In Vitro Study to Identify a Novel Combination Treatment for Ewing Sarcoma

Introduction: Ewing sarcoma (ES) is an aggressive tumor that predominantly occurs in young adolescent populations. Chemotherapy used to treat ES is associated with long-term morbidity. The objective of this study was to identify an effective combination treatment option for treating ES. A fusion protein EWS-FLI1 is associated with [greater than] 85% of ES incidences and mithramycin (Mit) is known to target this fusion protein and is currently in clinical trials. We investigated the combination of Mit to induce the efficacy of Doxorubicin (Dox), a widely used chemotherapeutic agent for treating ES patients. Methods: ES cell lines CHLA9, CHLA10, and TC71 were cultured in laboratory and treated with increasing concentration of Mit or Dox. Cell viability was assessed using CellTiter-Glo luminescent assay at 48 hour post-treatment. Optimized doses for Mit and Dox were used for combination treatment. Western blot analysis was used to evaluate the apoptotic markers, caspase 3, cleaved PARP and Bcl2. Real-Time PCR was used to evaluate the expression of EWS-FL1 downstream targets. RNA was extracted using Trizol reagent (Invitrogen) and subjected to cDNA synthesis using Superscript III reverse transcriptase. PCR was carried out with cDNA using TaqMan primer-probes specific for ID2, LDB2, NROB1 and NCOR1. Results: The results of current study illustrated that Mit or Dox treatment resulted in a dose and time dependent decrease in ES cell proliferation. Mit down-regulated the EWS-FLI1 downstream targets (ID2, LDB2, NROB1 and RCOR1) in TC71 cells. Combination of Mit+Dox enhancesd the anti-proliferative effect in ES cell lines which was accompanied by modulation of apoptotic markers. Conclusion: This investigation provides evidence for the use of Mithramycin for enhancing the efficacy of chemotherapeutic agents. In vivo assays are underway to confirm in vitro results. These pre-clinical studies will have clinical implications for treating Ewing Sarcoma patients. The proposed combination to induce chemotherapy response is highly helpful since it can be tested to reducing chemotherapy dose(s) and addressing the issues related to side-effects.