The mitochondrial Ca2+ uptake regulator,MICU1, is involved in cold stress‐induced ferroptosis

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia‐reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation‐dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome‐wide CRISPR screening, we found that a mitochondrial Ca²⁺ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress‐induced ferroptosis. MICU1 is required for mitochondrial Ca²⁺ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1‐dependent mitochondrial Ca²⁺ homeostasis‐MMP hyperpolarization axis is involved in cold stress‐induced lipid peroxidation and ferroptosis.     

Metabolic regulation of the PMCA: Role in cell death and survival

The plasma membrane Ca²⁺-ATPase (PMCA) is a ubiquitously expressed, ATP-driven Ca²⁺ pump that is critical for maintaining low resting cytosolic Ca²⁺ ([Ca²⁺]_i) in all eukaryotic cells. Since cytotoxic Ca²⁺ overload has such a central role in cell death, the PMCA represents an essential “linchpin” for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca²⁺ overload and Ca²⁺ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca²⁺-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.     

MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

Ferroptosis is an iron-dependent form of cell death driven by biochemical processes that promote oxidation within the lipid compartment. Calcium (Ca²⁺) is a signaling molecule in diverse cellular processes such as migration, neurotransmission, and cell death. Here, we uncover a crucial link between ferroptosis and Ca²⁺ through the identification of the novel tetraspanin MS4A15. MS4A15 localizes to the endoplasmic reticulum, where it blocks ferroptosis by depleting luminal Ca²⁺ stores and reprogramming membrane phospholipids to ferroptosis-resistant species. Specifically, prolonged Ca²⁺ depletion inhibits lipid elongation and desaturation, driving lipid droplet dispersion and formation of shorter, more saturated ether lipids that protect phospholipids from ferroptotic reactive species. We further demonstrate that increasing luminal Ca²⁺ levels can preferentially sensitize refractory cancer cell lines. In summary, MS4A15 regulation of anti-ferroptotic lipid reservoirs provides a key resistance mechanism that is distinct from antioxidant and lipid detoxification pathways. Manipulating Ca²⁺ homeostasis offers a compelling strategy to balance cellular lipids and cell survival in ferroptosis-associated diseases.Subject terms: Lipidomics, Cancer genetics, Cell biology     

Lipid storage and lipophagy regulates ferroptosis

The synthesis, storage, and degradation of lipids are highly regulated processes. Impaired lipid metabolism is implicated in inflammation and cell death. Although ferroptosis is a recently described form of regulated cell death driven by lipid peroxidation, the impact of lipid droplets on ferroptosis remains unidentified. Here, we demonstrate that lipophagy, the autophagic degradation of intracellular lipid droplets, promotes RSL3-induced ferroptotic cell death in hepatocytes. Lipid droplet accumulation is increased at the early stage but decreased at the late stage of ferroptosis in mouse or human hepatocytes. Importantly, either genetically enhancing TPD52-dependent lipid storage or blocking ATG5-and RAB7A-dependent lipid degradation prevents RSL3-induced lipid peroxidation and subsequent ferroptosis in vitro and in vivo. These studies support an antioxidant role for lipid droplets in cell death and suggest novel strategies for the inhibition of ferroptosis by targeting the lipophagy pathway.     

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD⁺ precursor supplement, rescued the imbalanced NAD⁺/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.     

Enhanced lipid peroxidation by extracellular ATP in PC12 cells

Recently we have demonstrated that extracellular ATP acts as an excitatory neurotransmitter and enhances cell death in the presence of ferrous ions. By using a newly developed cis-parinaric acid fluorescence technique, we demonstrated that ATP, in a dose dependent manner, enhanced the increased membrane lipid peroxidation in PC12 cells when cells were incubated with micromolar FeCl₂/DTP. P₂ purinoceptor agonists, α,β-methylene ATP and 2-methylthio-ATP, induced PC12 cell lipid peroxidation, but to a lesser extent than ATP. ATP-induced Ca²⁺ influx via P₂ purinoceptor activation significantly increased the intracellular Ca²⁺ concentration, which may have triggered a free radical generating cascade(s), and led to membrane lipid peroxidation and cell death. Since oxidative stress has been implicated in certain neurodegenerative diseases such as aging, extracellular ATP may contribute to neuronal cell death by an oxidative mechanism involving lipid peroxidation.     

