatally (in the diet of the mother) and postnatally (e.g., in soy-based infant formulas; Ref. 17). The known divergent chemical and biologic properties already identified of the phytoestrogens make it important to evaluate each individually and in the context of delivery as food or dietary supplement.

Phytoestrogens have the ability to bind the ER due to their biphenolic structure, which is required for the ligand-receptor association (18). They can act like partial ER agonists or antagonists. Several phytoestrogens show a pattern of differential binding to the two ER subtypes, ER{alpha} and ERß, with stronger binding to ERß as evaluated by radioligand-binding assay (19). The ER is part of the steroid/retinoid receptor gene superfamily, a class of soluble DNA-binding proteins that acts as ligand-activated enhancer factors (20). On ligand binding, the ER initiates transcriptional activation by binding to specific palindromic sequences called estrogen-response elements (EREs) in the promoters of target genes (21). However, non–ERE-driven transcriptional activation (e.g., via AP-1) has also been described (22). The differential effects of agonists and antagonists on receptor activity in a given cell context have been ascribed to different conformations of the receptor-ligand complex, as well as by differences in interaction with transcriptional coactivator and corepressor proteins and other transcription factors (23). Compounds showing varying degrees of agonist-antagonist activity, depending on the cell type and target gene, are referred to as selective ER modulators (22, 24). Many phytoestrogenic compounds have additional cellular activities not ascribed to activation of the ERs, such as regulation of cell-signaling pathways, and can have activity in inhibiting proliferation and inducing apoptosis in ER-negative breast cancer cell lines (e.g., MDA-MB-231), as well as in ER-positive lines (e.g., MCF-7; Ref. 25).

The two ER subtypes have significant sequence homology in amino acids in their DNA-binding domains (96%), but lesser in the ligand-binding domain (53%). Based on these homologies it is predicted that ERß recognizes the same EREs as ER{alpha}, but that the receptors bind distinct sets of ligands and, indeed, ER{alpha}- and ERß-specific ligands have been identified (genistein and coumestrol are considered ERß-selective agonist ligands; Refs. 20, 26). The "classical" ER, ER{alpha}, has been detected in the uterus, mammary glands, liver, central nervous system, cardiovascular system, bones, and urogenital tract in humans (27, 28). Originally cloned from rat prostate, ERß has been found to have a wide tissue distribution with the richest expression in the central nervous system, cardiovascular system, lungs, kidneys, urogenital tract, mammary glands, colon, immune system, and reproductive organs (27, 29). Estrogen receptor ß may negatively regulate cellular proliferation and have a protective role in normal breast (30). Currently, there is great interest in identifying ERß-specific agonists for therapeutic uses in several diseases, including cancer (31). In addition, as use of pharmacologic hormone-replacement therapy for the treatment of menopausal symptoms is waning, interest in the identification of natural sources of estrogens has increased (24).

We have adapted a transfected breast cancer bioassay system that has sensitivity up to 1 pM for 17ß-estradiol. In this assay system, either ER{alpha} or ERß is overexpressed by transient transfection. When the receptor is activated by the test ligand, the receptor forms a dimer that binds the estrogen response element, activating transcription of the luciferase reporter gene. In the studies presented we compared the abilities of genistein, daidzein, equol, apigenin, naringenin, kaempferol, quercetin, coumestrol, and resveratrol to activate a luciferase gene reporter system in two sets of transfected MCF-7 breast cancer cells. By altering the expression of ER{alpha} and ERß in a breast cancer environment, this assay may be useful to evaluate the activity of not only individual isolated compounds, but also of undefined extracts of whole foods and dietary supplements in a physiologically relevant model. Because of the endogenous expression of both ERs (weaker expression ERß), it is expected that necessary ER coactivators and corepressors are present in this cell line, something that may not be found in ER-negative cell lines. Conducting the assay with ligand alone versus against a background level of 17ß-estradiol allowed assessment of estrogenic versus antiestrogenic activity of this panel of phytoestrogens. This assay represents a flexible, sensitive, reproducible, and selective screening assay for transactivation capacity of various naturally derived estrogenic ligands in which activity on ER{alpha} and ERß can be compared in a parallel system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals.
Genistein, daidzein, equol, apigenin, naringenin, kaempferol, quercetin, resveratrol, and 17ß-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO); coumestrol was purchased from Indofine (Somerville, NJ). Phytoestrogens were dissolved in 100% ethanol (EtOH; Aldrich Chemical Co., Milwaukee, WI) and stored at –80°C. Purity of the stock solutions was verified for most of the phytoestrogens by high-performance liquid chromatography in the UCLA/NIH Clinical Nutrition Research Unit Nutritional Biomarker Core Laboratory. Concentrations were confirmed by absorbance measurements at the appropriate wavelength where the extinction coefficient was known.

