Arsenic trioxide inhibits DNA methyltransferase and restores methylation-silenced genes in human liver cancer cells.
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.
Cui
Xing
X
Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan. xing.cui@nies.go.jp
Wakai
Toshifumi
T
Shirai
Yoshio
Y
Yokoyama
Naoyuki
N
Hatakeyama
Katsuyoshi
K
Hirano
Seishiro
S
D000368:Aged; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000494:pharmacology*; D015820:Cadherins / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D020868:Gene Silencing / Q000187:drug effects*; D052138:Genes, Neoplasm / Q000187:drug effects; D019942:Genes, p16 / Q000187:drug effects; D051549:Glutathione S-Transferase pi / Q000235:genetics; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D010087:Oxides / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics
D000970:Antineoplastic Agents; D001152:Arsenicals; D015820:Cadherins; D004791:Enzyme Inhibitors; D010087:Oxides; C411762:RASSF1 protein, human; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D004248:DNA (Cytosine-5-)-Methyltransferases; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D000077237:Arsenic Trioxide