Mathematical Modelling of Ca2+ Oscillations in Airway Smooth Muscle Cells

The action of different agonists such as acetylcholine on the membrane of airway smooth muscle cells may induce cytosolic Ca²⁺ oscillations which can be a part of the Ca²⁺ signalling pathway, eventually leading to cell contraction. The aim of the present study is to present a mathematical model of the possible effect of the initial Ca²⁺ distribution within the cell on the form and frequency of induced Ca²⁺ oscillations. It takes into account intracellular Ca²⁺ stores such as sarcoplasmic reticulum and cytosolic proteins as well as Ca²⁺ exchange across the plasma membrane. We are able to demonstrate a closer agreement of model predictions with observed Ca²⁺ traces for a significantly wider range of parameter values, as was previously reported. We show also that the total cellular Ca²⁺ content is an important system parameter especially because of the content in sarcoplasmic reticulum. At a total Ca²⁺ increase of about 20%, the oscillation frequency increases by 25%; also, damped oscillations become sustained. Cases are indicated in which such a situation could occur.     

Role of phospholipases in cytosolic calcium overload and cardiomyocytes death in the presence of activated fatty acid derivatives

Ten to fifty micromoles of palmitoyl-L-carnitine (PC) or myristoyl-D,L-carnitine (MC) evoke a high-amplitude elevation of cytosolic calcium level ([Ca²⁺]_i), hypercontraction and cell death in the primary culture of rat ventricular myocytes. The lag period of this effect varies within 2–8 min and depends on the mitochondrial capacity to accumulate Ca²⁺. Maximal level of Ca²⁺, attainable at the end of the lag period, depends on calcium concentration in the external medium and is mediated by plasma membrane nonspecific permeability. Preincubation of cardiomyocytes with the inhibitors of phospholipase C, cytosolic phospholipase A₂ and/or Ca²⁺/calmodulin-dependent protein kinase II prevents cell death, increases lag period duration and reduces maximal [Ca²⁺]_i. Both PC and MC, even at low concentrations (1–5 μM), dramatically increase the frequency of Ca²⁺-sparks and Ca²⁺-waves in cardiomyocytes and promote the formation of sustained microdomains with elevated calcium concentration. We discuss possible mechanisms of Ca²⁺-microdomain formation, where the “vicious circle” of Ca²⁺-dependent phospholipases activation may arise. The “vicious circle” with combined autocatalytic action of Ca²⁺-dependent phospholipases may be implicated in hydrolysis of membrane phosphatidylcholine and subsequent induction of nonselective permeability for Na⁺ and Ca²⁺ (lipid pore).     

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis

Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe²⁺)‐dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron‐dependent, non‐apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.     

Mitochondrial permeability transition in Ca(2+)-dependent apoptosis and necrosis

A variety of stimuli utilize an increase of cytosolic free Ca²⁺ concentration as a second messenger to transmit signals, through Ca²⁺ release from the endoplasmic reticulum or opening of plasma membrane Ca²⁺ channels. Mitochondria contribute to the tight spatiotemporal control of this process by accumulating Ca²⁺, thus shaping the return of cytosolic Ca²⁺ to resting levels. The rise of mitochondrial matrix free Ca²⁺ concentration stimulates oxidative metabolism; yet, in the presence of a variety of sensitizing factors of pathophysiological relevance, the matrix Ca²⁺ increase can also lead to opening of the permeability transition pore (PTP), a high conductance inner membrane channel. While transient openings may serve the purpose of providing a fast Ca²⁺ release mechanism, persistent PTP opening is followed by deregulated release of matrix Ca²⁺, termination of oxidative phosphorylation, matrix swelling with inner membrane unfolding and eventually outer membrane rupture with release of apoptogenic proteins and cell death. Thus, a rise in mitochondrial Ca²⁺ can convey both apoptotic and necrotic death signals by inducing opening of the PTP. Understanding the signalling networks that govern changes in mitochondrial free Ca²⁺ concentration, their interplay with Ca²⁺ signalling in other subcellular compartments, and regulation of PTP has important implications in the fine comprehension of the main biological routines of the cell and in disease pathogenesis.     

Role of Caspases in N-Methyl-d-Aspartate-Induced Apoptosis in Cerebrocortical Neurons

Abstract: Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-d -aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1β-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICE_inh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD·fmk) that inhibits caspase activity. Pretreatment with either V-ICE_inh or Z-VAD·fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca²⁺ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca²⁺ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICE_inh or Z-VAD·fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICE_inh and Z-VAD·fmk. Taken together, our results suggest that Ca²⁺ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.     

Ferroptosis: Final destination for cancer?