Plasmids.
The laboratory of Thomas Scanlan at the University of California, San Francisco, graciously provided plasmids containing either ER{alpha} (psG5-HE0), ERß (psG5-hERß, 485 aa), or an estrogen-response element (ERE; EREII-Luciferase GL450) linked to the luciferase reporter gene. The HE0 plasmid contains a point mutation that elicits a lower background response. Use of these plasmids in cell transient transfection systems, including in HeLa and MCF-7 cells, has been previously described (22). We transformed Escherichia coli strain DH5{alpha} with these plasmids and used bacteria to amplify the plasmids to obtain microgram quantities. An additional ß-galactosidase–expression plasmid (CMV-ß; Promega, Madison, WI) was obtained from a commercial source and co-transfected in the assay system as an internal standard. Plasmid DNA was prepared using a commercially available kit (Qiagen Mega-kit; Qiagen, Valencia, CA).

Cell Culture, Transfection, and Luciferase Assays.
The MCF-7 cells (ATCC HTB-133; American Type Culture Collection, Rockville, MD) were maintained and subcultured in media containing Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12), with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies, Grand Island, NY), and bovine insulin (10 µg/ml; Sigma Chemical). They were kept in nongelatinized 75 cm2 flasks (Corning Incorporated, Corning, NY) under humidified conditions at 5% CO2. One day before transfection, the cells were plated in 24-well plates (Corning Incorporated) at a concentration of 8 x 104 cells per well. For transfection, maintenance media was aspirated and 100 µl of transfection mix was added. Transfection mix consisted of 0.5 µg DNA divided over the three plasmids (i.e., ER{alpha} or ERß, ERE-Luc, and CMV-ß), cationic liposome-based TransFast transfection reagent (1.5 µl/well; Promega), and phenol red-free DMEM/F12 medium. After an incubation of 1 hr for transfection, experimental medium was added without removal of the transfection media. The experimental medium was prepared with phenol red-free DMEM/F12, 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT), 10 µg/ml insulin (Sigma Chemical), and the purified phytoestrogens. Phytoestrogen stocks were prepared and stored at concentrations of 1–6 x 10–3 M, depending on the compound. Phytoestrogen stocks were diluted at 10-fold serial dilutions using cell-culture media to produce treatment media containing compounds at 10–5 M to 10–10 M. A 17ß-estradiol standard was also added in every plate at 10–6 M, and every experiment included an estradiol standard curve with 10-fold dilutions from 10–6 M to 10–12 M. A vehicle control containing 0.3% ethanol, which was the maximal level of ethanol used in the treatment media, was included for each dose-response set. Each level of compound was assayed in triplicate. Cells were incubated for 48 hrs at 37°C, then media was aspirated and cells were lysed with 100 µl of Reporter Gene Lysis Buffer (Promega). After a freeze-thaw cycle to enhance lysis, lysates were transferred to microcentrifuge tubes and stored at –80°C. For the luciferase assay, 20 µl of lysate was added to 100 µl of Luciferase Assay System (Promega). Luminescence was measured with a single tube luminometer (Type TD 20/20; Turner Designs, Sunnyvale, CA). For the internal standard, a ß-galactosidase assay kit (Promega) was used to assay enzyme activity, using a 50:50 ratio of lysate to reagent.

To evaluate the antiestrogenic activity of the various ligands, the previously described protocol was used in competition experiments whereby a background dose of 5 x 10–10 M 17ß-estradiol was included in the full range of phytoestrogen doses. The chosen level of estradiol represents approximately 80% of the maximal activation of the bioassay system (a subsaturating level).

