Necroptosis Inhibitor Research Articles

Necroptosis Contributes to LPS-Induced Activation of the Hypothalamic-Pituitary-Adrenal Axis in a Piglet Model.

Stressors cause activation of the hypothalamic-pituitary-adrenal (HPA) axis and a systemic inflammatory response. As a newly proposed cell death manner in recent years, necroptosis occurs in a variety of tissue damage and inflammation. However, the role of necroptosis in HPA axis activation remains to be elucidated. The aim of this study was to investigate the occurrence of necroptosis and its role in HPA activation in a porcine stress model induced by Escherichia coli lipopolysaccharide (LPS). Several typical stress behaviors like fever, anorexia, shivering and vomiting were observed in piglets after LPS injection. HPA axis was activated as shown by increased plasma cortisol concentration and mRNA expression of pituitary corticotropin-releasing hormone receptor 1 (CRHR1) and adrenal steroidogenic acute regulatory protein (StAR). The mRNA expression of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 in the hypothalamus, pituitary gland and adrenal gland was elevated by LPS, accompanied by the activation of necroptosis indicated by higher mRNA expression of necroptosis signals including receptor-interacting protein kinase (RIP) 1, RIP3, and phosphorylated mixed-lineage kinase domain-like protein (MLKL). Furthermore, necrostatin-1 (Nec-1), an inhibitor of necroptosis, inhibited necroptosis indicated by decreased mRNA levels of RIP1, RIP3, MLKL, and phosphoglycerate mutase family member 5 (PGAM5) in the hypothalamus, pituitary gland and adrenal gland. Nec-1 also decreased the mRNA expression of TNF-α and IL-β and inhibited the activation of the HPA axis indicated by lower plasma cortisol concentration and mRNA expression of adrenal type 2 melanocortin receptor (MC2R) and StAR. These findings suggest that necroptosis is present and contributes to HPA axis activation induced by LPS. These findings provide a potential possibility for necroptosis as an intervention target for alleviating HPA axis activation and stress responses.

Therapeutic effects of mesenchymal stem cells-conditioned medium derived from suspension cultivation or silymarin on liver failure mice.

Common treatments of liver disease failed to meet all the needs in this important medical field. It results in an urgent need for proper some new adjuvant therapies. Mesenchymal stem cells (MSCs) and their derivatives are promising tools in this regard. We aimed to compare the Silymarin, as traditional treatment with mesenchymal stem cell conditioned medium (MSC-CM), as a novel strategy, both with therapeutic potentialities in term of liver failure (LF) treatment. Mice models with liver failure were induced with CCl4 and were treated in the groups as follows: normal mice receiving DMEM-LG medium as control, LF-mice receiving DMEM-LG medium as sham, LF-mice receiving Silymarin as LF-SM, and LF-mice receiving MSC sphere CM as LF-MSC-CM. Biochemical, histopathological, molecular and protein level parameters were evaluated using blood and liver samples. Liver enzymes, MicroRNA-122 values as well as necrotic score were significantly lower in the LF-SM and LF-MSC-CM groups compared to sham. LF-SM showed significantly higher level of total antioxidant capacity and malondialdehyde than that of LF-MSC-CM groups. Sph-MSC-CM not only induced more down-regulated expression of fibrinogen-like protein 1 and receptor interacting protein kinases1 but also led to higher expression level of keratinocyte growth factor. LF-MSC-CM showed less mortality rate compared to other groups. Hepato-protective potentialities of Sph-MSC-CM are comparable to those of Silymarin. More inhibition of necroptosis/ necrosis and inflammation might result in rapid liver repair in case of MSC-CM administration.

Metabolic Maturation Increases Susceptibility to Hypoxia-induced Damage in Human iPSC-derived Cardiomyocytes.

