Autophagic cell death unraveled: Pharmacological inhibition of apoptosis and autophagy enables necrosis

Apoptosis is a well-characterized pathway to cell death, yet how it is related to other forms of cell death such as necrosis, and possibly also autophagic cell death has not been entirely clear. Difficulties arise because necrotic cell death is poorly characterized at the molecular level, and also because autophagy is primarily a survival pathway that has been associated with cell death induction in some circumstances. A common theme appears to be now emerging where autophagy promotes survival of apoptosis-defective cells, and inhibition of the autophagy survival function in this setting represents a means to divert cells into a necrotic cell fate. In cells denied the ability to commit suicide by apoptosis, and that are also unable to access the autophagy survival mechanism to sustain homeostasis, necrosis is the default activity. This was most recently illustrated with the discovery that the caspase and apoptosis inhibitor, zVAD, also inhibits a lysosomal protease, and thereby autophagy, and it is this dual inhibition that is responsible for induction of necrotic cell death.(1) This radically alters the interpretation of earlier findings reporting induction of autophagic cell death by zVAD,(2) instead, suggests that autophagy functions to promote cell survival.     

The variability of autophagy and cell death susceptibility

Impaired autophagic machinery is implicated in a number of diseases such as heart disease, neurodegeneration and cancer. A common denominator in these pathologies is a dysregulation of autophagy that has been linked to a change in susceptibility to cell death. Although we have progressed in understanding the molecular machinery and regulation of the autophagic pathway, many unanswered questions remain. How does the metabolic contribution of autophagy connect with the cell’s history and how does its current autophagic flux affect metabolic status and susceptibility to undergo cell death? How does autophagic flux operate to switch metabolic direction and what are the underlying mechanisms in metabolite and energetic sensing, metabolite substrate provision and metabolic integration during the cellular stress response? In this article we focus on unresolved questions that address issues around the role of autophagy in sensing the energetic environment and its role in actively generating metabolite substrates. We attempt to provide answers by explaining how and when a change in autophagic pathway activity such as primary stress response is able to affect cell viability and when not. By addressing the dynamic metabolic relationship between autophagy, apoptosis and necrosis we provide a new perspective on the parameters that connect autophagic activity, severity of injury and cellular history in a logical manner. Last, by evaluating the cell’s condition and autophagic activity in a clear context of regulatory parameters in the intra- and extracellular environment, this review provides new concepts that set autophagy into an energetic feedback loop, that may assist in our understanding of autophagy in maintaining healthy cells or when it controls the threshold between cell death and cell survival.     

Synergistic induction of apoptosis and caspase-independent autophagic cell death by a combination of nitroxide Tempo and heat shock in human leukemia U937 cells

We have shown that heat stress or a superoxide dismutase mimic nitroxide, Tempo, induces apoptosis, while their combination causes nonapoptotic cell death; however, the underlying mechanism for this switch remains unclear. Here we identified for the first time that 10 mM Tempo present during heating at 44°C for 30 min rapidly induced autophagy in U937 leukemic cells in spite of Bax activation and mitochondrial outer membrane (MOM) permeabilization. This co-treatment inhibited the processing of heat-activated procaspases-2, -8, -9 and -3 into active small subunits, leading to the inhibition of caspase-dependent apoptosis, and instead caused the induction of autophagy. The inactivation of caspases, a key event, could result from oxidation of active-site-CysSH of all caspases by a prooxidant oxo-ammonium cation, an intermediate derived Tempo during dismutation of heat-induced superoxide anion. In addition, the co-treatment caused mitochondrial calcium overloads, the mitochondrial inner membrane permeabilization, profound mitochondrial dysfunction, and liberation of Beclin 1 from the Bcl-2/Beclin 1 complex, all of which contributed to induction of autophagy. These autophagic cells underwent propidium iodide-positive necrosis in a delayed fashion, leading to the complete proliferative inhibition. Remarkably, ruthenium red and BAPTA, which interfere with mitochondrial calcium uptake, facilitated autophagic necrotic death. Cyclosporin A, which binds to cyclophilin D, had a similar necrotic effect. 3-Methyladenine facilitated the necrosis of autophagic cells. In contrast, 5 mM Tempo-44°C/10 min or 44°C/30 min induced Bax-mediated MOM permeabilization and caspase-dependent apoptosis more potently than Tempo alone. Thus, Tempo is a unique thermosensitizer to synergistically induce apoptosis and autophagic cell death.     

