Epigenetic therapy in rhabdomyosarcoma -

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, and occurs as one of two major histologic subtypes: embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS). ERMS and ARMS have different underlying oncogenic pathway aberrations, but are currently treated similarly. With current treatment strategies not curing most children with recurrent or high-risk disease there is a need for novel therapeutic approaches. We attempted to investigate the therapeutic potential of epigenetic modulating drugs, using two well-characterized drugs that have activity as histone deacetylase inhibitors (HDAC i): Valproic Acid (VPA) and Vorinostat (SAHA). We first used different concentrations of the two drugs, to investigate the efficacy of different concentrations of the two compounds. To this effect, cell accumulation and MTT assays were performed. Results showed that, using both ERMS cell lines (JR1, Rh18 and Rh36) and ARMS cell lines (Rh28, Rh30 and Rh41) and treatment with 0.3 mM VPA, 0.5 mM VPA, 0.5 µM SAHA and 1 µM SAHA, the latter drug and concentration exhibited the greatest effect. Subsequently 1 µM SAHA was used in all further experiments. Cell cycle analysis of both ERMS and ARMS cell lines after treatment with 1 µM SAHA resulted in a predominant increase in the pre-G0 cell population indicative of cell death starting at 48 hours, with some changes in G0-G1, S and G2-M phases. Cells treated with the drug also exhibited marked morphological changes, suggestive of enhanced differentiation. Further analysis of cell cycle changes using BrdU incorporation assays demonstrated a marked decrease in S-phase entry upon treatment with SAHA. Similarly, apoptosis after treatment with SAHA was confirmed by TUNEL assay. Further investigation of the mechanism of SAHA-induced effects, by use of western blotting of protein lysates, revealed in some of the cell lines, changes in the expression of proteins of the apoptotic machinery at day two of treatment, and an upregul