The development of new cardioprotective approaches using in vivo models of ischemic heart disease remains challenging as differences in cardiac physiology, phenotype, and disease progression between humans and animals influence model validity and prognostic value. Furthermore, economical and ethical considerations have to be taken into account, especially when using large animal models with relevance for conducting preclinical studies. The development of human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has opened new opportunities for in vitro studies on cardioprotective compounds. However, the immature cellular phenotype of iPSC-CMs remains a roadblock for disease modeling. Here, we show that metabolic maturation renders the susceptibility of iPSC-CMs to hypoxia further toward a clinically representative phenotype. iPSC-CMs cultured in a conventional medium did not show significant cell death after exposure to hypoxia. In contrast, metabolically matured (MM) iPSC-CMs showed inhibited mitochondrial respiration after exposure to hypoxia and increased cell death upon increased durations of hypoxia. Furthermore, we confirmed the applicability of MM iPSC-CMs for in vitro studies of hypoxic damage by validating the known cardioprotective effect of necroptosis inhibitor necrostatin-1. Our results provide important steps to improving and developing valid and predictive human in vitro models of ischemic heart disease.

The regulation of necroptosis and perspectives for the development of new drugs preventing ischemic/reperfusion of cardiac injury

In the last 10years, mortality from acute myocardial infarction (AMI) has not significantly decreased. This situation is associated with the absence in clinical practice of highly effective drugs capable of preventing the occurrence of reperfusion injury of the heart. Necroptosis inhibitors may become prototypes for the creation of highly effective drugs that increase cardiac tolerance to ischemic/reperfusion (I/R) and reduce the mortality rate in patients with AMI. Necroptosis is involved in I/R cardiac injury and inhibition of RIPK1 or RIPK3 contributes to an increase in cardiac tolerance to I/R. Necroptosis could also be involved in the development of adverse remodeling of the heart. It is unclear whether pre- and postconditioning could inhibit necroptosis of cardiomyocytes and endothelial cells. The role of necroptosis in coronary microvascular obstruction and the no-reflow phenomenon also needs to be studied. MicroRNAs and LncRNAs can regulate necroptotic cell death. Ca2+ overload and reactive oxygen species could be the triggers of necroptosis. Activation of kinases (p38, JNK1, Akt, and mTOR) could promote necroptotic cell death. The interaction of necroptosis, apoptosis, autophagy, ferroptosis, and pyroptosis is discussed. The water-soluble necroptosis inhibitors may be highly effective drugs for treatment of AMI or stroke. It is possible that microRNAs may become the basis for creating drugs for treatment of diseases triggered by I/R of organs.

Acidosis induces RIPK1-dependent death of glioblastoma stem cells via acid-sensing ion channel 1a

Eliciting regulated cell death, like necroptosis, is a potential cancer treatment. However, pathways eliciting necroptosis are poorly understood. It has been reported that prolonged activation of acid-sensing ion channel 1a (ASIC1a) induces necroptosis in mouse neurons. Glioblastoma stem cells (GSCs) also express functional ASIC1a, but whether prolonged activation of ASIC1a induces necroptosis in GSCs is unknown. Here we used a tumorsphere formation assay to show that slight acidosis (pH 6.6) induces necrotic cell death in a manner that was sensitive to the necroptosis inhibitor Nec-1 and to the ASIC1a antagonist PcTx1. In addition, genetic knockout of ASIC1a rendered GSCs resistant to acid-induced reduction in tumorsphere formation, while the ASIC1 agonist MitTx1 reduced tumorsphere formation also at neutral pH. Finally, a 20 amino acid fragment of the ASIC1 C-terminus, thought to interact with the necroptosis kinase RIPK1, was sufficient to reduce the formation of tumorspheres. Meanwhile, the genetic knockout of MLKL, the executive protein in the necroptosis cascade, did not prevent a reduction in tumor sphere formation, suggesting that ASIC1a induced an alternative cell death pathway. These findings demonstrate that ASIC1a is a death receptor on GSCs that induces cell death during prolonged acidosis. We propose that this pathway shapes the evolution of a tumor in its acidic microenvironment and that pharmacological activation of ASIC1a might be a potential new strategy in tumor therapy.

Ameliorative effects of epigallocatechin-3-gallate nanoparticles on 2,4-dinitrochlorobenzene induced atopic dermatitis: A potential mechanism of inflammation-related necroptosis.

