Autophagy plays a protective role during zVAD-induced necrotic cell death.
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 1 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.
Wu
You-Tong
YT
Department of Community, Occupational and Family Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Tan
Hui-Ling
HL
Huang
Qing
Q
Kim
You-Sun
YS
Pan
Ning
N
Ong
Wei-Yi
WY
Liu
Zheng-Gang
ZG
Ong
Choon-Nam
CN
Shen
Han-Ming
HM
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism*; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001343:Autophagy / Q000502:physiology*; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D000071186:Beclin-1; D015415:Biomarkers / Q000378:metabolism; D061945:Caspase Inhibitors; D002403:Cathepsins / Q000378:metabolism; D002460:Cell Line; D002738:Chloroquine / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009336:Necrosis; D010588:Phagosomes / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; C495773:Atg5 protein, mouse; C502661:Atg7 protein, mouse; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D015415:Biomarkers; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008869:Microtubule-Associated Proteins; D011506:Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D002738:Chloroquine; D002403:Cathepsins; D000071193:Autophagy-Related Protein 7; D020123:Sirolimus