Elsevier

Nitric Oxide

Volume 115, 1 October 2021, Pages 34-43
Nitric Oxide

Nitric oxide protects against ferroptosis by aborting the lipid peroxidation chain reaction

https://doi.org/10.1016/j.niox.2021.07.003Get rights and content

Highlights

  • A NO donor treatment inhibits ferroptosis induced by Cys starvation.

  • A NO donor treatment inhibits ferroptosis induced by GPX4 inhibition.

  • A NO donor treatment inhibits the ferroptosis induced by TBHP treatment.

  • A NO donor treatment does not alter iron status during ferroptosis.

  • A NO donor treatment aborts ferroptosis by terminating the lipid peroxidation chain reaction.

Abstract

Ferroptosis is a type of iron-dependent necrotic cell death, which is typically triggered by the depletion of intracellular glutathione (GSH), which is associated with increased lipid peroxidation. Nitric oxide (NO) is a highly reactive gaseous radical mediator with anti-oxidation properties that terminates lipid peroxidation reactions. In the current study, we report the anti-ferroptotic action of NOC18, an NO donor that spontaneously releases NO, in cells under various ferroptotic conditions in vitro. Our results indicate that, when mouse hepatoma Hepa 1–6 cells are incubated with NOC18, cell death induced by various ferroptotic stimuli such as cysteine (Cys) starvation, the inhibition of glutathione peroxidase 4 (GPX4) and treatment with tertiary-butyl hydroperoxide (TBHP) is significantly reduced. Treatment with NOC18 failed to improve the decrease in the levels of Cys or GSH and the accumulation of ferrous iron upon ferroptotic stimuli. The fluorescent intensity of C11-BODIPY581/591, a probe that is used to detect lipid peroxidation products, was increased somewhat by treatment with NOC18 under conditions of Cys starvation, and the accumulation of lipid peroxidation end-products, as evidenced by the levels of 4-hydroxynonenal, were effectively suppressed. The pre-incubation of TBHP with NOC7, a short-lived NO donor completely eliminated its ability to trigger ferroptosis. These collective results indicate that NO exerts a cytoprotective action against various ferroptotic stimuli 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.

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      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].

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