Synergism of non-thermal plasma and low concentration RSL3 triggers ferroptosis via promoting xCT lysosomal degradation through ROS/AMPK/mTOR axis in lung cancer cells.

BACKGROUND: Though (1S, 3R)-RSL3 has been used widely in basic research as a small molecular inducer of ferroptosis, the toxicity on normal cells and poor pharmacokinetic properties of RSL3 limited its clinical application. Here, we investigated the synergism of non-thermal plasma (NTP) and low-concentration RSL3 and attempted to rise the sensitivity of NSCLC cells on RSL3. METHODS: CCK-8 assay was employed to detect the change of cell viability. Microscopy and flowcytometry were applied to identify lipid peroxidation, cell death and reactive oxygen species (ROS) level respectively. The molecular mechanism was inspected with western blot and RT-qPCR. A xenograft mice model was adopted to investigate the effect of NTP and RSL3. RESULTS: We found the synergism of NTP and low-concentration RSL3 triggered severe mitochondria damage, more cell death and rapid ferroptosis occurrence in vitro and in vivo. NTP and RSL3 synergistically induced xCT lysosomal degradation through ROS/AMPK/mTOR signaling. Furthermore, we revealed mitochondrial ROS was the main executor for ferroptosis induced by the combined treatment. CONCLUSION: Our research shows NTP treatment promoted the toxic effect of RSL3 by inducing more ferroptosis rapidly and provided possibility of RSL3 clinical application.

ß-Carbolines norharman and harman change neurobehavior causing neurological damage in Caenorhabditis elegans.

ß-Carbolines norharman and harman, belonging to the class of heterocyclic aromatic amines (HAAs), are typical hazardous substances produced during the thermal processing of food. Compared to other HAAs, there have been limited reports on the toxicity of ß-carbolines. Nevertheless, the current studies are concerned with the neurotoxic effects of norharman and harman at high doses. It is still unknown whether the relatively low dose of ß-carbolines in foods induces neurotoxicity and the mechanism of the toxicity. In this study, C. elegans was exposed to a series of gradients of norharman and harman (0, 0.05, 5, and 10 mg L-1). The survival rate and indicators of ethology (locomotor behaviors, foraging behavior, and chemotaxis ability) were assessed. The antioxidant system and the contents of neurotransmitters, as well as the activity of acetylcholinesterase (AChE), were evaluated. Additionally, the RNA-seq screening of differentially expressed genes (DEGs) revealed the potential molecular mechanisms of norharman- and harman-induced toxic effects. Our results indicated that the risk of long-term exposure to norharman and harman at low doses (food-related doses) should be emphasized. Moreover, ß-carbolines might induce neurotoxicity by causing oxidative damage, regulating the content of neurotransmitters, and interfering with cytochrome P450 metabolism. This study would provide a toxicological basis for the neurotoxicity of ß-carbolines and lay the foundation for the risk assessment of endogenous pollutants in food.

Health effects of exposure to ß-carboline heterocyclic amines: insight into metabolic perturbations and biochemical analysis.

As a class of bioactive and toxic compounds widely present in foodstuffs, the health effects of dietary exposure to ß-carboline heterocyclic amines (HAs) have not been elucidated. Based on our previous research that a typical ß-carboline HA (harmane) affects blood glucose metabolism and organ dysfunction, the present study mainly focused on the health effects of dietary exposure to harmane in diabetic Goto-Kakizaki (GK) rats. Twenty-four GK rats were administered daily with harmane (0.1 mg per kg body weight) for eight weeks. A comprehensive evaluation of the health effects of harmane was conducted on serum biochemistry, histopathology, and GC-TOF-MS-based metabolomics. The results showed that harmane exerts non-significant effects on the blood glucose metabolism of GK rats. However, it did cause pathological damage to gastrocnemius nerves and showed adverse effects on brain neurons by significantly activating astrocytes and downregulating brain-derived neurotrophic factor (BDNF), which are potential mechanisms related to the disruption of the normal glutamine-glutamate/γ-aminobutyric acid cycle. Moreover, an increased value of AST and urea, alterations in the amino acid, carbohydrate, purine, pyrimidine, and gut microbiota metabolism as well as the tricarboxylic acid (TCA) cycle could be associated with kidney, liver, and gut dysfunction. Our results suggest that given the role of harmane in nerve injury in GK rats, reducing the production and consumption of ß-carboline heterocyclic amines in our daily diets should be considered.

