PROTECTIVE EFFECT OF CREATINE AGAINST 6-HYDROXYDOPAMINE-INDUCED CELL DEATH IN HUMAN NEUROBLASTOMA SH-SY5Y CELLS: INVOLVEMENT OF INTRACELLULAR SIGNALING PATHWAYS

The guanidine-like compound creatine exerts bioenergetic, antiexcitotoxic, antioxidant and neuroprotective properties; however, the intracellular mechanisms responsible for these effects are still not well established. The purpose of this study was to investigate the protective effect of creatine against 6-hydroxydopamine (6-OHDA)-induced cell death in neuroblastoma SH-SY5Y cells and the possible intracellular signaling pathways involved in such effect. Exposure of SH-SY5Y cells to 100-300 mu M of 6-OHDA for 24 h caused a significant concentration-dependent cell death measured as a diminution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction and as an increase in the extracellular release of lactate dehydrogenase. SH-SY5Y cells incubated for 24 or 48 h with creatine (10-5000 mu M) was not cytotoxic. However, pre and co-treatment with creatine (0.3-1000 mu M) for 24 h reduced 6-OHDA-induced toxicity. The protective effect afforded by creatine against 6-OHDA-induced toxicity was reversed by inhibitors of different protein kinases, i.e. phosphatidylinositol-3 kinase (PI3K) (LY294002), Ca2+/calmodulin-dependent protein kinase II (CaMKII) (KN-93), protein kinase A (H-89), mitogen-activated protein kinase kinase 1/2 (MEK1/2) (PD98059) and protein kinase C (PKC) (chelerythrine). Furthermore, creatine prevented the 6-OHDA-induced dephosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta) at the serine 9 residue. In conclusion, the results of this study show that creatine can protect against 6-OHDA-induced toxicity and its protective mechanism is related to a signaling pathway that involves PI3K, PKC, PKA, CaMKII, MEK1/2 and GSK-3 beta. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.