Estrogen Receptor-Mediated Actions of Polyphenolic Catechins in Vivo and in Vitro

M. G. Goodin*, K. C. Fertuck{dagger}, T. R. Zacharewski{dagger} and R. J. Rosengren*,1

* Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand; and {dagger} Department of Biochemistry and Molecular Biology, and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824

Received May 29, 2002; accepted July 16, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent investigations have demonstrated that polyphenolic catechins inhibit breast cancer cell proliferation and tumor growth. However, the ER-mediated effects of the three predominant catechins (EGCG, ECG, and EGC) have not been extensively examined in vitro or in vivo. Therefore, EGCG, ECG, and EGC were examined for their ability to compete with [3H]-17ß-estradiol ([3H]-E2) for binding to ER{alpha} and ERß and to elicit reporter gene activity in MCF-7 human breast cancer cells transiently transfected with either chimeric ER{alpha} or ERß. EGCG and ECG displaced [3H]-E2 from GST-hER{alpha}def (D, E, and F domains of human ER{alpha} fused to GST) or from full-length human ERß. Additionally, only EGCG elicited Gal4-hER{alpha}def and Gal4-mERßdef-mediated reporter gene expression (EC50 values: 28 and 19 µM, respectively) in MCF-7 cells cotransfected with a Gal4-regulated luciferase reporter gene. In cotreatment experiments, EGCG (1–50 µM) and ECG (1 µM) decreased E2-induced (1 nM) ERß-mediated gene expression 35–50%. In vivo, no catechin induced estrogenic responses (uterine weight or uterine peroxidase activity) in immature C57BL/6 mice. However, when mice were cotreated with E2 (10 µg/kg/day, 3 days) and either EGCG (30 and 50 mg/kg/day, 3 days) or ECG (50 mg/kg/day, 3 days), uterine peroxidase activity was increased 2.3-fold above that elicited by E2 alone. In conclusion, EGCG and ECG bind to ER{alpha} and ERß, but only EGCG elicited ER-mediated gene expression in vitro. However, both of these compounds moderately increased E2-inducible responses in vivo.

Key Words: catechins; EGCG; ECG; EGC; ER{alpha}; ERß; human breast cancer cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Catechins are a group of polyphenolic compounds found in a variety of foods, such as green tea (Mukhtar et al., 1992Go), red wine (Damianaki et al., 2000Go), chocolate (Arts et al., 1999Go), and certain fruits (Arts et al., 2000Go). The predominant catechins contained in these sources include epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and epigallocatechin (EGC). There is scientific interest in members of this chemical family partly due to their chemopreventative (Stoner and Mukhtar, 1995Go; Suganuma et al., 1999Go) and antitumor properties (Hirose et al., 1994Go; Kavanagh et al., 2001Go; Liao et al., 1995Go). For example, the catechins inhibit the proliferation of human breast cancer cells (Morre et al., 2000Go; Valcic et al., 1996Go) and epidemiological studies have suggested that green tea consumption is linked to a decrease in the rate of development and recurrence of breast cancer (Nakachi et al., 1998Go). Additionally, treatment with EGCG (50 mg/kg/day, 14 days) reduced the growth of MCF-7 implanted breast tumors in athymic nude mice by 40% (Liao et al., 1995Go). Therefore, the catechins may have beneficial characteristics that could be exploited in the treatment of breast cancer. However, the mechanism of catechin-mediated inhibition of tumor growth and human breast cancer cell proliferation has yet to be defined. Since the catechin family is structurally similar to isoflavones (Fig. 1Go), it is possible that catechins act as estrogen receptor (ER) antagonists.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 1. Chemical structures depicting the catechin and isoflavone backbones and EC, EGC, ECG, and EGCG.