Data Analysis.
The software program Prism 4.0 (GraphPad Software Inc., San Diego, CA) was used for data analyses. Levels of luciferase transcription for each phytoestrogen tested were compared with 17ß-estradiol (E2) at 10–6 M (positive) and vehicle (0.3% ethanol; negative) controls. The levels of relative luciferase activity were divided by the ß-galactosidase values to correct for well-to-well variation. The maximal achievable response of the native ligand (17ß-estradiol) was set at 100%, and responses for individual phytoestrogens were calculated as a percentage of the estradiol response. Mean values were calculated from triplicate wells averaged over three experiments. The effective concentration that elicits a half-maximal response (EC50) was estimated for each compound using nonlinear regression analysis with a sigmoidal dose-response model provided by the software package. Inter-and intraassay coefficients of variation (CVs) were calculated based on the error on multiple measurements of the 17ß-estradiol positive control included in three wells on every plate. For the competition assays, the response of 1 µM 17ß-estradiol was set at 100%, and responses for different levels of added phytoestrogens were calculated as a percentage of the estradiol response. A one-way ANOVA was performed for each compound at each ER, with Dunnett’s posttest used to compare each treatment level to untreated control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The aim of the study was to evaluate differential transcriptional activation of the two ERs, ER{alpha} and ERß, by phytoestrogens commonly found in foods. Cells transiently transfected with either ER{alpha} or ERß and the ERE-reporter plasmid were incubated with either 17ß-estradiol or one of a panel of phytoestrogens/metabolites. An overview of the chemical names, chemical class, common natural source of the phytoestrogens tested, and reported plasma values is given in Table 1Go.

The natural ligand 17ß-estradiol induced a reproducible response in our system in a range from 10–6 to 10–12M; the response with either ER{alpha} or ERß activation was equivalent (Fig. 1Go). The sensitivity of the assay under the given conditions reaches 1 pM for 17ß-estradiol, and the 17ß-estradiol response reaches a plateau beginning at 1 nM. The interassay CV was calculated to be 8%, and the intraassay CV was calculated to be 7% for the ER{alpha} system and 6% for the ERß system. A validation experiment conducted with reporter gene only, devoid of supplemented ER, showed that the maximal response at the highest dose of 17ß-estradiol was only 38% that of the response with ER{alpha} (data not shown).



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  		Figure 1. Dose-response curve for 17ß-estradiol. The MCF-7 cells were transiently transfected with one of two expression plasmids containing ER{alpha} (circle) and ERß (triangle) plus a luciferase reporter plasmid containing the ERE. Stock 17ß-estradiol was diluted 10-fold from 10–6 M to 10–12 M and included in the experimental media. Transcriptional activity is represented as relative light units (RLUs) calculated as percentage of the maximal induction by estradiol (10–6 M) and presented normalized to ß-galactosidase activity. Nonlinear regression analysis was performed using a sigmoidal dose-response model. Data presented are the means + SEM of three experiments, with each dose assayed in triplicate wells.

 
Activation of luciferase transcription after treatment with the phytoestrogens genistein, daidzein, equol, apigenin, naringenin, kaempferol, quercetin, resveratrol, or coumestrol at log doses from 10–10 M to 10–5 M was compared with the maximal response of luciferase transcription at 1 µM 17ß-estradiol. Transcriptional activity at each dose is shown in Figure 2Go as a percentage of the maximal response with 17ß-estradiol treatment. The baseline response, representing background responsiveness in the absence of ligand, was <20% of the estradiol response in almost all cases. We found that genistein, daidzein, coumestrol, and apigenin each elicited a robust full dose–response in ERE-mediated gene transcription in the range of 10–10 M to 10–5 M, especially at ERß (Fig. 2Go). However, coumestrol did not fully stimulate the system relative to estradiol (Vmax was 90%), and the major metabolite of daidzein, equol, was only a partial agonist (Vmax was 57%). Naringenin and quercetin were also weak agonists, as was resveratrol (except for the 10–5 M dose at ERß).



