Lipid rafts mediate EGCG- and GTE-stimulated viability of human colon adenocarcinoma COLO 205 cells; role of clusterin and lipid rafts-associated signaling pathways
Pajak Beata
Beata Pajak
Department of Cell Ultrastructure
Mossakowski Medical Research Centre Polish Academy of Sciences
5 Pawinskiego Str., 02-106 Warsaw, POLAND
Abstract:
Purpose: Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea extract (GTE) that efficiently reduces proliferation of many cancer cell lines. The objective of this study was to verify the possible pro-apoptotic action of GTE/EGCG on human colon adenocarcinoma COLO 205 cells.
Methods: The effect of EGCG/GTE treatments on cell viability was studied using MTT assay. Cell proliferation was assessed with crystal violet staining, whereas protein expression levels were evaluated by Western-blot followed by densitometric analysis. Furthermore, clusterin cellular localization was visualized by transmission electron microscopy (TEM). Obtained results were statistically evaluated.
Results: Surprisingly, EGCG/GTE dose-dependently up-regulated COLO 205 cells survival and proliferation. Observed biological effects were mediated by lipid rafts, by dint of cholesterol depletion significantly prevented EGCG/GTE-dependent cell stimulation. Furthermore, treatment of COLO 205 cells with EGCG/GTE resulted in activation of MEK/ERK1/2 as well as Akt1/2/GSK-3b signaling pathways. The presence of MEK inhibitor - PD98059 but not PI3-K inhibitor - LY294002, reduced both EGCG/GTE-induced ERK1/2 activation and the proliferative effects of those substances. Furthermore, EGCG/GTE stimulated secretory clusterin (sClu) expression, which seems to be regulated by complex cellular signaling pathway including lipid rafts/PKC/Wnt/b-catenin system.
Conclusions: Our studies demonstrated that in COLO 205 cells EGCG and GTE stimulate cell survival and proliferation in a lipid rafts-dependent manner via at least two signaling pathways: MEK/ERK1/2 and Akt1/2/GSK-3b. Furthermore, EGCG/GTE mediated positive effects on viability and mitogenicity of COLO 205 even during suppression of b-catenin transcriptional activity, which was positively correlated with sClu clusterin expression.