 
Only a few investigations have examined catechin interactions with the ER and most have primarily focused on the effects of (-)-catechin and epicatechin (EC). High ER{alpha} binding affinities have been reported for (-)-catechin and EC in MCF-7 and T47-D human breast cancer cells (Damianaki et al., 2000Go). However, (-)-catechin (1 nM–1 mM) failed to bind to the rat uterine cytosolic ER (Fang et al., 2001Go) and these two catechins lacked activity in a yeast screen assay (Breinholt and Larsen, 1998Go). In the catechin family EGCG, ECG, and EGC are the most potent inhibitors of human breast cancer cell proliferation (Morre et al., 2000Go; Valcic et al., 1996Go), but only one investigation has examined their ER-mediated effects. Kuruto-Niwa et al.(2000) demonstrated that these catechins were unable to elicit an ER-mediated response in ER{alpha} and ERß reporter gene assays. However, 5 µM of EGCG and ECG antagonized the E2-induced response via ER{alpha}, while lower concentrations of EGCG increased the E2-induced response. In contrast, 5 nM–5 µM concentrations of EGCG, EGC, and EC increased the E2-induced response mediated through ERß (Kuruto-Niwa et al., 2000Go). These results indicate that catechin-mediated responses via ERE-regulated reporter genes are both concentration and ER isoform-dependent. However, the reported responses have not been correlated with either binding affinities for ER{alpha} and ERß or in vivo responses. Therefore, there is insufficient evidence to evaluate the ER-mediated activities of the catechins. The aim of the present study was to comprehensively determine the in vivo and in vitro ER-mediated actions of EGCG, ECG, and EGC. Receptor binding and reporter gene assays were used to determine ER{alpha} and ERß-mediated responses in vitro while uterotropic responses were examined in immature female mice to determine in vivo ER-mediated responses.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
17ß-Estradiol (E2), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), guaiacol, dimethyl sulfoxide (DMSO), trizma hydrochloride (Tris-HCl), alanine aminotransferase (ALT) diagnostic kit, o-nitrophenyl-ß-D-galactopyranoside (ONPG), and calcium chloride were purchased from Sigma Chemical Co. (St. Louis, MO). [2,4,6,7,16,17–3H]-E2 ([3H]E2; 118 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). Fetal bovine serum (FBS) was obtained from Intergen (Purchase, NY). Phenol red-free Dulbecco’s Modified Eagle Medium (DMEM) and antibiotics were purchased from Life Technologies (Rockville, MD). D-Luciferin was purchased from Molecular Probes (Eugene, OR). Hydroxyapatite was purchased from Bio-Rad (Auckland, New Zealand). All other chemicals were of the highest purity available.

Competitive ligand binding assay.
The method used for the competitive binding assay has recently been described in detail (Matthews and Zacharewski, 2000Go), but is outlined briefly as follows. Experiments were performed using either a bacterially expressed fusion protein consisting of glutathione-S-transferase and the D, E, and F domains of human ER{alpha} (GST-hER{alpha}def, > 85% purity; Matthews and Zacharewski, 2000Go) or full-length human ERß (hERß, > 80% purity; Panvera, Madison, WI; Fertuck et al., 2001Go). The receptor was first diluted in TEGD buffer (10 mM Tris pH 7.6, 1.5 mM EDTA, 1 mM DTT, and 10% [v/v] glycerol) containing 1 mg/ml BSA as a carrier protein. An aliquot (240 µl) was incubated at 4°C for 12 h with 5 µl of 2.5 nM [3H]-E2 and 5 µl of unlabeled competitor (10 pM to 1 µM final concentration of E2, or 0.1 µM to 2 mM final concentration of the catechins). [3H]-E2 and all competitor compounds were dissolved in DMSO and the final solvent concentration did not exceed 4%. Each concentration was tested in quadruplicate and at least three independent experiments were performed. Results are expressed as percent [3H]-E2 bound versus log of competitor concentration. Analysis was performed using nonlinear regression and the single-site competitive binding option of GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Reported IC50 values denote the calculated concentration of test compound required to displace 50% of the [3H]-E2 from the receptor protein. Relative binding affinity was then determined from the following equation (IC50 17ß-estradiol/IC50 of the catechin) x 100.

Cell culture and viability.
MCF-7 human breast cancer cells were kindly provided by Dr. L. Murphy (University of Manitoba, Winnipeg, Manitoba, Canada). Cells were maintained in DMEM supplemented with 10% FBS and with 20 mM HEPES, 2 mM L-glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, 2.5 mg/ml amphotericin B, and 50 mg/ml gentamicin. Cells were grown at 37°C in a 4% CO2 humidified environment. Cell viability was determined by the Sulforhodamine-B assay as described (Villalobos et al., 1995Go).

Transfection and reporter gene assays.
Cells were plated in 6-well culture dishes in 2 ml DMEM supplemented with 5% FBS that had earlier been dextran-coated charcoal-stripped (Clarke et al., 1989Go). Transfections were performed by the calcium phosphate coprecipitation method, which has a transfection efficiency of up to 20% (Sambrook et al., 1989Go), as earlier described (Fertuck et al., 2001Go) using the following three plasmids: (1) 1.5 µg 17m5-G-Luc (provided by Dr. P. Chambon, IGBMC CNRS-LGME, Illkirch Cedex C.U. de Strasbourg, France), (2) 0.2 µg Gal4-hER{alpha}def (Gal4 linked to D, E, and F domains of the hER{alpha}; also known as Gal4-HEG0) or Gal4-mERßdef (Gal4 linked to D, E, and F domains of mouse ERß), and (3) 0.2 µg pCMV-lacZ, a ß-galactosidase expression vector (Amersham Pharmacia) used for normalizing transfection efficiency across wells. Eighteen h posttransfection, cells were treated with test compound dissolved in DMSO so that the total solvent concentration did not exceed 0.1%. In cotreatment experiments, cells were treated with catechins (0.1 µM to 0.2 mM) and E2 (0.1 or 1 nM), and the DMSO concentration did not exceed 0.2%. The cells were harvested in 100 µl of lysis buffer after 24 h of treatment and reporter gene activity was measured using standard protocols (Brasier et al., 1989Go; Sambrook et al., 1989Go). Luciferase activity of a 10 µl aliquot was measured using a Luminoskan luminometer (Lab-systems, Frankin, MA) in the presence of 9 µM D-luciferin and 2 mM ATP. ß-Galactosidase activity was measured at 420 nm in the presence of 2.5 mM ONPG. Each treatment was performed in duplicate, and two aliquots were assayed from each well. Independent experiments were performed at least three times, and results are expressed as % of the maximum E2 response, normalized for ß-galactosidase activity. GraphPad Prism 3.0 software was used for graphical analyses, including the calculation of EC50 values, which denote the concentration of test compound required to cause 50% of the maximal response induced by E2.

