Cancer Res Treat.  2004 Oct;36(5):315-323.

Activity of Green Tea Polyphenol Epigallocatechin-3-gallate Against Ovarian Carcinoma Cell Lines

• Kim YW
• Bae SM
• Lee JM
• Namkoong S
• Han SJ
• Lee BR
• Lee IP
• Kim SH
• Lee YJ
• Kim CK
• Kim YW
• Ahn WS

Abstract

PURPOSE
A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG), is known to possess anti-cancer properties. In this study, the time-course of the anticancer effects of EGCG on human ovarian cancer cells were investigated to provide insights into the molecular-level understanding of the growth suppression mechanism involved in EGCG-mediated apoptosis and cell cycle arrest. MATERIALS AND METHODS: Three human ovarian cancer cell lines (p53 negative, SKOV-3 cells; mutant type p53, OVCAR-3 cells; and wild type p53, PA-1 cells) were used. The effect of EGCG treatment was studied via a cell count assay, cell cycle analysis, FACS, Western blot and macroarray assay. RESULTS: EGCG exerts a significant role in suppressing ovarian cancer cell growth, showed dose dependent growth inhibitory effects in each cell line and induced apoptosis and cell cycle arrest. The cell cycle was arrested at the G1 phase by EGCG in SKOV-3 and OVCAR-3 cells. In contrast, the cell cycle was arrested in the G1/S phase in PA-1 cells. EGCG differentially regulated the expression of genes and proteins (Bax, p21, Retinoblastoma, cyclin D1, CDK4 and Bcl-XL) more than 2 fold, showing a possible gene regulatory role for EGCG. The continual expression in p21WAF1 suggests that EGCG acts in the same way with p53 proteins to facilitate apoptosis after EGCG treatment. Bax, PCNA and Bcl-X are also important in EGCG-mediated apoptosis. In contrast, CDK4 and Rb are not important in ovarian cancer cell growth inhibition. CONCLUSION: EGCG can inhibit ovarian cancer cell growth through the induction of apoptosis and cell cycle arrest, as well as in the regulation of cell cycle related proteins. Therefore, EGCG-mediated apoptosis could be applied to an advanced strategy in the development of a potential drug against ovarian cancer.

Keyword

(-)-epigallocatechin-3-gallate (EGCG); Ovarian Cancer; Apoptosis; Cell Cycle

MeSH Terms

Apoptosis
Blotting, Western
Cell Count
Cell Cycle
Cell Cycle Checkpoints
Cell Line*
Cyclin D1
G1 Phase
Humans
Ovarian Neoplasms
Proliferating Cell Nuclear Antigen
Retinoblastoma
Tea*
Cyclin D1
Proliferating Cell Nuclear Antigen
Tea

Figure

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  Fig. 1 Growth inhibition effects of EGCG on various ovarian cancer cell lines, SKOV-3, OVCAR-3 and PA-1 cells, at different concentrations. Cells (105 cells/well) were cultured in 12-well plates, in triplicate, overnight and treated with EGCG at increasing concentrations. After EGCG treatment, cells were cultured for 6 days and then trypsinized for counting under a microscope. The mean cell count values from triplicate measurements were plotted. Values and bars represent the mean and SD, respectively.

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  Fig. 2 Detection of EGCG-induced cell death in ovarian cancer cells. Quantitative analysis of the apoptotic cells using annexin V-FITC and PI in exponentially growing SKOV-3, OVCAR-3 and PA-1 cells Each cell line was incubated for 2 days. Flow cytometric analysis was performed on 105 cells and the percentages apoptotic, live and dead cells measured. The graph shows the percentage of the lower right (LR; annexin+/PI-) cells represents early apoptotic cells, and the upper right (UR; annexin+/PI+) dead (late apoptotic plus necrosis) cells. Data are the mean and SD of three separate experiments. Quadrant statistics for labeled cells are displayed for ■ control, □ 25 µmol, ▒ 50 µmol and ▓ 100 µmol.

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  Fig. 3 Confirmation of the macroarray assay by RT-PCR analysis. Total RNA obtained from SKOV-3 cells without and with EGCG treatment (lane -) and (lane +), respectively, were subjected to RT-PCR analysis, as described in Materials and methods.

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  Fig. 4 Western blot analyses for cell cycle-related proteins. Total extracts obtained from SKOV-3 cells without and with EGCG treatment (lane -) and (lane +), respectively, were subjected to Western blot analysis, as described in Materials and methods.

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