DHA exhibits synergistic therapeutic efficacy with cisplatin to induce ferroptosis in pancreatic ductal adenocarcinoma via modulation of iron metabolism

Pancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with limited treatment options. Cisplatin (DDP) is used as a mainstay of chemotherapeutic agents in combination with other drugs or radiotherapy for PDAC therapy. However, DDP exhibits severe side-effects that can lead to discontinuation of therapy, and the acquired drug resistance of tumor cells presents serious clinical obstacles. Therefore, it is imperative to develop a more effective and less toxic therapeutic strategy. We and others have previously discovered that dihydroartemisinin (DHA) represents a safe and promising therapeutic agent to preferentially induce cancer cell ferroptosis. In the present study, we find that DHA could intensively strengthen the cytotoxicity of DDP and significantly reduce its effective concentrations both in vitro and in vivo. Combination of DHA and DDP synergistically inhibits the proliferation and induces DNA damage of PDAC cells. Mechanically, the combinative treatment impairs mitochondrial homeostasis, characterized by destroyed mitochondrial morphology, decreased respiratory capacity, reduced ATP production, and accumulated mitochondria-derived ROS. Further studies show that ferroptosis contributes to the cytotoxic effects in PDAC cells under the challenge of DHA and DDP, together with catastrophic accumulation of free iron and unrestricted lipid peroxidation. Moreover, pharmacologic depleting of the free iron reservoir or reconstituted expression of FTH contributes to the tolerance of DHA/DDP-induced ferroptosis, while iron addition accelerates the ferroptotic cell death. In summary, these results provide experimental evidence that DHA acts synergistically with DDP and renders PDAC cells vulnerable to ferroptosis, which may act as a promising therapeutic strategy.Subject terms: Chemotherapy, Preclinical research     

Liproxstatin-1 attenuates unilateral ureteral obstruction-induced renal fibrosis by inhibiting renal tubular epithelial cells ferroptosis

Renal fibrosis is a common pathological process that occurs with diverse etiologies in chronic kidney disease. However, its regulatory mechanisms have not yet been fully elucidated. Ferroptosis is a form of non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. It is currently unknown whether ferroptosis is initiated during unilateral ureteral obstruction (UUO)-induced renal fibrosis and its role has not been determined. In this study, we demonstrated that ureteral obstruction induced ferroptosis in renal tubular epithelial cells (TECs) in vivo. The ferroptosis inhibitor liproxstatin-1 (Lip-1) reduced iron deposition, cell death, lipid peroxidation, and inhibited the downregulation of GPX4 expression induced by UUO, ultimately inhibiting ferroptosis in TECs. We found that Lip-1 significantly attenuated UUO-induced morphological and pathological changes and collagen deposition of renal fibrosis in mice. In addition, Lip-1 attenuated the expression of profibrotic factors in the UUO model. In vitro, we used RSL3 treatment and knocked down of GPX4 level by RNAi in HK2 cells to induce ferroptosis. Our results indicated HK2 cells secreted various profibrotic factors during ferroptosis. Lip-1 was able to inhibit ferroptosis and thereby inhibit the secretion of the profibrotic factors during the process. Incubation of kidney fibroblasts with culture medium from RSL3-induced HK2 cells promoted fibroblast proliferation and activation, whereas Lip-1 impeded the profibrotic effects. Our study found that Lip-1 may relieve renal fibrosis by inhibiting ferroptosis in TECs. Mechanistically, Lip-1 could reduce the activation of surrounding fibroblasts by inhibiting the paracrine of profibrotic factors in HK2 cells. Lip-1 may potentially be used as a therapeutic approach for the treatment of UUO-induced renal fibrosis.Subject terms: Cell death, RNAi, Urinary tract obstruction     

Role of ACSL4 in the chemical-induced cell death in human proximal tubule epithelial HK-2 cells

