Dose dependent paradoxical increases in DNA methylation, reductions in p16 expression, and changes in histone modifications in gastric cancer cells treated with DNMT inhibitors

DNA methyltransferases (DNMT) methylate DNA which can silence the expression of genes. Abnormal promoter CpG DNA methylation silences expression of the tumor suppressor cyclin-dependent kinase inhibitor CDKN2A (p16) and the lack of p16 causes some gastric cancers. DNMT inhibitors are cancer therapeutics that can reduce p16 promotor methylation, increasing p16 expression, and reducing gastric cancer cell proliferation. Here we measured changes in total and p16 promoter CpG DNA methylation, p16 and associated gene expression, and histone modifications in gastric cancer cells treated with increasing doses of DNMT inhibitors decitabine, RG108, and GSK3685032. At low doses, DNMT inhibitors decrease total and p16 promoter CpG DNA methylation to a minimum and increased p16 protein and mRNA. However, at higher doses, total and p16 CpG promoter DNA methylation increased from its minimum, and p16 expression (mRNA and protein) decreased, sometimes back to pre-treatment levels. Increased DNA methylation at high doses of DNMT is attributed to increased DNMT3B and TET1/2 mRNA and protein. GSK3685032 induced higher p16 expression than decitabine whereas decitabine induced higher p21 expression than GSK3685032 such that GSK3685032 induced G0/G1 cell cycle arrest, whereas decitabine induced G2/M arrest. Decitabine and GSK3685032 reduced histone lysine acetylation and methylated-lysines whereas GSK3685032 had minimal effects on histone modifications in cells. Our results suggest that some doses of DNMT inhibitors may increase p16 expression making them potentially effective in gastric cancer, while higher doses will decrease p16 expression possibly reducing efficacy.