Animals.
Immature female C57BL/6 mice (20 days old) were purchased from Department of Laboratory Animal Sciences, Dunedin. The animals were housed in microisolator cages with shredded paper bedding and had free access to rodent diet and water. They were maintained at 21–24°C with a 12-h light/dark cycle and allowed to acclimatize for 1 day before experimentation. Mice were dosed with either E2 (10 µg/kg/day), EGCG, ECG, EGC (30 or 50 mg/kg/day, ip), or E2 + catechins. Sesame oil served as the vehicle and there were 8 mice in each of the treatment groups. Each animal was dosed ip at a volume of 5 ml/kg for 3 consecutive days, and sacrificed on Day 4 by CO2 inhalation 20 h following the final dose. The doses selected were based on published work, which demonstrated that EGCG (50 mg/kg/day, 14 days, ip) inhibited tumor growth in an MCF-7 cell implant model in mice (Liao et al., 1995Go).

Evaluation of hepatic injury.
Immediately following euthanasia, blood was collected from the inferior vena cava and stored on ice. Plasma was separated and alanine amiontransferase (ALT) activity was determined kinetically using a Sigma diagnostic kit. Results are expressed as IU/l.

Rodent uterotrophic assay.
The rodent uterotrophic assay was performed as described previously (Patel and Rosengren, 2001Go). Uteri were removed just above the junction with the cervix and below the junction with each ovary. After removal, fat was trimmed off and the uteri were blotted on filter paper and weighed. Blotted uterine weight is expressed as mg of uterine tissue per g of body weight.

Uterine peroxidase activity.
Uterine peroxidase activity was performed as described previously (Patel and Rosengren, 2001Go) and is briefly outlined as follows. Upon removal, the uteri were placed in ice-cold 10 mM Tris-HCl buffer, pH 7.2. Uteri were pooled from 2 mice to ensure a sufficient amount of protein for each measurement and then homogenized in 10 mM Tris-HCl buffer (pH 7.2). The homogenate was centrifuged at 39,000 x g for 45 min at 2°C and the pellet resuspended in 1 ml 10 mM Tris-HCl buffer containing 0.5 M CaCl2, pH 7.2. After another centrifugation at 39,000 x g for 45 min at 2°C, the protein concentration of the supernatant was determined (Bradford, 1976Go). Oxidation of guaiacol was used as a measure of peroxidase activity. Extract (0.4 mg/ml) was added to guaiacol buffer (13 mM guaiacol, 0.3 mM H2O2 in 10 mM Tris-HCl buffer containing 0.5 M CaCl2) and the increase in absorbance was read at 470 nm at 25°C. Results are expressed as percent of control.

