Autophagy plays a protective role during zVAD-induced necrotic cell death

Abstract

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 zVADinduced 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.

zVAD-induced necroptosis in L929 cells depends on autocrine production of TNFα mediated by the PKC–MAPKs–AP-1 pathway

“…Interestingly, although zVAD has been demonstrated to be of low cytotoxicity to most of cell lines in vitro, 18 it can induce robust necrotic cell death in certain cell lines, particularly in L929 cells. 19,20 Such necrosis was originally recognized as an autophagic cell death evidenced by a massive accumulation of the autophagic markers in the dying cells. 19 However, our earlier works have provided compelling evidence that zVAD-induced cell death in L929 cells is not autophagy-dependent; instead, autophagy has a protective function for cell survival.…”

“…19 However, our earlier works have provided compelling evidence that zVAD-induced cell death in L929 cells is not autophagy-dependent; instead, autophagy has a protective function for cell survival. 20,21 Therefore, the pro-death mechanism in zVAD-mediated necroptosis remains to be examined.…”

“…Intriguingly, zVAD is also capable of efficiently inducing necrotic cell death in a selected group of cell types, particularly in L929 cells. 19,20 In this study, we first asked whether other caspase inhibitors possess the similar activity as zVAD for induction of necrosis. As shown in Figure 1a, among the caspase inhibitors tested, only zVAD-fmk and BocD-fmk were able to induce evident cell death in L929 cells.…”

New frontiers in promoting tumour cell death: targeting apoptosis, necroptosis and autophagy

“…As mentioned before, autophagy is regulated by energy availability within the cell. Autophagy is often activated as an adaptive response to ATP depletion to replenish energy reserves to help counteract necrosis/ necroptosis (Lum et al, 2005;Amaravadi and Thompson, 2007;Wu et al, 2008). Likewise, the inhibition of autophagy can trigger a bioenergetic crisis, promoting necroptosis (Degenhardt et al, 2006).…”

Retrospective electron microscopy: Preservation of fine structure by freezing and aldehyde fixation

“…2,3 Autophagy is triggered by nutrient starvation or other environmental stress conditions and appears to be a preponderantly cytoprotective mechanism. [4][5][6] A widelyused marker of autophagy activity is monitoring the conversion of LC3-I to the electrophoretically more mobile LC3-II by immunoblot analysis. Levels of the autophagy marker LC3-II correlate well with the formation of autophagosomes.…”