LUHMES Dopaminergic Neurons Are Uniquely Susceptible to Ferroptosis.

Ferroptosis is a necrotic cell death caused by lipid oxidation that may be responsible for neural degeneration in Parkinson's disease. We assessed whether three neuronal cell lines are sensitive to killing by ferroptosis. Ferroptosis inducer erastin killed LUHMES neurons at sub-micromolar concentrations, whereas neuronal cells derived from SH-SY5Y cells or neural stem cells were at least 50-fold less sensitive. LUHMES differentiated neurons were likewise sensitive to killing by RSL3 or ML210, inhibitors of the glutathione peroxidase 4 enzyme (GPX4) that consumes GSH to detoxify lipid peroxides. Additional assays showed that erastin, RSL3, and ML210 increased lipid peroxide levels, and that LUHMES neurons were protected from both peroxide accumulation and cell death by ferrostatin-1. A possible role of iron was assessed by evaluating the effects of five metal chelators on cytotoxicity of erastin and RSL3. LUHMES neurons were protected from RSL3 by three of the chelators, 2,3-dimercapto-1-propanesulfonic acid (DMPS), deferoxiprone (DFX), and deferiprone (DFP). Collectively, these results demonstrate the vulnerability of LUHMES neurons to ferroptosis by chemical treatments that disrupt glutathione synthesis, lipid peroxide detoxification, or iron metabolism. The same vulnerabilities may occur in CNS neurons, which reportedly generate low levels of GSH and metallothioneins, limiting their ability to neutralize oxidative stresses and toxic metals. These results suggest a rationale and methods to search for environmental toxicants that may exploit these vulnerabilities and promote neurodegenerative diseases.

Baicalein inhibits RLS3-induced ferroptosis in melanocytes.

We explored the effect of baicalein on the ferroptosis of melanocytes in vitiligo. Melanocytes were treated with single RSL3 or combined RSL3 with FAC for 24 h and the effect of baicalein on RSL3 toxicity was further evaluated. Cell viability was examined by CCK8 assay. The mitochondrial membrane potential and the level of iron ion were measured by assay kit. Intracellular and lipid ROS production was detected by flow cytometry. The results indicated that RSL3 induced cell death, mitochondrial dysfunction, ROS production, and iron ion accumulation in melanocytes, which was aggravated by the addition of FAC. The damage induced by RSL3 was significantly relieved by baicalein treatment. Besides, baicalein up-regulated GPX4 and reduced TFR1 level in melanocytes treated with RSL3+FAC. Baicalein protected melanocytes against ferroptosis through up-regulating GPX4. Ferroptosis might be pervasive in the occurrence and development of vitiligo, and could be proposed as the potential therapeutic target.

Cytoglobin promotes sensitivity to ferroptosis by regulating p53-YAP1 axis in colon cancer cells.

Ferroptosis is an iron-dependent mode of non-apoptotic cell death characterized by accumulation of lipid reactive oxygen species (ROS). As a regulator of ROS, cytoglobin (CYGB) plays an important role in oxygen homeostasis and acts as a tumour suppressor. However, the mechanism by which CYGB regulates cell death is largely unknown. Here, we show that CYGB overexpression increased ROS accumulation and disrupted mitochondrial function as determined by the oxygen consumption rate and membrane potential. Importantly, ferroptotic features with accumulated lipid ROS and malondialdehyde were observed in CYGB-overexpressing colorectal cancer cells. Moreover, CYGB significantly increased the sensitivity of cancer cells to RSL3- and erastin-induced ferroptotic cell death. Mechanically, both YAP1 and p53 were significantly increased based on the RNA sequencing. The knock-down of YAP1 alleviated production of lipid ROS and sensitivity to ferroptosis in CYGB overexpressed cells. Furthermore, YAP1 was identified to be inhibited by p53 knock-down. Finally, high expression level of CYGB had the close correlation with key genes YAP1 and ACSL4 in ferroptosis pathway in colon cancer based on analysis from TCGA data. Collectively, our results demonstrated a novel tumour suppressor role of CYGB through p53-YAP1 axis in regulating ferroptosis and suggested a potential therapeutic approach for colon cancer.