Uterine cytosolic ER binding.
Uterine cytosolic extract preparation and competitive binding has recently been described in detail (Patel and Rosengren, 2001Go), but is outlined briefly as follows. After separation of uterine cytosolic extract by centrifugation, 980 µl of cytosol (2 mg/ml) was incubated at 30°C for 30 min with 10 µl of 10 nM [3H]-E2 and 10 µl of unlabeled competitor (10 pM to 0.1 µM final concentration of E2, or 1 µM to 1 mM final concentration of the catechins). [3H]-E2 and all competitor compounds were dissolved in DMSO and the final solvent concentration did not exceed 2%. Following the incubation, 200 µl was added to 200 µl of hydroxyapatite slurry (1:3 in TEGD buffer) and incubated on ice for 30 min with vortexing every 10 min. The pellets were washed twice with 1 ml TEGD buffer and then dissolved in 1 ml absolute ethanol that was then transferred to scintillation vials and counted on a Beckman LS3801 scintillation counter. Each concentration was tested in quadruplicate and at least three independent experiments were performed. Results are expressed as percent [3H]-E2 bound versus log of competitor concentration. Analysis was performed using nonlinear regression and the single-site competitive binding option of GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego, CA). Reported IC50 values denote the calculated concentration of test compound required to displace 50% of the [3H]-E2 from the ER.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Results from binding experiments demonstrated that EGC was the only catechin unable to compete with [3H]-E2 for binding to either GST-hER{alpha}def or full-length hERß (Fig. 2Go). Both EGCG and ECG elicited similar potencies for GST-hER{alpha}def (relative binding affinities of 0.0012 and 0.0010 for EGCG and ECG, respectively) while EGCG had a greater affinity for hERß than ECG (relative binding affinities of 0.0085 and 0.0004 for EGCG and ECG, respectively; Fig. 2Go). Similar results were obtained using mouse uterine cytosol, as EGCG and ECG competed with [3H]-E2 while EGC did not (Fig. 3Go). The IC50 values obtained were similar to those obtained with GST-hER{alpha}def (460 ± 84 and 580 ± 96 µM for EGCG and ECG, respectively). However, all IC50 values were significantly higher than E2, which elicited similar IC50 values with both GST-hER{alpha}def and hERß (5.7 ± 1.1 and 8.2 ± 0.8 nM, respectively). Reporter gene assays demonstrated that E2 elicited a concentration-dependent increase in luciferase activity. The EC50 values for this response were similar for both chimeric receptors (59 ± 13 and 68 ± 12 pM for Gal4-hER{alpha}def and Gal4-mERßdef, respectively). Maximum induction occurred at 10 nM E2 and was increased 35- and 25-fold for Gal4-hER{alpha}def and Gal4-mERßdef, respectively. However, only EGCG elicited both Gal4-hER{alpha}def and Gal4-mERßdef-mediated gene expression (Fig. 4Go). While a strong response was elicited, the response only occurred at high concentrations, with EC50 values of 28 ± 15 and 19 ± 13 µM for ER{alpha} and ERß, respectively. Cotreatment of the catechins and 0.1 or 1 nM E2 was conducted to test for antiestrogenicity and none of the catechins attenuated ER{alpha}-mediated gene expression (Fig. 5Go). However, cotreatment with 0.5 µM EGCG plus 0.1 nM E2 elevated ERß-mediated gene expression to the level elicited by 1 nM E2 (a 200% increase over that produced by 0.1 nM E2) (Fig. 5Go). In contrast, cotreatment of either 1–50 µM EGCG or 1 µM ECG with 1 nM E2 decreased the ERß-mediated gene expression 35–50% compared to 1 nM E2 alone (Fig. 5Go).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 2. Competitive displacement curves for E2, EGCG, ECG, and EGC from GST-hER{alpha}def and full-length hERß as described in the Materials and Methods section. Results are the mean ± SD of three independent experiments performed in quadruplicate.

 


View larger version (16K):
[in this window]
[in a new window]
 
FIG. 3. Competitive displacement curves for E2, EGCG, ECG, and EGC from mouse uterine cytosolic ER as described in the Materials and Methods section. Results are the mean ± SD of three independent experiments performed in quadruplicate.

 


View larger version (12K):
[in this window]
[in a new window]
 
FIG. 4. Induction of (A) Gal4-hER{alpha}def- and (B) Gal4-mERßdef-mediated reporter gene expression in transiently transfected MCF-7 cells treated with E2, EGCG, ECG and EGC for 24 h. Results are expressed as percent induction of luciferase activity relative to the maximal level elicited by E2. Results are the mean ± SD of three independent experiments performed in duplicate.

 


View larger version (58K):
[in this window]
[in a new window]
 
FIG. 5. Induction of Gal4-hER{alpha}def- and Gal4-mERßdef-mediated reporter gene expression in transiently transfected MCF-7 cells following cotreatment of either 0.1 nM E2 or 1 nM E2 and EGCG, ECG, or EGC. Results are expressed as percent induction of luciferase activity relative to 1 nM E2. The results are the mean ± SD from three independent experiments performed in duplicate. **Significantly increased compared to 0.1 nM E2 at a level of p < 0.05. *Significantly decreased compared to 1 nM E2 at a level of p < 0.05.

