Background: The Ewing’s Sarcoma Family Tumours (ESFT) is an aggressive group of poorly differentiated pedriatic and young adult cancers of bone and soft tissue. It is characterized by the presence of a chromosomal translocation that produces a fusion protein. The 85 % of this tumours have the fusion protein EWS-FLI-1 t(11;22)(q24;q12), that acts as an aberrant transcription factor and modulate specific genes involved in oncogenesis. Melatonin (N-acetyl-5-methoxytryptamine) is an endogenous indoleamine derived from the tryptophan that has been know to exerts wide range of physiological and pathophysiological effects including antitumoral actions. Methods: cell lines used were: Ewing Sarcoma SK-N-MC, TC-71, TC-32, A673 (EWS-FLI1 type 1), SK-ES1 (type 2), A4573 (type 3); neuroblastoma SK-N-SH, SH-SY5Y, SK-N-AS and EWS-FLI1 transformed murine embryonic fibroblasts (MEF). Cell proliferation and cytotoxicity was measured by MTT and LDH assays. Apoptosis was evaluated using DAPI staining and caspase-3 activity. The direct interaction of melatonin with recombinant EWS-FLI1 was measured using surface plasmon resonance (SPR) (Biacore T100, GE) on the surface of a dextran coated surface (CM5 chip) and levels of phospho and total proteins were evaluated by western blot. Results: Melatonin induces apoptotic cell death in all the Ewing Sarcoma cell lines in spite of the primary origin or the type of the fusion protein present, with the same efficiency as we had previously shown in SK-N-MC cells. During the apoptotic process we also observed an associated decrease of the intracellular glucogen accumulations that do not correspond with an activation of the GSK3B, furthermore, there is an increase of the inactive form of the enzyme (P-GSK3B). On the other hand, melatonin doesn’t induce apoptosis in the neuroblastoma cells but inhibits the cellular growth in a significant way. The analysis of the direct interaction between EWS-FLI1 and melatonin by the SPR technology shows a specific binding affinity between EWS-FLI1 and melatonin with a Kd around 85 µM, very close to the melatonin’s range of action (100µM-1mM). Unfortunately melatonin was not able to kill EWS-FLI1 transformed MEF cells, even at the highest concentration (1mM), and only inhibits cell growth. Conclusions: The present work extends our previous observations in the human SK-N-MC cell line and demonstrated a specific pro-apoptotic effect of melatonin in the ESFT that is not shared in other similar cancer types like neuroblastoma. Despite that melatonin don’t induces apoptosis in transformed fibroblast, probably due to a not suitable cellular model, the protein interaction data between melatonin and EWS-FLI1 led us to think that melatonin could target the fusion protein in some critical way that leads to a cellular disbalance and induction of the apoptotic process.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA