Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing's sarcoma cells

Purpose The present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing's sarcoma. Methods Cytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot. Results All three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewing's sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis. Conclusions These results suggest that HDIs may be considered as a novel treatment strategy for Ewing's sarcoma either applied as monotherapy or in combination with TRAIL.