Combination of Mithramycin and Standard Chemotherapeutic Agents Induces Anti-proliferative activity in Ewing Sarcoma cell lines

Background/Hypothesis: Ewing sarcoma (ES) is a small, round, blue cell tumor found primarily in bones of adolescents. The EWS-FLI1 transcription factor is associated with proliferation of cancer cells and is over-expressed in [greater than] 85% of Ewing sarcoma cases. Mithramycin (MIT) is an antibiotic with antineoplastic properties and has been shown to inhibit EWS-FLI1. A recent trial of MIT treatment in ES patients found that hepatotoxicity precluded the administration of MIT at a dose required to inhibit EWS-FLI1 ([greater than]50nmol/L). We hypothesize that the efficacy of adjunct treatment can be enhanced if MIT is used along with standard chemotherapeutic agents such as Vincristine (VIN) and Etoposide (ETO). Combination treatment will reduce the effective dose of both Mithramycin and the standard agent thereby decreasing the therapeutic dose range and side effects. Methods: ES cells, CHLA10 and TC205 were cultured in the presence of vehicle or MIT or VIN or ETO or in combinations (MIT+VIN or MIT+ETO). After 2 days, cell viability was measured using The CellTiter-Glo® Luminescent Cell Viability Assay kit. The apoptosis induced by each of the above-mentioned treatments on the ES cells was measured by Flow cytometry using Annexin V Apoptosis Detection Kit. The expression of cleaved-Poly (ADP-ribose) polymerase (c-PARP), a marker for apoptosis was determined by Western blot analysis. Results: While all treatments showed ES cell growth inhibition, the combination treatment of MIT+ETO was more effective (significant at p Conclusion: The combination MIT+ETO caused more cell growth inhibition when compared to individual treatments in the TC205 and CHLA10 cell lines. These results demonstrate that MIT in combination with standard chemotherapeutic agents potentially increases therapeutic efficacy in ES. However, these results are limited to in vitro studies and need to be tested in an animal model to determine reproducibility and assess the toxicity.