4.4 Article

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

Journal

WORLD JOURNAL OF GASTROINTESTINAL ONCOLOGY
Volume 14, Issue 3, Pages 664-677

Publisher

BAISHIDENG PUBLISHING GROUP INC
DOI: 10.4251/wjgo.v14.i3.664

Keywords

O-6-methylguanine-DNA methyltransferase; DNA methylation; Malignant transformation; Gastric carcinogenesis; Epigenetic regulation

Funding

  1. National Natural Science Foundation of China [81472543, 81772919]
  2. Zhejiang Provincial Natural Science Foundation of China [LY18H160024, LY20H160040]

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The aim of this study was to investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis. The results found that the upregulation of MGMT was induced through DNA promoter hypomethylation. The highly expressed MGMT prevented the NOCs-induced cell malignant transformation and tumorigenesis.
BACKGROUND O-6-methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the mispairing base O-6-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 O-6-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. AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis. METHODS We 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. RESULTS We 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 O-6-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. CONCLUSION Our 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.

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