Tolfenamic Acid Enhances the Anti-Proliferative Activity of Vincristine in Medulloblastoma Cell Lines
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 (VCR), 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. The aim of this study was to test a combination treatment involving VCR and an anti-cancer non-steroidal anti-inflammatory drug (NSAID), Tolfenamic acid (TA) against MB cell lines. Previously, we showed that TA inhibited MB cell proliferation and tumor growth in mice by targeting an inhibitor of apoptosis protein, survivin. The overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers, including MB. DAOY and D283 cells were treated with vehicle (DMSO) or low dose of VCR (DAOY: 2ng/ml; D283: 1ng/ml) or TA (10 μg/ml) or combination of VCR + 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 [cleaved Poly ADP-ribose polymerase (c-PARP) and survivin] by Western blot (WB) analysis. The expression of key proteins associated with cell cycle [Cyclin A, B, D and CDK4/6] was also determined by WB analysis. When compared to individual agents, the combination of TA and VCR increased MB cell growth inhibition by targeting survivin expression. This growth inhibitory effect is accompanied by an induction of apoptotic markers and the modulation of proteins associated with cell cycle phase distribution. These results suggest that VCR and TA combination treatment is effective for inducing enhanced anti-proliferative response in MB cells. We also proposed that the survivin inhibition by TA contributes towards the increased efficacy of VCR by sensitizing MB cells. As future prospects for this preliminary study, we will evaluate the effect of TA+VCR combination in patient-derived primary cultures and mouse xenograft model for MB.