Signaling unmasked: Autophagy and catalase promote programmed cell death

Autophagy contributes to the removal of harmful cellular refuse, whereas catalase plays an important protective role by detoxifying reactive oxygen species. We recently found that autophagy and catalase are also required for promoting programmed cell death induced during plant immune responses. Here we discuss the difficulties in identifying cell death effectors, which are also required to maintain cellular homeostasis, and how their prodeath roles were unmasked using an unbiased forward genetics approach.     

Oxidative stress induces reactivation of Kaposi's sarcoma-associated herpesvirus and death of primary effusion lymphoma cells

Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) cells are predominantly infected with latent Kaposi's sarcoma-associated herpesvirus (KSHV), presenting a barrier to the destruction of tumor cells. Latent KSHV can be reactivated to undergo lytic replication. Here we report that in PEL cells, oxidative stress induced by upregulated reactive oxygen species (ROS) can lead to KSHV reactivation or cell death. ROS are upregulated by NF-κB inhibition and are required for subsequent KSHV reactivation. Disruption of the intracellular redox balance through depletion of the antioxidant glutathione or inhibition of the antioxidant enzyme catalase also induces KSHV reactivation, suggesting that hydrogen peroxide induces reactivation. In addition, p38 signaling is required for KSHV reactivation induced by ROS. Furthermore, treatment of PEL cells with a higher concentration of the NF-κB inhibitor than that used for inducing KSHV reactivation further upregulates ROS and induces massive cell death. ROS, but not p38 signaling, are required for PEL cell death induced by NF-κB inhibition as well as by glutathione depletion. Importantly, anticancer drugs, such as cisplatin and arsenic trioxide, also induce KSHV reactivation and PEL cell death in a ROS-dependent manner. Our study thus establishes a critical role for ROS and oxidative stress in the regulation of KSHV reactivation and PEL cell death. Disrupting the cellular redox balance may be a potential strategy for treating KSHV-associated lymphoma.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.     

Catalase effect on cell death for the improvement of recombinant protein production in baculovirus-insect cell system

Baculovirus expression vector system (BEVS) in host insect cells is a powerful technology to produce recombinant proteins, as well as virus-like particles (VLP). However, BEVS is based on baculovirus infection, which limits the recombinant protein production by inducing insect cell death. Herein a new strategy to enhance cell life span and to increase recombinant protein production was developed. As baculovirus infection induces cellular oxidative stress, the ability of several antioxidants to inhibit cell death was tested during infection. The production of rotavirus structural proteins was used as model to analyse this new strategy. We found that only catalase is able to partially prevent cell death triggered by baculovirus infection and to inhibit lipid peroxidation. An increase in recombinant protein production was coupled with the partial cell death inhibition. In summary, the addition of catalase is a promising strategy to improve recombinant protein production in BEVS, by delaying insect cell death.     

Regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer

Autophagy, a lysosomal degradation pathway for cellular constituents and organelles, is an adaptive and essential process required for cellular homeostasis. Although autophagy functions as a survival mechanism in response to cellular stressors such as nutrient or growth factor deprivation, it can also lead to a non-apoptotic form of programmed cell death (PCD) called autophagy-induced cell death or autophagy-associated cell death (type II PCD). Current evidence suggests that cell death through autophagy can be induced as an alternative to apoptosis (type I PCD), with therapeutic purpose in cancer cells that are resistant to apoptosis. Thus, modulating autophagy is of great interest in cancer research and therapy. Natural polyphenolic compounds that are present in our diet, such as rottlerin, genistein, quercetin, curcumin, and resveratrol, can trigger type II PCD via various mechanisms through the canonical (Beclin-1 dependent) and non-canonical (Beclin-1 independent) routes of autophagy. The capacity of these compounds to provide a means of cancer cell death that enhances the effects of standard therapies should be taken into consideration for designing novel therapeutic strategies. This review focuses on the autophagy- and cell death-inducing effects of these polyphenolic compounds in cancer.     

