Experimental Study On EGCG Alone Or In Combination With TSA Reversing Hypermethylation Of P16 And Activating Its Transcription In Malignant Lymphoma Cell Line CA46

Objective To measure methylation level of gene p16 in the malignant lymphoma cell line -CA46 by DNA sequencing and Modified Methylation Specific PCR(MSP), i.e. Nested Methylation Specific PCR(nMSP);and to investigate the mechanism that how Epigallocatechin-3-gallate (EGCG) alone or in combination with Trichostatin A (TSA)would reverse hypermethylation of p16 and exert its function of transcription regulation in the malignant lymphoma cell line --CA46. Methods The CA46 cell line were cultured in exposure to varied doses of EGCG and TSA,and th cell viability was obtained by the MTT assay;Both methylation levels of CA46 before and after EGCG alone or in combination with TSA disposal were analyzed by nMSP;The expression of p16, DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B were analyzed by RT-PCR; DNA ploid analytical method was used to analyze the effect that EGCG alone or in combination with TSA would bring on the cell life cycle of the target cell line. Results 1. In comparison to the control, CA46 cell growth was arrested by treatment with EGCG alone or in combination with TSA, the cell cycle test showed that most of the cells were arrested at G0/G1 phrase. 2. P16 gene in CA46 cell was found hypermethylated and the methylation level was apparently attenuated after 48h disposal of EGCG alone or in combination with TSA, and hypermethylation of p16 had been successfully reversed.;3. Expression of p16 gene in untreated group was mild, after treate with EGCG it had been greatly strengthened in a dose-dependent manner, exhibiting a significant difference (p<0.05), There were differences between the combination of the two drugs and the EGCG 12μg/ml group; 4.Compared with the untreated group, after 48h disposal of EGCG alone or in combination with TSA, the expression of DNMT3A and DNMT3B was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 stood nearly unchanged. Conclusions 1. EGCG might have exerted its function of cell cycle control and cell growth retardation in malignancy through inhibiting the expression of DNMT3A and DNMT3B, as well as inhibiting the demethylation of p16, which results in activating and upregulating its expression, and finally blocking the cell at G0 or G1 phase. 2. TSA might have synergistic effect on demethylation of EGCG through changing the chromatin configuration.