* Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand; and
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
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ABSTRACT |
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Key Words: catechins; EGCG; ECG; EGC; ER; ERß; human breast cancer cells.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Competitive ligand binding assay.
The method used for the competitive binding assay has recently been described in detail (Matthews and Zacharewski, 2000), 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
(GST-hER
def, > 85% purity; Matthews and Zacharewski, 2000
) or full-length human ERß (hERß, > 80% purity; Panvera, Madison, WI; Fertuck et al., 2001
). 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., 1995).
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., 1989). Transfections were performed by the calcium phosphate coprecipitation method, which has a transfection efficiency of up to 20% (Sambrook et al., 1989
), as earlier described (Fertuck et al., 2001
) 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
def (Gal4 linked to D, E, and F domains of the hER
; 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., 1989
; Sambrook et al., 1989
). 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 2124°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., 1995).
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, 2001). 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, 2001) 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, 1976
). 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, 2001), 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.
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RESULTS |
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DISCUSSION |
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At concentrations from 10 to 50 µM EGCG acted as an ER agonist by inducing luciferase activity in both Gal4-hER and Gal4-mERß systems. Higher concentrations of EGCG were cytotoxic, as cell viability was decreased 30% at 200 µM. In previous ER
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., 2000
). 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
. 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
or ERß. Additionally, no catechin modulated ER
-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., 1998
). 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., 1997
). CYP450 activity is also cell line specific, as HeLa cells lack CYP450 (Nouso et al., 1993
) while MCF-7 cells express CYP1A1, CYP1A2, and CYP2B1 (Spink et al., 1998
). 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 50200% (Kuruto-Niwa et al., 2000
). 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., 1995
).
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., 1995). 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., 1994
; Kao et al., 2000
; Liao et al., 1995
). 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., 1995
).
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., 1996).
EGCG and ECGs 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., 1993; Nakayama et al., 2000
). 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, 1998
), 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 8090% (Wang et al., 1988
), and human CYP3A4, CYP2A6, CYP2C19, and CYP2E1 (Muto et al., 2001
). Additionally, EGCG and ECG inhibited the glucuronidation of E2 in vitro (Zhu and Conney, 1998
).
In conclusion, EGC did not bind to ER 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
and ERß in vitro and high concentrations of EGCG elicited ER
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.
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ACKNOWLEDGMENTS |
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NOTES |
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