Zhe Hou

Mechanism of Action of (−)-Epigallocatechin-3-Gallate: Auto-oxidation–Dependent Inactivation of Epidermal Growth Factor Receptor and Direct Effects on Growth Inhibition in Human Esophageal Cancer KYSE 150 Cells

(-)-Epigallocatechin-3-gallate (EGCG), the principal polyphenol in green tea, has been shown to inhibit the growth of many cancer cell lines and to suppress the phosphorylation of epidermal growth factor receptor (EGFR). We observed similar effects of EGCG in esophageal squamous cell carcinoma KYSE 150 cells and epidermoid squamous cell carcinoma A431 cells. Pretreatment of KYSE 150 cells with EGCG (20 micromol/L) for 0.5 to 24 hours in HAMs F12 and RPMI 1640 mixed medium at 37 degrees C, before the addition of EGF, resulted in a decreased level of phosphorylated EGFR (by 32-85%). Prolonged treatment with EGCG (8 or 24 hours) also decreased EGFR protein level (both by 80%). EGCG treatment for 24 hours also caused decreased signals of HER-2/neu in esophageal adenocarcinoma OE19 cells. These effects of EGCG were prevented or diminished by the addition of superoxide dismutase (SOD, 5 units/mL), or SOD plus catalase (30 units/mL), to the cell culture medium. A similar phenomenon on inactivation of EGFR was observed in A431 cells as well. Under culture conditions for KYSE 150 cells, EGCG was unstable, with a half-life of approximately 30 minutes; EGCG dimers and other oxidative products were formed. The presence of SOD in the culture medium stabilized EGCG and increased its half-life to longer than 24 hours and some EGCG epimerized to (+)-gallocatechin-3-gallate. A mechanism of superoxide radical-mediated dimerization of EGCG and H2O2 formation is proposed. The stabilization of EGCG by SOD in the culture medium potentiated the activity of EGCG in inhibiting KYSE 150 cell growth. The results suggest that in cell culture conditions, the auto-oxidation of EGCG leads to EGFR inactivation, but the inhibition of cell growth is due to other mechanisms. It remains to be determined whether the presently observed auto-oxidation of EGCG occurs in vivo. In future studies of EGCG and other polyphenolic compounds in cell culture, SOD may be added to stabilize EGCG and to avoid possible artifacts.

Inhibition of intestinal tumorigenesis in Apcmin/+ mice by (-)-epigallocatechin-3-gallate, the major catechin in green tea

The present study was designed to investigate the effects of two main constituents of green tea, (-)-epigallocatechin-3-gallate (EGCG) and caffeine, on intestinal tumorigenesis in Apc(min/+) mice, a recognized mouse model for human intestinal cancer, and to elucidate possible mechanisms involved in the inhibitory action of the active constituent. We found that p.o. administration of EGCG at doses of 0.08% or 0.16% in drinking fluid significantly decreased small intestinal tumor formation by 37% or 47%, respectively, whereas caffeine at a dose of 0.044% in drinking fluid had no inhibitory activity against intestinal tumorigenesis. In another experiment, small intestinal tumorigenesis was inhibited in a dose-dependent manner by p.o. administration of EGCG in a dose range of 0.02% to 0.32%. P.o. administration of EGCG resulted in increased levels of E-cadherin and decreased levels of nuclear beta-catenin, c-Myc, phospho-Akt, and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) in small intestinal tumors. Treatment of HT29 human colon cancer cells with EGCG (12.5 or 20 micromol/L at different times) also increased protein levels of E-cadherin by 27% to 58%, induced the translocation of beta-catenin from nucleus to cytoplasm and plasma membrane, and decreased c-Myc and cyclin D1 (20 micromol/L EGCG for 24 hours). These results indicate that EGCG effectively inhibited intestinal tumorigenesis in Apc(min/+) mice, possibly through the attenuation of the carcinogenic events, which include aberrant nuclear beta-catenin and activated Akt and ERK signaling.

Molecular targets for the cancer preventive activity of tea polyphenols

Inhibition of carcinogenesis by tea and tea polyphenols has been demonstrated in many animal models. The mechanisms of action have been extensively investigated mostly in cell culture systems with (‐)‐epigallocatechin‐3‐gallate (EGCG), the most active and major polyphenolic compound from green tea. However, the mechanisms of cancer preventive activity by tea and tea polyphenols are not clearly understood. This article discusses some of the reported mechanisms and possible targets for the action of EGCG. The difficulties and major issues in extrapolating data from studies in cancer cell lines to cancer prevention mechanisms are discussed. Activities observed in cell culture with high concentrations of EGCG may not be relevant because of the limited systemic bioavailability of EGCG. In addition, possible artifacts due to the auto‐oxidation of EGCG may complicate this issue. Some recent studies revealed high‐affinity EGCG binding proteins as possible direct targets for the action of EGCG. Validating the related cancer preventive mechanisms found in in vitro studies in animal models and human samples would be exciting.