Inhibition of PI3k Class III-Dependent Autophagy Prevents Apoptosis and Necrosis by Oxidative Stress in Dopaminergic Neuroblastoma Cells
Inhibition of PI3k Class III-Dependent Autophagy Prevents Apoptosis and Necrosis by Oxidative Stress in Dopaminergic Neuroblastoma Cells
复制标题
DOI：
10.1093/toxsci/kfq170
复制
发表时间：
2010-09-01
影响因子：
3.8
通讯作者：
Isidoro, Ciro
中科院分区：
文献类型：
--
作者：
Castino, Roberta;Bellio, Natascia;Isidoro, Ciro
过氧化氢（H2O2）是一种极具活性的氧化剂，通常作为线粒体活性的副产品产生，也在几种代谢应激条件下产生。自噬是一种溶酶体降解途径，是由氧化应激触发的一种防御反应。细胞在氧化应激下自噬和死亡途径是如何协调的尚不清楚。在人神经母细胞瘤SH-SY5Y细胞中，200 μ M H2O2可快速诱导lc3阳性的自噬液泡和beclin1-Vps34双阳性大聚集体的形成。当用Vps34抑制剂3-甲基腺嘌氨酸（3MA）预处理或用表达Vps34显性阴性突变体的重组腺病毒感染的细胞进行氧化应激时，液泡LC3和beclin1聚集体不形成。H2O2引起溶酶体（30分钟）和线粒体的通透性，伴随bax的寡聚，并最终（2小时）导致约50%的细胞死亡。在氧化应激细胞中，vsv34依赖的自噬失活消除了溶酶体渗漏、bax活化和caspase依赖的凋亡，并提供长达16小时的保护。尽管氧化自由基不断产生，自噬液泡和泄漏的溶酶体在细胞中积累，但抑制caspase活性（通过zvd -fmk）并没有触发另一种细胞死亡途径，而是提供了完全的氧化毒性保护。在长期（16小时）暴露于H2O2时，lc3阳性细胞出现明显的坏死细胞死亡迹象，zad -fmk可以阻止这一现象。目前的数据强调了自噬在h2o2诱导的多巴胺能神经母细胞瘤细胞死亡中的关键作用。
Hydrogen peroxide (H2O2) 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, 200 mu M H2O2 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. H2O2 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 H2O2, 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 H2O2-induced cell death in dopaminergic neuroblastoma cells.