Demethylation effects of tanshinone II A on SMMC-7721 cells

Objective: To investigate the demethylation effects of tanshinone II A on tumor suppressor genes of hepatocellular carcinoma cells. Methods: SMMC-7721 cells were devided into three groups during the culture course in vitro. In tanshinone II A group, cells were treated with tanshinone II A at different doses in the culture media. Cells in the positive control group were cultured in the media with 5-Aza-2′-deoxycytidine at 10 μmol/L. Cells in the negative group were cultured with blank media. The methylation status of tumor suppressor gene profile including TFPI2, SPARC, DKK3, P16, APC and MGMT was analyzed in different groups by methylation-specific polymerase chain reaction (PCR). The mRNA expression of genes was examined by reverse transcription PCR and the protein expression was tested by immunocytochemistry staining. By bisulfite sequencing, methylation status of CpG sites in SPARC promoter region was estimated in different groups. Results: Both methylated and unmethylated status of TFPI2, P16 and APC could be detected in the three groups by methylation-specific PCR. In the negative control group, methylated segments of SPARC, DKK3 and MGMT were only detected, however, both methylated and unmethylated segments of these genes were found in tanshinone II A group and 5-Aza-2′-deoxycytidine group. SPARC, DKK3 and MGMT were totally methylated while TFPI2, P16 and APC were partially methylated in SMMC-7721 cells. The mRNA expression of SPARC, DKK3 and MGMT could be restored in tanshinone II A group compared with negative control group. Immunocytochemistry showed positive expression of SPARC in tanshinone II A group. Methylation frequencies of CpG sites in SPARC promoter region were 96.53% (139/144) in the negative control group, 18.75% (27/144) in 5-Aza-2′-deoxycytidine group and 45.14% (65/144) in tanshinone II A group, respectively. There was a significant difference in methylation frequencies of CpG sites among the three groups (P < 0.001). Parts of methylated CpGs were converted to unmethylation status in both tanshinone II A group and 5-Aza-2′-deoxycytidine group. Compared with that in the negative control group, the mRNA expression of DNA methytransferase I was downregulated by 0.36-0.78 folds in tanshinone II A group. Conclusion: Tanshinone II A might convert the methylation status of some tumor suppressor genes in SMMC-7721 cells by downregulating DNA methytransferase I.