Enhancement of In vitro and In vivo Tumor Cell Radiosensitivity by the DNA Methylation Inhibitor Zebularine

Abstract

Aberrant DNA hypermethylation is a frequent finding in tumor cells, which has suggested that inhibition of DNA methylation may be an effective cancer treatment strategy. Because DNA methylation affects gene expression and chromatin structure, parameters considered to influence radioresponse, we investigated the effects of the DNA methylation inhibitor zebularine on the radiosensitivity of human tumor cells. Three human tumor cell lines were used in this study (MiaPaCa, DU145, and U251) and the methylation status of three genes frequently hypermethylated in tumor cells (RASSF1A, HIC-1, and14-3-3r) was determined as a function of zebularine exposure. Zebularine resulted in DNA demethylation in a time-dependent manner, with the maximum loss of methylation detected by 48 hours. Treatment of cells with zebularine for 48 hours also resulted in an increase in radiosensitivity with dose enhancement factors of >1.5. As a measure of radiation-induced DNA damage, gH2AX expression was determined.Whereas zebularine had no effect on radiation-induced gH2AX foci at 1hour, the number of gH2AX foci per cell was significantly greater in the zebularine-treated cells at 24 hours after irradiation, suggesting the presence of unrepaired DNA damage. Zebularine administration to mice reactivated gene expression in U251xenografts; irradiation of U251tumors in mice treated with zebularine resulted in an increase in radiation-induced tumor growth delay.These results indicate that zebularine can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect may involve an inhibition of DNA repair.

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer

“…Consequently, it is possible that zebularine acts to impede nucleotide excision repair or restore p53 activity by unknown mechanisms unrelated to activity of hMLH1. In support of this conjecture, a recent study showed that zebularine increased radiosensitivity of tumor cells, likely by inhibition of DNA repair (57). Although 5-aza-dC treatment resulted in sensitization to cisplatin in our system (similar to zebularine), clinical trials of that combination have been quite discouraging, resulting in significant toxicity (58,59).…”

Epigenetic Therapies for Cancer

“…Although this drug works in a manner similar to 5-Aza-CR and 5-Aza-CdR, it is more stable and less toxic than 5-Aza-CR and 5-Aza-CdR DNMTis (Zhou et al, 2002;Cheng et al, 2003). In line with these findings, zebularine has been shown to reactivate tumor suppressor genes (Flotho et al, 2009;Billam et al, 2010), enhance tumor cells' chemotherapy and radiation sensitivity (Dote et al, 2005), exert angiostatic and antimitogenic activities (Balch et al, 2005;Hellebrekers et al, 2006) and to be stable enough for oral administration (Zhou et al, 2002;Cheng et al, 2003). In addition, at low doses, zebularine can be given to patients continuously without the overt cytoxicity associated with 5-Aza-CR and 5-Aza-CdR.…”

The pivotal role of DNA methylation in the radio‐sensitivity of tumor radiotherapy

“…Dote et al23 found the enhancement of tumor cell radio‐sensitivity by the DNMT inhibitor (zebularine) in vitro and in vivo. Treating cells with zebularine, the radio‐sensitivity of 3 human tumor cells, including pancreatic carcinoma, glioblastoma, and prostate carcinoma, were more sensitive than control groups.…”

“…To investigate the role of DNA methylation inhibitor in tumor radio‐sensitivity, researchers studied 3 human tumor cell line (pancreatic carcinoma, glioblastoma and prostate carcinoma) 23. Hyper‐methylated in cancer 1 ( HIC1 ) gene is a growth regulatory and tumor repressor gene.…”