Novel Carboline Fungal Histone Deacetylase (HDAC) Inhibitors for Combinational Treatment of Azole-Resistant Candidiasis.

Due to the evolution and development of antifungal drug resistance, limited efficacy of existing drugs has led to high mortality in patients with serious fungal infections. To develop novel antifungal therapeutic strategies, herein a series of carboline fungal histone deacetylase (HDAC) inhibitors were designed and synthesized, which had potent synergistic effects with fluconazole against resistant Candida albicans infection. In particular, compound D12 showed excellent in vitro and in vivo synergistic antifungal efficacy with fluconazole to treat azole-resistant candidiasis. It cooperated with fluconazole in reducing the virulence of C. albicans by blocking morphological mutual transformation and inhibiting biofilm formation. Mechanism studies revealed that the reversion of drug resistance was due to downregulation of the expression of the azole target gene ERG11 and efflux gene CDR1. Taken together, fungal HDAC inhibitor D12 offered a promising lead compound for combinational treatment of azole-resistant candidiasis.

Involvement of oxidative stress-induced annulus fibrosus cell and nucleus pulposus cell ferroptosis in intervertebral disc degeneration pathogenesis.

Ferroptosis is a necrotic form of regulated cell death that was associated with lipid peroxidation and free iron-mediated Fenton reactions. It has been reported that iron deficiency had been implicated in the pathogenesis of intervertebral disc degeneration (IVDD) by activating apoptosis. However, the role of ferroptosis in the process of IVDD has not been illuminated. Here, we demonstrate the involvement of ferroptosis in IVDD pathogenesis. Our in vitro models show the changes in protein levels of ferroptosis marker and enhanced lipid peroxidation level during oxidative stress. Safranin O staining, hematoxylin-eosin staining, and immunohistochemical were used to assess the IVDD after 8 weeks of surgical procedure in vivo. Treatment with ferrostatin-1, deferoxamine, and RSL3 demonstrate the role of ferroptosis in tert-butyl hydroperoxide (TBHP)-treated annulus fibrosus cells (AFCs) and nucleus pulposus cells (NPCs). Ferritinophagy, nuclear receptor coactivator 4 (NCOA4)-mediated ferritin selective autophagy, is originated during the process of ferroptosis in response to TBHP treatment. Knockdown and overexpression NCOA4 further prove TBHP may induce ferroptosis of AFCs and NPCs in an autophagy-dependent way. These findings support a role for oxidative stress-induced ferroptosis in the pathogenesis of IVDD.

Biological significance of aminophenyl-ß-carboline derivatives formed from co-mutagenic action of ß-carbolines and aniline and o-toluidine and its effect on tumorigenesis in humans: A review.