Acyl-CoA synthetase long-chain family member 4 (ACSL4) activates polyunsaturated fatty acids (PUFAs) to produce PUFA-derived acyl-CoAs, which are utilised for the synthesis of various biological components, including phospholipids (PLs). Although the roles of ACSL4 in non-apoptotic programmed cell death ferroptosis are well-characterised, its role in the other types of cell death is not fully understood. In the present study, we investigated the effects of ACSL4 knockdown on the levels of acyl-CoA, PL, and ferroptosis in the human normal kidney proximal tubule epithelial (HK-2) cells. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses revealed that the knockdown of ACSL4 markedly reduced the levels of PUFA-derived acyl-CoA, but not those of other acyl-CoAs. In contrast with acyl-CoA levels, the docosahexaenoic acid (DHA)-containing PL levels were preferentially decreased in the ACSL4-knockdown cells compared with the control cells. Cell death induced by the ferroptosis inducers RSL3 and FIN56 was significantly suppressed by treatment with ferrostatin-1 or ACSL4 knockdown, and, unexpectedly, upon treating with a necroptosis inhibitor. In contrast, ACSL4 knockdown failed to suppress the other oxidative stress-induced cell deaths initiated by cadmium chloride and sodium arsenite. In conclusion, ACSL4 is involved in the biosynthesis of DHA-containing PLs in HK-2 cells and is specifically involved in the cell death induced by ferroptosis inducers.     

NMDA receptor activation induces damage of alveolar type II cells and lung fibrogenesis through ferroptosis

Ferroptosis, a newly discovered type of regulated cell death, has been implicated in numerous human diseases. Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease with poor prognosis and limited treatment options. Emerging evidence has linked ferroptosis and glutamate-determined cell fate which is considered a new light on the etiology of pulmonary fibrosis. Here, we observed that N-methyl d-aspartate receptor (NMDAR) activation promoted cell damage and iron deposition in MLE-12 cells in a dose-, time-, and receptor-dependent manner. This mediated substantial Ca²⁺ influx, upregulated the expression levels of nNOS and IRP1, and affected intracellular iron homeostasis by regulating the expression of iron transport-related proteins (i.e., TFR1, DMT1, and FPN). Excessive iron load promoted the continuous accumulation of total intracellular and mitochondrial reactive oxygen species, which ultimately led to ferroptosis. NMDAR inhibition reduced lung injury and pulmonary fibrosis in bleomycin-induced mice. Bleomycin stimulation upregulated the expression of NMDAR1, nNOS, and IRP1 in mouse lung tissues, which ultimately led to iron deposition via regulation of the expression of various iron metabolism-related genes. NMDAR activation initiated the pulmonary fibrosis process by inducing iron deposition in lung tissues and ferroptosis of alveolar type II cells. Our data suggest that NMDAR activation regulates the expression of iron metabolism-related genes by promoting calcium influx, increasing nNOS and IRP1 expression, and increasing iron deposition by affecting cellular iron homeostasis, ultimately leading to mitochondrial damage, mitochondrial dysfunction, and ferroptosis. NMDAR activation-induced ferroptosis of alveolar type II cells might be a key event to the initiation of pulmonary fibrosis.     

Benefits of Iron Chelators in the Treatment of Parkinson’s Disease

As a novel discovered regulated cell death pattern, ferroptosis has been associated with the development of Parkinson’s disease (PD) and has attracted widespread attention. Nevertheless, the relationship between ferroptosis and PD pathogenesis is still unclear. This study aims to investigate the effect of iron overload on dopaminergic (DA) neurons and its correlation with ferroptosis. Here we use nerve growth factor (NGF) induced PC12 cells which are derived from pheochromocytoma of the rat adrenal to establish a classical PD in vitro model. We found significantly decreased cell viability in NGF-PC12 cell under ammonium ferric citrate (FAC) administration. Moreover, excessive intracellular iron ions induced the increase of (reactive oxygen species) ROS release as well as the decrease of mitochondrial membrane potential in PC12-NGF cells. In addition, we also found that overloaded iron can activate cell apoptosis and ferroptosis pathways, which led to cell death. Furthermore, MPP-induced PD cells were characterized by mitochondrial shrinkage, decreased expression of glutathione peroxidase 4 (Gpx4) and ferritin heavy chain (FTH1), and increased divalent metal transporter (DMT1) and transferrin receptor 1 (TfR1) expression level. In contrast, Lip-1 and DFO increased the expression level of GPX4 and FTH1 compared to MPP-induced PD cell. In conclusion, we indicated that overloaded intracellular iron contributes to neurons death via apoptosis and ferroptosis pathways, while DFO, an iron chelator, can inhibit ferroptosis in order to protect the neurons in vitro.

