Epigallocatechin-3-gallate (EGCG) Combined Of TRAIL Effects In Inducing Apoptosis Of Human Melanoma A375 Cell

Objective1. To study Epigallocatechin-3-gallate,Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),combined use of EGCG and TRAIL effects on human melanoma A375 cell line inhibitory growth and apoptosis.2. To study death receptor 4 (DR4) and death receptor 5 (DR5) expression in human melanoma A375 cell line.3. To study Epigallocatechin-3-gallate effects on mRNA Level and Protein Expression of death receptor 4 (DR4)and death receptor 5 (DR5) in human melanoma A375 cell line.4. To study Epigallocatechin-3-gallate effects on Caspase-3 Expression in human melanoma A375 cell line.5. To study combined use of Epigallocatechin-3-gallate and TRAIL or EGCG only effects on Caspase-8 Expression in human melanoma A375 cell line.Methods1. Cultured human melanoma A375 cell line and A875 were treated with a serial concentration of EGCG or TRAIL in vitro. 2. MTT assay was employed to determine the growth regulatory effects of EGCG or TRAIL or combination of EGCG and TRAIL, and Flow Cytometry Analysis was used to determine apoptosis rate in different groups.3. Death receptor 4 and death receptor 5 protein expression was identified by Direct Immunofluorescence Assay in cultured human malignant melanoma A375 cell line in vitro.4. After human melanoma A375 cell line were treated with different concentration of EGCG,DR4 and DR5 mRNA and protein expression were determined by RT-PCR and Flow Cytometry Analysis or Western blot Assay.5. After human melanoma A375 cell line were treated with different concentration of EGCG, Caspase-3 expression were determined by Colorimetric Assay.6. After human melanoma A375 cell line were treated with different concentration of EGCG or combination EGCG and TRAIL, Caspase-8 protein expression were determined by Colorimetric Assay.Results1. Effect of EGCG or TRAIL or combination EGCG and TRAIL inhibitory growth and apoptosis in A375 Cell line.1) EGCG inhibited the proliferation of A375 cells in a time and concentration-dependant manner (0-40μg/ml)2) TRAIL inhibited the proliferation of A375 cells in a concentration-dependant manner (0-150ng/ml), the IC50 of TRAIL was 132.47 ng/ml.3) The apoptosis rate was 11.8% in the TRAIL group,5.3-8.1% in the EGCG group and 48.9%-59.1% in the combined group. Significant difference was found in the apoptosis between the combined group and the EGCG or TRAIL group (P<0.01 for each).2. Expression and the role of TRAIL receptor 1 and TRAIL receptor, EGCG effects on TRAIL receptor 1 and TRAIL receptor 2 expression in melanoma A375 cell line in vitro1) Melanoma A375 cell line is both death receptor 4 and death receptor 5-positive.2) DR4 expression was increased in the EGCG-treated cells as compared with that in the non-treated cells.3) However, there was no significant change in the protein levels of DR5 in the cells after EGCG treatment.4) Effect of EGCG on Caspase-3 and Caspase-8 Expression5) EGCG significantly increased the expression of activated caspase-3 in a concentration-dependent manner in A375 cells.6) However, EGCG did not cause any change in the expression of caspase-8 in A375 cells. In contrast to EGCG alone, the combination of EGCG and TRAIL was more effective in activating caspase-8.Conclusions1. Our results found that combined application of TRAIL and EGCG was more effective on the cell growth of A375 than treatment with EGCG, or TRAIL alone.2. EGCG was found to up-regulate the expression of DR4 and increased the expression of activated Caspase-3 in A375 cells, these may one of mechanism that EGCG can strength the TRAIL-induced apoptosis.