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  		Figure 2. Dose-response curves for isolated phytoestrogens. Cells containing either ER{alpha} (circle) or ERß (triangle) and the ERE reporter plasmid were incubated with either genistein, daidzein, equol, apigenin, naringenin, kaempferol, quercetin, resveratrol, or coumestrol at levels between 10–5 M and 10–10 M. Transcriptional activity is represented as RLUs calculated as percentage of the maximal induction by estradiol (10–6 M) and presented normalized to ß-galactosidase activity. Nonlinear regression analysis was performed using a sigmoidal dose-response model. Data points represent the means + SEM of three experiments, with each dose assayed in triplicate wells.

 
The EC50 was estimated for each of the phytoestrogens with a full dose-response curve and these values are summarized in Table 2Go. The EC50s ranged from 10–6 to 10–7M and from 10–7 to 10–11M for ER{alpha} and ERß, respectively. For ER{alpha}, we were only able to calculate an EC50 for the response to coumestrol and genistein; each had an approximately 100-fold greater sensitivity for activating transcription via ERß compared with ER{alpha}.


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  		Table 2. The EC50 Values for 17ß-Estradiol of Various Compounds for ER{alpha} and ERßa
 
When a background of estradiol was present, individual phytoestrogens displayed differing activities (Figure 3Go). In the case of kaempferol and quercetin, which had minimal agonist activity, the highest doses were able to compete with estradiol for receptor binding and transcriptional activation (about 42% inhibition relative to 0 M phytoestrogen for both kaempferol and quercetin at ERß). However, the compounds genistein and daidzein, which had robust estrogenic-activating capacity especially at ERß, at the highest doses showed activity well above the maximal activation capacity of 10–6 M estradiol alone (up to 218% for genistein and 151% for daidzein at ERß), indicating that estradiol alone does not maximally stimulate the system. Resveratrol also exhibited additive agonist activity when presented with estradiol. The other phytoestrogens (i.e., apigenin, naringenin, coumestrol) did not compete with estrogen activity, although each had agonist activity when tested in the system alone.



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  		Figure 3. Phytoestrogen response in the presence of estradiol. Cells containing either ER{alpha} (light shading) or ERß (dark shading) and the ERE reporter plasmid were incubated with either genistein, daidzein, apigenin, naringenin, resveratrol, kaempferol, quercetin, or coumestrol, at doses between 10–5 M and 10–9 M in media containing 0.5 nM estradiol. Transcriptional activity is represented as RLUs calculated as a percentage of the maximal induction by estradiol (10–6 M) and presented normalized to ß-galactosidase activity. The 0 M treatment contains estradiol media but no ligand, and the control (ctrl) is a vehicle control (0.3% EtOH). Bars represent the means + SEM of three experiments, with each dose assayed in triplicate wells. One-way ANOVA with Dunnett’s posttest was performed for each compound at each ER. Treatment means that are significantly different from control are indicated as *P < 0.05 and **P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we present a breast cancer bioassay system in which MCF-7 cells are transiently transfected with ER{alpha} or ERß and the estrogenic response was measured through an ERE-response element linked to a luciferase reporter gene. The MCF-7 cell line was chosen because endogenous expression of ERs in MCF-7 cells is predominantly ER{alpha} with weak expression of ERß, suggesting that the full complement of coactivators and corepressors necessary for full ER activity are present in this line (32). The transactivational activity of nine commonly found phytoestrogens was determined in this assay. As others have shown in various transactivation assays in several mammalian cell types, we found that genistein, daidzein, apigenin, and coumestrol selectively promote ERß-mediated transcription (19, 33–36). We also assessed the agonist activity of equol, a metabolite of daidzein that is produced by the action of intestinal bacterial metabolism in a subset (approximately 33%) of humans (37, 38). In our assay system, equol also exhibited partial agonist activity; however, its maximal activation was only about half that of daidzein at ERß. The ability to produce equol in the gut has been associated with lower risk of breast cancer in premenopausal women; "equol producers" show plasma steroid hormone profiles associated with a lowered risk of breast cancer (39). Plasma levels up to 8 x 10–7 M of equol have been reported in equol producers (14), a value higher than the EC50 we found for activity at ERß (Table 2Go).