Autophagy-driven cell fate decision maker: Activated microglia induce specific death of glioma cells by a blockade of basal autophagic flux and secondary apoptosis/necrosis

Programmed cell death is classified into apoptosis and autophagic cell death. The extensive crosstalk that occurs between these two types of death often prevents a clear identification of the leading death mechanism in a given experimental system. An accurate assessment of the type of death at work is of crucial relevance for the design of efficient cancer therapies aiming at eliminating tumor cells. Indeed, accumulating evidence indicates that resistance of tumor cells to apoptosis can be overcome by induction of autophagy. The latter would thus seem to represent an ideal strategy for eliminating certain tumor cells, except for the fact that autophagy induction may also contribute to cell survival.

It therefore is of paramount importance to clarify the mechanistic links between autophagy and apoptosis as well as the nature of autophagy-dependent cell death. We recently reported that glioma cells resistant to death ligands were killed by the supernatant of activated microglia. What at first glance seemed to be apoptosis turned out to be autophagy-dependent cell death resulting from a blockade in the autophagic flux. This blockade most likely occurs at the level of lysosome recycling. We hypothesize that this autophagy-dependent process leads to either apoptosis or necrosis depending on the extent of lysosomal permeabilization and on the relative contribution of other cellular compartments. Autophagy therefore appears in our model as a cell-fate decision maker, not as a cell death execution pathway.     

A specific pathway inducing autophagic cell death is marked by an IP3R mutation

Three main advantages make Dictyostelium a very favorable model to study the induction of autophagic cell death in vitro. First, its small, sequenced and haploid genome facilitates genetic approaches. Second, the Dictyostelium genome does not encode the two main molecular families involved in apoptosis (caspases and bcl-2 family), which therefore cannot interfere in this case with autophagic cell death. Third, induction of autophagic cell death follows in this case a two-step process, namely starvation-induced sensitization leading to autophagy but not to death, followed by a DIF-1-induced pathway leading to cell death proper. The latter, DIF-1-induced pathway is defined experimentally, through sequential additions, and most important also genetically, through random mutagenesis leading in particular to the preparation and study of an iplA mutant. The iplA gene encodes the IP3 Receptor, and its mutation leads to the absence of vacuolization and of death when autophagic cell death is triggered. Further study of the DIF-1 pathway should shed additional light on the induction of autophagic cell death (as opposed to that of just autophagy) in Dictyostelium and by extension perhaps in other organisms.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.     

Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis

The effectiveness of ischemic preconditioning (IP) against hepatic ischemia/reperfusion injury during human liver surgery is linked to decreased apoptotic cell death as well as preservation of the ATP content in liver tissue. Overproduction of Bcl-2 is reported in preconditioned organs. In human liver biopsies exhibiting steatosis and/or vascular injuries (mainly peliosis) induced by chemotherapy, we find that the expression of Bcl-2 in centrolobular and peliotic areas colocalizes with the autophagy protein Beclin 1 in IP livers. Increased expression of phosphorylated Bcl-2 in preconditioned livers is associated with a decreased immunoprecipitation of Beclin 1 and increased expression of LC3-II. The increased number of autophagic vacuoles seen by electron microscopy confirmed that IP could trigger autophagy in chemotherapy-injured livers, probably to reduce the pro-inflammatory necrotic cell death of hepatocytes or endothelial cells and to increase ATP levels. Indeed, necrosis is less frequent (p = 0.04) in IP livers than in the others although no change in apoptosis as assessed by TUNEL assay or caspase-3, -8 and -9 expressions is observed. In conclusion, Bcl-2 and Beclin 1 could be major targets in the regulation of cell death during ischemia/reperfusion injury modulating autophagy to switch on/off necrosis and/or apoptosis.     

