Superoxide is the major reactive oxygen species regulating autophagy

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O2•−) and hydrogen peroxide (H2O2). However, the relative functions of O2•− and H2O2 in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H2O2. We found that O2•− was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O2•− and H2O2. Both types of starvation induced autophagy and autophagy was inhibited by overexpression of SOD2 (manganese superoxide dismutase, Mn-SOD), which reduced O2•− levels but increased H2O2 levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O2•− and H2O2 levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors 3-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the SOD inhibitor 2-methoxyestradiol (2-ME) increased O2•− levels, lowered H2O2 levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H2O2 induced autophagy and increased intracellular O2•− but failed to increase intracellular H2O2. Taken together, these findings indicate that O2•− is the major ROS-regulating autophagy.

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title: The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.
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title: Dopamine induces autophagic cell death and α‐synuclein increase in human neuroblastoma SH‐SY5Y cells
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title: Oxidative stress and autophagy.
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title: Protective effects of vacuolar H+-ATPase c on hydrogen peroxide-induced cell death in C6 glioma cells
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title: Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells.
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title: Apoptosis and Autophagy in Photoreceptors Exposed to Oxidative Stress
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title: Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis1[W][OA]
abstract: Upon encountering oxidative stress, proteins are oxidized extensively by highly reactive and toxic reactive oxidative species, and these damaged, oxidized proteins need to be degraded rapidly and effectively. There are two major proteolytic systems for bulk degradation in eukaryotes, the proteasome and vacuolar autophagy. In mammalian cells, the 20S proteasome and a specific type of vacuolar autophagy, chaperone-mediated autophagy, are involved in the degradation of oxidized proteins in mild oxidative stress. However, little is known about how cells remove oxidized proteins when under severe oxidative stress. Using two macroautophagy markers, monodansylcadaverine and green fluorescent protein-AtATG8e, we here show that application of hydrogen peroxide or the reactive oxidative species inducer methyl viologen can induce macroautophagy in Arabidopsis (Arabidopsis thaliana) plants. Macroautophagy-defective RNAi-AtATG18a transgenic plants are more sensitive to methyl viologen treatment than wild-type plants and accumulate a higher level of oxidized proteins due to a lower degradation rate. In the presence of a vacuolar H+-ATPase inhibitor, concanamycin A, oxidized proteins were detected in the vacuole of wild-type root cells but not RNAi-AtATG18a root cells. Together, our results indicate that autophagy is involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis.

title: Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species
abstract: Autophagy is a self-digestion process important for cell survival during starvation. It has also been described as a form of programmed cell death. Mitochondria are important regulators of autophagy-induced cell death and damaged mitochondria are often degraded by autophagosomes. Inhibition of the mitochondrial electron transport chain (mETC) induces cell death through generating reactive oxygen species (ROS). The role of mETC inhibitors in autophagy-induced cell death is unknown. Herein, we determined that inhibitors of complex I (rotenone) and complex II (TTFA) induce cell death and autophagy in the transformed cell line HEK 293, and in cancer cell lines U87 and HeLa. Blocking the expression of autophagic genes (beclin 1 and ATG5) by siRNAs or using the autophagy inhibitor 3-methyladenine (3-MA) decreased cell death that was induced by rotenone or TTFA. Rotenone and TTFA induce ROS production, and the ROS scavenger tiron decreased autophagy and cell death induced by rotenone and TTFA. Overexpression of manganese-superoxide dismutase (SOD2) in HeLa cells decreased autophagy and cell death induced by rotenone and TTFA. Furthermore, blocking SOD2 expression by siRNA in HeLa cells increased ROS generation, autophagy and cell death induced by rotenone and TTFA. Rotenone- and TTFA-induced ROS generation was not affected by 3-MA, or by beclin 1 and ATG5 siRNAs. By contrast, treatment of non-transformed primary mouse astrocytes with rotenone or TTFA failed to significantly increase levels of ROS or autophagy. These results indicate that targeting mETC complex I and II selectively induces autophagic cell death through a ROS-mediated mechanism.

title: Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells
abstract: Autophagy is a self-digestion process that degrades intracellular structures in response to stresses leading to cell survival. When autophagy is prolonged, this could lead to cell death. Generation of reactive oxygen species (ROS) through oxidative stress causes cell death. The role of autophagy in oxidative stress-induced cell death is unknown. In this study, we report that two ROS-generating agents, hydrogen peroxide (H2O2) and 2-methoxyestradiol (2-ME), induced autophagy in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa. Blocking this autophagy response using inhibitor 3-methyladenine or small interfering RNAs against autophagy genes, beclin-1, atg-5 and atg-7 inhibited H2O2 or 2-ME-induced cell death. H2O2 and 2-ME also induced apoptosis but blocking apoptosis using the caspase inhibitor zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) failed to inhibit autophagy and cell death suggesting that autophagy-induced cell death occurred independent of apoptosis. Blocking ROS production induced by H2O2 or 2-ME through overexpression of manganese-superoxide dismutase or using ROS scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid-disodium salt decreased autophagy and cell death. Blocking autophagy did not affect H2O2- or 2-ME-induced ROS generation, suggesting that ROS generation occurs upstream of autophagy. In contrast, H2O2 or 2-ME failed to significantly increase autophagy in mouse astrocytes. Taken together, ROS induced autophagic cell death in transformed and cancer cells but failed to induce autophagic cell death in non-transformed cells.

title: Hyperthermia in combination with oxidative stress induces autophagic cell death in HT‐29 colon cancer cells
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title: The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation
abstract: TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.

title: Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death
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title: Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells
abstract: The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-xL ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.

title: Autophagy in cell death: an innocent convict?
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title: Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3
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title: How to Interpret LC3 Immunoblotting
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title: Oxidative stress and apoptosis: impact on cancer therapy.
abstract: It is well established that some chemotherapeutic agents and radiation therapy generate reactive oxygen species (ROS) in patients during cancer therapy. Free radicals, particularly ROS have been proposed as common mediators for apoptosis. Recent studies have demonstrated that the mode of cell death depends on the severity of the oxidative damage. This review will address some of the current paradigms of oxidative stress, and antioxidants on apoptosis, and discuss the potential mechanisms by which oxidants can regulate apoptotic pathways. It will also review new developments in eliminating cancer cells by selectively inducing apoptosis.

title: Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment.
abstract: Reactive oxygen species (ROS) have been identified as signaling molecules in various pathways regulating both cell survival and cell death. Autophagy, a self-digestion process that degrades intracellular structures in response to stress, such as nutrient starvation, is also involved in both cell survival and cell death. Alterations in both ROS and autophagy regulation contribute to cancer initiation and progression, and both are targets for developing therapies to induce cell death selectively in cancer cells. Many stimuli that induce ROS generation also induce autophagy, including nutrient starvation, mitochondrial toxins, hypoxia, and oxidative stress. Some of these stimuli are under clinical investigation as cancer treatments, such as 2-methoxyestrodial and arsenic trioxide. Recently, it was demonstrated that ROS can induce autophagy through several distinct mechanisms involving Atg4, catalase, and the mitochondrial electron transport chain (mETC). This leads to both cell-survival and cell-death responses and could be selective toward cancer cells. In this review, we give an overview of the roles ROS and autophagy play in cell survival and cell death, and their importance to cancer. Furthermore, we describe how autophagy is mediated by ROS and the implications of this regulation to cancer treatments.

title: Autophagy fights disease through cellular self-digestion
abstract: Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology. For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing. Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.

title: 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process
abstract: The effects of 2-Methoxyestradiol (2ME)-induced apoptosis was examined in human leukemia cells (U937 and Jurkat) in relation to mitochondrial injury, oxidative damage, and perturbations in signaling pathways. 2ME induced apoptosis in these cells in a dose-dependent manner associated with release of mitochondrial proteins (cytochrome c, AIF), generation of reactive oxygen species (ROS), downregulation of Mcl-1 and XIAP, and inactivation (dephosphorylation) of Akt accompanied by activation of JNK. In these cells, enforced activation of Akt by a constitutively active myristolated Akt construct prevented 2ME-mediated mitochondrial injury, XIAP and Mcl-1 downregulation, JNK activation, and apoptosis, but not ROS generation. Conversely, 2ME lethality was potentiated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Furthermore, in U937 cells, the hydrogen peroxide scavenger catalase and a superoxide dismutase (SOD) mimetic, TBAP, blocked these events, as well as Akt inactivation. Interruption of the JNK pathway by pharmacologic or genetic (e.g. siRNA) means attenuated 2ME-induced mitochondrial injury, XIAP and Mcl-1 downregulation, and apoptosis. Collectively, these findings suggest a hierarchical model of 2ME-related apoptosis induction in human leukemia cells in which 2ME-induced oxidative injury represents a primary event resulting in Akt inactivation, leading, in turn, to JNK activation, and culminating in XIAP and Mcl-1 downregulation, mitochondrial injury, and apoptosis. They also suggest that in human leukemia cells, the Akt pathway plays a critical role in mediating the response to oxidative stress induced by 2ME.

title: Detection of catalase in rat heart mitochondria.
abstract: The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.