The greater affinity of the phytoestrogens for ERß is thought to be significant in cancer prevention, as ERß has been proposed to exert a protective role against cancer. Gustafsson (27) hypothesized that the ratio of ERß relative to ER{alpha} in a given tissue may be important to determine the susceptibility of a tissue to estrogen-induced carcinogenesis. It is also postulated that ER{alpha} and ERß have different or even opposite biologic actions, demonstrating a yin-yang relationship (40). The ERß may negatively regulate cellular proliferation and have a protective role in normal breast (30); therefore, there is substantial interest in ERß-selective ligands (27, 40). One molecular mechanism for ERß selectivity by isoflavones appears to involve their capacity to create an activation function-2 surface of ERß that has a greater affinity for coregulators than ER{alpha} (35). In addition, ER{alpha} and ERß can heterodimerize and, when coexpressed in the same cell, ERß exerts an inhibitory action on ER{alpha}-mediated gene expression (41). We did not complete an ER{alpha} and ERß cotransfection in our system, but it is possible that endogenous ER{alpha} or ERß expression could alter the activity of the exogenously introduced receptor and could account for some differences in results relative to other systems using ER-negative cell types. However, because we are overexpressing the ERs artificially, one might assume that the contribution of the exogenous receptors would supersede that of the endogenous.

The activity of the phytoestrogens is mediated not only through activation of the ER, but also via extragenomic actions. For example, other cellular actions of genistein have been described, such as inhibition of tyrosine kinase activity, modulation of transforming growth factor ß1 signaling pathways, regulation of cell-cycle checkpoints including G2M arrest, inhibition of DNA topoisomerase II activity, and antioxidant activity (42–44). The mechanisms of these actions are not well understood, but there is increasing evidence that distinct pools of ERs localize to the plasma membrane and play roles in some of the responses due to estrogenic ligands (41).

An interesting finding of our system is that when genistein and daidzein were coincubated with a background dose of 17ß-estradiol, the activity of these phytoestrogens with 17ß-estradiol was additive. As other competitive binding assays of these isoflavones have shown that they can compete with 17ß-estradiol for binding the receptor, albeit with lower affinity, this result might have been unexpected (45, 46). However this "superagonist" effect has been previously seen in stably transfected T47T breast cancer cells treated with genistein (47) and in MVLN cells (an MCF-7 derivative, which has been stably transfected with a vitellogenin-A2 promoter/luciferase reporter construct) treated with genistein, daidzein, or resveratrol (48). The additive agonist activity shows that the capacity of the assay is not maximized by the highest dose of 17ß-estradiol. The mechanisms for this effect is unknown, but because the binding affinities of these compounds to the ERs are thought to be only a fraction of that of 17ß-estradiol (especially at ER{alpha}; Ref. 19), this activity may potentially be due to extragenomic actions of these ligands, as previously described.

Resveratrol had no agonist activity except at the very highest dose at ERß; our data are in agreement with receptor-binding data showing weak affinity of resveratrol for both ER{alpha} and ERß (49). However, resveratrol also exhibited an additive agonist activity in conjunction with 17ß-estradiol. Gehm et al. (50, 51) studied this effect in MCF cells transiently and stably transfected with a luciferase reporter gene and treated with resveratrol. The additive action was seen at micromolar doses of resveratrol in MCF cells, but not in BG-1 ovarian carcinoma cells. Suboptimal doses of 17ß-estradiol and resveratrol were additive, but maximal activation by resveratrol was not further increased by 17ß-estradiol, suggesting that both compounds activate the same receptor (50). Conversely, coumestrol, which had strong agonist activity especially at ERß, had no inhibitory activity when presented with 17ß-estradiol.

The other flavonoid compounds naringenin, kaempferol, and quercetin exhibited weak agonist activity at either receptor. The relative magnitude of the maximal agonist response for these three compounds roughly parallels the relative transactivation activity found in HEK293 cells transiently transfected with ER{alpha} and ERß (19). In addition, naringenin has been previously shown to be only weakly estrogenic in the MVLN assay (52). However, in the competition experiments, kaempferol and quercetin, but not naringenin, inhibited 17ß-estradiol activity by some 42% at the highest doses at both receptors, indicating that these two compounds may have estrogen antagonistic activity. Anti-estrogen activity of quercetin has been reported previously; for example, 1 µM quercetin inhibits estrogen-stimulated growth of MCF-7 cells (53).