Integrin–ECM interactions and membrane-associated Catalase cooperate to promote resilience of the Drosophila intestinal epithelium

Balancing cellular demise and survival constitutes a key feature of resilience mechanisms that underlie the control of epithelial tissue damage. These resilience mechanisms often limit the burden of adaptive cellular stress responses to internal or external threats. We recently identified Diedel, a secreted protein/cytokine, as a potent antagonist of apoptosis-induced regulated cell death in the Drosophila intestinal midgut epithelium during aging. Here, we show that Diedel is a ligand for RGD-binding Integrins and is thus required for maintaining midgut epithelial cell attachment to the extracellular matrix (ECM)-derived basement membrane. Exploiting this function of Diedel, we uncovered a resilience mechanism of epithelial tissues, mediated by Integrin–ECM interactions, which shapes cell death spreading through the regulation of cell detachment and thus cell survival. Moreover, we found that resilient epithelial cells, enriched for Diedel–Integrin–ECM interactions, are characterized by membrane association of Catalase, thus preserving extracellular reactive oxygen species (ROS) balance to maintain epithelial integrity. Intracellular Catalase can relocalize to the extracellular membrane to limit cell death spreading and repair Integrin–ECM interactions induced by the amplification of extracellular ROS, which is a critical adaptive stress response. Membrane-associated Catalase, synergized with Integrin–ECM interactions, likely constitutes a resilience mechanism that helps balance cellular demise and survival within epithelial tissues.

A key feature of the resilience mechanisms that underlie the control of epithelial tissue damage is the balance between cell death and survival. This study shows that the anti-oxidant enzyme catalase can relocate to membranes in order to promote the resilience of the Drosophila midgut epithelium, synergizing with integrin-ECM interactions to prevent the spread of cell death.     

Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury.

HepG2 cells were transfected with vectors containing human catalase cDNA and catalase cDNA with a mitochondrial leader sequence to allow comparison of the effectiveness of catalase overexpressed in the cytosolic or mitochondrial compartments to protect against oxidant-induced injury. Overexpression of catalase in cytosol and in mitochondria was confirmed by Western blot, and activity measurement and stable cell lines were established. The intracellular level of H(2)O(2) induced by exogenously added H(2)O(2) or antimycin A was lower in C33 cell lines overexpressing catalase in the cytosol and mC5 cell lines overexpressing catalase in the mitochondria as compared with Hp cell lines transfected with empty vector. Cell death caused by H(2)O(2), antimycin A, and menadione was considerably suppressed in both the mC5 and C33 cell lines. C33 and mC5 cells were also more resistant to apoptosis induced by H(2)O(2) and to the loss of mitochondrial membrane potential induced by H(2)O(2) and antimycin A. In view of the comparable protection by catalase overexpressed in the cytosol versus the mitochondria, catalase produced in both cellular compartments might act as a sink to decompose H(2)O(2) and move diffusable H(2)O(2) down its concentration gradient. The present study suggests that catalase in cytosol and catalase in mitochondria are capable of protecting HepG2 cells against cytotoxicity or apoptosis induced by oxidative stress.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

Protective effects of a Rhodiola crenulata extract and salidroside on hippocampal neurogenesis against streptozotocin-induced neural injury in the rat

Previously we have demonstrated that a Rhodiola crenulata extract (RCE), containing a potent antioxidant salidroside, promotes neurogenesis in the hippocampus of depressive rats. The current study was designed to further investigate the protective effect of the RCE on neurogenesis in a rat model of Alzheimer's disease (AD) induced by an intracerebroventricular injection of streptozotocin (STZ), and to determine whether this neuroprotective effect is induced by the antioxidative activity of salidroside. Our results showed that pretreatment with the RCE significantly improved the impaired neurogenesis and simultaneously reduced the oxidative stress in the hippocampus of AD rats. In vitro studies revealed that (1) exposure of neural stem cells (NSCs) from the hippocampus to STZ strikingly increased intracellular reactive oxygen species (ROS) levels, induced cell death and perturbed cell proliferation and differentiation, (2) hydrogen peroxide induced similar cellular activities as STZ, (3) pre-incubation of STZ-treated NSCs with catalase, an antioxidant, suppressed all these cellular activities induced by STZ, and (4) likewise, pre-incubation of STZ-treated NSCs with salidroside, also an antioxidant, suppressed all these activities as catalase: reduction of ROS levels and NSC death with simultaneous increases in proliferation and differentiation. Our findings indicated that the RCE improved the impaired hippocampal neurogenesis in the rat model of AD through protecting NSCs by its main ingredient salidroside which scavenged intracellular ROS.     