Norharman exists in cigarette smoke and cooked foods and is non-mutagenic among Salmonella strains but mutagenic to S. typhimurium TA98 and YG1024 in the presence of S9 mix and aniline and o-toluidine. Co-mutagenesis of ß-carbolines and aniline and o-toluidine occurs through the formation of novel mutagenic aminophenyl-ß-carboline derivatives including 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole [aminophenylnorharman] (APNH)] and 9-(4'- amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole [aminomethylphenylnorharman] (AMPNH)]. Since humans are often simultaneously exposed to ß-carbolines and aniline and o-toluidine, their effects on humans should be clarified. The most potent of these, APNH, induced both point mutations and small deletions in the liver and colon of gpt delta transgenic mice. Major APNH-induced mutations in the liver occurred at a G:C base pair, suggesting that APNH-DNA adducts (dG-C8-APNH) are potentially involved in these mutations. Furthermore, APNH induced hepatic and colon tumors harboring K-ras, ß-catenin, and Apc mutations in F344 rats, with high incidence. Mutations at G:C base pairs were predominant, similar to those in the in vivo mutation assay using gpt delta mice. Moreover, APNH detected in human urine samples obtained from both healthy volunteers on a normal diet and inpatients receiving parenteral alimentation; therefore, APNH can be considered an endogenous carcinogen contributing to tumorigenesis. Exposure levels of these aminophenyl-ß-carboline derivatives may be lower than those of carcinogenic heterocyclic amines (HCAs) including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); however, their health risks in terms of tumorigenesis may be comparable owing to stronger genotoxic effects of APNH rather than HCAs. This review summarized APNH mutagenicity/carcinogenicity, and its in vivo formation. Moreover, the effect on tumorigenesis in humans also discussed.

Tryptoline-based benzothiazoles re-sensitize MRSA to ß-lactam antibiotics.

Resistance-modifying agents (RMAs) offer a promising solution to combat bacterial antibiotic resistance. Here we report the discovery and structure-activity relationships of a new class of RMAs with a novel tryptoline-based benzothiazole scaffold. Our most potent compound in this series (4ad) re-sensitizes multiple MRSA strains to cephalosporins at low concentrations (2 µg/mL) and has low mammalian cytotoxicity with a half growth inhibitory concentration (GI50) > 100 µg/mL in human cervical carcinoma (HeLa) cells. In addition, the same core scaffold with different substitutions also gives good antibacterial activity against MRSA.

Bioactivation of the tobacco carcinogens 4-aminobiphenyl (4-ABP) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in human bladder RT4 cells.

Occupational and tobacco exposure to aromatic amines (AAs) including 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA) are associated with bladder cancer (BC) risk. Several epidemiological studies have also reported a possible role for structurally related heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meats with BC risk. We had screened for DNA adducts of 4-ABP, 2-NA, and several prominent HAAs formed in tobacco smoke or grilled meats including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-9H-pyrido[2,3-b]indole (AαC) in the bladder DNA of BC patients, using liquid chromatography/mass spectrometry. We detected DNA adducts of 4-ABP, but not adducts of the other carcinogens. In this study, we have examined the capacity of RT4 cells, an epithelial human bladder cell line, to bioactivate AAs and HAAs to DNA damaging agents, which may contribute to BC. 4-ABP and AαC formed DNA adducts, but DNA adducts of 2-NA, PhIP, and MeIQx were not detected. 4-ABP DNA adducts were formed at tenfold higher levels than AαC adducts. Pretreatment of RT4 cells with α-naphthoflavone (1-10 µM), a specific cytochrome P450 1 (CYP1) inhibitor, decreased AαC adduct formation by 50% but did not affect the level of 4-ABP adducts. However, cell pretreatment with 8-methoxypsoralen (0.1-1 µM), a potent inhibitor of CYP2A, resulted in a 90% decrease of 4-ABP DNA adducts levels. These data signify that CYP2A and CYP1A isoforms expressed in the target urothelium bioactivate 4-ABP and AαC, respectively, and may be a critical feature of aromatic amine-induced urinary bladder carcinogenesis. The bioactivation of other tobacco and environmental AAs by bladder CYPs and their ensuing bladder DNA damage warrants further study.

In-silico design and synthesis of N9-substituted ß-Carbolines as PLK-1 inhibitors and their in-vitro/in-vivo tumor suppressing evaluation.

A new series of ß-Carboline/Schiff bases was designed, synthesized, characterised and biologically evaluated as inhibitors of PLK-1. The synthesized compounds exhibited strong to moderate cytotoxic activities against NCI-60 panel cell assay. Compound SB-2 was the most potent, particularly against colon with GI50 of 3-45 µM on NCI-60 panel cell lines. SB-2 selectively inhibited PLK-1 at 15 µM on KinomeScan screening. It also showed a dose-dependent cell cycle arrest at S/G2 phase on HCT-116 and induced apoptosis by the activation of procaspase-3 and cleaved PARP. Further, the antitumor studies on DLA and EAC model revealed that SB-2, at 100 mg/kg/bd.wt significantly increased their average lifespan. Further, a decrease in the body weight of the tumor-bearing mice was also observed when compared to the tumor controlled mice. SB-2 thus shows good potential as antitumor agent.

