Inhibition of PI3k class III-dependent autophagy prevents apoptosis and necrosis by oxidative stress in dopaminergic neuroblastoma cells.
Hydrogen peroxide (H(2)O(2)) is an extremely reactive oxidoradical that is normally produced as a by-product of the mitochondrial activity and also under several metabolic stress conditions. Autophagy, a lysosomal degradation pathway, is triggered by oxidative stress as a defensive response. How autophagy and death pathways are coordinated in cells subjected to oxidative stress is still poorly understood. In human neuroblastoma SH-SY5Y cells, 200microM H(2)O(2) rapidly induced the formation of LC3-positive autophagic vacuoles and of beclin1-Vps34 double-positive macroaggregates. Vacuolar LC3 and beclin1 aggregates did not form when oxidative stress was performed in cells pretreated with 3-methyladenine (3MA), an inhibitor of Vps34, or infected with a recombinant adenovirus expressing a dominant-negative mutant of Vps34. H(2)O(2) provoked the permeabilization of lysosomes (at 30 min) and of mitochondria, the concomitant oligomerization of bax, and eventually (at 2 h), cell death in about 50% of the cell culture. Inactivation of Vps34-dependent autophagy in oxidative-stressed cells abrogated lysosome leakage, bax activation, and caspase-dependent apoptosis and conferred protection for as long as 16 h. Inhibition of caspase activity (by ZVAD-fmk) did not trigger an alternative cell death pathway but rather afforded complete protection from oxidative toxicity, despite the ongoing generation of oxidoradicals and the cellular accumulation of autophagic vacuoles and of leaking lysosomes. On long-term (16 h) exposure to H(2)O(2), signs of necrotic cell death became apparent in LC3-positive cells, which could be prevented by ZVAD-fmk. The present data highlight the pivotal role of autophagy in H(2)O(2)-induced cell death in dopaminergic neuroblastoma cells.
Laboratorio di Patologia Molecolare, Dipartimento di Scienze Mediche, Università del Piemonte Orientale A. Avogadro, Novara, Italy.
Roberta
R
Castino
Natascia
N
Bellio
Carlo
C
Follo
David
D
Murphy
Ciro
C
Isidoro
D017209:Apoptosis; D001343:Autophagy; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004298:Dopamine / Q000378:metabolism*; D006801:Humans; D009336:Necrosis; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D018384:Oxidative Stress; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors
D020169:Caspases; D004298:Dopamine; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors