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
micromol/L of AS2O3 and/or 1 micromol/L of 5-aza-2'-deoxycytidine for 24, 48, and
72 hours. The methylation status of the CpG island around the promoter regions of
p161NK4a, 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 p16INK4a, 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 p16INK4a, 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 micromol/L) of As2O3 through
demethylation, as well as 1 micromol/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 micromol/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.