Abstract B11: Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells

Radiation (XRT) is a part of standard therapy for treating medulloblastoma (MB) in children. Morbidities associated with irradiation often cause long-term side-effects. Here we are testing a strategy to enhance the response of radiation using pre-clinical models for MB. XRT-induced cell death is partially associated with the generation of reactive oxygen species (ROS). Since ROS production is implicated in cell apoptosis, induction of increased ROS may serve as a promising strategy for inhibiting cancer cell growth. Nifurtimox (Nfx), a nitrofuran compound used to treat the parasitic infection Chagas9 disease, is known to induce ROS in pre-clinical models for neuroblastoma and medulloblastoma and is currently in clinical trials for treating these malignancies in children. We investigated the efficacy of Nfx in augmenting the effect of XRT using MB cell lines (DAOY and D283) and a patient-derived primary culture (003BN cells). DAOY, D283, and 003BN cells were treated with vehicle (DMSO) or increasing concentrations/doses of Nfx (5-70 µg/ml) or XRT (2-10 Gy) and cell viability was measured up to 3 (MB cell lines) or 5 (primary cultures) days post-treatment. The optimized concentration/dose of Nfx and XRT were used for combination experiments. MB cells were treated with vehicle, Nfx (20 μg/ml), XRT (5 or 10 Gy) or both and the effect on cell viability, apoptosis, ROS levels and catalase activity was measured. Cell viability was measured with CellTiter Glo kit (Promega). Apoptosis was analyzed by flow cytometry (Annexin-V staining) and caspase 3/7 activity was measured using Caspase-Glo Assay (Promega). The expression of c-PARP was determined by Western blot analysis. Results revealed that Nfx and XRT combination significantly increased MB cell growth inhibition when compared to the effect of either Nfx or XRT. The anti-proliferative effect of Nfx and XRT combination was accompanied by the activation of apoptosis as determined by increased annexin V staining and caspase 3/7 activity. An increase in levels of ROS was also observed following combination treatment with Nfx and XRT. Notably, catalase activity was unaltered following the treatment with single or double agents suggesting a contribution of impairment in the redox system in causing MB cell growth inhibition. This pre-clinical study demonstrates that Nfx potentially enhances the therapeutic efficacy of radiation in MB cell lines and primary cultures. To further delineate the underlying mechanisms and to identify other potential candidates modulated by this combination therapy we are undertaking a molecular profiling approach using Affimetrix gene expression arrays. Citation Format: Don Eslin, Chris M. Lee, Giselle Sholler, Amy Smith, Robert M. Sutphin, Dennis A. Steindler, Ping Zhao, Shanshan Wang, Umesh T. Sankpal, Sarah Connelly, Riyaz Basha. Nifurtimox and radiation combination increases reactive oxygen species and induces apoptosis in medulloblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B11.