 
To examine the in vivo effects of the three catechins, immature female C57BL/6 mice were dosed with individual catechins with and without E2 to test for both estrogenic and anti-estrogenic responses. Catechin administration was well tolerated by the mice with the exception of 50 mg/kg of EGCG. All mice receiving this concentration significantly lost between 0.1 and 0.8 g of body weight, while vehicle control mice gained 1.2 ± 0.2 g (Table 1Go). Additionally, 50 mg/kg of EGCG produced mild hepatotoxicity as indicated by ALT activities that ranged from 200–400 IU/l (Table 1Go). Histologically, liver slices from mice treated with EGCG (50 mg/kg) showed single necrotic hepatocytes randomly distributed throughout the entire section.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Body Weight Change, Liver Weight, and ALT Activity following Catechin Treatment
 
Uterine wet weight and uterine peroxidase were used as indicators of ER-mediated responses in vivo. While E2 (10 µg/kg) produced a 4-fold increase in uterine weight compared to vehicle control, none of the catechins administered alone increased uterine weight above control (Fig. 6Go). However, when mice were cotreated with 50 mg/kg of ECG and E2 (10 µg/kg), uterine weight induced by E2 was significantly increased (blotted uterine weight values of 2.43 ± 0.06 vs. 3.08 ± 0.13, for E2 and ECG + E2, respectively, p < 0.05; Fig. 6Go). The immature mouse uterus contained minimally detectable uterine peroxidase activity following exposure to sesame oil, while E2-induced a 10.4-fold increase in uterine peroxidase (Table 2Go). Similar to the uterine weight results, none of the individual catechins altered uterine peroxidase activity compared to control (Table 2Go). However, EGCG (30 and 50 mg/kg) and ECG (50 mg/kg) when combined with 10 µg/kg E2 resulted in a 2.3-fold increase in uterine peroxidase compared to that elicited by E2 alone.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 6. Uterine wet weight in immature C57BL/6 mice following catechin treatment. Mice were administered either E2 (10 µg/kg/day, ip), EGCG, ECG, EGC (30 or 50 mg/kg/day, ip), E2 + catechins, or vehicle for 3 consecutive days. Necropsies were performed on Day 4 and uteri were removed, blotted, and weighed. Uterine weight is expressed as mg/g of body weight. The bars represent the mean ± SD of 8 mice. Significance was determined with an ANOVA coupled with the Student-Newman Keuls post-hoc test in which p < 0.05 was required for a statistically significant difference. **Significantly increased compared to E2 at a level of p < 0.05.

 

View this table:
[in this window]
[in a new window]
 
TABLE 2 Uterine Peroxidase Activity following Catechin Treatment
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Binding studies with uterine tissue and purified ER{alpha} and ERß demonstrated that EGCG and ECG were able to compete with [3H]-E2 for the ER. These two catechins contain a gallate group in their structure, while EGC lacks this group (Fig. 1Go). Structure-activity studies have shown that substitutions at positions, which are equivalent to the 7{alpha}- and 11ß-positions of E2, often improve binding to the ER (Fang et al., 2001Go; Tedesco et al., 1997Go) and the 3-gallate group of the catechins corresponds to the 7{alpha}-position of E2. It is likely that this substitution facilitates ER binding since catechins containing a 3-gallate group bind, while catechins lacking this group (EGC and (-)-catechin) do not (Fang et al., 2001Go). EGCG and ECG exhibited a similar affinity for ER{alpha} but EGCG had a 21-fold greater relative binding affinity for ERß than ECG. ER isoform-specific binding has been demonstrated with phyto-estrogens such as genistein, daidzein, apigenin, quercetin, and naringenin, which have a higher relative binding affinity for ERß than for ER{alpha} (Kuiper et al., 1998Go). This trend was also true for EGCG, which exhibited a 7-fold higher relative binding affinity for ERß than ER{alpha}. However, compared to genistein, daidzein, and coumestrol, EGCG produced 117 to 16,000-fold lower relative binding affinities for ERß and 83 to 17,000-fold lower relative binding affinities for ER{alpha} (Kuiper et al., 1998Go). Therefore, while EGCG and ECG compete with [3H]-E2 for both isoforms of the ER, they are significantly weaker than classical phyto-estrogens.