Serine proteases increase oxidative stress in lung cells

Several serine proteases are directly cytotoxic. We investigated whether the cytotoxic effects of proteases are associated with increased levels of reactive oxygen species (ROS) in cells. We found that treatment of lung fibroblasts or bronchial epithelial cells with relatively high concentrations (0.1--100 U/ml) of neutrophil elastase, trypsin, and Pronase increased ROS levels in the mitochondria and cytoplasm. The protease-induced increase in ROS was associated with oxidative cellular injury as determined by generation of 8-hydroxy-2'-deoxyguanosine and malonaldehyde plus 4-hydroxyalkenal. The protease-induced increase in ROS was not merely due to cell detachment because the proteases also caused an increase in ROS in suspended cells, which precluded attachment to the extracellular matrix. The protease-induced increase in ROS appears to contribute to cytotoxicity because cell death induced by proteases was attenuated by treatment with catalase, a decomposer of H(2)O(2), and accelerated by treatment with aminotriazole, a catalase inhibitor. These results suggest that several proteases increase oxidative stress, indicating a direct interaction between proteases and ROS in mediating cytotoxicity.     

Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. The mechanisms involved in heat-induced cell killing are currently unknown. Hyperthermia may increase oxidative stress in cells, and thus, oxidative stress could have a role in the mechanism of cell death. We use hydrogen peroxide as a model oxidant to improve understanding of interactions between heat and oxidative stress. Heat increased cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells. Altered levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus modifying cytotoxic responses to heat and to oxidants. We determine the involvement of the two cellular antioxidant defenses against peroxides, catalase and the glutathione redox cycle, in cellular sensitivity to heat, to hydrogen peroxide, and to heat combined with the oxidant. Defense systems were either inhibited or increased. For inhibition studies, intracellular glutathione was diminished to less than 15% of its initial level by treatment with L-buthionine sulfoximine (1 mM, 24 h). Inhibition of catalase was achieved with 3-amino-1,2,4-triazole (20 mM, 2 h), which caused a 80% decrease in endogenous enzyme activity. To increase antioxidants, cells were pretreated with the thiol-containing reducing agents, N-acetyl-L-cysteine, 2-oxo-4-thiazolidine carboxylate, and 2-mercaptoethane sulfonate. These compounds increased intracellular glutathione levels by 30%. Catalase activity was increased by addition of exogenous enzyme to cells. We show that levels of glutathione and catalase affect cellular cytotoxic responses to heat and hydrogen peroxide, either used separately or in combination. These findings are relevant to mechanisms of cell killing at elevated temperatures and suggest the involvement of oxidative stress.     

Deregulation of catalase, not MnSOD, is associated with necrotic death of p53-defective DF-1 cells under antimycin A-induced oxidative stress

One of distinct genetic alterations in spontaneously immortalized DF-1 cells was found to be dysfunction of p53 and E2F-1 as well as altered antioxidant gene expression (upregulation of MnSOD and downregulation of catalase). We have characterized the cellular responses of primary and immortal DF-1 cells to oxidative stress and found that DF-1 cells were more sensitive to oxidative stress than their primary counterparts when treated with antimycin A. The increased DF-1 cell death by oxidative stress was accompanied by an increase in the levels of intracellular superoxide anions and hydrogen peroxide. The cell death in DF-1 cells by antimycin A showed none of the hallmarks of apoptosis, but displayed a significantly increased necrotic cell population. Anti-apoptotic Bcl-2 failed to inhibit oxidative-induced necrotic cell death in the DF-1 cells. However, this necrotic cell death was significantly decreased by treatment with hydrogen peroxide scavengers such as sodium pyruvate and N-acetyl-cysteine. Interestingly, overexpression of human catalase in DF-1 cells endowed cells resistant to the oxidative stress by antimycin A treatment, although the downregulation of MnSOD by an antisense strategy showed no evident change in the cytotoxic effect caused by antimycin A. Taken together, the present study might provide new therapeutic approach for tumor cells having the loss of p53 function and the altered antioxidant functions.     