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor's Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor's Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

Autophagic cell death exists

The term autophagic cell death (ACD) initially referred to cell death with greatly enhanced autophagy, but is increasingly used to imply a death-mediating role of autophagy, as shown by a protective effect of autophagy inhibition. In addition, many authors require that autophagic cell death must not involve apoptosis or necrosis. Adopting these new and restrictive criteria, and emphasizing their own failure to protect human osteosarcoma cells by autophagy inhibition, the authors of a recent Editor’s Corner article in this journal argued for the extreme rarity or nonexistence of autophagic cell death. We here maintain that, even with the more stringent recent criteria, autophagic cell death exists in several situations, some of which were ignored by the Editor’s Corner authors. We reject their additional criterion that the autophagy in ACD must be the agent of ultimate cell dismantlement. And we argue that rapidly dividing mammalian cells such as cancer cells are not the most likely situation for finding pure ACD.     

Targeting autophagy to prevent neonatal stroke damage

Cell death due to cerebral ischemia has been attributed to necrosis and apoptosis, but autophagic mechanisms have recently been implicated as well. Using rats exposed to neonatal focal cerebral ischemia, we have shown that lysosomal and autophagic activities are increased in ischemic neurons, and have obtained strong neuroprotection by post-ischemic inhibition of autophagy.     

Autophagy in hypoxia-ischemia induced brain injury: Evidences and speculations

The interaction among autophagy, apoptosis and necrosis is complex and still a matter of debate. We have recently studied this interaction after neonatal hypoxia-ischemia (HI) in rats. We found that autophagic and apoptotic pathways were significantly increased at short times after HI in neuronal cells. 3-Methyladenine (3-MA) and wortmannin (WM), that inhibit autophagy, significantly reduced autophagic pathways activation and switched the mechanism of cell death from apoptotic to necrotic. Rapamycin, conversely, that increases autophagy, reduced necrotic cell death, and decreased brain injury. A prophylactic treatment with simvastatin or hypoxic preconditioning also caused up-regulation of autophagic pathways. In this Addendum, we summarize these findings and speculate on the possible physiological role of autophagy during hypoxia-ischemia induced neurodegeneration.     

Autophagy functions in programmed cell death

Autophagic cell death is a prominent morphological form of cell death that occurs in diverse animals. Autophagosomes are abundant during autophagic cell death, yet the functional role of autophagy in cell death has been enigmatic. We find that autophagy and the Atg genes are required for autophagic cell death of Drosophila salivary glands. Although caspases are present in dying salivary glands, autophagy is required for complete cell degradation. Further, induction of high levels of autophagy results in caspase-independent autophagic cell death. Our results provide the first in vivo evidence that autophagy and the Atg genes are required for autophagic cell death and confirm that autophagic cell death is a physiological death program that occurs during development.

Addendum to: Berry DL, Baehrecke EH. Growth arrest and autophagy are required for programmed salivary gland cell degradation in Drosophila. Cell 2007; 131:1137-48.     

Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts

The previous studies by this author group has shown that paclitaxel, a mitotic inhibitor used in breast cancer chemotherapy, inhibits cell growth via induction of Raf-1-dependent apoptosis. In this article, the role of autophagy in paclitaxel anticancer action was investigated using v-Ha-ras-transformed NIH 3T3 cells. Paclitaxel induced a notable increase in the number of fluorescent particles labeled with monodansylcadaverine (MDC), a specific marker for autophagic vacuoles. MDC-labeled vacuoles clearly exhibited the fluorescent-tagged LC3 in cells transiently overexpressing GFP-LC3 (a protein that associates with autophagosome membranes). However, autophagy inhibition with 3-methyladenine (3-MA) failed to rescue v-Ha-ras-transformed NIH 3T3 cells from paclitaxel-induced cell death. More interestingly, the apoptosis inhibition by overexpression of the X-linked inhibitor of apoptosis (XIAP) did not fully block the cell death by paclitaxel, implying that apoptosis inhibition might accelerate the autophagic components of the paclitaxel response. Conversely, Raf-1 shRNA expression protected against paclitaxel-induced cell death through the simultaneous inhibition of both autophagy and apoptosis. These results suggest that both autophagy and apoptosis act as cooperative partners to induce cell death in v-Ha-ras-transformed NIH 3T3 cells treated with paclitaxel.     