At concentrations from 10 to 50 µM EGCG acted as an ER agonist by inducing luciferase activity in both Gal4-hER{alpha} and Gal4-mERß systems. Higher concentrations of EGCG were cytotoxic, as cell viability was decreased 30% at 200 µM. In previous ER{alpha} and ERß reporter gene studies conducted in HeLa cells, EGCG, ECG, and EGC failed to elicit a response via either receptor subtype (Kuruto-Niwa et al., 2000Go). However, the highest concentration tested was 5 µM, which may explain the lack of response. In cotreatment experiments, Kuruto-Niwa and coworkers reported antagonism of 1 nM E2 by 5 µM of both EGCG and ECG in HeLa cells transfected with ER{alpha}. Additionally, EGCG, EGC, and EC increased the E2-induced response elicited by 1 nM E2 via ERß. Our results do not support ER-mediated activity via EGC as this catechin did not alter E2-induced luciferase activity and did not compete with [3H]-E2 for either ER{alpha} or ERß. Additionally, no catechin modulated ER{alpha}-mediated luciferase activity induced by E2 and cotreatments with 1 nM E2 and EGCG (1 to 50 µM) or ECG (1 µM) antagonized ERß-medated luciferase activity. Therefore, there are major discrepancies between our results and those previously reported. The variation in the results may be due to the type of cell transfected. While both reporter gene assays measured ERE-regulated luciferase activity, a cell-dependent expression of coactivators and corepressors could alter the expression of luciferase activity. For example, SRC1 isoforms differ in both their ER-binding properties and in their ability to increase the transcriptional activity of the ER in transfected cells, as demonstrated by the decreased activity of the SRC1e isoform in HeLa cells compared to COS-1 cells (Kalkhoven et al., 1998Go). Additionally, MCF-7 cells contain a greater than 20-fold amplification of AlB1 (a member of the SRC1 family) and increasing amounts of this coactivator resulted in a dose-dependent increase in E2-dependent transcription (Anzick et al., 1997Go). CYP450 activity is also cell line specific, as HeLa cells lack CYP450 (Nouso et al., 1993Go) while MCF-7 cells express CYP1A1, CYP1A2, and CYP2B1 (Spink et al., 1998Go). Therefore metabolism of the catechins would not occur in HeLa cells and this could affect the response produced. However, in both HeLa and MCF-7 cells high concentrations of the catechins modulated E2-induced gene expression by 50–200% (Kuruto-Niwa et al., 2000Go). Therefore, in vivo examinations were performed as compounds such as coumestrol are several thousand-fold less potent than E2 in vitro but induce uterine weight to a similar extent as E2 (Sheehan et al., 1995Go).

The ability of EGCG, ECG, and EGC to elicit ER-mediated responses in vivo was examined at doses relevant to the tumor inhibitory properties reported for EGCG (Liao et al., 1995Go). Catechins were well tolerated by the mice with the exception of EGCG, which was minimally hepatotoxic when administered at 50 mg/kg/day for 3 days, as indicated by a significant increase in ALT activity, the appearance of single necrotic cells and a decrease in body weight. Kao et al.(2000) is the only other group to report a significant decrease in body weight of female Sprague Dawley rats following EGCG (85 mg/kg/day, 7 days) administration. However, there have been no reports of catechin-induced hepatotoxicity following doses as high as 85 mg/kg (Hirose et al., 1994Go; Kao et al., 2000Go; Liao et al., 1995Go). Since our study was conducted in immature female mice, it is possible that the mild hepatotoxicity produced was an age-specific response and therefore when 50 mg/kg of EGCG was administered to adult mice for 14 days no hepatotoxicity was produced (Liao et al., 1995Go).

Despite EGCG and ECG competing with E2 for the ER in uterine tissue, none of the catechins tested increased blotted uterine weight or uterine peroxidase. However, cotreatment with ECG (50 mg/kg) and E2 elicited a 1.25-fold increase in uterine weight compared to E2 alone. Additionally, cotreatment of E2 and either EGCG (30 or 50 mg/kg) or ECG (50 mg/kg) increased uterine peroxidase activity 2.3-fold above than that elicited by E2 alone. While these increased responses were statistically significant, the overall increases were quite modest. Since the catechins alone did not elicit uterotropic effects, the moderate increase in the E2-induced response is not likely to be due to a direct interaction between catechins and the ER. Similar conclusions have been drawn for atrazine and simazine, compounds that were not ER agonists in vivo but when administered with E2 produced a 1.08 to 1.25-fold increase in E2-induced uterine peroxidase (Connor et al., 1996Go).

EGCG and ECG’s increase in E2-induced responses in vivo may involve alterations in the absorption and metabolism of E2 that could increase the concentration of E2 in the uterine tissue. Two possible mechanisms could be responsible for this effect. High concentrations of EGCG and ECG, but not EGC, have been shown to disrupt the liposome membrane structure (Ikigai et al., 1993Go; Nakayama et al., 2000Go). This destabilization of membranes could result in an increased absorption of E2 through the plasma membrane and result in an increase in the cellular concentration of E2. Alternatively, EGCG and ECG may be inhibiting the metabolism of E2 to 2- and 4-hydroxyestradiol. In humans, E2 is metabolized by CYP1A1, CYP1A2, and CYP3A4 (Zhu and Conney, 1998Go), and several reports have demonstrated that EGCG and ECG (but not EGC) inhibit CYP450 isoforms. Specifically, EGCG and ECG inhibited both rat CYP1A1/2 by 80–90% (Wang et al., 1988Go), and human CYP3A4, CYP2A6, CYP2C19, and CYP2E1 (Muto et al., 2001Go). Additionally, EGCG and ECG inhibited the glucuronidation of E2 in vitro (Zhu and Conney, 1998Go).

