Abstract 12: Evaluation of metformin and clotam combination for medulloblastoma

Abstract

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children. Standard treatment, which includes surgery, chemotherapy, and radiation, is successful for most patients, but survivors often suffer from significant long-term side effects affecting their neurocognitive and growth potential. Therefore, there is a critical need to understand the molecular processes that regulate MB growth and find less toxic therapies. Survivin (BIRC5) was identified as a protein belonging to the Inhibitor of Apoptosis Protein family that inhibits apoptosis by inhibiting caspase activation. Survivin expression is also associated with increased resistance of tumor cells to radiation and chemotherapy. In MB, several studies suggest that increased expression of survivin serves as a marker of tumor morphology and is associated with poor prognosis. Specificity protein 1 (Sp1) is one of the transcription factors that regulates the expression of survivin gene. Sp1 is also shown to be overexpressed in some cancer cells and is associated with poor prognosis. In this study, we are testing a strategy of targeting survivin in MB using anti-diabetic drug Metformin, which targets Sp1, and the NSAID Clotam (Tolfenamic acid, TA), which targets Sp1 and survivin. Dose response curves were established by treating MB cell lines DAOY and D283 with increasing concentrations of metformin or TA and measuring cell viability at 24 and 48h post-treatment using the CellTiter-Glo Cell Viability Assay. A time and dose dependent inhibition of cell proliferation was observed for both the drugs. Metformin dose response was then repeated in the presence of 25 or 50 µM TA. Our results indicate that TA significantly increased the growth inhibitory response of metformin. To further characterize this response, we determined the combination index (CI) using the Chou-Talalay method. We found that the CI values were between 0.85 to 0.61 for various combinations of metformin and TA, strongly suggesting a synergistic effect of the two drugs on cell proliferation. Combination of metformin and TA was also accompanied by a 3-4 fold increase in apoptotic cells at 48h post-treatment, as determined by AnnexinV staining of DAOY cells. Western blot analysis of DAOY cells treated with the two drugs also revealed a decrease in survivin and Bcl-2 protein levels, and an increase in cleaved-PARP. In conclusion, the anticancer activity of metformin in MB cells is enhanced in the presence of TA. Both drugs synergistically combine to inhibit MB cell proliferation and increase apoptosis, which in part may be the result of a decrease in survivin levels. This combination strategy may therefore represent a novel targeted therapy for MB. Studies are underway to determine whether this drug combination can enhance the response of chemotherapy in MB cells, since both TA and metformin are known to sensitize cancer cells to chemotherapy. Citation Format: Umesh T. Sankpal, W Paul Bowman, Jeffrey C. Murray, Riyaz Basha. Evaluation of metformin and clotam combination for medulloblastoma [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 12. doi:10.1158/1538-7445.AM2017-12