title: Protective effect of creatine against 6-hydroxydopamine-induced cell death in human neuroblastoma SH-SY5Y cells: Involvement of intracellular signaling pathways.
abstract: 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 100-300μM of 6-OHDA for 24h 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 48h with creatine (10-5000μM) was not cytotoxic. However, pre and co-treatment with creatine (0.3-1000μM) for 24h 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), Ca(2+)/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-3β (GSK-3β) 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-3β.
authors.affiliation: Instituto Teófilo Hernando, Facultad de Medicina, Universidad Autónoma de Madrid, 4-28029 Madrid, Spain. mauricio.personal@gmail.com
authors.forename: M P
authors.initials: MP
authors.lastname: Cunha
authors.forename: M D
authors.initials: MD
authors.lastname: Martín-de-Saavedra
authors.forename: A
authors.initials: A
authors.lastname: Romero
authors.forename: E
authors.initials: E
authors.lastname: Parada
authors.forename: J
authors.initials: J
authors.lastname: Egea
authors.forename: L
authors.initials: L
authors.lastname: Del Barrio
authors.forename: A L S
authors.initials: AL
authors.lastname: Rodrigues
authors.forename: M G
authors.initials: MG
authors.lastname: López
mesh_terms: D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003401:Creatine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016627:Oxidopamine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D016627:Oxidopamine; D003401:Creatine