     

Therapeutic potentials of cell death inhibitors in rats with cardiac ischaemia/reperfusion injury

Growing evidence demonstrated that cell death pathways including ferroptosis, apoptosis and necroptosis contribute to cardiac ischaemia/reperfusion (I/R) injury. We hypothesized that ferroptosis, apoptosis and necroptosis contribute differently to myocardial damage during acute cardiac I/R injury. Rats underwent cardiac I/R or sham operation. I/R‐operated rats were divided into 4 groups: vehicle, apoptosis (Z‐vad), ferroptosis (Fer‐1) and necroptosis (Nec‐1) inhibition. Rats in each cell death inhibitor group were subdivided into 3 different dose regimens: low, medium and high. Infarct size, left ventricular (LV) function, arrhythmias and molecular mechanism were investigated. Cardiac I/R caused myocardial infarction, LV dysfunction, arrhythmias, mitochondrial dysfunction, mitochondrial dynamic imbalance, inflammation, apoptosis and ferroptosis. Infarct size, LV dysfunction, mitochondrial dysfunction, apoptosis and ferroptosis were all reduced to a similar extent in rats treated with Z‐vad (low and medium doses) or Fer‐1 (medium and high doses). Fer‐1 treatment also reduced mitochondrial dynamic imbalance and inflammation. No evidence of necroptosis was found in association with acute I/R injury, therefore Nec‐1 treatment could not be assessed. Apoptosis and ferroptosis, not necroptosis, contributed to myocardial damage in acute I/R injury. Inhibitors of these 2 pathways provided effective cardioprotection in rats with I/R injury though modulation of mitochondrial function and attenuated apoptosis and ferroptosis.     

HucMSC-derived exosomes delivered BECN1 induces ferroptosis of hepatic stellate cells via regulating the xCT/GPX4 axis

Activated hepatic stellate cells (HSCs) are significant in liver fibrosis. Our past investigations have shown that human umbilical cord mesenchymal stem cells (hucMSCs) and their secreted exosomes (MSC-ex) could alleviate liver fibrosis via restraining HSCs activation. However, the mechanisms underlying the efficacy were not clear. Ferroptosis is a regulatory cell death caused by excessive lipid peroxidation, and it plays a vital role in the occurrence and development of liver fibrosis. In the present study, we aimed to study the proferroptosis effect and mechanism of MSC-ex in HSCs. MSC-ex were collected and purified from human umbilical cord MSCs. Proferroptosis effect of MSC-ex was examined in HSCs line LX-2 and CCl4 induced liver fibrosis in mice. Gene knockdown or overexpression approaches were used to investigate the biofactors in MSC-ex-mediated ferroptosis regulation. Results: MSC-ex could trigger HSCs ferroptosis by promoting ferroptosis-like cell death, ROS formation, mitochondrial dysfunction, Fe²⁺ release, and lipid peroxidation in human HSCs line LX-2. Glutathione peroxidase 4 (GPX4) is a crucial regulator of ferroptosis. We found that intravenous injection of MSC-ex significantly decreased glutathione peroxidase 4 (GPX4) expression in activated HSCs and collagen deposition in experimental mouse fibrotic livers. Mechanistically, MSC-ex derived BECN1 promoted HSCs ferroptosis by suppressing xCT-driven GPX4 expression. In addition, ferritinophagy and necroptosis might also play a role in MSC-ex-promoted LX-2 cell death. Knockdown of BECN1 in MSC diminished proferroptosis and anti-fibrosis effects of MSC-ex in LX-2 and fibrotic livers. MSC-ex may promote xCT/GPX4 mediated HSCs ferroptosis through the delivery of BECN1 and highlights BECN1 as a potential biofactor for alleviating liver fibrosis.Subject terms: Translational research, Stem-cell research     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

