Abstract 5431: Tolfenamic acid enhances the therapeutic efficacy of certain chemotherapeutic agents in medulloblastoma cell lines

Abstract

Abstract Tolfenamic acid (TA), a small molecule NSAID exhibits anti-cancer response in several malignancies. Recently we demonstrated for the first time that TA inhibits medulloblastoma (MB) cell proliferation and tumor growth in mice xenografts. Since MB requires intensive therapy that often causes long-term side-effects, we tested the efficacy of TA for enhancing the response of chemotherapy. Initial experiments were performed using MB cell lines, DAOY and D283 to test the anti-proliferative response of individual agents, TA, Irinotecan (IRI), Topotecan (TOPO), Temozolomide (TZM) and Doxorubicin (DOXO). Subsequently, the combination of TA with TOPO was tested using the optimized doses. DAOY and D283 cells were treated with DMSO or increasing concentrations of TA (5-50 μg/ml) or chemotherapeutic agents TOPO (10-500 nM), TZM (100-500 μM), IRI (0.5-5 μM) and DOXO (10-500 nM). Cell viability was measured at 24, 48, and 72 h post-treatment using CellTiter Glo kit and all agents caused a dose and time-dependent inhibition of cell viability. To assess the effect of combination therapy, MB cells were treated with optimized doses of TA (10 μg/ml) or TOPO (DAOY: 20 nM; D283: 25 nM) or both and the cell viability was measured at 48 h and 72 h post-treatment. The combination of TA and TOPO caused significantly higher inhibition when compared to either TA or TOPO alone confirming the efficacy of this combination therapy in pre-clinical models for MB. The effect on cell apoptosis was evaluated at 48 h post-treatment. Apoptotic cells were measured by flow cytometry using Annexin V-PE/7-AAD kit. The results showed a significant increase in the apoptotic fraction (annexin V positive) of both MB cell lines following the combination therapy when compared to individual treatment of TA or TOPO. These results were further supported by determining the activity of effector caspases. Consistent with Annexin V staining, combination therapy significantly upregulated caspase 3/7 levels when compared to individual treatment. DAOY and D283 were treated with DMSO or 10 or 20 µg/ml TA for 48 h. Whole cell lysates were prepared and the expression of Sp1, survivin and c-PARP was determined by Western blot analysis. TA inhibited both Sp1 and survivin in MB cell lines. Consistent with annexin V staining and caspase 3/7 results, TA augmented the PARP cleavage confirming the activation of apoptotic pathways. In order to evaluate the precise molecular markers/pathways associated with the beneficial (higher) efficacy of TA and TOPO combination therapy, we are conducting molecular profiling analysis. Overall, these results demonstrate that the combination of anti-cancer small molecule, TA and standard chemotherapeutic agents such as TOPO effectively inhibits MB cell growth. These preliminary results strongly demonstrate the efficacy of the proposed combination therapy in enhancing therapeutic response in pre-clinical models of MB. Citation Format: Don Eslin, Umesh T. Sankpal, Chris M. Lee, Robert M. Sutphin, W. Paul Bowman, Jeffrey C. Murray, Riyaz Basha. Tolfenamic acid enhances the therapeutic efficacy of certain chemotherapeutic agents in medulloblastoma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5431. doi:10.1158/1538-7445.AM2014-5431