Autophagy plays a protective role during zVAD-induced necrotic cell death
Autophagy plays a protective role during zVAD-induced necrotic cell death
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DOI：
10.4161/auto.5662
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发表时间：
2008-05-16
期刊：
影响因子：
13.3
通讯作者：
Shen, Han-Ming
中科院分区：
文献类型：
--
作者：
Wu, You-Tong;Tan, Hui-Ling;Shen, Han-Ming
本研究的目的是研究自噬在细胞死亡中的作用，通过使用一个完善的系统，其中zVAD，一种泛caspase抑制剂，诱导L929小鼠纤维肉瘤细胞坏死细胞死亡。首先，我们观察到自噬标志的存在，包括zad处理的L929细胞中自噬体数量的增加和LC3-II的积累。由于这种自噬标志的存在可能是自噬通量增加或自噬体成熟阻塞（溶酶体融合和降解）的结果，我们接下来测试了雷帕霉素（mTOR的特异性抑制剂）和氯喹（溶酶体酶抑制剂）对zvad诱导的细胞死亡的影响。令我们惊讶的是，已知的自噬诱导剂雷帕霉素可以阻断zvd诱导的细胞死亡，而氯喹则可以使zvd诱导的L929细胞死亡致敏。此外，雷帕霉素和氯喹在U937细胞中也观察到类似的结果。在L929细胞中，通过血清饥饿诱导自噬对zvd诱导的细胞死亡具有显著的保护作用，而敲低Atg5、Atg7或Beclin I可显著致敏zvd诱导的细胞死亡。更重要的是，Atg基因敲除完全消除了血清饥饿对zvad诱导的细胞死亡的保护作用。最后，我们证明了zVAD能够抑制溶酶体酶组织蛋白酶B的活性，并随后阻断自噬体的成熟。综上所述，与之前认为zVAD诱导自噬细胞死亡的观点相反，本研究提供了令人信服的证据，表明自噬是一种细胞存活机制，通过抑制溶酶体功能抑制自噬有助于zVAD诱导的坏死细胞死亡。
The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin I markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.