二氢青蒿素通过诱导铁死亡抑制肝癌细胞生长

二氢青蒿素（dihydroartemisinin,DHA）是青蒿素的一种衍生物,在多种肿瘤中表现出明显的抗肿瘤活性,但其具体机制不详。本文探讨了DHA对肝癌细胞的毒性作用机制。利用CCK-8试剂检测DHA对肝癌细胞株活力的影响,通过荧光探针染色及流式细胞术分析细胞内ROS及脂质过氧化物水平的变化;通过谷胱甘肽测定试剂盒检测细胞内还原型谷胱甘肽含量的变化,并通过免疫印迹分析DHA作用下细胞内铁死亡通路蛋白质中GPX4的变化。结果发现,DHA能显著抑制SMMC-7721及Huh-7细胞活力,其半数抑制浓度分别为23.74 μmol/L及26.92 μmol/L。 在35 μmol/L DHA 处理下,SMMC-7721及Huh-7细胞内ROS分别升高2.6倍和2.1倍,脂质过氧化物升高2.3倍和1.7倍。DHA可诱导细胞内GSH含量下降,并能下调铁死亡相关蛋白质GPX4蛋白水平。通过利用小分子抑制剂进行功能恢复实验发现,ROS抑制剂、铁螯合剂及铁死亡抑制剂都可不同程度恢复DHA引起的细胞活力下降。进一步检测发现,铁死亡抑制剂可抑制DHA诱导的脂质过氧化,并恢复GSH含量及GPX4蛋白水平。结果表明,在肝癌细胞中,DHA可通过诱导细胞发生铁死亡抑制肝癌细胞生长。     

Role of ferroptosis in cisplatin-induced acute nephrotoxicity in mice

BackgroundCisplatin is widely used as an antitumor drug for the treatment of solid tumors. However, its use has been limited owing to nephrotoxicity, a major side effect. The mechanism of cisplatin-induced nephrotoxicity (CIN) has long been investigated in order to develop preventive/therapeutic drugs. Ferroptosis is a newly identified form of non-apoptotic regulated cell death induced by iron-mediated lipid peroxidation and is involved in the pathophysiology of various diseases. In this study, we examined the role of ferroptosis in CIN.MethodsWe evaluated the role of ferroptosis in CIN by in vivo experiments in a mouse model.ResultsCisplatin increased the protein expressions of transferrin receptor-1 and ferritin, and iron content in the kidney of mice. In addition, treatment with cisplatin augmented renal ferrous iron and hydroxyl radical levels with co-localization. Mice administered cisplatin demonstrated kidney injury, with renal dysfunction and increased inflammatory cytokine expression; these changes were ameliorated by Ferrostatin-1 (Fer-1), an inhibitor of ferroptosis. The expression of the ferroptosis markers, COX2 and 4-hydroxynonenal (4-HNE), increased with cisplatin administration, and decreased with the administration of Fer-1. By contrast, cisplatin-induced apoptosis and necroptosis were inhibited by treatment with Fer-1. Moreover, deferoxamine, an iron chelator, also inhibited CIN, with a decrease in the expression of COX-2 and 4-HNE.ConclusionFerroptosis is involved in the pathogenesis of CIN and might be used as a new preventive target for CIN.     

Ferroptosis,a new form of cell death,and its relationships with tumourous diseases

Ferroptosis is a newly discovered type of cell death that differs from traditional apoptosis and necrosis and results from iron‐dependent lipid peroxide accumulation. Ferroptotic cell death is characterized by cytological changes, including cell volume shrinkage and increased mitochondrial membrane density. Ferroptosis can be induced by two classes of small‐molecule substances known as class 1 (system X _c ^? inhibitors) and class 2 ferroptosis inducers [glutathione peroxidase 4 (GPx4) inhibitors]. In addition to these small‐molecule substances, a number of drugs (e.g. sorafenib, artemisinin and its derivatives) can induce ferroptosis. Various factors, such as the mevalonate (MVA) and sulphur‐transfer pathways, play pivotal roles in the regulation of ferroptosis. Ferroptosis plays an unneglectable role in regulating the growth and proliferation of some types of tumour cells, such as lymphocytoma, ductal cell cancer of the pancreas, renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Here, we will first introduce the discovery of and research pertaining to ferroptosis; then summarize the induction mechanisms and regulatory pathways of ferroptosis; and finally, further elucidate the roles of ferroptosis in human tumourous diseases.     

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.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

α-Lipoic acid alleviates ferroptosis in the MPP+-induced PC12 cells via activating the PI3K/Akt/Nrf2 pathway