Ferroptosis is a recently defined, non‐apoptotic, regulated cell death (RCD) process that comprises abnormal metabolism of cellular lipid oxides catalysed by iron ions or iron‐containing enzymes. In this process, a variety of inducers destroy the cell redox balance and produce a large number of lipid peroxidation products, eventually triggering cell death. However, in terms of morphology, biochemistry and genetics, ferroptosis is quite different from apoptosis, necrosis, autophagy‐dependent cell death and other RCD processes. A growing number of studies suggest that the relationship between ferroptosis and cancer is extremely complicated and that ferroptosis promises to be a novel approach for the cancer treatment. This article primarily focuses on the mechanism of ferroptosis and discusses the potential application of ferroptosis in cancer therapy.     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis

Ferroptosis, a newly defined mode of regulated cell death caused by unbalanced lipid redox metabolism, is implicated in various tissue injuries and tumorigenesis. However, the role of ferroptosis in stem cells has not yet been investigated. Glutathione peroxidase 4 (GPX4) is a critical suppressor of lipid peroxidation and ferroptosis. Here, we study the function of GPX4 and ferroptosis in hematopoietic stem and progenitor cells (HSPCs) in mice with Gpx4 deficiency in the hematopoietic system. We find that Gpx4 deletion solely in the hematopoietic system has no significant effect on the number and function of HSPCs in mice. Notably, hematopoietic stem cells (HSCs) and hematopoietic progenitor cells lacking Gpx4 accumulated lipid peroxidation and underwent ferroptosis in vitro. α-Tocopherol, the main component of vitamin E, was shown to rescue the Gpx4-deficient HSPCs from ferroptosis in vitro. When Gpx4 knockout mice were fed a vitamin E-depleted diet, a reduced number of HSPCs and impaired function of HSCs were found. Furthermore, increased levels of lipid peroxidation and cell death indicated that HSPCs undergo ferroptosis. Collectively, we demonstrate that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs.Subject terms: Cell biology, Physiology, Stem-cell research     

Hydrogen sulfide guards myoblasts from ferroptosis by inhibiting ALOX12 acetylation

Recognized as a novel and important gasotransmitter, hydrogen sulfide (H₂S) is widely present in various tissues and organs. Cystathionine gamma-lyase (CSE)-derived H₂S has been shown to regulate oxidative stress and lipid metabolism. The aim of the present study is to examine the role of H₂S in ferroptosis and lipid peroxidation in mouse myoblasts and skeletal muscles. Ferroptosis agonist RSL3 inhibited the expressions of Gpx4 and reduced CSE/H₂S signaling, which lead to increased oxidative stress, lipid peroxidation, and ferroptotic cell death. In addition, ferroptosis antagonist ferrostatin-1 (Fer-1) up-regulated the expression of CSE, scavenged the generation of reactive oxygen species (ROS) and lipid peroxidation, and improved cell viability. Exogenously applied NaHS was also able to block RSL3-induced ferroptotic cell death. Neither RSL3 nor H₂S affected cell apoptosis. Furthermore, H₂S reversed RSL3-induced Drp1 expression and mitochondrial damage, which lead to abnormal lipid metabolism as evidenced by altered expressions of ACSL4, FAS, ACC and CPT1 as well as higher acetyl-CoA contents in both cytoplasm and mitochondria. RSL3 promoted the protein expression and acetylation of ALOX12, a key protein in initiating membrane phospholipid oxidation, while the addition of NaHS attenuated ALOX12 acetylation and protected from membrane lipid peroxidation. Moreover, we observed that CSE deficiency alters the expressions of ferroptosis and lipid peroxidation-related proteins and enhances global protein acetylation in mouse skeletal muscles under aging or injury conditions. These results indicate that downregulation of CSE/H₂S signaling would contribute to mitochondrial damage, abnormal lipid metabolism, membrane lipid peroxidation, and ferroptotic cell death. CSE/H₂S system can be a target for preventing ferroptosis in skeletal muscle.     

Plasma-activated medium induces ferroptosis by depleting FSP1 in human lung cancer cells

Cold atmospheric plasma (CAP) that generates reactive oxygen species (ROS) has received considerable scientific attentions as a new type of anticancer. In particular, an indirect treatment method of inducing cancer cell death through plasma-activated medium (PAM), rather than direct plasma treatment has been well established. Although various cell death pathways such as apoptosis, necroptosis, and autophagy have been suggested to be involved in PAM-induced cell death, the involvement of ferroptosis, another type of cell death regulated by lipid ROS is largely unknown. This study reports, that PAM promotes cell death via ferroptosis in human lung cancer cells, and PAM increases intracellular and lipid ROS, thereby resulting in mitochondrial dysfunction. The treatment of cells with N-acetylcysteine, an ROS scavenging agent, or ferrostatin-1, a ferroptosis inhibitor, protects cells against PAM-induced cell death. Interestingly, ferroptosis suppressor protein 1 (FSP1) is downregulated upon PAM treatment. Furthermore, the treatment of cells with iFSP1, an inhibitor of FSP1, further enhances PAM-induced ferroptosis. Finally, this study demonstrates that PAM inhibits tumor growth in a xenograft model with an increase in 4-hydroxynoneal and PTGS2, a byproduct of lipid peroxidation, and a decrease in FSP1 expression. This study will provide new insights into the underlying mechanism and therapeutic strategies of PAM-mediated cancer treatment.Subject terms: Non-small-cell lung cancer, Drug development