Our results also support the structure-activity relationships that have been shown to be essential for estrogenic activity. Structural similarity of the phytoestrogens with 17ß-estradiol is based on the phenolic ring required for binding to the ER, as well as the presence of two hydroxy groups. The ER-binding ability and transcriptional activation appear to be dependent on the position of the hydroxyl groups, where maximal potency is achieved at positions 4' hydroxy on the phenolic B ring, in combination with the 7-position hydroxy on the A ring (54). For antagonistic properties, however, the 5' hydroxy/4'-keto grouping on the A/C ring has been suggested to be of importance (54). The structural difference between genistein and kaempferol (shifting of the phenolic B ring from the 2' to the 3' position) results in a 10-fold lower potency in ER binding and in our assay, transcriptional activity. Likewise, oxidation of kaempferol to quercetin results in another 10-fold lower potency in ER binding (54). Our results support a similar relationship.

This system proves to be a sensitive assay to differentiate response to ER{alpha} and ERß by estrogen-like compounds. The sensitivity of the assay under the given conditions reaches the picomolar level for 17ß-estradiol. This sensitivity is comparable or greater than reported elsewhere for estradiol response at both ER{alpha} and ERß in several transfected mammalian cell lines (19, 34–36, 55). The reported sensitivity of the MVLN assay is approximately the same for the prototype agonist 17ß-estradiol (subnanomolar levels of estradiol), as we found in our assay (56).

Our system is unique in that transcriptional activation by the ERß can be directly compared with that of the classical receptor ER{alpha}. This bioassay system was validated to be specific for the ER in preliminary agonist and antagonist experiments comparing 17ß-estradiol (0.1 µM) with diethylstilbestrol (DES; 1 µM), tamoxifen (5 µM), and ICI 182,780 (1 µM). As expected from previous reports, we found robust stimulation of both ER{alpha} and ERß with single doses of 17ß-estradiol and DES, with activity at or below control levels with tamoxifen and ICI 182,780 (data not shown). These results were confirmatory of the original report of Paech et al. (22) showing activation of both ER{alpha} and ERß in HeLa cells by 17ß-estradiol and DES and antagonism by raloxifene, tamoxifen, and ICI 164,384 at the same doses. As previously seen, 17ß-estradiol was able to activate both ER isomers with similar potency, as shown in Figure 1Go. Table 2Go shows that the EC50s for activation by 17ß-estradiol at both ERs are comparable. The EC50s for 17ß-estradiol in similar transfection systems using ER{alpha} and ERß have not generally been reported; therefore, making comparisons to previously published values is difficult. Using a radioligand receptor-binding assay, Kuiper et al. (57) reported dissociation constant (Kd) values for 17ß-estradiol binding in the nanomolar range, with similar values for both ER{alpha} and ERß. Similarly, Tremblay et al. (58) found that Cos-1 cells transfected with murine ER{alpha} and ERß showed similar luciferase activation curves; ligand-binding analysis calculated similar Kds of 0.2 nM for ER{alpha} vs. 0.5 nM for ERß. In a system using HepG2 cells transfected with both rat receptors, calculated EC50s were 5-fold different between ER{alpha} and ERß (0.32 nM and 1.5 nM, respectively; Ref. 36). Our system exhibited greater sensitivity by order of magnitude, as our calculated EC50s were 0.06 nM and 0.04 nM (Table 2Go).

The phytoestrogens coumestrol, genistein, daidzein, naringenin, apigenin, and kaempferol were tested in the radioligand-binding system and showed an overall stronger affinity for ERß (19). The bioassay presented here extends these observations, is unique in offering a direct comparison of the responses of isolated phytoestrogens for ER{alpha} and ERß in a breast cancer context, and incorporates the entire biologic phenomena of activation of transcription. The estrogenic response of compounds has been shown to depend heavily on the cell or tissue type due to the cell-specific presence of corepressors and coactivators (22, 27, 31, 40, 59).

