Epigallocatechin-3-gallate and trichostatin A synergistically inhibit human lymphoma cell proliferation through epigenetic modification of p16INK4a

DNA methylation and histone deacetylation play important roles in the occurrence and development of cancers by inactivating the expression of tumor suppressors, including p16(INK4a), a cyclin-dependent kinase inhibitor. The present study investigated the effect of epigallocatechin-3-gallate (EGCG) alone or in combination with trichostatin A (TSA) on p16(INK4a) gene expression and growth in human malignant lymphoma CA46 cells. CA46 cell viability and cell cycle were analyzed; methylation of the p16(INK4a) gene was assessed by nested methylation-specific PCR (n-MSP). p16(INK4a) mRNA and protein expression was determined by real-time quantitative PCR and western blot analyses, respectively. Both EGCG and TSA alone inhibited CA46 cell proliferation; the combined treatment (6 g/ml EGCG and 15 ng/ml TSA) significantly reduced CA46 cell proliferation from 24 to 96 h (all P<0.001). Cells treated with 24 g/ml EGCG or the combination treatment (6 g/ml EGCG and 15 ng/ml TSA) had lower proliferative indices when compared to the other groups. Co-treatment with EGCG and TSA decreased p16(INK4a) gene methylation, which coincided with increased p16(INK4a) mRNA and protein expression. Thus, EGCG and TSA synergistically reactivate p16(INK4a) gene expression in part through reducing promoter methylation, which may decrease CA46 cell proliferation.