Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction
Introduction
Ferroptosis is a type of iron-dependent, regulated cell death that occurs as a consequence of lipid peroxidation [1,2]. The reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) in the presence of peroxides triggers the production of hydroxyl radicals generally through Fenton-type reactions [3], leading to the initiation of lipid peroxidation. The resulting lipid peroxides that are generated in membrane phospholipids cause ferroptotic cell death by disrupting the integrity of the plasma membrane. Glutathione (GSH), a cysteine (Cys)-centered tripeptidyl redox molecule, plays a pivotal role in protecting against the accelerated lipid peroxidation associated with ferroptosis. The anti-ferroptotic action of GSH can be largely attributed to glutathione peroxidase 4 (GPX4), which reductively detoxifies lipid peroxides by utilizing GSH as an electron donor in the ferroptotic process [4]. xCT, the core transporter protein of system xc−, is responsible for the cellular uptake of cystine, an oxidized Cys dimer linked by a disulfide bridge [5]. Because the availability of free Cys is the major determinant of GSH synthesis, ferroptosis can be readily induced by either Cys starvation (e.g. the deprivation of cystine from the culture medium or the inhibition of xCT by erastin) or GPX4 inhibition. Thus, the Cys–GSH–GPX4 axis appears to be the primary protective system for coping with ferroptosis [2,6].
Nitric oxide (NO) interacts with reactive oxygen species (ROS) and is converted into several reactive nitrogen oxide species, which can irreversibly modify DNA, proteins, lipids and other biomolecules. For example, NO interacts with superoxide at a similar or even faster rate than the dismutation reaction catalyzed by superoxide dismutase (SOD) and results in the formation of peroxynitrite (ONOO−) [7]. Peroxynitrite has been shown to induce cellular injury through the oxidative modification of DNA, lipids, and proteins [8,9]. The initiation of lipid peroxidation by the action of peroxynitrite appears to play major roles in variety of diseases, as has been suggested for atherosclerosis [10]. For this (and other) reason, NO is often considered to be a toxic species. However, NO has also been shown to abate oxidative injury in several experimental models [11,12]. Indeed, it has been reported that NO potently inhibits lipid peroxidation in low-density lipoprotein and liposome membranes [[13], [14], [15]]. This is primarily a consequence of NO reacting with lipid-derived peroxy radicals (LOO·) to terminate lipid peroxidation propagation reactions [16,17]. NO has also been shown to form an iron-nitrosyl complex and inhibit the reaction between a peroxide and a metal ion, thereby preventing ROS production [18].
The issue of whether NO exerts either harmful or beneficial to cells has been a subject of considerable debate, and may also depend on the experimental conditions being used. In the current study, we examined the anti-ferroptotic properties of NOC18, a long-lasting NO donor that spontaneously releases NO under various ferroptotic conditions in cultured cells. Herein we report on the potential roles of NO in coping with ferroptotic cell death, which appears to be achieved by the suppression of ROS production and the termination of the lipid peroxidation reaction by NO.
Section snippets
Cell culture and chemicals
Hepa 1–6 cells, a mouse hepatoma-derived cell line, were obtained from the RIKEN Bioresource Center (Tsukuba, Japan). The human cervical carcinoma HeLa cells and mouse melanoma B16–F1 cells were obtained from the American Type Culture Collection (ATCC). Mouse embryonic fibroblasts capable of undergoing tamoxifen-inducible GPX4 disruption (Pfa1 cells) were described in a previous report [19]. In all cases, the cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; FUJIFILM Wako Pure
NOC18 treatment inhibits ferroptosis induced by Cys starvation
To investigate the potential role of NO in protecting cells against ferroptosis, we treated mouse hepatoma-derived Hepa 1–6 cells with a long-lasting NO donor, NOC18 (half-life >1200 min). A CellTiter-Blue® assay showed that depriving cystine from the culture media resulted in a decreased cell viability, while the administration of NOC18 improved cell viability at concentrations higher than 25 μM (Fig. 1A). An assay for the release of LDH confirmed that NOC18 suppressed the ferroptotic cell
Discussion
In the current study, we showed that NO produced by an NO donor NOC18 exerts protective effects against ferroposis caused by diverse stimuli, including Cys starvation, GPX4 inhibition and a TBHP treatment (Fig. 1, Fig. 5, Fig. 6). NO failed to prevent the decrease in cellular Cys or GSH levels but still suppressed ferroptosis under Cys starvation (Fig. 2). Although the levels of the GPX4 protein, which were decreased under Cys starvation, were partly rescued by the NOC18 treatment, it failed to
Declaration of competing interest
M.C. is co-founder and shareholder of ROSCUE Therapeutics GmbH.