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro- trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per- oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time- and dose-dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.     

Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1α expression by HIF-1α specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1α expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.     

Antioxidant enzyme inhibitors enhance singlet oxygen-induced cell death in HL-60 cells

Singlet oxygen is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules and it also promotes deleterious processes such as cell death. The protective role of antioxidant enzymes against singlet oxygen-induced oxidative damage in HL-60 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole and oxlalomalate, specific inhibitors of superoxide dismutase, catalase and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to rose bengal (20 μM)/light (15 min), which generates singlet oxygen, to HL-60 cells, the viability was lower and the lipid peroxidation and oxidative DNA damage were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species as well as the significant decrease in the intracellular GSH level in inhibitor-treated HL-60 cells exposed to singlet oxygen. Upon exposure to rose bengal (3 μM)/light (15 min), which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated HL-60 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against singlet oxygen-induced cell death including necrosis and apoptosis.     

LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy

The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H(2)O(2). A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H(2)O(2) accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.     

Induction of apoptosis in fetal pulmonary arterial smooth muscle cells by a combined superoxide dismutase/catalase mimetic

Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are known to play an important role in the proliferation and viability of vascular smooth muscle cells. In this study, we determined the effects of increased superoxide dismutase and catalase activity on fetal pulmonary arterial smooth muscle cell (FPASMC) proliferation and viability using EUK-134, a superoxide dismutase/catalase mimetic. Treatment of FPASMC with EUK-134 or with a combination of superoxide dismutase and catalase enzymes decreased superoxide and hydrogen peroxide levels as detected by the fluorescent dyes dihydroethidium and dichlorodihydrofluorescein diacetate, respectively. EUK-134 (5 microM) attenuated serum-induced FPASMC proliferation, whereas 50 microM EUK-134 decreased the number of viable cells, suggesting cell death. Conversely, combined superoxide dismutase and catalase enzyme activity equivalent to 50 microM EUK-134 prevented proliferation but did not reduce the number of viable FPASMC. The loss of mitochondrial membrane potential after 18 h, an increase in caspase-9 and caspase-3 activity after 24 h, and the subsequent appearance of TdT-mediated dUTP nick end labeling-positive nuclei were detected in FPASMC after treatment with 50 microM EUK-134. This indicates an induction of programmed rather than necrotic cell death and suggests that prolonged removal of ROS is required to stimulate apoptosis. Compounds such as EUK-134 may, therefore, prove more effective than enzymic antioxidants over longer periods, especially when the aim is to decrease the number of smooth muscle cells in diseases resulting from excessive muscularization.     

Catalase and NO CATALASE ACTIVITY1 Promote Autophagy-Dependent Cell Death in Arabidopsis

Programmed cell death often depends on generation of reactive oxygen species, which can be detoxified by antioxidative enzymes, including catalases. We previously isolated catalase-deficient mutants (cat2) in a screen for resistance to hydroxyurea-induced cell death. Here, we identify an Arabidopsis thaliana hydroxyurea-resistant autophagy mutant, atg2, which also shows reduced sensitivity to cell death triggered by the bacterial effector avrRpm1. To test if catalase deficiency likewise affected both hydroxyurea and avrRpm1 sensitivity, we selected mutants with extremely low catalase activities and showed that they carried mutations in a gene that we named NO CATALASE ACTIVITY1 (NCA1). nca1 mutants showed severely reduced activities of all three catalase isoforms in Arabidopsis, and loss of NCA1 function led to strong suppression of RPM1-triggered cell death. Basal and starvation-induced autophagy appeared normal in the nca1 and cat2 mutants. By contrast, autophagic degradation induced by avrRpm1 challenge was compromised, indicating that catalase acted upstream of immunity-triggered autophagy. The direct interaction of catalase with reactive oxygen species could allow catalase to act as a molecular link between reactive oxygen species and the promotion of autophagy-dependent cell death.