Think of cell death and you probably think of caspase-executed apoptosis. Cells going through tough times can also undergo self-digestion — autophagy — but only now have Li et al. discovered that this directly causes cell death and found some clues to how it does so.

Several factors cause mouse fibroblasts to die independently of caspase activity. Rather unexpectedly, though, a broad-spectrum caspase inhibitor zVAD also induced death in these, and other, cells. Cellular post-mortem examination uncovered death traits — in particular, membrane-bound vacuoles — that were different from those seen in apoptosis and, instead, resembled autophagic traits. Could autophagy condemn cells to die?

Although two inhibitors of autophagy prevented zVAD-induced death, these compounds inhibit phosphatidylinositol 3-kinase, and therefore might kill cells by other means. So Li et al. directly assessed the effect on cell death of inhibiting genes that are required for autophagy (ATG genes) using RNA inhibition (RNAi). Reduced expression of ATG7 virtually inhibited zVAD-induced death, as did reducing the levels of another ATG gene, beclin-1. In both cases, the lack of cell death correlated with an inhibition of autophagic vacuole formation, so ATG7 and beclin-1 are necessary for zVAD-induced non-apoptotic cell death.

Although the mechanisms of autophagy aren't well understood, non-apoptotic death can arise from death receptors through the serine/threonine kinase RIP (receptor-interacting protein). Inhibiting RIP by RNAi decreased both autophagy and death. RIP can activate JUN amino-terminal kinase (JNK), and JNK was activated in response to zVAD treatment. RNAi treatment of JNK or its upstream kinase MKK7 (mitogen-activated protein kinase kinase-7) blocked zVAD-induced autophagy and death. Inhibiting protein synthesis in zVAD-treated cells also blocked cell death, implying that new protein (or proteins) needs to be made, possibly through the transcription of target genes. One transcription factor that lies downstream of JNK is JUN, which, when inhibited, blocked autophagy and cell death by 45–50%.

So a RIP–MKK7–JNK–JUN pathway is involved in autophagy and cell death, but how does zVAD induce this? Because caspase-8 is involved in lymphocyte-receptor signalling in non-cell-death situations, Li et al. studied its role in autophagy. Depleting caspase-8, but not caspases-1, -2, -3, -9 or -12, using RNAi increased cell death with autophagic traits, and, because caspase-8 is potently inhibited by zVAD, it seems likely that zVAD induces cell death by inhibiting caspase-8.

So this caspase-8-mediated suppression of autophagic death points to caspases being regulators of apoptotic and non-apoptotic cell death — which has important implications for the development of caspase inhibitors for therapeutic treatment.