Cyclosporine A Induces Apoptotic and Autophagic Cell Death in Rat Pituitary GH3 Cells

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.     

A specific pathway inducing autophagic cell death is marked by an IP3R mutation

Three main advantages make Dictyostelium a very favorable model to study the induction of autophagic cell death in vitro. First, its small, sequenced and haploid genome facilitates genetic approaches. Second, the Dictyostelium genome does not encode the two main molecular families involved in apoptosis (caspases and bcl-2 family), which therefore cannot interfere in this case with autophagic cell death. Third, induction of autophagic cell death follows in this case a two-step process, namely starvation-induced sensitization leading to autophagy but not to death, followed by a DIF-1-induced pathway leading to cell death proper. The latter, DIF-1-induced pathway is defined experimentally, through sequential additions, and most important also genetically, through random mutagenesis leading in particular to the preparation and study of an iplA mutant. The iplA gene encodes the IP3 Receptor, and its mutation leads to the absence of vacuolization and of death when autophagic cell death is triggered. Further study of the DIF-1 pathway should shed additional light on the induction of autophagic cell death (as opposed to that of just autophagy) in Dictyostelium and by extension perhaps in other organisms.

Addendum to: Lam D, Kosta A, Luciani MF, Golstein P. The IP3 receptor is required to signal autophagic cell death. Mol Biol Cell 2008;10.1091/mbc.E07-08-0823.     

zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.     

Oxyresveratrol activates parallel apoptotic and autophagic cell death pathways in neuroblastoma cells

BackgroundDrug resistance from apoptosis is a challenging issue with different cancer types, and there is an interest in identifying other means of inducing cytotoxicity. Here, treatment of neuroblastoma cells with oxyresveratrol (OXYRES), a natural antioxidant, led to dose-dependent cell death and increased autophagic flux along with activation of caspase-dependent apoptosis.MethodsFor cell viability, we performed the CCK-8 assay. Protein expression changes were with Western blot and immunocytochemistry. Silencing of proteins was with siRNA. The readouts for cell cycle, mitochondria membrane potential, caspase-3, autophagy and apoptosis were performed with flow cytometry.ResultsPhosphorylation of p38 MAPK increased with OXYRES treatment and inhibition of p38 reduced autophagy and cell death from OXYRES. In contrast, PI3K/AKT/mTOR signaling decreased in the target cells with OXYRES and inhibition of PI3K or mTOR enhanced OXYRES-mediated cytotoxicity with increased levels of autophagy. Modulation of either of the apoptosis and autophagy flux pathways affected the extent of cell death by OXYRES, but did not affect the indicators of these pathways with respect to each other. Both pathways were independent of ROS generation or p53 activation.ConclusionOXYRES led to cell death from autophagy, which was independent of apoptosis induction. The OXYRES effects were due to changes in the activity levels of p38 MAPK and PI3K/AKT/mTOR.General significanceWith two independent and parallel pathways for cytotoxicity induction in target cells, this study puts forward a potential utility for OXYRES or the pathways it represents as novel means of inducing cell death in neuroblastoma cells.     

Amino acid starvation induced autophagic cell death in PC-12 cells: Evidence for activation of caspase-3 but not calpain-1

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis. Shankar Sadasivan and Anu Waghray have contributed equally to this work.     

The role of cell signalling in the crosstalk between autophagy and apoptosis

Not surprisingly, the death of a cell is a complex and well controlled process. For several decades, apoptosis, the first genetically programmed death process to be identified has taken centre stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Apoptosis has been extensively studied and its contribution to the pathogenesis of disease well documented. However, apoptosis does not function alone in determining the fate of a cell. More recently, autophagy, a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components, has been shown to engage in complex interplay with apoptosis. As a result, cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II cell death is not completely clear and as we will discuss in this review and perhaps a discrete difference does not exist, due to intrinsic factors among different cell types and crosstalk among organelles within each cell type. Apoptosis may begin with autophagy and autophagy can often end with apoptosis, inhibition or a blockade of caspase activity may lead a cell to default into Type II cell death from Type I.