gms | German Medical Science

Objective: The fatal prognosis of glioblastoma multiforme (GBM) is based on strong radio- and chemoresistance. Nitric oxide (NO) release of the diazeniumdiolate JS-K is mediated by glutathione-S-transferases (GSTs). GBM cells overexpress GSTs, thus JS-K facilitates a targeted intracellular release of NO in these tumor cells. The aim of this project is to investigate the sensitizing effect of JS-K on U87 cells to repetitive irradiation and its underlying molecular mechanisms in vitro.

Methods: U87 cells (p53 wt) were used as in vitro model system. Cells were exposed to increasing JS-K concentrations (0 - 15 μM) for 4 hours and/or to repetitive irradiation (3 x 2 Gy). Cell viability was assessed 0 - 72h after the end of treatment by MTT assay. The total number of induced DNA double strand breaks (DSB) was determined by imunocytochemistry against H2AX and confocal microscopy. The effect on proliferation activity was analyzed using Bromodeoxyuridine (BrdU) incorporation assay. Accumulation and nuclear import of the tumor suppressor p53 was investigated by Western Blot (WB) and imunocytochemistry. Increased induction of apoptosis was assessed by WB analysis of inactivated Poly(ADP-ribose)-Polymerase 1 (PARP) and activated caspases. All experiments were performed in triplicates with 3 repetitions and analyzed by t-test.

Results: Repetitive irradiation has no significant effect on cell viability of U87 cells. Their proliferation activity and the number of induced DSB remained unchanged confirming their strong radioresistance. In contrast, JS-K treatment showed a dose- and time-dependent cytotoxic effect. Inhibition of cell proliferation was already detectable after exposure to lower JS-K concentrations. Repetitive irradiation combined with JS-K treatment resulted in a significant enhancement of these cellular effects compared to repetitive irradiation or JS-K treatment alone. Biochemical analysis revealed significantly increased accumulation of p53 after JS-K treatment and its translocation into the nucleus. JS-K induced activation of caspases and cleavage of PARP confirming the induction of apoptosis.

Conclusions: JS-K sensitizes U87 cells to repetitive irradiation, mediated by the increased induction of DSB and of proapoptotic mechanisms, including accumulation of p53 and inactivation of PARP. The advantage of cell type-specific NO-release warrants further investigation of JS-K as a promising adjunct in multimodal GBM treatment.