In conclusion, EGC did not bind to ER{alpha} or ERß and did not elicit ER-mediated responses in vivo or in vitro. While, EGCG and ECG did not produce ER-mediated responses in vivo, they competed with [3H]-E2 for ER{alpha} and ERß in vitro and high concentrations of EGCG elicited ER{alpha} and ERß reporter gene activity. Additionally, neither EGCG nor ECG antagonized E2-mediated responses in vivo. Therefore, the mechanism by which catechins inhibit breast cancer cell proliferation and ER-dependent tumor growth is not likely to be via ER antagonism. However, EGCG and ECG may inhibit estradiol metabolism and/or enhance uptake in vivo, resulting in a moderate increase in E2-induced responses at high doses.


    ACKNOWLEDGMENTS
 
The authors would like to thank S. M. Samy for his technical assistance. This project was funded in part by a grant from the Deans’ Bequest Fund at the University of Otago.


    NOTES
 
1 To whom correspondence should be addressed at Dept. of Pharmacology and Toxicology, University of Otago Medical School, 18 Frederick St., Rm. 238 Adams Bldg., Dunedin, New Zealand. Fax: +64 3 479 9140. E-mail: rhonda.rosengren{at}stonebow.otago.ac.nz. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Anzick, S. L., Kononen, J. K., Walker, R. L., Azorsa, D. O., Tanner, M. M., Guan, X.-Y., Sauter, G., Kallioniemi, O.-P., Trent, J. M., and Meltzer, P. S. (1997). AlB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277, 965–968.[Abstract/Free Full Text]

Arts, I. C., Hollman, P. C., and Kromhout, D. (1999). Chocolate as a source of tea flavonoids. Lancet 354, 488.[ISI][Medline]

Arts, I. C., van de Putte, B., and Hollman, P. C. (2000). Catechin contents of foods commonly consumed in the Netherlands. 1. Fruits, vegetables, staple foods, and processed foods. J. Agric. Food Chem. 48, 1746–1751.[ISI][Medline]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.[ISI][Medline]

Brasier, A. R., Tate, J. E., and Habener, J. F. (1989). Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7, 1116–1122.[ISI][Medline]

Breinholt, V., and Larsen, J. C. (1998). Detection of weak estrogenic flavonoids using a recombinant yeast strain and a modified MCF7 cell proliferation assay. Chem. Res. Toxicol. 11, 622–629.[ISI][Medline]

Clarke, R., Brunner, N., Thompson, E. W., Glanz, P., Zatz, D., Dickson, R. B., and Lippman, M. E. (1989). The inter-relationships between ovarian-independent growth, tumorigenicity, invasiveness and antioestrogen resistance in the malignant progression of human breast cancer. J. Endocrinol. 122, 331–340.[Abstract]

Connor, K., Howell, J., Chen, I., Liu, H., Berhane, K., Sciarretta, S., Safe, S., and Zacharewski, T. (1996). Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro. Fundam. Appl. Toxicol. 30, 93–101.[ISI][Medline]

Damianaki, A., Bakogeorgou, E., Kampa, M., Notas, G., Hatzoglou, A., Panagiotou, S., Gemetzi, C., Kouroumalis, E., Martin, P. M., and Castanas, E. (2000). Potent inhibitory action of red wine polyphenols on human breast cancer cells. J. Cell. Biochem. 78, 429–441.[ISI][Medline]

Fang, H., Tong, W., Shi, L. M., Blair, R., Perkins, R., Branham, W., Hass, B. S., Xie, Q., Dial, S. L., Moland, C. L., and Sheehan, D. M. (2001). Structure-activity relationships for a large diverse set of natural, synthetic, and environmental estrogens. Chem. Res. Toxicol. 14, 280–294.[ISI][Medline]

Fertuck, K. C., Matthews, J. B., and Zacharewski, T. R. (2001). Hydroxylated benzo[a]pyrene are responsible for in vitro estrogen receptor-mediated gene expression of benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. Toxicol. Sci. 59, 231–240.[Abstract/Free Full Text]

Hirose, M., Hoshiya, T., Akagi, K., Futakuchi, M., and Ito, N. (1994). Inhibition of mammary gland carcinogenesis by green tea catechins and other naturally occurring antioxidants in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz[a]anthracene. Cancer Lett. 83, 149–156.[ISI][Medline]

Ikigai, H., Nakae, T., Hara, Y., and Shimamura, T. (1993). Bactericidal catechins damage the lipid bilayer. Biochim. Biophys. Acta 1147, 132–136.[ISI][Medline]

Kalkhoven, E., Valentine, J. E., Heery, D. M., and Parker, M. G. (1998). Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. Embo J. 17, 232–243.[Abstract/Free Full Text]

Kao, Y.-H., Hiipakka, R. A., and Liao, S. (2000). Modulation of endocrine systems and food intake by green tea epigallocatechin gallate. Endocrinology 141, 980–987.[Abstract/Free Full Text]

