Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Resistance to caspase-8 and -9 fragments in a malignant pleural mesothelioma cell line with acquired cisplatin-resistance

Apoptotic cysteine–aspartate proteases (caspases) are essential for the progression and execution of apoptosis, and detection of caspase fragmentation or activity is often used as markers of apoptosis. Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic drug that is clinically used for the treatment of solid tumours. We compared a cisplatin-resistant pleural malignant mesothelioma cell line (P31res1.2) with its parental cell line (P31) regarding the consequences of in vitro acquired cisplatin-resistance on basal and cisplatin-induced (equitoxic and equiapoptotic cisplatin concentrations) caspase-3, -8 and -9 fragmentation and proteolytic activity. Acquisition of cisplatin-resistance resulted in basal fragmentation of caspase-8 and -9 without a concomitant increase in proteolytic activity, and there was an increased basal caspase-3/7 activity. Similarly, cisplatin-resistant non-small-cell lung cancer cells, H1299res, had increased caspase-3 and -9 content compared with the parental H1299 cells. In P31 cells, cisplatin exposure resulted in caspase-9-mediated caspase-3/7 activation, but in P31res1.2 cells the cisplatin-induced caspase-3/7 activation occurred before caspase-8 or -9 activation. We therefore concluded that in vitro acquisition of cisplatin-resistance rendered P31res1.2 cells resistant to caspase-8 and caspase-9 fragments and that cisplatin-induced, initiator-caspase independent caspase-3/7 activation was necessary to overcome this resistance. Finally, the results demonstrated that detection of cleaved caspase fragments alone might be insufficient as a marker of caspase activity and ensuing apoptosis induction.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

Integrated activation of MAP3Ks balances cell fate in response to stress

In vivo, tissues and organs are exposed to numerous stressors that require cells to respond appropriately for viability and homeostasis. Cells respond to these stressors, which range from UV irradiation, heat shock, chemicals, and changes in osmolality, to oxidative stress and inflammatory cytokines, by activating pathways that protect cells from damage. If the stress is too great, cells commit to undergo apoptosis. Such cell fate decisions involve the stress-mediated activation of mitogen-activated protein kinase (MAPK) networks, ultimately under the control of MAPK kinase kinases, or MAP3Ks. It is the MAP3Ks that coordinate the localization, duration and magnitude of MAPK activation in response to cell stress. A single stressor may activate several MAP3Ks, each of which impacts the balance between survival and apoptotic signaling. In this prospect article, we review the specific MAP3Ks that integrate the physiological response to cell stressors. The interrelationships among different stressors are discussed, with an emphasis on how the balance of signaling through MAP3Ks controls the MAPK response to determine cell fate.     

Regulation and execution of programmed cell death in response to pathogens, stress and developmental cues

Recent studies have expanded our view of the interactions between small molecule signals that regulate the hypersensitive response and other forms of cell suicide in plants. The mitochondrion has received increasing support as a mediator of at least some forms of programmed cell death in plants. In addition, new information provides a glimpse of how plant hormone signaling may be integrated with extensive autolysis, sensitivity to reactive oxygen intermediates and cell death.     

New anti-cancer molecules targeting HSPA5/BIP to induce endoplasmic reticulum stress,autophagy and apoptosis

Treatment of melanoma has significantly advanced over the last decade, with the development of targeted therapies against the MAPK pathway and immunotherapies to reactivate antitumor immunity. Unfortunately, currently more than 50% of patients are in treatment failure. Thus, identification of new common cellular vulnerability among melanoma cells is an urgent need and will help in the discovery of more efficient treatments against melanoma. We have focused our study on protein processing and have identified a new compound, HA15, targeting HSPA5/BiP, the master regulator of the unfolded protein response (UPR). By inhibiting HSPA5 specifically, our molecule increases the UPR and leads to the death of cancer cells by concomitant induction of autophagy and apoptosis, an effect seen both in vitro and in vivo. Our study provides compelling evidence to support the idea that endoplasmic reticulum (ER) stress inducers could be useful as a new therapeutic approach in melanoma treatment.     

