The Isopeptidase Inhibitor G5 Triggers a Caspase-independent Necrotic Death in Cells Resistant to Apoptosis

Abstract

Inhibitors of the ubiquitin-proteasome system (UPSIs) promote apoptosis of cancer cells and show encouraging anti-tumor activities in vivo. In this study, we evaluated the death activities of two different UPSIs: bortezomib and the isopeptidase inhibitor G5. To unveil whether these compounds elicit different types of death, we compared their effect both on apoptosis-proficient wild type mouse embryo fibroblasts and on cells defective for apoptosis (double-deficient Bax/Bak mouse embryo fibroblasts) (double knockout; DKO). We have discovered that (i) both inhibitors induce apoptosis in a Bax and Bak-dependent manner, (ii) both inhibitors elicit autophagy in WT and DKO cells, and (iii) only G5 can kill apoptosis-resistant DKO cells by activating a necrotic response. The induction of necrosis was confirmed by different experimental approaches, including time lapse analysis, HMGB1 release, and electron microscopy studies. Neither treatment with antinecrotic agents, such as antioxidants, poly(ADP-ribose) polymerase and JNK inhibitors, necrostatin, and the intracellular Ca 2؉ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester, nor overexpression of Bcl-2 and Bcl-xL prevented necrosis induced by G5. This necrotic death is characterized by the absence of protein oxidation and by the rapid cyclosporin A-independent dissipation of the mitochondrial membrane potential. Notably, a peculiar feature of the G5-induced necrosis is an early and dramatic reorganization of the actin cytoskeleton, coupled to an alteration of cell adhesion. The importance of cell adhesion impairment in the G5-induced necrotic death of DKO cells was confirmed by the antagonist effect of the extracellular matrixadhesive components, collagen and fibronectin.

Inhibitors of the ubiquitin-proteasome system are not all alike: Identification of a new necrotic pathway

“…Autophagy was an early response to both UPSIs and was detectable also in wild-type cells. 4 Broad and commonly used inhibitors of the autophagic process did not influence death induced by G5 in DKO cells, thus excluding the possibility that the observed death is mediated by autophagy. It is plausible that, thanks to the autophagic machinery, cells could compensate for the block of protein turnover induced by the UPSIs.…”

“…By contrast, strengthening the adhesion to the extracellular matrix restrains G5-induced necrosis, thus further confirming a major role for the cytoskeletal changes. 4 All these features indicate that G5 activates a rather peculiar form of necrosis. We are in an initial age in the necrosis field; nevertheless examples of necrotic death involving microfilament changes are emerging.…”

“…In contrast, under the time-frame of our analysis similar treatments with bortezomib failed to efficiently induce necrosis. 4 This result indicates that the block in protein turnover and the consequent accumulation of misfolded proteins was insufficient to robustly trigger necrosis. Indeed a low percentage of death in DKO cells was observed in the presence of high concentrations of bortezomib, thus not excluding the possibility that low levels of necrosis can be induced by the accumulation of misfolded proteins.…”

“…8 We have shown that during necrosis, cell swelling occurs approximately 5 min after mitochondrial depolarization, thus suggesting the existence of a temporal window, enough to activate caspases before the rupture of the plasma membrane. 4 Perhaps the rapid decline of ATP levels during necrosis could be the limiting step to promote caspase activation. It is interesting to note that we observed a very rapid and exhaustive deterioration of the mitochondrial membrane potential during G5-induced necrosis.…”

“…These cytoskeletal changes are detectable approximately 5 hours before the Δ ψm collapse and the appearance of necrotic markers. 4 MAPK inhibitors, including JNK inhibitors, the RIP inhibitor or a calcium chelator, cannot suppress this necrotic death. Furthermore this necrosis is not accompanied by the induction of oxidative stress.…”