Article
NO donor JS-K enhances cytotoxic effects of radiotherapy in U87 gliomas in vitro
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Published: | April 28, 2011 |
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Objective: The dismal prognosis of glioblastoma patients is largely caused by a high level of drug and radioresistance. The nitric oxide donor JS-K releases nitric oxide in GST-overexpressing tumor cells after enzymatic activation by glutathione-S-transferase (GST) resulting in growth inhibition, chemosensitization and anti-migration. The objective of this work was to analyze potential radiosensitizing effects of JS-K in U87 glioma cells in vitro.
Methods: U87 cells were exposed to increasing JS-K-concentrations (5–50 µM) for 4 h and were irradiated with 2, 5 or 10 Gray (Gy) 2h after JS-K-application. Cell viability and proliferative activity were assessed by MTT and colony formation assays at the end of treatment and in 48 h intervals up to 216 h. The induction of DNA-damage was analyzed by immunocytochemical staining of H2AX-Foci directly and 24 h after treatment. The influence of JS-K on cell cycle distribution and arrest was assessed by FACS analysis. All experiments were done in triplicates and repeated three times. Statistical analysis was either performed by Jonckheere-Terpstra-Test or by student’s T-Test.
Results: U87 were highly radioresistant and showed only a small dose- and time dependent reduction of cell viability up to 216 h after exposure. JS-K showed a strong dose-dependent antiproliferative effect with an IC50 of for 15 µM. Colony formation was abolished after JS-K treatment. Concomitant treatment with JS-K significantly enhanced the efficacy of irradiation with 2 Gy as late as 216h after treatment. Cell death was the result of JS-K-induced enhanced DNA-damage and decreased repair of DNA-double-strand-breaks. JS-K caused incorrect G1-arrest and a reentry of incompletely repaired U87-cells at an early stage.
Conclusions: JS-K sensitizes U87 glioma cell to irradiation and has potent antiproliferative and DNA damaging effects. This warrants further investigation of NO-releasing compounds as radiosensitizers in multimodal glioblastoma therapy.