Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells☆

Summary 

In the present study, we investigated methylation status of the CpG islands of some major tumor suppressor genes both in human hepatocellular carcinoma and liver cancer cell lines and examined whether demethylation by arsenic trioxide (As₂O₃) could restore their expression in the cell lines. HepG2 and Huh-7 cells were treated with 2 to 10 μmol/L of As₂O₃ and/or 1 μmol/L of 5-aza-2′-deoxycytidine for 24, 48, and 72 hours. The methylation status of the CpG island around the promoter regions of p16^INK4a, RASSF1A, E cadherin, and GSTP1 was detected by a methylation-specific polymerase chain reaction (MSP). The messenger RNA (mRNA) and protein levels of these genes were determined by quantitative real-time reverse transcriptase–polymerase chain reaction, Western blot, and immunohistochemical analyses. The DNA methyltransferase (DNMT) mRNA levels and enzyme activity were also examined. The hypermethylated status of the promoter regions of p16^INK4a, RASSF1A, E cadherin, and GSTP1 was observed in 10 (40%), 14 (56%), 6 (24%), and 12 (48%) of 25 patients with hepatocellular carcinoma, respectively. CpG methylation of the p16^INK4a, RASSF1A, E cadherin, and GSTP1 genes was correlated to the reduction of mRNA levels in the cell lines, and mRNA expression of these 4 genes were indeed restored by low concentrations (2-6 μmol/L) of As₂O₃ through demethylation, as well as 1 μmol/L of 5-aza-2′-deoxycytidine. Western blot and immunohistochemical analyses confirmed that each protein was markedly enhanced after treatment with a low concentration of As₂O₃. In contrast, As₂O₃ at a high concentration (10 μmol/L) damaged cell membranes and remarkably suppressed these 4 protein levels. As₂O₃ decreased the mRNA expression of DNMT 1 and also dose-dependently inhibited DNMT activity. In conclusion, a low concentration of As₂O₃ induces CpG island demethylation of tumor suppressor genes by inhibition of DNMT and reactivates the partially/fully silenced genes in liver cancer cells.

Keywords: Arsenite, Epigenetics, DNA demethylation, Hepatoma, Gene reactivation