4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Induces Retinoic Acid Receptor β Hypermethylation through DNA Methyltransferase 1 Accumulation in Esophageal Squamous Epithelial Cells

Overexpression of DNA methyltransferase 1 (DNMT1) has been detected in many cancers. Tobacco exposureis known to induce genetic and epigenetic changes in the pathogenesis of malignancy. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important carcinogen present in tobacco smoke; however the detailedmolecular mechanism of how NNK induces esophageal carcinogenesis is still unclear. We found that DNMT1was overexpressed in ESCC tissues compared with paired non-cancerous tissues, the overexpression beingcorrelated with smoking status and low expression of RARβ. The latter could be upregulated by NNKtreatment in Het-1A cells, and the increased DNMT1 expression level reflected promoter hypermethylation anddownregulation of retinoic acid receptor β(RARβ). RNA interference mediated knockdown of DNMT1 resultedin promoter demethylation and upregulation of RARβ in KYSE30 and TE-1 cells. 3-(4,5-Dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometric analysis demonstrated that NNK treatment in Het-1A cells could enhance cell proliferation and inhibit cell apoptosis in a dose-dependent manner. In conclusion,DNMT1 overexpression is correlated with smoking status and low expression of RARβ in esophageal SCCpatients. NNK could induce RARβ promoter hypermethylation through upregulation of DNMT1 in esophagealsquamous epithelial cells, finally leading to enhancement of cell proliferation and inhibition of apoptosis.