Parkinson's disease (PD) is a typical neurodegenerative disease. α-Lipoic acid (α-LA) can reduce the incidence of neuropathy. The present study explored the role and mechanism of α-LA in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell model of PD. The PD model was induced via treating PC12 cells with MPP⁺ at different concentrations. MPP⁺ and α-LA effects on PC12 cells were assessed from cell viability and ferroptosis. Cell viability was detected using the cell counting kit-8 assay. Malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), iron, reactive xygen species (ROS), and glutathione (GSH) concentrations, and ferroptosis-related protein SLC7A11 and GPx4 expressions were used for ferroptosis evaluation. p-PI3K, p-Akt, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels were detected. The PI3K/Akt/Nrf2 pathway inhibitors were applied to verify the role of the PI3K/Akt/Nrf2 pathway in α-LA protection against MPP⁺-induced decreased cell viability and ferroptosis. MPP⁺-reduced cell viability and induced ferroptosis as presented by increased MDA, 4-HNE, iron, and ROS concentrations, and reduced levels of GSH and ferroptosis marker proteins (SLC7A11 and GPx4). α-LA attenuated MPP⁺-induced cell viability decline and ferroptosis. The PI3K/Akt/Nrf2 pathway was activated after α-LA treatment. Inhibiting the PI3K/Akt/Nrf2 pathway weakened the protection of α-LA against MPP⁺ treatment. We highlighted that α-LA alleviated MPP⁺-induced cell viability decrease and ferroptosis in PC12 cells via activating the PI3K/Akt/Nrf2 pathway.     

Baicalein attenuates oxidant stress in cardiomyocytes

Flavonoids within Scutellaria baicalensis may be potent antioxidants on the basis of our studies of S. baicalensis extract. To further this work, we studied the antioxidative effects of baicalein, a flavonoid component of S. baicalensis, in a chick cardiomyocyte model of reactive oxygen species (ROS) generation during hypoxia, simulated ischemia-reperfusion, or mitochondrial complex III inhibition with antimycin A. Oxidant stress was measured by oxidation of the intracellular probes 2',7'-dichlorofluorescin diacetate and dihydroethidium. Viability was assessed by propidium iodide uptake. Baicalein attenuated oxidant stress during all conditions studied and acted within minutes of treatment. For example, baicalein given only at reperfusion dose dependently attenuated the ROS burst at 5 min after 1 h of simulated ischemia. It also decreased subsequent cell death at 3 h of reperfusion from 52.3 +/- 2.5% in untreated cells to 29.4 +/- 3.0% (with return of contractions; P < 0.001). In vitro studies using electron paramagnetic resonance spectroscopy with the spin trap 5-methoxycarbonyl-5-methyl-1-pyrroline-N-oxide revealed that baicalein scavenges superoxide but does not mimic the effects of superoxide dismutase. We conclude that baicalein can scavenge ROS generation in cardiomyocytes and that it protects against cell death in an ischemia-reperfusion model when given only at reperfusion.     

Multiple metabolic hits converge on CD36 as novel mediator of tubular epithelial apoptosis in diabetic nephropathy

Background

Diabetic nephropathy (DNP) is a common complication of type 1 and type 2 diabetes mellitus and the most common cause of kidney failure. While DNP manifests with albuminuria and diabetic glomerulopathy, its progression correlates best with tubular epithelial degeneration (TED) and interstitial fibrosis. However, mechanisms leading to TED in DNP remain poorly understood.

Methods and Findings

We found that expression of scavenger receptor CD36 coincided with proximal tubular epithelial cell (PTEC) apoptosis and TED specifically in human DNP. High glucose stimulated cell surface expression of CD36 in PTECs. CD36 expression was necessary and sufficient to mediate PTEC apoptosis induced by glycated albumins (AGE-BSA and CML-BSA) and free fatty acid palmitate through sequential activation of src kinase, and proapoptotic p38 MAPK and caspase 3. In contrast, paucity of expression of CD36 in PTECs in diabetic mice with diabetic glomerulopathy was associated with normal tubular epithelium and the absence of tubular apoptosis. Mouse PTECs lacked CD36 and were resistant to AGE-BSA-induced apoptosis. Recombinant expression of CD36 in mouse PTECs conferred susceptibility to AGE-BSA-induced apoptosis.

Conclusion

Our findings suggest a novel role for CD36 as an essential mediator of proximal tubular apoptosis in human DNP. Because CD36 expression was induced by glucose in PTECs, and because increased CD36 mediated AGE-BSA-, CML-BSA-, and palmitate-induced PTEC apoptosis, we propose a two-step metabolic hit model for TED, a hallmark of progression in DNP.     

