Green Tea Extract (EGCG) Inhibits Proliferation of Human Leiomyoma Cells.

Uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. These tumors occur in up to 70% of adult women, and their prevalence is especially high in Africa-American women. Currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. Green, black and oolong tea are the three major commercial types of tea. About 20% of tea consumed world wide is in the form of green tea. Epigallocatechin gallate (EGCG) constitutes the main solid extract of green tea and is believed to contribute most of the antioxidant and anti-inflammation capacity of green tea. EGCG has been shown to have beneficial effects on prostate cancer, breast cancer and other cancer types by inducing apoptosis and inhibiting proliferation of cancer cells. In this study, we investigated the ability of EGCG to inhibit proliferation of human leiomyoma cells (HLM) in vitro. Human leiomyoma cell line (HLM) were cultured at 37ºC in a humidified atmosphere of 5% CO2-95% air in SmBM medium supplied with 5%FBS, 0.1% insulin, 0.2% hFGF-B, 0.1% GA-1000 and 0.1% hEGF(Lonza). For cell proliferation assay, the cells were plated at density of 2×105cells/well in 6-well plates and grown under the same conditions as above. The monolayer cultures at approximately 70% confluence were treated with various concentrations (0, 0.1, 1.0, 10, 50, 100 and 200µm) of EGCG (Sigma) for 7 days. A fluorometric assay using Hoechst 33258 dye (Sigma) for DNA quantitation was conducted at the following designed time points, day 1, 3, 5, and 7 post EGCG treatment. The cells were lysed and DNA content was determined using Hoechst dye solution (1µg/ml). Fluorescence was measured after excitation at 355nm and emission at 458nm. For western blotting, HLM cells were seeded into 10cm dish in a range of 1x106 cells/dish. After 24 h culture, the cells were treated with indicated concentrations of EGCG as above. After 48 h, the cells were lysed and proteins were harvested. Equivalent amounts of protein (30ug) were separated by NuPAGE Novex 10% Bis-Tris Gel (Invitrogen Life Technologies, Carlsbad, CA) and the proteins were then electrophoretically transferred onto PVDF membranes (Millipore Corp., Billerica, MA). After blocking nonspecific binding sites, the membranes were blotted with the primary antibodies overnight at 4C. Immunologic detection was performed using the following primary antibodies: human proliferation cell nuclear antigen (PCNA) (1:500 dilution), Bax (1:500 dilution), Bcl-2 (1:200 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and Cdk4 (1:1000 dilution, Sigma, St. Louis, MO, USA). The antigen-antibody complexes were detected with the ECL chemiluminescence detection system (Amersham Bioscience, Poscataway, NJ, USA). Blots were scaned and optical densities of the band were quantitated. EGCG exhibited marked anti-proliferation effect on HLM cells. Compared with untreated control, the inhibitory effect of EGCG on HLM cells was observed at 10 µM starting at day 3 while with 50 µM and higher concentrations, it started as early as at day1. High concentrations significantly reduced the cell proliferation by 45% after 24 h treatment, compared with the untreated control (p<0.05). Effects of graded concentrations of EGCG for 48 h on PCNA, Cdk4, bcl-2 and Bax expression in cultured leiomyoma cells were assessed by western blot analysis. Compared with untreated control cultures, treatment with concentrations of 50uM and higher significantly decreased PCNA, Cdk4 and Bcl-2 protein expression (p<0.05). Additionally, EGCG increased the expression of of the proapoptotic Bax protein at 100 µM and 200 µM treated cells, compared with untreated control cultures (p<0.05). EGCG significantly inhibited the proliferation of human leiomyoma cells in a dose-depended and time-depended pattern. The possible mechanism of the inhibition is via down-regulation of the expression of the anti-apoptotic protein Bcl-2 and the cyclin-dependent kinase Ckd4 of G1 phase, while up-regulating the expression of pro-apoptotic protein Bax. EGCG may present a potential effective and safe medicinal treatment for uterine fibroids.