Combination of Vincristine and Tolfenamic Acid induces Anti-proliferative activity in Medulloblastoma Cells.

Background: 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, a commonly used chemotherapeutic agent for MB treatment, is known to induce some toxic effects including peripheral neuropathy. Reducing the dose of the drug to minimize the toxic effect also reduces the cytotoxic efficacy of the drug. Purpose: The aim of this study was to test a combination treatment involving vincristine and an anti-cancer non-steroidal anti-inflammatory drug, Tolfenamic acid (TA) against MB cell lines. Previously, we showed that TA inhibited MB cell proliferation and tumor growth in mice by targeting the transcription factor specificity protein-1 (Sp1) and an inhibitor of apoptosis protein, survivin. The overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers. Methods: DAOY and D283 cells were treated with vehicle (DMSO) or low dose of vincristine (DAOY: 2ng/ml; D283: 1ng/ml) or TA (10 µg/ml) or combination of vincristine + TA and the cell viability was measured at one and two 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 Caspase 3/7-Glo kit and the expression of apoptotic markers [c-PARP, Bcl2, and survivin] by Western blot (WB) analysis. The expression of key proteins associated with cell cycle [Cyclin A, B, D CDK4/6 and p21] was also determined by WB analysis. Results: When compared to individual agents, the combination of TA and vincristine increased MB cell growth inhibition which is accompanied by an induction of apoptotic markers and the modulation of proteins associated with cell cycle phase distribution. Conclusions: These results suggest that vincristine and TA combination treatment is effective for inducing anti-proliferative response in MB cells. The experiments to evaluate the effect of this combination in animal model for MB are currently under study.