Increased mitochondrial superoxide generation in neurons from trisomy 16 mice: a model of Down's syndrome

Increased neuronal cell death in neurodegenerative diseases has been suggested to result from an increased mitochondrial generation of radical oxygen species (ROS). To test this hypothesis, we investigated superoxide formation in cultured hippocampal neurons from diploid and trisomy 16 mice (Ts16), a model of Down's syndrome. Microflurometric techniques were used to measure superoxide-induced oxidation rate of hydroethidine (HEt) to ethidium and reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) autofluorescence signal to monitor changes in neuronal energy metabolism. We found an increase in superoxide formation by more than 50% in Ts16 neurons in comparison with diploid control neurons. In the presence of the mitochondrial respiratory chain complex I inhibitor rotenone superoxide production was blocked in diploid neurons, but the increased superoxide generation in Ts16 neurons remained. Uncoupling of mitochondrial oxidative phosphorylation using carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused irreversible deficiency in the energy metabolism, monitored by NAD(P)H autofluorescence in Ts16 neurons, but not in diploid control neurons. These results suggest an increased basal generation of superoxide in Ts16 neurons, probably caused by a deficient complex I of mitochondrial electron transport chain, which leads to an impaired mitochondrial energy metabolism and finally neuronal cell death.     

Mechanisms of nucleophosmin (NPM)-mediated regulated cell death elucidated by Hsp70 during renal ischemia

Nucleophosmin (NPM), a nucleolar-based protein chaperone, promotes Bax-mediated mitochondrial injury and regulates cell death during acute kidney injury. However, the steps that transform NPM from an essential to a toxic protein during stress are unknown. To localize NPM-mediated events causing regulated cell death during ischemia, wild type (WT) and Hsp70 mutant proteins with characterized intracellular trafficking defects that restrict movement to either the nucleolar region (M45) or cytosol (985A) were expressed in primary murine proximal tubule epithelial cells (PTEC) harvested from Hsp70 null mice. After ischemia in vitro, PTEC survival was significantly improved and apoptosis reduced in rank order by selectively overexpressing WT?>?M45?>?985A Hsp70 proteins. Only Hsp70 with nuclear access (WT and M45) inhibited T95 NPM phosphorylation responsible for NPM translocation and also reduced cytosolic NPM accumulation. In contrast, WT or 985A?>?M45 significantly improved survival in Hsp70 null PTEC that expressed a cytosol-restricted NPM mutant, more effectively bound NPM, and also reduced NPM-Bax complex formation required for mitochondrial injury and cell death. Hsp70 knockout prevented the cytoprotective effect of suppressing NPM in ischemic PTEC and also increased cytosolic NPM accumulation after acute renal ischemia in vivo, emphasizing the inhibitory effect of Hsp70 on NPM-mediated toxicity. Distinct cytoprotective mechanisms by wild type and mutant Hsp70 proteins identify dual nuclear and cytosolic events that mediate NPM toxicity during stress-induced apoptosis and are rational targets for therapeutic AKI interventions. Antagonizing these early events in regulated cell death promotes renal cell survival during experimental AKI.

     

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     

Iron deposition-induced ferroptosis in alveolar type II cells promotes the development of pulmonary fibrosis

Ferroptosis is a newly discovered type of regulated cell death, characterized by the iron-dependent accumulation of lipid reactive oxygen species, which has been implicated in numerous human diseases. However, its role in pulmonary fibrosis, a fatal lung disease with unknown etiology, is largely unknown. Here, we investigated the role of ferroptosis in pulmonary fibrosis. We found a large amount of iron deposition in the lung tissue of patients with pulmonary fibrosis. We observed ferroptosis in alveolar type II (ATII) cells, fibrotic lung tissues of BLM-induced pulmonary fibrosis mice. BLM-induced increase in iron level was accompanied by pathological changes, collagen deposition, and ferroptosis in ATII cells, indicating iron deposition-induced ferroptosis, which promoted the development of pulmonary fibrosis. Moreover, deferoxamine (DFO) completely prevented the pro-fibrosis effects of BLM by reducing iron deposition and ferroptosis in ATII cells. Genes associated with intracellular iron metabolism and homeostasis, such as transferrin receptor 1, divalent metal transporter 1, and ferroportin-1, and showed abnormal expression levels in animal tissues and lung epithelial MLE-12 cells, which responded to BLM stimulation. Overall, we demonstrated that BLM-induced iron deposition in MLE-12 cells is prone to both mitochondrial dysfunction and ferroptosis and that DFO reverses this phenotype. In the future, understanding the role of ferroptosis may shed new light on the etiology of pulmonary fibrosis. Ferroptosis inhibitors or genetic engineering of ferroptosis-related genes might offer potential targets to treat pulmonary fibrosis.