Inhibition of PI3k Class III–Dependent Autophagy Prevents Apoptosis and Necrosis by Oxidative Stress in Dopaminergic Neuroblastoma Cells

Abstract

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.

Inhibition of Autophagy as a Strategy to Augment Radiosensitization by the Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235

“…To determine whether inhibiting the transit of cells through autophagy would influence radiosensitivity we first used the drug 3-MA, which blocks autophagy at an early stage by inhibiting the autophagy protein Vps34 (Castino et al, 2010). Figure 6A shows that pretreatment of SQ20B cells with 3-MA before NVP-BEZ235 treatment led to a decrease in the LC3-II band compared with NVP-BEZ235 treatment alone (compare lanes 8 and 9), which is consistent with the inhibition of autophagy.…”

Application and interpretation of current autophagy inhibitors and activators

“…Several studies have demonstrated that PI3K inhibitors interfere with the formation of autophagosomes [75][76][77] . The PI3K inhibitor 3-methyladenine (3-MA) was the first identified and is the most widely used autophagy inhibitor [78] .…”

Autophagy: for better or for worse

“…In contrast, autophagy activity can also provoke non-apoptotic cell death. For example, in C. elegans, autophagy is required for necrotic neuron destruction, and both 3-MA and knockdown of key Atg proteins (Bec-1, Igg-1, Atg18) reduce necrotic cell death [151,152]. Often, autophagy-associated necrotic cell death occurs when apoptosis is defective.…”