O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUNDO⁶-methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the mispairing base O⁶-methyl-guanine induced by environmental and experimental carcinogens. It can transfer the alkyl group to a cysteine residue in its active site and became inactive. The chemical carcinogen N-nitroso compounds (NOCs) can directly bind to the DNA and induce the O⁶-methylguanine adducts, which is an important cause of gene mutation and tumorigenesis. However, the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis, especially in the initiation phase, remains largely unclear.AIMTo investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODSWe established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitroso-urea (MNU) treatment. Cell proliferation, colony formation, soft agar, cell migration, and xenograft assays were used to verify the malignant phenotype. By using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis, we detected the MGMT expression in malignant transformed cells. We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry. MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR. The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays. RESULTSWe observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment. Moreover, we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells. Inhibition of the MGMT expression by O⁶-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes. Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU. Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues, but downregulated in the gastric cancer tissues.CONCLUSIONOur finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation. The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis, which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.