Kavanagh, K. T., Hafer, L. J., Kim, D. W., Man, K. K., Sherr, D. H., Rogers, A. E., and Sonenshein, G. E. (2001). Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. J. Cell. Biochem. 82, 387–398.[ISI][Medline]

Kuiper, G. G., Lemmen, J. G., Carlsson, B., Corton, J. C., Safe, S. H., van der Saag, P. T., van der Burg, B., and Gustafsson, J. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ß. Endocrinology 139, 4252–4263.[Abstract/Free Full Text]

Kuruto-Niwa, R., Inoue, S., Ogawa, S., Muramatsu, M., and Nozawa, R. (2000). Effects of tea catechins on the ERE-regulated estrogenic activity. J. Agric. Food Chem. 48, 6355–6361.[ISI][Medline]

Liao, S., Umekita, Y., Guo, J., Kokontis, J. M., and Hiipakka, R. A. (1995). Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate. Cancer Lett. 96, 239–243.[ISI][Medline]

Matthews, J., and Zacharewski, T. (2000). Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss) and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay. Toxicol. Sci. 53, 326–339.[Abstract/Free Full Text]

Morre, D. J., Bridge, A., Wu, L.-Y., and Morre, D. M. (2000). Preferential inhibition by (-)-epigallocatechin-3-gallate of the cell surface NADH oxidase and growth transformed cells in culture. Biochem. Pharmacol. 60, 937–946.[ISI][Medline]

Mukhtar, H., Wang, Z. Y., Katiyar, S. K., and Agarwal, R. (1992). Tea components: Antimutagenic and anticarcinogenic effects. Prev. Med. 21, 351–360.[ISI][Medline]

Muto, S., Fujita, K., Yamazaki, Y., and Kamataki, T. (2001). Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450. Mutat. Res. 479, 197–206.[ISI][Medline]

Nakachi, K., Suemasu, K., Suga, K., Takeo, T., Imai, K., and Higashi, Y. (1998). Influence of drinking tea on breast cancer malignancy among Japenese patients. Japan. J. Cancer Res. 89, 254–261.[ISI][Medline]

Nakayama, T., Hashimoto, T., Kajiya, K., and Kumazawa, S. (2000). Affinity of polyphenols for lipid bilayers. Biofact. 13, 147–151.

Nouso, K., Battula, N., Thorgeirsson, S. S., Higashi, T., and Tsuji, T. (1993). Recombinant mouse cytochrome P1-450 and P3-450: Enzymatic characterization of the hemoprotein expressed in human cells infected with recombinant vaccinia virus. Acta Med. Okayama 47, 377–382.[ISI][Medline]

Patel, R., and Rosengren, R. J. (2001). Acetaminophen elicits anti-estrogenic but not estrogenic responses in the immature mouse model. Toxicol. Lett. 122, 89–96.[ISI][Medline]

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Plainview, NY.

Sheehan, D. M., Branham, W. S., and Medlock, K. L. (1995). An estrogen development potency and toxicity assay for environmental estrogens. Teratology 51, 196–197.

Spink, D. C., Spink, B. C., Cao, J. Q., DePasquale, J. A., Pentecost, B. T., Fasco, M. J., Li, Y., and Sutter, T. R. (1998). Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19, 291–298.[Abstract]

Stoner, G. D., and Mukhtar, H. (1995). Polyphenols as cancer chemopreventive agents. J. Cell. Biochem. 22, 169–180.

Suganuma, M., Okabe, S., Sueoka, N., Sueoka, E., Matsuyama, S., Imai, K., Nakachi, K., and Fujiki, H. (1999). Green tea and cancer chemoprevention. Mutat. Res. 428, 339–344.[ISI][Medline]

Tedesco, R., Katzenellenbogen, J. A., and Napolitano, E. (1997). 7{alpha},11ß-Disubstituted estrogens: Probes for the shape of the ligand binding pocket in the estrogen receptor. Bioorgan. Med. Chem. Lett. 7, 2919–2924.[ISI]

Valcic, S., Timmermann, B. N., Alberts, D. S., Wachter, G. A., Krutzsch, M., Wymer, J., and Guillen, J. M. (1996). Inhibitory effect of six green tea catechins and caffeine on the growth of four selected human tumor cell lines. AntiCancer Drugs 7, 461–468.[ISI][Medline]

Villalobos, M., Olea, N., Brotons, J. A., Olea-Serrano, M. F., Ruiz de Almodovar, J. M., and Pedraza, V. (1995). The E-screen assay: A comparison of different MCF7 cell stocks. Environ. Health Perspect. 103, 844–450.[ISI][Medline]

Wang, Z. Y., Das, M., Bickers, D. R., and Mukhtar, H. (1988). Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450. Drug. Metab. Disp. 16, 98–103.[Abstract]

Zhu, B. T., and Conney, A. H. (1998). Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer Res. 58, 2269–2277.[Abstract]