5-Azacytidine Enhances the Radiosensitivity of CNE2 and SUNE1 Cells In Vitro and In Vivo Possibly by Altering DNA Methylation

Wei Jiang,Jun Ma,Ying Sun,Lei Chen,Qing-Mei He,Na Liu,Ya-Fei Xu,Ying-Qin Li,Ning Jiang,Pierre Busson

doi:10.1371/journal.pone.0093273

Wei Jiang, Jun Ma + Show 8 more

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https://doi.org/10.1371/journal.pone.0093273

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Abstract

The radioresistance of tumor cells remains a major cause of treatment failure in nasopharyngeal carcinoma (NPC). Recently, several reports have highlighted the importance of epigenetic changes in radiation-induced responses. Here, we investigated whether the demethylating agent 5-azacytidine (5-azaC) enhances the radiosensitivity of NPC cells. The NPC cell lines CNE2 and SUNE1 were treated with 1 μmol/L 5-azaC for 24 h before irradiation (IR); clonogenic survival was then assessed. Tumor growth was investigated in a mouse xenograft model in vivo. The apoptosis, cell cycle progression and DNA damage repair were examined using flow cytometry, immunofluorescent staining and western blotting. Promoter methylation and the expression of four genes epigenetically silenced during the development of NPC were evaluated by pyrosequencing and real-time PCR. We found that pretreatment with 5-azaC significantly decreased clonogenic survival after IR compared to IR alone; the sensitivity-enhancement ratio of 5-azaC was 1.4 and 1.2 for CNE2 and SUNE1 cells, respectively. The combined administration of 5-azaC and IR significantly inhibited tumor growth in the mouse xenograft model, and enhanced radiation-induced apoptosis in vitro compared to 5-azaC alone or IR alone. 5-AzaC also decreased promoter methylation and upregulated the expression of genes which are epigenetically silenced both in vitro and in vivo in NPC. Thus, 5-azaC enhance the radiosensitivity of both the CNE2 and SUNE1 cell lines, possibly by altering DNA methylation levels and increasing the ability of irradiated cells to undergo apoptosis. The use of 5-azaC combined with IR maybe represent an attractive strategy for the treatment of NPC.

Highlights

Nasopharyngeal carcinoma (NPC) is prevalent in southeastern Asia, especially in southern China where the incidence is approximately 25-50 per 100,000 population per year [1]
Epigenetic modifications, DNA hypermethylation that leads to the aberrant silencing of multiple tumor suppressor genes, are believed to play a pivotal role in variety of cellular events [7,8], including alterations in apoptosis, cell cycle progression, mitotic checkpoint regulation and DNA repair; all of these mechanisms have been considered to mediate radiosensitizing effects [4,9]
Compared with the control group, no significant differences were observed in the survival rates of CNE2 and SUNE1 cells treated with 50 nmol/L to 1 mmol/L 5-azaC for 72 h (p.0.05)

Summary

Introduction

Nasopharyngeal carcinoma (NPC) is prevalent in southeastern Asia, especially in southern China where the incidence is approximately 25-50 per 100,000 population per year [1]. Radiation resistance is one of the major obstacles that leads to locoregional recurrence of NPC during treatment [3].the identification of effective radiosensitizing agents to enhance the radiosensitivity of NPC cells may help to decrease both tumor recurrence and radiation-associated morbidity. Increasing evidence supports the suggestion that genome-wide changes in methylation levels are associated with the radiosensitivity of cancer cells [4,5,6]. Epigenetic modifications, DNA hypermethylation that leads to the aberrant silencing of multiple tumor suppressor genes, are believed to play a pivotal role in variety of cellular events [7,8], including alterations in apoptosis, cell cycle progression, mitotic checkpoint regulation and DNA repair; all of these mechanisms have been considered to mediate radiosensitizing effects [4,9]

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