Nerve growth factor-mediated inhibition of apoptosis post-caspase activation is due to removal of active caspase-3 in a lysosome-dependent manner

Nerve growth factor (NGF) is well characterised as an important pro-survival factor in neuronal cells that can inhibit apoptotic cell death upstream of mitochondrial outer membrane permeabilisation. Here we addressed the question of whether NGF can also protect against apoptosis downstream of caspase activation. NGF treatment promoted a rapid reduction in the level of the p17 subunit of active caspase-3 in PC12 cells that had been induced to undergo apoptosis by various cytotoxins. The mechanism involved TrkA-dependent activation of extracellular signal-regulated kinase (ERK1/2) but not phosphatidylinositol 3-kinase (PI3K)/Akt, and de novo protein synthesis. Involvement of inhibitor of apoptosis proteins (IAPs) and proteasomal degradation were ruled out. In contrast, inhibition of lysosome function using chloroquine and concanamycin A reversed NGF-induced removal of p17. Moreover, in NGF-treated cells, active caspases were found to be localised to lysosomes. The involvement of macroautophagy and chaperone-mediated autophagy were ruled out. Taken together, these findings suggest an anti-apoptotic mechanism by which NGF induces removal of active caspase-3 in a lysosome-dependent manner.     

ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis

The molecular interactions between Arabidopsis and the pathogenic powdery mildew Golovinomyces cichoracearum were studied by characterizing a disease-resistant Arabidopsis mutant atg2-2. The atg2-2 mutant showed enhanced resistance to powdery mildew and dramatic mildew-induced cell death as well as early senescence phenotypes in the absence of pathogens. Defense-related genes were constitutively activated in atg2-2. In atg2-2 mutants, spontaneous cell death, early senescence and disease resistance required the salicylic acid (SA) pathway, but interestingly, mildew-induced cell death was not fully suppressed by inactivation of SA signaling. Thus, cell death could be uncoupled from disease resistance, suggesting that cell death is not sufficient for resistance to powdery mildew. ATG2 encodes autophagy-related 2, a protein known to be involved in the early steps of autophagosome biogenesis. The atg2-2 mutant exhibited typical autophagy defects in autophagosome formation. Furthermore, mutations in several other ATG genes, including ATG5, ATG7 and ATG10, exhibited similar powdery mildew resistance and mildew-induced cell death phenotypes. Taken together, our findings provide insights into the role of autophagy in cell death and disease resistance, and may indicate general links between autophagy, senescence, programmed cell death and defense responses in plants.     

OMA1-mediated integrated stress response protects against ferroptosis in mitochondrial cardiomyopathy

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Hydrogen peroxide treatment in Atlantic salmon induces stress and detoxification response in a daily manner

Daily variation in the absorption, metabolism and excretion of toxic substances will ultimately determine the actual concentration to which the cells and tissues are exposed. In aquaculture, Atlantic salmon (Salmo salar) can be frequently exposed to hydrogen peroxide (H₂O₂) to treat topical skin and gill infections, particularly in relation to parasitic infections (e.g. sea lice Lepeophtheirus salmonis and amoebic gill disease caused by Neoparamoeba perurans). It is well accepted that the time of administration influences pharmacodynamics and pharmacokinetics of drugs which in turn affects their efficacy and toxicity. Consequently, a better understanding of drug side effects as a function of time of day exposure would help to improve treatment efficacy and fish welfare. To this end, salmon were exposed to H₂O₂ (1500 mg/L) for 20 min at six different times of the day during a 24-h cycle and we investigated the time-dependent effects of exposure on physiological stress (glucose, lactate and cortisol) and antioxidant enzyme expression (gpx1, cat, Mn-sod and hsp70) in liver and gills. In addition, at each sampling point, 8 control fish were also sampled. Our results revealed that the time of administration of H₂O₂ caused significant differences in the induction of both physiological and oxidative stress responses. Glucose and lactate were higher in the treated fish during daytime whereas cortisol levels appeared to be systematically increased (>1000 ng/mL) after H₂O₂ treatment irrespective of exposure time, although differences with control levels were higher during the day. In liver, gene expression of antioxidant enzymes displayed daily rhythmicity in both treated and control groups and showed higher mRNA expression levels in salmon treated with H₂O₂ at ZT6 (6 h after lights onset). In gills, rhythmic expression was only found for gpx1 in the control fish and for hsp70 and Mn-sod in the treated groups. However, in the treated salmon, higher gene expression levels of all the investigated enzymes were also observed at ZT6-10. Clock gene expression showed rhythmicity only in the liver in accordance with the daily rhythm of enzyme expression observed in this tissue. Altogether, this study provides first evidence of chronotoxicity in Atlantic salmon treated with H₂O₂ and suggests increased sublethal toxic effect during the first half of the day. These results have direct relevance to the salmon and broader aquaculture industry by optimising the timing of treatment administration, opening the door to chronotherapy to treat fish diseases.     

