Abstract A02: Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid
Authors: Eslin Don , Sankpal Umesh T. , Murray Jeffrey C. , Bowman W. Paul , Sutphin Robert , Basha Riyaz 
Published: May 2015

Introduction: Medulloblastomas (MB) are the most common malignant brain tumors in children and constitute 20% of all pediatric brain tumors. Significant developments in the areas of neurosurgical techniques, dosing and delivery of radiation and optimized chemotherapy have limited response on improving the five year survival rates. Because of the aggressive multimodality therapy, survivors frequently suffer numerous long-term side effects. There is an urgent need for novel strategies for enhancing and optimizing the therapeutic efficacy of current MB treatments. Studies have shown that the Specificity Protein (Sp) transcription factors regulate critical genes that are involved in regulation of proliferation, survival, angiogenesis and resistance to chemo-/radiation therapy.

Rationale: Research from our laboratory and others showed the anti-cancer activity of Tolfenamic acid (TA), a small molecule and non-steroidal anti-inflammatory drug in several adult malignancies. Recently, we demonstrated for the first time that TA inhibits MB cell proliferation and tumor growth in mice xenografts. Mechanistic studies revealed that TA acts via downregulation of Sp proteins and its downstream targets like survivin and VEGF. In this study, we tested the efficacy of TA for enhancing the response of chemotherapeutic agents used as standard therapy for MB. We hypothesize that TA sensitizes MB cells to chemotherapeutic drugs via down-regulation of Sp1 and survivin.

Methods: Human MB cell lines, DAOY and D283 were used to test the anti-proliferative response of TA, Irinotecan, Topotecan (TOPO), Temozolomide (TMZ) and Doxorubicin (DOXO). Subsequently, the combination of TA with TOPO was tested using the optimized doses of TA (10 µg/ml) or TOPO (20 nM for DAOY; 25 nM for D283). Cell viability was measured at 24, 48, and 72h post-treatment using CellTiter-Glo kit. To correlate the anti-proliferative response with the induction of apoptotic pathways we analyzed apoptosis by flow cytometric analysis of Annexin V stained cells, PARP cleavage, and the activation of caspase3/7. The expression of Sp1, survivin, and cleaved-PARP was determined by Western blot. qPCR analysis using TaqMan assays was used to detect microRNAs (miR-20a and miR-27a), which act as Sp repressor proteins.

Results: TA and chemotherapeutic agents caused a dose and time-dependent inhibition of cell viability. 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. Annexin V staining for apoptotic cells showed a significant increase in the apoptotic fraction (annexin V positive) following combination therapy when compared to individual treatment of TA or TOPO. These results were further supported by significantly upregulated caspase 3/7 activity when compared to individual treatment. DAOY and D283 were treated with vehicle (DMSO) or 10 or 20 µg/ml TA for 48 h. Whole cell lysates were prepared and analyzed 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 PARP cleavage confirming the activation of apoptotic pathways. TA treatment also downregulated mirR-20a and miR-27a.

Conclusion: Overall, these results demonstrate that the combination of TA with standard chemotherapeutic agents effectively inhibits MB cell growth. This chemo-sensitization can be correlated with the downregulation of survivin by TA, in part due to its effects on Sp1 transcription factor either directly or through miRNAs. These preliminary results strongly demonstrate the potential of targeting Sp1 and survivin using TA to enhance the therapeutic response in pre-clinical model of MB.

Financial Support: HyundaiHopeOnWheels; UFHCC, Orlando Health, Orlando, FL and Department of Pediatrics, UNT Health Science Center, Fort Worth, TX.

Citation Format: Don Eslin, Umesh T. Sankpal, Jeffrey C. Murray, W. Paul Bowman, Robert Sutphin, Riyaz Basha. Chemo-sensitization of medulloblastoma cells using small molecule tolfenamic acid. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A02.