Fluorescent Canthin-6-one Alkaloids from Simaba bahiensis: Isolation, Identification, and Cell-Labeling Properties.

Canthin-6-one alkaloids, which are present in plants of the genus Simaba, are natural compounds that are capable of acting as fluorescent probes. However, the chemical composition and fluorescent properties of most species of this genus have not been analyzed. The objective of this study was to characterize the fluorescent properties of an extract of S. bahiensis and identify the chemical entities responsible for these properties. In addition, the cell-labeling properties of the fluorescent dye from A and of the isolated compounds were characterized by confocal fluorescence microscopy and flow cytometry. One quassinoid and three fluorescent alkaloids were isolated from S. bahiensis, all compounds were identified by using NMR spectroscopy and high-resolution mass spectrometry. Staining experiments and HPLC-FL analysis shown that canthin-6-one alkaloids are the main green fluorescent compounds in the analyzed dyes. All compounds evaluated showed a cytoplasmic marker with a residence time of 24 h. The present study is the first to describe the presence of canthin-6-one alkaloids in S. bahiensis, in addition to demonstrating promising cell-labeling properties of fluorescent compounds from S. bahiensis with broad emission wavelengths.

Assessment of toxicity and differential antimicrobial activity of methanol extract of rhizome of Simaba ferruginea A. St.-Hil. and its isolate canthin-6-one.

ETHNOPHARMACOLOGICAL RELEVANCE: Simaba ferruginea A. St.-Hil., Simaroubaceae, popularly known as "calunga" is a typical subtropical shrub used in Central Brazil mainly for infection, anti-inflammatory, analgesic and gastric duodenal-ulcers. It presents in its composition the alkaloid canthin-6-one, an alkaloid indole ß-carboxylic. AIM: This study aims to investigate the toxicity, antimicrobial activities of methanol extract of Simaba ferruginea (MESf) and canthin-6-one by using different experimental models. METHODS: The present study evaluated the phytochemical analysis by high performance liquid chromatography (HPLC), toxicological potential of MESf and canthin-6-one, using the cytotoxicity, genotoxicity assays with CHO-K1 cells and in vivo acute test in mice. Antimicrobial activity was evaluated by the broth microdilution assays, while the antimicrobial mechanism of action was also assessed using different in vitro bacterial and fungal models. RESULTS: The HPLC analysis of MESf revealed the presence of canthin-6-one, kaempferol and morin. Differential in vitro toxicities were observed between MESf and canthin-6-one. In the cytotoxicity assay, MESf presented toxicity against CHO-K1, while canthin-6-one did not. In the case of in vitro genotoxicity, both showed to be potentially genotoxic. In the in vivo toxicity study, both MESf (up to 1000 mg/kg) and cantin-6-one (up to 100 mg/kg) caused no toxicologically relevant alterations and are thus considered not to be toxic. MESf was shown to be relatively safe with NOAEL (100 mg/kg) when administrate in mice. Both MESf and canthin-6-one also showed differential antimicrobial activities. On one hand, MESf demonstrated good spectrum of antibacterial action against Staphylococcus aureus (MIC 12.5 µg/mL) and Escherichia coli (MIC 25 µg/mL) and moderate activity against Enterococcus faecalis and Shigella flexneri (MIC 200 µg/mL) but no antifungal effect. On the hand, canthin-6-one showed no antibacterial activity, except against Staphylococcus aureus (100 µg/mL), but potent in vitro fungicidal activity against clinically important Aspergillus niger and Candida species at MFC intervals ranging from 3.12 to 25 µg/mL. Both MESf and canthin-6-one were bacteriostatic in action. MESf antimicrobial mechanism of actions are associated with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in Shigella flexneri, intense K+ efflux (Shigella flexneri, Staphylococcus aureus) and nucleotides leakage (Staphylococcus aureus). In the antifungal mode of action, canthin-6-one inhibited Saccharomyces cerevisiae growth and including alteration in the cell membrane of Neurospora crassa. CONCLUSION: The results of this work demonstrated the differential antimicrobial activities of MESf and its alkaloid isolate, canthin-6-one with antibacterial and antifungal activities, respectively. The present study support the popular use of Simaba ferruginea in combatting afflictions related to bacterial infections, and demonstrate that canthin-6-one as a promising antifungal agent. Both MESf and canthin-6-one are considered non-toxic based on the in vitro toxicological study.

