Original article
(−)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells

https://doi.org/10.1016/j.cdp.2007.10.018Get rights and content

Abstract

Background: (−)-Epigallocatechin-3-gallate (EGCG) is the most active catechin present in green tea, demonstrated to have chemopreventive action and to kill cancer cells selectively. As a previous study found that catechins could compete with 17-β-estradiol for binding to estrogen receptor alpha (ERα), we asked whether EGCG could regulate ERα action. Methods: We used MCF-7, a breast carcinoma cell line having a high level of ERα expression. The cells were treated with various EGCG concentrations and cell viability was evaluated by MTT assay. ERα and pS2 expression were analyzed by RT-PCR after RNA extraction. To better define EGCG action in relation to ERα, we studied EGCG cytotoxicity on MCF-7 resistant to tamoxifen (MCF-7tam), MCF-7 treated with 10−7 M ICI 182,780 for 8 days and on MDA-MB-231, a cell line that lacked ERα by flow cytometry (FCM). Results: Both ERα and pS2 mRNA were expressed in samples treated with low EGCG concentration (30 μg/ml). At this concentration, no cell change was detectable. In contrast, pS2 expression was lost in samples treated with 100 μg/ml EGCG for 24 h, indicating ERα alteration. EGCG cytotoxicity was lower when ERα was not present (MDA-MB-231) or inactivated (by tamoxifen or ICI 182,780). Conclusions: Functionally active ERα may have a role in EGCG cytotoxicity, increasing the sensitivity to the drug. As higher EGCG concentrations also killed cells resistant to tamoxifen or treated by 10−7 M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy.

Introduction

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most important catechins present in green tea (Camellia sinensis), a beverage widely diffused and consumed in the Asian population, which shows powerful antioxidant and anti-inflammatory properties. Epidemiological studies indicate that green tea consumption protects against cancer (stomach, lung, colorectal, liver and breast) [1]. In several animal models, EGCG treatment reduced the tumor incidence, the number of tumor masses and metastasis [2], [3]. Numerous in vitro studies detected cell cycle arrest and apoptosis after EGCG treatment. EGCG also reduced angiogenesis and both local and distant invasion, and it could inhibit DNA methyltransferas and reactivate methylation-silenced genes in cancer cell lines [4], but the mechanisms underlying green tea effects and the molecular targets are still under investigation [5], [6]. Among the numerous molecules proposed to be target of EGCG action, two studies demonstrated that green tea catechins can bind and downregulate ERα and ERβ [7], [8]. These findings can explain why various epidemiological studies found a correlation between green tea assumption and low incidence of breast cancer [9]. Estrogens are key molecules in breast cancer development that can influence breast cancer cell growth [10]. ERα is a fundamental molecular target of therapy in breast cancer patients expressing the steroid receptors, although a consistent number of treated patients develop resistance to therapy and relapse [11]. We considered that the interaction between EGCG and ERα might be potentially important and may have therapeutic implications. To verify this hypothesis, we used MCF-7 cells, which express a high level of ERα, and we studied ERα and pS2 expression after EGCG treatment. pS2 (also known as Trefoil factor 1, TFF1) is an estrogen-inducible protein [12], widely used as a marker of ERα functional activity. The detection of estrogen target gene expression, like pS2, represents a valuable physiologic assay for tissue specific estrogenicity or antiestrogenicity of a drug [13].

We also asked whether EGCG cytotoxicity changed after inactivation of ERα signaling pathway. To this aim we used two different drugs, tamoxifen and ICI 182,780, having different mechanisms of ERα inactivation. Tamoxifen is the most commonly used drug to treat ER-positive breast cancer patients: it binds to ERα and it blocks the estrogen action, but it also possesses ER-agonist properties. Long-term use of tamoxifen is associated with several important side effects and risk of relapse with resistant disease [14]. ICI 182,780 is a new ER antagonist that destroys the ER signaling pathway by decreasing the level of ERα through their ubiquitinylation and targeting to the proteasome and it is not associated with tamoxifen-like agonist effects. To investigate EGCG cytotoxicity in MCF-7 cells after inactivation of ERα signaling pathway, EGCG was given to MCF-7 cell line resistant to tamoxifen and to MCF-7 cells after 8 days of treatment with 10−7 M 182,780. As a comparison, we also treated MDA-MB-231 cells, which lack of ERα, with EGCG.

Section snippets

Cell lines

MCF-7 and MDA-MB-231 cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in E-MEM and D-MEM, respectively, supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin/streptomycin, 2 mM l-glutamine and 1 mM Na pyruvate (all from Cambrex Bio Science, Rockland, MA, USA). MCF-7tam cell line was developed by growing MCF-7 cells in α-MEM medium (Invitrogen, Carlsbad, CA, USA) without phenol red, supplemented by 10% charcoal-stripped FCS serum, 100 

MCF-7 cell viability following EGCG treatment

At the concentration of 100 μg/ml, the OD value decreased of 24% in treated cells with respect to the control cells (Fig. 1). Treatment with higher concentrations of EGCG (120–160 μg/ml) resulted in higher dose-dependent reduction OD value. Therefore we considered that 100 μg/ml might be an EGCG concentration suitable to our aims.

pS2 expression was lost in MCF-7 cells treated with 100 μg/ml EGCG for 24 h

MCF-7 cells were treated with EGCG at 30 and 100 μg/ml for 2 and 24 h, in order to detect early changes in ERα transcription. After short time treatment (2 h), the cells did

Discussion

In the present study, we found that EGCG treatment downregulated ERα pathway in MCF-7 cells, as demonstrated by loss of pS2 transcription after 100 μg/ml treatment for 24 h. Loss of pS2 was not detectable after 2 h treatment, or after EGCG treatment at a low concentration (30 μg/ml). It only occurred at an early stage of EGCG action, when EGCG was given at a concentration able to induce cell death in a limited number of cells. As 70–80% cells were still viable, these findings suggest that ERα

Conflict of interest

None.

Acknowledgment

The authors wish to thank Dr. Mariella Chiricolo for suggestions and help.

References (25)

  • R. Kuruto-Niwa et al.

    Effect of tea catechins on the ERE-regulated estrogenic activity

    J Agric Food Chem

    (2000)
  • A.H. Wu et al.

    Tea intake, COMT genotype, and breast cancer in Asian–American women

    Cancer Res

    (2003)
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