Cellular responses to a prolonged delay in mitosis are determined by a DNA damage response controlled by Bcl-2 family proteins

Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-x_L by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.     

Squamous cell carcinoma antigen 1 promotes caspase-8-mediated apoptosis in response to endoplasmic reticulum stress while inhibiting necrosis induced by lysosomal injury

Squamous cell carcinoma antigen 1 (SCCA1) is a member of the serine protease inhibitor (serpin) family of proteins, whose target proteases include the cathepsins. Initially identified as a serological marker for advanced squamous cell carcinomas of the cervix, SCCA1 has also been found to be associated with other cancer types of epithelial or endodermal origins such as lung cancer, head and neck cancer, melanoma, and hepatocellular carcinoma. While the biological function of SCCA1 remains largely unclear, it is believed to limit cellular damage resulting from lysosomal cathepsin release. Here, we show that SCCA1 acts as a molecular switch that inhibits cell death induced by lysosomal injury resulting from DNA alkylating agents and hypotonic shock, whereas it promotes a caspase-8-mediated apoptosis in response to endoplasmic reticulum (ER) stress. In response to ER stress, SCCA1 blocks both lysosomal and proteasomal protein degradation pathways and enhances the interaction between sequestosome 1/p62 and caspase-8, which leads to the aggregation of intracellular caspase-8 and its subsequent cleavage and activation. Hence, on one hand, SCCA1 inhibits cell death induced by lysosomal injury while, on the other hand, it sensitizes cells to ER stress by activating caspase-8 independently of the death receptor apoptotic pathway.     

Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response

Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl‐prolyl‐tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild‐type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF‐induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2‐to‐eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic.     

Icariside II enhances cisplatin-induced apoptosis by promoting endoplasmic reticulum stress signalling in non-small cell lung cancer cells

Although cisplatin is the most effective first-line drug in the management of advanced non-small cell lung cancer (NSCLC), drug resistance remains a major clinical challenge. There is increasing evidence that icariside II (IS) exhibits antitumour activity in a variety of cancers. In the current study, we investigated the anticancer effects of icariside II combined with cisplatin and elucidated the underlying mechanism in NSCLC. Here, we showed that cotreatment with IS and cisplatin inhibited cell proliferation and induced cellular apoptosis. Using mRNA sequencing (mRNA-seq), we identified differentially expressed genes (DEGs) in which there was an enrichment in PERK-mediated unfolded protein response (UPR) signalling. The western blot results revealed that IS activated endoplasmic reticulum (ER) stress, including three branches of UPR signalling, PERK, IRE1 and ATF6, and the downstream PERK-eIF2α-ATF4-CHOP pathway, thus potentiating the apoptosis induced by cisplatin. In addition, the combination of IS with cisplatin significantly reduced xenograft tumour growth in C57BL/6 and BALB/c nude mice in vivo. Notably, the combination therapy displayed no evident toxicity. Taken together, IS enhances cisplatin-induced apoptosis partially by promoting ER stress signalling in NSCLC, suggesting that combination treatment with IS and cisplatin is a novel potential therapeutic strategy for NSCLC.     

Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia–ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress.