Abstract
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.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- AA:
-
amino acids and serum
- AO:
-
acridine orange
- AVOs:
-
acidic vesicular organelles
- CM-H2DCFDA:
-
5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester
- DHE:
-
dihydroethidium
- 2-ME:
-
2-methoxyestradiol
- 3-MA:
-
3-methyladenine
- GFP:
-
green fluorescent protein
- GP:
-
glucose, L-glutamine, pyruvate and serum
- GPx:
-
glutathione peroxidase
- H2O2:
-
hydrogen peroxide
- LC3:
-
microtubule-associated protein 1 light chain 3
- mETC:
-
mitochondrial electron transport chain
- NAC:
-
N-acetyl-cysteine
- O2•−:
-
superoxide
- •OH:
-
hydroxyl radical
- PCD:
-
programmed cell death
- PrxIII:
-
peroxiredoxin III
- ROS:
-
reactive oxygen species
- SOD:
-
superoxide dismutase
- SOD1:
-
copper-zinc superoxide dismutase (Cu, Zn-SOD)
- SOD2:
-
manganese superoxide dismutase (Mn-SOD)
- TTFA:
-
2-thenoyltrifluoroacetone
References
Levine B, Yuan J . Autophagy in cell death: an innocent convict? J Clin Invest 2005; 115: 2679–2688.
Azad MB, Chen Y, Gibson SB . Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal 2008; 11: 777–790.
Chen Y, Gibson SB . Is mitochondrial generation of reactive oxygen species a trigger for autophagy? Autophagy 2008; 4: 246–248.
Ozben T . Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci 2007; 96: 2181–2196.
Bridges KR . Ascorbic acid inhibits lysosomal autophagy of ferritin. J Biol Chem 1987; 262: 14773–14778.
Lemasters JJ, Nieminen AL, Qian T, Trost LC, Elmore SP, Nishimura Y et al. The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1998; 1366: 177–196.
Kirkland RA, Adibhatla RM, Hatcher JF, Franklin JL . Loss of cardiolipin and mitochondria during programmed neuronal death: evidence of a role for lipid peroxidation and autophagy. Neuroscience 2002; 115: 587–602.
Gómez-Santos C, Ferrer I, Santidrián AF, Barrachina M, Gil J, Ambrosio S . Dopamine induces autophagic cell death and alpha-synuclein increase in human neuroblastoma SH-SY5Y cells. J Neurosci Res 2003; 73: 341–350.
Djavaheri-Mergny M, Amelotti M, Mathieu J, Besançon F, Bauvy C, Souquère S et al. NF-κB activation represses tumor necrosis factor-α-induced autophagy. J Biol Chem 2006; 281: 30373–30382.
Kiffin R, Bandyopadhyay U, Cuervo AM . Oxidative stress and autophagy. Antioxid Redox Signal 2006; 8: 152–162.
Kissová I, Deffieu M, Samokhvalov V, Velours G, Bessoule JJ, Manon S et al. Lipid oxidation and autophagy in yeast. Free Radic Biol Med 2006; 41: 1655–1661.
Byun YJ, Lee SB, Kim DJ, Lee HO, Son MJ, Yang CW et al. Protective effects of vacuolar H+-ATPase c on hydrogen peroxide-induced cell death in C6 glioma cells. Neurosci Lett 2007; 425: 183–187.
Kim EH, Sohn S, Kwon HJ, Kim SU, Kim MJ, Lee SJ et al. Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells. Cancer Res 2007; 67: 6314–6324.
Kunchithapautham K, Rohrer B . Apoptosis and autophagy in photoreceptors exposed to oxidative stress. Autophagy 2007; 3: 433–441.
Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z . Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO J 2007; 26: 1749–1760.
Xiong Y, Contento AL, Nguyen PQ, Bassham DC . Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol 2007; 143: 291–299.
Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB . Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species. J Cell Sci 2007; 120: 4155–4166.
Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB . Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ 2008; 15: 171–182.
Chen F, Wang CC, Kim E, Harrison LE . Hyperthermia in combination with oxidative stress induces autophagic cell death in HT-29 colon cancer cells. Cell Biol Int 2008; 32: 715–723.
