Molecular mechanisms and gene regulation of melphalan- and hyperthermia-induced apoptosis in Ewing sarcoma cells.

The prognosis of high-risk Ewing tumours (HR-ET) remains poor. Melphalan-containing chemotherapy regimens are commonly applied for HR-ET patients. Moreover, melphalan (Mel) is a promising agent in thermochemotherapy. Therefore, we investigated the single effects, the synergism and the gene regulation of Mel and hyperthermia (HT) in an ET cell line (RD-ES). Dose-dependent cytotoxicity by Mel was demonstrated, which was enhanced by the concomitant application of HT (42 degrees C for 2 h). Mel, HT and their combination caused a significant activation of caspase-3. Using the pan-caspase inhibitor z-VAD-fmk, we demonstrated that both stimuli mediated predominantly caspase-dependent cytotoxicity. With cDNA array analysis, 20 out of 198 apoptosis-related genes were identified to be differentially expressed by Mel and/or HT. Although a significant enhancement of three selected genes could not be proven at the protein level in subsequent experiments, this study gives insight into the complex molecular and genetic response of tumour cells to cytotoxic stimulation.