Abstract
BackgroundHistone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process.MethodsA549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry.ResultsPretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone.ConclusionsPsammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair.
Highlights
Histone modifications and DNA methylation are two major factors in epigenetic phenomenon
Western blot analysis revealed a drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2’-deoxycytidine, and zebularine in both A549 and U373MG cell lines
No depletion of DNMT3B by the three DNA methyltransferase (DNMT) inhibitors was observed in either of the cell lines (Figure 2). These results indicate that DNMT inhibitors induce selective demethylation in each of the evaluated tumor cell lines
Summary
Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. Epigenetic events are not changes in gene function that occur in conjunction with DNA sequence changes. DNA methylation and histone modifications are two principal factors in epigenetic phenomena. These two mechanisms perform a crucial function in carcinogenesis and tumor progression. DNMT overexpression has been detected in a variety of malignancies, including lung, prostate, and colorectal tumors [2,3,4]
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