Evaluation of Mithramycin in Combination with Chemotherapeutic Agents Against Ewing Sarcoma Cell Lines

Ewing Sarcoma (ES) is a rare, malignant bone neoplasm that is primarily diagnosed in childhood and adolescence. The aggressive nature of this neoplasm requires the use of surgery, radiation and a rigorous chemotherapy regimen. Metastatic ES carries a poor prognosis, which necessitates the development of new therapeutic agents. Mithramycin was tested in targeted therapy due to its specific inhibitory effects on the EWS-FLI1 fusion protein which is present in >85% of ES tumors. We tested the combination of Mithramycin with chemotherapeutic agents vincristine (VCR) and Etoposide (Eto) for inducing higher cytotoxicity against ES cells, CHLA10 and TC205. Cardiomyocyte cell line, H9C2 was used to test the effect on non-malignant cells. Cell viability was measured using the CellTiter-Glo kit, and the combination index was evaluated to determine the type of combination response (antagonistic, additive, or synergistic). Apoptotic cells were measured post-treatment with vehicle (DMSO, control), monotherapy (mithramycin or etoposide), or combination therapy (mithramycin + etoposide) using BD LSRII flow cytometer and analyzed utilizing FlowJo software V8.0. The apoptotic protein marker c-PARP in both treatment and control groups was analyzed using Western blot analysis. The results showed higher cytotoxicity for combination treatment when compared to individual agents, and the combination index confirmed the response as synergistic. H9C2 cells did not demonstrate significant decreases in cell viability when treated with combination therapy, highlighting the specificity of the treatment toward its target tissue. Flow cytometry confirmed the underlying mechanism as upregulation of apoptosis which is further supported by an increase in effector caspases 3/7 and elevated expression of c-PARP. These in vitro assays using ES cells provide preliminary evidence for the benefit of chemotherapy and mithramycin combination.