The levels of phytochemicals included in our assay are thought to be achievable in vivo, as plasma concentrations of daidzein, genistein, and equol have been determined to be in the µM range after acute soy ingestion (Table 1Go; Ref. 1). In another study, the plasma concentrations of total phytoestrogens were estimated to be 2–4 µM following the moderate consumption of soy products (60). These levels are dependent on rates of metabolism and clearance, which show considerable individual variation as seen with equol production from daidzein (61). A relevant question is to what levels of phytoestrogens, and in which form, are cells exposed in vivo? Most of the phytoestrogens are converted to glucuronide or sulfate conjugates in the small intestinal cell and further metabolized in the liver. Therefore, the main circulating form is the conjugated form (62, 63). However, information in the literature is limited in regards to tissue uptake and metabolism. Most likely the conjugated form is reversed before absorption into the tissue. Based on previous findings outlined in Table 1Go, which summarizes maximal blood levels of the various phytoestrogens reported in the literature (where available), the concentrations used in our experiments could be physiologically relevant. Note that in many cases these plasma values exceed the EC50 for activation of ERß. Granted, serum levels do not predict local metabolism at the level of the breast, but we believe most of the conversion occurs at the level of the gut and liver, which gives us some indication that target tissue levels may be somewhat reflected by plasma levels. One study that evaluated breast tissue levels of phytoestrogens in soy-supplemented individuals before aesthetic breast surgery is present in the literature (64). It was found that genistein was undetectable in breast tissue homogenate, but daidzein was present in approximately 100-fold lower levels than serum (64). Equol, however, was present in higher concentrations in breast tissue (and undetectable in serum). This study was run in only nine women, and interindividual variability was great. Also, the time between last supplement ingestion and breast tissue sampling was 12 hrs or more, probably exceeding the half-life of the compounds in blood and making interpretation of the study still complex. In mice, gavage of tritiated naringenin showed that 8 hrs after treatment the compound accumulated in a multitude of tissues, including the uterus and ovary reproductive organs and, to a lesser extent, in the mammary glands (65). These studies suggest that considering their abundance in the diet, phytoestrogens may reach sufficiently high levels in tissues, including breast, to exert biologic effects and that the levels evaluated in our system are physiologically achievable. However, the question of tissue-level bioavailability of phytoestrogens is an issue that merits further study.

This assay is useful not only to evaluate isolated compounds, but also to evaluate extracts of whole foods and herbal supplements. Future studies will identify actions and interactions of sets of compounds as found in natural products. A system that evaluates only ER{alpha} activity in transfected yeast identified estrogenic activity in a number of herbs including hops, licorice, soy, red clover, and fo-ti at levels up to one eightieth the activity of estradiol (for soy aglycone; Ref. 66). The system presented here could be useful to detect the estrogenicity of complex mixtures of bioactive compounds as found in dietary supplements, food, serum, and from environmental sources (67, 68). Additional applications could include use in standardizing herbal products before medicinal compounding, which will aid in identifying variability among acquisitions.

In summary, our bioassay system distinguishes differential effects of phytoestrogens on the {alpha} and ß isoforms of the ER in a breast cancer cell environment. The differential effects of individual phytoestrogens show that the bioactivity of each must be independently evaluated. The direct comparison between responses to the two ER isomers and in the presence or absence of an estradiol background makes this assay an important screening tool for natural estrogen surrogate compounds (to prevent symptoms associated with estrogen deficiency or identify environmental endocrine disruptors), or for compounds that work antagonistically (which may be important for natural selective ER modulator activity in a given tissue). This provides insight into one facet of phytoestrogen action, with the full picture requiring multiple levels of study in vitro and in vivo.


   Acknowledgments
 
The authors gratefully acknowledge the technical support of Eric Genosian and Randolph Madrid.


   Footnotes
 
These studies were funded by the UCLA Center for Dietary Supplement Research: Botanicals (The National Institutes of Health Grant AT00151). Support was also provided by the American Gastroenterological Association/Miles & Shirley Fiterman Foundation, as well as the Dr. H. Muller’s Foundation in the Netherlands (for E.B.).

Received for publication December 15, 2004. Accepted for publication May 31, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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