Lipid rafts mediate epigallocatechin-3-gallate- and green tea extract-dependent viability of human colon adenocarcinoma COLO 205 cells; clusterin affects lipid rafts-associated signaling pathways.

Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea extract (GTE) that was widely believed to reduce proliferation of many cancer cell lines. The purpose of this study was to verify the possible pro-apoptotic action of GTE/EGCG in human colon adenocarcinoma COLO 205 cells. The effect of EGCG/GTE treatments on cell viability was studied using methyl thiazolyl tetrazolium (MTT) assay. Cell proliferation was assessed with crystal violet staining, whereas protein expression levels were evaluated by western blotting followed by densitometric analysis. Obtained results were analyzed statistically. Surprisingly, EGCG/GTE dose-dependently up-regulated COLO 205 cells viability and proliferation. Observed effects were mediated by lipid rafts, as cholesterol depletion significantly prevented EGCG/GTE-dependent cell survival. Furthermore, treatment of COLO 205 cells with EGCG/GTE resulted in activation of MEK/ERK1/2, but not Akt1/2/GSK-3ß signaling pathway. The presence of MEK inhibitor - PD98059 but not PI3-K inhibitor - LY294002, both reduced EGCG/GTE-induced ERK1/2 activation and the proliferative effect of catechins. Furthermore, EGCG/GTE stimulated secretory clusterin (sClu) expression level, which underwent complex control through lipid rafts/PKC/Wnt/ß-catenin system. Our studies demonstrated that EGCG and GTE stimulate cell survival and proliferation of COLO 205 cells in a lipid rafts-dependent manner via at least MEK/ERK1/2 signaling pathway. Furthermore, EGCG/GTE mediated positive effects on viability and mitogenicity of COLO 205, while suppression of ß-catenin activity was positively correlated with sClu clusterin expression.

Benzene metabolism in rodent hepatocytes: role of sulphate conjugation.

1. Hepatocytes isolated from the adult male NMRI mouse or Wistar rat were incubated for 1 h with 0.5 mM 14C-benzene, the supernatant was separated from the cells, and analysed for benzene metabolites. Separately, formation of sulphate conjugates during benzene metabolism was studied in hepatocytes in the presence of 35S-sulphate. In addition sulphate conjugation of the benzene metabolites hydroquinone and 1,2,4-trihydroxybenzene was investigated in mouse liver cytosol supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate. 2. Two novel metabolites, not detectable in rat hepatocyte incubations, were found in mouse hepatocytes, and were identified as 1,2,4-trihydroxybenzene sulphate and hydroquinone sulphate. Formation of the 35S-labelled conjugates could be demonstrated in incubations of mouse liver cytosol with hydroquinone or 1,2,4-trihydroxybenzene supplemented with 3'-phosphoadenosine-5'-phospho-35S-sulphate, and in mouse hepatocytes incubated with benzene and 35S-sulphate. 3. In comparison with hepatocytes from the Wistar rat, hepatocytes from the NMRI mouse were almost three times more effective in metabolizing benzene. The higher formation of hydroquinone, and the formation of trihydroxybenzene sulphate and hydroquinone sulphate, mainly contributed to the higher rate of benzene metabolism. 4. In conclusion, qualitative and quantitative differences in benzene metabolism may contribute to the higher susceptibility of mouse towards the myelotoxic and leucaemogenic action of benzene.