Medulloblastoma (MB) is the most common pediatric malignant brain tumor and usually originates in the cerebellum. These tumors have the propensity to disseminate throughout the central nervous system and are often difficult to treat. Chemotherapy is widely accepted as part of the multimodality treatment approach for MB. However, it is associated with debilitating toxicity and potential long term disabilities. Vincristine is a commonly used chemotherapeutic agent for MB treatment. This drug is known to induce some toxic effects including peripheral neuropathy. The aim of this study was to test a combination treatment involving Vincristine and an anti-cancer non-steroidal anti-inflammatory drug, Clotam (Tolfenamic acid) against MB cell lines. Previously, we showed that Clotam inhibited MB cell proliferation and tumor growth in mice by targeting the transcription factor, Specificity protein1 (Sp1) and an inhibitor of apoptosis protein, BIRC5 (baculoviral inhibitor of apoptosis repeat-containing 5). The overexpression of BIRC5 is associated with aggressiveness and poor prognosis in several cancers. MB cells, DAOY and D283 cells were treated with vehicle (dimethyl sulfoxide) or low dose of Vincristine (DAOY: 2ng/ml; D283: 1ng/ml) or Clotam (DAOY & D283: 10 µg/ml) or combination of Vincristine + Clotam and the cell viability was measured at 1 and 2 days post-treatment using Cell-TiterGlo kit. Flow cytometry was employed to analyze apoptotic cells using Annexin-V staining and cell cycle phase distribution using propidium iodide staining. The activation of apoptotic pathways was further investigated by assessing the levels of effector caspases with CaspaseGlo kit and the expression of apoptotic markers [cleaved Poly (ADP-ribose) polymerase (c-PARP), B-cell lymphoma 2, and BIRC5] by Western blot analysis. The expression of key proteins associated with cell cycle (Cyclin A, B, D, CDK4/6, and p21) was also determined by Western blot analysis. When compared to individual agents, the combination of Clotam and Vincristine increased MB cell growth inhibition which is accompanied by an induction of apoptotic markers and the modulation of (Cyclin A, B, D and CDK4/6: down-regulation; p21: up-regulation) proteins associated with cell cycle phase distribution. These results suggest that Vincristine and Clotam combination treatment is effective for inducing anti-proliferative effect in MB cells. The experiments to evaluate the effect of this combination in animal model for MB are currently under study.

Citation Format: Shruti V. Patil, Don Eslin, Robert Sutphin, Umesh T. Sankpal, Yazmin Hernandez, Areeba Hafeez, W. Paul Bowman, Riyaz Basha. Combination of Vincristine and Clotam induces antiproliferative response in medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1947. doi:10.1158/1538-7445.AM2017-1947