Dadakhujaev S, Noh HS, Jung EJ, Hah YS, Kim CJ, Kim DR . The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation. Exp Cell Res 2008; 314: 3094–3106.
Pyo JO, Nah J, Kim HJ, Lee HJ, Heo J, Lee H et al. Compensatory activation of ERK1/2 in Atg5-deficient mouse embryo fibroblasts suppresses oxidative stress-induced cell death. Autophagy 2008; 4: 315–321.
Yang J, Wu LJ, Tashino S, Onodera S, Ikejima T . Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells. Free Radic Res 2008; 42: 492–504.
Huang P, Feng L, Oldham EA, Keating MJ, Plunkett W . Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2000; 407: 390–395.
Azad MB, Chen Y, Henson ES, Cizeau J, McMillan-Ward E, Israels SJ et al. Hypoxia induces autophagic cell death in apoptosis-competent cells through a mechanism involving BNIP3. Autophagy 2008; 4: 195–204.
Mizushima N, Yoshimori T . How to interpret LC3 immunoblotting. Autophagy 2007; 3: 542–545.
Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4: 151–175.
Gao N, Rahmani M, Dent P, Grant S . 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt-dependent process. Oncogene 2005; 24: 3797–3809.
Hagen T, D'Amico G, Quintero M, Palacios-Callender M, Hollis V, Lam F et al. Inhibition of mitochondrial respiration by the anticancer agent 2-methoxyestradiol. Biochem Biophys Res Commun 2004; 322: 923–929.
Kulisz A, Chen N, Chandel NS, Shao Z, Schumacker PT . Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes. Am J Physiol Lung Cell Mol Physiol 2002; 282: L1324–L1329.
Nonn L, Berggren M, Powis G . Increased expression of mitochondrial peroxiredoxin-3 (thioredoxin peroxidase-2) protects cancer cells against hypoxia and drug-induced hydrogen peroxide-dependent apoptosis. Mol Cancer Res 2003; 1: 682–689.
Li J, Li Q, Xie C, Zhou H, Wang Y, Zhang N et al. Beta-actin is required for mitochondria clustering and ROS generation in TNF-induced, caspase-independent cell death. J Cell Sci 2004; 117: 4673–4680.
Mizushima N, Levine B, Cuervo AM, Klionsky DJ . Autophagy fights disease through cellular self-digestion. Nature 2008; 451: 1069–1075.
Radi R, Turrens JF, Chang LY, Bush KM, Crapo JD, Freeman BA . Detection of catalase in rat heart mitochondria. J Biol Chem 1991; 266: 22028–22034.
Acknowledgements
The work was supported by a grant from CancerCare Manitoba Foundation. YC was supported by a post-doctoral fellowship from Manitoba Health Research Council (MHRC). MBA was supported by a US Army Department of Defense Breast Cancer Research Program Predoctoral Traineeship Award. SBG is a Manitoba Research Chair supported by MHRC.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by D Klionsky
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Supplementary information
Rights and permissions
About this article
Cite this article
Chen, Y., Azad, M. & Gibson, S. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 16, 1040–1052 (2009). https://doi.org/10.1038/cdd.2009.49
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2009.49
Keywords
This article is cited by
-
The birth of death, 30 years ago
Cell Death & Differentiation (2024)
-
Abamectin induces mortality, inhibits food consumption, and causes histological changes in the midgut of the velvetbean caterpillar Anticarsia gemmatalis (Lepidoptera: Noctuidae)
Journal of Pest Science (2024)
-
Effects of pyrrolopyrimidine derivatives on cancer cells cultured in vitro and potential mechanism
Naunyn-Schmiedeberg's Archives of Pharmacology (2024)
-
Apoptotic vesicles resist oxidative damage in noise-induced hearing loss through activation of FOXO3a-SOD2 pathway
Stem Cell Research & Therapy (2023)
-
Disrupting iron homeostasis can potentiate colistin activity and overcome colistin resistance mechanisms in Gram-Negative Bacteria
Communications Biology (2023)
