Anales RANF

P.42 INVESTIGATING THE ROLE OF GUANOSINE IN HUMAN NEUROBLASTOMA CELL DIFFERENTIATION. P. Giuliani, M. Zuccarini, M. Carluccio, S. Ziberi, R. Ciccarelli, F. Caciagli, P. Di Iorio. University of Chieti-Pescara, Chieti, Italy. Neuroblastoma is the most common solid extracranial tumor of infancy. It arises from the neural crest cell precursors that fail to complete the process of differentiation; therefore, agents able to help tumor cells to differentiate into normal cells represent a valid therapeutic approach. To date, only few differentiating agents are available for therapy. Since guanosine, a natural purine nucleoside, increases the NGF-induced cell differentiation in rat hippocampal neurons and rat pheochromocytoma cells, the aim of this study was to evaluate whether extracellular guanosine was involved in the human neuroblastoma SH-SY5Y cell line differentiation and to investigate the molecular mechanisms involved. For this purpose, guanosine was added to cell medium and the percentage of differentiated cells (showing neurite greater than one cell body length ) was evaluated. The analysis of signaling pathways involved was carried out using western blot and cGMP enzyme immunoassay while guanosine metabolism was evaluated by HPLC. We found that guanosine promoted neuronal differentiation in a concentration-dependent manner, an effect already evident after 24h and mainly induced by its extracellular action as demonstrated using transporter inhibitors. Guanosine increased phosphorylated ERK1/2 and Akt in a concentration-dependent manner and enhanced cGMP level via heme-oxygenase. The neuritogenic activity of Guanosine was significantly reduced by specific inhibitors of ERK1/2, heme- oxygenase or soluble guanylate cyclase but not by Akt inhibitors. Interestingly, in the medium, guanosine was largely metabolized into guanine by purine nucleoside phosphorylase (PNP) enzyme released from cells. Thus, our study suggests that guanosine, promoting neuroblastoma differentiation, may represent a potential therapeutic agent. However, due to its spontaneous extracellular metabolism, the role played by guanosine-PNP-guanine system needs to be further investigated.