Acknowledgments
This work was supported, in part, by the YU-COE (C) (C31-3) program of Yamagata University. We thank Prof. Makoto Arita and Dr. Ryohei Aoyagi of Graduate School of Pharmaceutical Sciences, Keio University for their technical advice.
References (50)
- et al.
Ferroptosis: an iron-dependent form of nonapoptotic cell death
Cell
(2012) - et al.
Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease
Cell
(2017) - et al.
Iron and thiol redox signaling in cancer: an exquisite balance to escape ferroptosis
Free Radic. Biol. Med.
(2017) - et al.
Regulation of ferroptotic cancer cell death by GPX4
Cell
(2014) Peroxynitrite, a stealthy biological oxidant
J. Biol. Chem.
(2013)- et al.
DNA damage induced by peroxynitrite: subsequent biological effects
Nitric Oxide Biol. Chem.
(1997) - et al.
The participation of nitric oxide in cell free- and its restriction of macrophage-mediated oxidation of low-density lipoprotein
Biochim. Biophys. Acta (BBA) - Mol. Basis Dis.
(1992) - et al.
Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of low density lipoprotein
FEBS Lett.
(1994) - et al.
Action of nitric oxide as an antioxidant against oxidation of soybean phosphatidylcholine liposomal membranes
FEBS Lett.
(1995) - et al.
Nitration of unsaturated fatty acids by nitric oxide-derived reactive species
Methods Enzymol.
(1999)
Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death
Cell Metabol.
Edaravone, a free radical scavenger, protects against ferroptotic cell death in vitro
Exp. Cell Res.
Characterization of a rat monoclonal antibody raised against ferroptotic cells
J. Immunol. Methods
Mechanism of radical production from the reaction of cytochrome c with organic hydroperoxides
J. Biol. Chem.
tert-Butyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids
Arch. Biochem. Biophys.
The Haber-Weiss reaction - the latest revival
Free Radic. Biol. Med.
Lipid peroxidation: physiological levels and dual biological effects
Free Radic. Biol. Med.
Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. Chemical aspects involved in the balance between oxidative and nitrosative stress
J. Biol. Chem.
Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives
J. Biol. Chem.
Nitric oxide produced by NOS2 copes with the cytotoxic effects of superoxide in macrophages
Biochem. Biophys. Reports.
C11-BODIPY581/591, an oxidation-sensitive fluorescent lipid peroxidation probe: (micro)spectroscopic characterization and validation of methodology
Free Radic. Biol. Med.
Superoxide produced by mitochondrial complex III plays a pivotal role in the execution of ferroptosis induced by cysteine starvation
Arch. Biochem. Biophys.
iNOS as a metabolic enzyme under stress conditions
Free Radic. Biol. Med.
Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide
Free Radic. Biol. Med.
Induction of ferroptosis by singlet oxygen generated from naphthalene endoperoxide
Biochem. Biophys. Res. Commun.
Cited by (27)
Sodium nitroprusside alleviates nanoplastics-induced developmental toxicity by suppressing apoptosis, ferroptosis and inflammation
2023, Journal of Environmental ManagementChemical biology of reactive nitrogen species (RNS) and its application in postharvest horticultural crops
2023, Oxygen, Nitrogen and Sulfur Species in Post-Harvest Physiology of Horticultural CropsRegulation of pleiotropic physiological roles of nitric oxide signaling
2023, Cellular SignallingCitation Excerpt :In addition, NOC18 treatment had no effect on the expression of genes involved in iron metabolism which suggests that NOC18 did not alter the iron status. Also, NOC18 exerted a protective effect against the ferroptosis induced by glutathione peroxidase 4 inhibition and cystine starvation [21]. Another study has reported that NOC12, a NO donor, suppressed the oxidation of soybean phosphatidylcholine liposome membranes which confirms the peroxyl radical scavenging property of NO. This may be due to the capacity of NO to penetrate multilamellar membranes in order to scavenge the lipid peroxyl radicals [22].