Marbostat-100 Defines a New Class of Potent and Selective Antiinflammatory and Antirheumatic Histone Deacetylase 6 Inhibitors.

Epigenetic modifiers of the histone deacetylase (HDAC) family contribute to autoimmunity, cancer, HIV infection, inflammation, and neurodegeneration. Hence, histone deacetylase inhibitors (HDACi), which alter protein acetylation, gene expression patterns, and cell fate decisions, represent promising new drugs for the therapy of these diseases. Whereas pan-HDACi inhibit all 11 Zn2+-dependent histone deacetylases (HDACs) and cause a broad spectrum of side effects, specific inhibitors of histone deacetylase 6 (HDAC6i) are supposed to have less side effects. We present the synthesis and biological evaluation of Marbostats, novel HDAC6i that contain the hydroxamic acid moiety linked to tetrahydro-ß-carboline derivatives. Our lead compound Marbostat-100 is a more potent and more selective HDAC6i than previously established well-characterized compounds in vitro as well as in cells. Moreover, Marbostat-100 is well tolerated by mice and effective against collagen type II induced arthritis. Thus, Marbostat-100 represents a most selective known HDAC6i and the possibility for clinical evaluation of a HDAC isoform-specific drug.

Selective dopaminergic neurotoxicity of three heterocyclic amine subclasses in primary rat midbrain neurons.

Heterocyclic amines (HCAs) are primarily produced during high temperature meat cooking. These compounds have been intensively investigated as mutagens and carcinogens. However, converging data suggest that HCAs may also be neurotoxic and potentially relevant to neurodegenerative diseases such as Parkinson's disease (PD). The identification of new potential etiological factors is important because most PD cases are sporadic. Our group previously showed that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was selectively neurotoxic to dopaminergic neurons. However, PhIP is one of many HCAs, a class of compounds that exhibits wide structural variability. The goal of this study was to determine the neurotoxicity of the most prevalent and best studied HCAs from three subclasses: aminoimidazoaazarenes (AIA), α-carbolines, and ß-carbolines. Using E17 rat primary midbrain cultures, we tested dopaminergic and non-dopaminergic neurotoxicity elicited by the following compounds: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), PhIP, 1-methyl-9H-pyrido[3,4-b]indole (harmane), 9H-pyrido[3,4-b]indole (norharmane) and 2-amino-9H-pyrido[2,3-b]indole (AαC) at concentrations ranging from 100 nM-5 µM. All tested HCAs were selectively neurotoxic, though the dose required to elicit selective loss of dopaminergic neurons or decreases in dopaminergic neurite length was compound specific. Non-dopaminergic neurons were unaffected at all tested doses. The sensitivity (determined by threshold dose required to elicit selective neurotoxicity) appears to be unrelated to published mutagenic potency. Both AIA and α/ß-carbolines produced oxidative damage, which was magnified in dopaminergic neurons vs. non-dopaminergic neurons as further evidence of selective neurotoxicity. These studies are expected to prompt clinical and mechanistic studies on the potential role of HCA exposure in PD.

Mitochondrial rescue prevents glutathione peroxidase-dependent ferroptosis.

