Ewing sarcoma (ES) is the second most common pediatric primary bone tumor, and affects children between the ages of 1 to 20 years. With the advancement in therapeutic options the 5-year event free survival rate of patients with localized tumor has gone up to 70%. However, in patients with metastases at diagnosis, this rate is less than 20%. One of the distinguishing hallmarks of ES is the presence of oncogenic fusion transcription factor formed by the translocation between EWS and ETS family transcription factor. The EWS-FLI1 translocation is most commonly found in ES patients. Therefore targeting EWS-FLI1 transcription factor or its downstream targets presents a novel therapeutic approach to treat ES. Research from our group demonstrated the anti-cancer activity of a small molecule, Tolfenamic acid (TA) in pediatric cancers, leukemia, neuroblastoma and medulloblastoma. TA has been shown to target transcription factor Sp1, which regulates the expression of several genes associated with cancer including survivin, a member of inhibitor of apoptosis protein (IAP) family and involved in chemoresistance. It is currently in phase I clinical trials for treating upper gastro-intestinal cancer patients along with radiation. In this study, we evaluated the therapeutic efficacy of TA alone and in combination with chemotherapeutic drugs, and its effect on the EWS-FLI1 downstream targets. ES cell lines CHLA9, CHLA10 and TC71 were treated with TA, Etoposide, Doxorubicin and Vincristine and cell viability was measured at 24 and 48h post-treatment. TA and chemotherapeutic drugs caused a dose and time dependent inhibition of cell proliferation. We then assessed the effects of TA in combination with these chemotherapeutic drugs. Results indicate that the combination treatment causes increased (2-3 fold) inhibition of cell viability compared to individual agents. To access the mode of action of combination therapy, cells were treated with TA and Vincristine and apoptosis was monitored at 48h post-treatment via western blot analysis (c-PARP), flow cytometry (Annexin V staining), and caspase 3/7 activity (Caspase3/7-Glo assay). We were able to demonstrate that the inhibition of cell viability in the combination treatment was the result of apoptotic mechanism as determined by increased cleavage of PARP and up-regulation of caspase 3/7activity. In addition we also studied the effects of TA on the regulation of EWS-FLI1 downstream targets NROB1, ID2, ROCO1 and LDB2 by real-time PCR. Results demonstrate that TA treatment leads to the downregulation of EWS-FLI1 genes. Taken together our results indicate that the combination of TA and chemotherapy drugs inhibits the proliferation of sarcoma cells. This investigation also suggests that the proposed combination therapy involving small molecule TA and standard chemotherapeutic agents could be a viable strategy for the treatment of Ewing sarcoma.
Citation Format: Umesh T. Sankpal, Anish Ray, Leslie Tabor-Simecka, W Paul Bowman, Riyaz M. Basha. Effect of tolfenamic acid on the therapeutic efficacy of chemotherapeutic drugs used in the treatment of Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2197. doi:10.1158/1538-7445.AM2015-2197