DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

Abstract

Background: 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 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.

Methods: A549 (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.

Results: Pretreatment 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.

Conclusions: Psammaplin 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.

Figure 1

The effects of DNMT inhibitors on tumor cell radiosensitivity. Survival curves of (A) A549 cells and (B) U373MG cells treated with the respective DNMT inhibitors prior to radiation and radiation were compared with those of radiation alone. Points, mean for three independent experiments; bars, SE.

Figure 2

Methylation status was determined after exposure to DNMT inhibitors using Western blot analysis of DNMT1, 3A/3B. The cells were treated with DNMT inhibitors for 18 hours. A drastic depletion of DNMT1 and DNMT3A by psammaplin A, 5-aza-2'deoxycytidine, and zebularine in both A549 and U373MG cell lines was observed. However, there was no depletion of DNMT3B by the three DNMT inhibitors in either of the cell lines. Each blot is representative of two independent experiments, with actin used as a loading control.

Figure 3

Influence of DNMT inhibitors on cell cycle phase distributions of A549 and U373MG cells. Cell cycle phase was measured using flow cytometry at 12 hours after 6 Gy of radiation. Columns, proportion of cell cycle phase; bars, SE.

Figure 4

The effect of zebularine on radiation-induced G2/M arrest in A549 cells. A549 cells were treated with 800 uM zebularine for 18 hours and then irradiated with 6 Gy. A549 cells were accumulated in G2/M phase after radiation. This radiation-induced G2/M arrest was abrogated by zebularine pretreatment at 6-12 hours, but this abrogation disappeared at 24 hours.

Figure 5

Western blot analysis of cleaved caspase-3. The cells were treated with a combination of three different DNMT inhibitors and 6 Gy of radiation. Increased cleaved caspase-3 protein level was observed in cells treated with a combination of psammaplin A and 6 Gy of radiation in the A549 cells.

Figure 6

Radiation-induced γH2AX foci. Representative micrographs obtained from (A) control cells and (B) cells that had received 6 Gy of radiation 1 hour earlier. (A) top panel, (B) bottom panel.

Figure 7

Influence of DNMT inhibitors on radiation-induced γH2AX foci. A549 and U373MG cells growing in chamber slides were exposed to DNMT inhibitors for 18 hours, irradiated, and fixed at specific times for immunocytochemical analyses of nuclear γH2AX foci. Open columns, data from cells receiving radiation alone; grey columns, data from cells that were exposed to psammaplin A and radiation; filled columns, data from cells that were exposed to 5-aza-2'-deoxycytidine and radiation; hatched columns, data from cells that were exposed to zebularine and radiation. Cells with more than five foci per nucleus were classified as positive for radiation-induced γH2AX. Foci were evaluated in 50 nuclei. Bars, SE. *p < 0.01 as determined by a logistic regression compared with radiation alone (6 Gy) group.