Research into oxidative cell death is producing exciting new mechanisms, such as ferroptosis, in the neuropathologies of cerebral ischemia and hemorrhagic brain insults. Ferroptosis is an oxidative form of regulated necrotic cell death featuring glutathione (GSH) depletion, disrupted glutathione peroxidase-4 (GPX4) redox defense and detrimental lipid reactive oxygen species (ROS) formation. Further, our recent findings identified mitochondrial damage in models of oxidative glutamate toxicity, glutathione peroxidase depletion, and ferroptosis. Despite knowledge on the signaling pathways of ferroptosis increasing, the particular role of mitochondrial damage requires more in depth investigation in order to achieve effective treatment options targeting mitochondria. In the present study, we applied RSL3 to induce ferroptosis in neuronal HT22 cells and mouse embryonic fibroblasts. In both cell types, RSL3 mediated concentration-dependent inhibition of GPX4, lipid peroxidation, enhanced mitochondrial fragmentation, loss of mitochondrial membrane potential, and reduced mitochondrial respiration. Ferroptosis inhibitors, such as deferoxamine, ferrostatin-1 and liproxstatin-1, but also CRISPR/Cas9 Bid knockout and the BID inhibitor BI-6c9 protected against RSL3 toxicity. We found compelling new information that the mitochondria-targeted ROS scavenger mitoquinone (MitoQ) preserved mitochondrial integrity and function, and cell viability despite significant loss of GPX4 expression and associated increases in general lipid peroxidation after exposure to RSL3. Our data demonstrate that rescuing mitochondrial integrity and function through the inhibition of BID or by the mitochondria-targeted ROS scavenger MitoQ serves as a most effective strategy in the prevention of ferroptosis in different cell types. These findings expose mitochondria as promising targets for novel therapeutic intervention strategies in oxidative cell death.

Protective effects of the mechanistic target of rapamycin against excess iron and ferroptosis in cardiomyocytes.

Clinical studies have suggested that myocardial iron is a risk factor for left ventricular remodeling in patients after myocardial infarction. Ferroptosis has recently been reported as a mechanism of iron-dependent nonapoptotic cell death. However, ferroptosis in the heart is not well understood. Mechanistic target of rapamycin (mTOR) protects the heart against pathological stimuli such as ischemia. To define the role of cardiac mTOR on cell survival in iron-mediated cell death, we examined cardiomyocyte (CM) cell viability under excess iron and ferroptosis conditions. Adult mouse CMs were isolated from cardiac-specific mTOR transgenic mice, cardiac-specific mTOR knockout mice, or control mice. CMs were treated with ferric iron [Fe(III)]-citrate, erastin, a class 1 ferroptosis inducer, or Ras-selective lethal 3 (RSL3), a class 2 ferroptosis inducer. Live/dead cell viability assays revealed that Fe(III)-citrate, erastin, and RSL3 induced cell death. Cotreatment with ferrostatin-1, a ferroptosis inhibitor, inhibited cell death in all conditions. mTOR overexpression suppressed Fe(III)-citrate, erastin, and RSL3-induced cell death, whereas mTOR deletion exaggerated cell death in these conditions. 2',7'-Dichlorodihydrofluorescein diacetate measurement of reactive oxygen species (ROS) production showed that erastin-induced ROS production was significantly lower in mTOR transgenic versus control CMs. These findings suggest that ferroptosis is a significant type of cell death in CMs and that mTOR plays an important role in protecting CMs against excess iron and ferroptosis, at least in part, by regulating ROS production. Understanding the effects of mTOR in preventing iron-mediated cell death will provide a new therapy for patients with myocardial infarction. NEW & NOTEWORTHY Ferroptosis has recently been reported as a new form of iron-dependent nonapoptotic cell death. However, ferroptosis in the heart is not well characterized. Using cultured adult mouse cardiomyocytes, we demonstrated that the mechanistic target of rapamycin plays an important role in protecting cardiomyocytes against excess iron and ferroptosis.

Heme oxygenase-1 mitigates ferroptosis in renal proximal tubule cells.

Ferroptosis is an iron-dependent form of regulated nonapoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI because of its antiapoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of antioxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared with vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Cotreatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-l-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important antiferroptotic role of HO-1 in renal epithelial cells.