Atopic dermatitis (AD) is a common autoimmune and chronic inflammatory cutaneous disease with a relapsing-remitting course. Necroptosis is a regulated necrotic cell death mediated by receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and mixed lineage kinase domain-like pseudokinase (MLKL), which is activated by tumor necrosis factor-α (TNF-α). However, the mechanism and the role of necroptosis have not been delineated in AD progression. (-)-Epigallocatechin-3-gallate (EGCG), the main biological activity of tea catechin, is well known for its beneficial effects in the treatment of skin diseases. Here, PEG-PLGA-EGCG nanoparticles (EGCG-NPs) were formulated to investigate the bioavailability of EGCG to rescue cellular injury following the inhibition of necroptosis after AD. 2,4-dinitrochlorobenzene (DNCB) was used to establish AD mouse models. As expected, topically applied EGCG-NPs elicited a significant amelioration of AD symptoms in skin lesions, including reductions in the ear and skin thickness, dermatitis score, and scratching behavior, which was accompanied by redox homeostasis restored early in the experiment. In addition, EGCG-NPs significantly decreased the expression of inflammatory cytokines like TNF-α, interferon-γ (IFN-γ), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-17A (IL-17A) in a time-dependent manner than those of in AD group. As a result, the overexpression of RIP1, RIP3, and MLKL in the entire epidermis layers was dramatically blocked by EGCG-NPs, as well as the expression ofphosphorylated p38 (p-p38), extracellular signal-regulated kinase 1 (ERK1), and extracellular signal-regulated kinase 2 (ERK2). These findings promote that EGCG-NPs formulation represents a promising drug-delivery strategy for the treatment of AD by maintaining the balance of Th1/Th2 inflammation response and targeting necroptosis.

PT-112 Induces Mitochondrial Stress and Immunogenic Cell Death, Targeting Tumor Cells with Mitochondrial Deficiencies.

Simple SummaryPT-112 is a novel pyrophosphate–platinum conjugate under Phase 1/2 clinical development for the treatment of several tumor types. In this study, using mouse tumor cells with well-characterized mitochondrial and metabolic status, we investigated the mechanisms underlying PT-112’s cancer cell death effects. Our results showed that cells with defective mitochondria were more sensitive to PT-112 when compared to cells with normal mitochondrial function. Moreover, PT-112 induced tumor cell death in those sensitive cells through non-conventional mechanisms, including increased mitochondrial stress, free radical generation and immunogenic cell death, a form of cell death that elicits an immune response. Taken together, the present findings suggest the potential for predictors of PT-112 sensitivity in the clinical setting on the basis of metabolic function.PT-112 is a novel pyrophosphate–platinum conjugate, with clinical activity reported in advanced pretreated solid tumors. While PT-112 has been shown to induce robust immunogenic cell death (ICD) in vivo but only minimally bind DNA, the molecular mechanism underlying PT-112 target disruption in cancer cells is still under elucidation. The murine L929 in vitro system was used to test whether differential metabolic status alters PT-112’s effects, including cell cytotoxicity. The results showed that tumor cells presenting mutations in mitochondrial DNA (mtDNA) (L929dt and L929dt cybrid cells) and reliant on glycolysis for survival were more sensitive to cell death induced by PT-112 compared to the parental and cybrid cells with an intact oxidative phosphorylation (OXPHOS) pathway (L929 and dtL929 cybrid cells). The type of cell death induced by PT-112 did not follow the classical apoptotic pathway: the general caspase inhibitor Z-VAD-fmk did not inhibit PT-112-induced cell death, alone or in combination with the necroptosis inhibitor necrostatin-1. Interestingly, PT-112 initiated autophagy in all cell lines, though this process was not complete. Autophagy is known to be associated with an integrated stress response in cancer cells and with subsequent ICD. PT-112 also induced a massive accumulation of mitochondrial reactive oxygen species, as well as changes in mitochondrial polarization—only in the sensitive cells harboring mitochondrial dysfunction—along with calreticulin cell-surface exposure consistent with ICD. PT-112 substantially reduced the amount of mitochondrial CoQ10 in L929 cells, while the basal CoQ10 levels were below our detection limits in L929dt cells, suggesting a potential relationship between a low basal level of CoQ10 and PT-112 sensitivity. Finally, the expression of HIF-1α was much higher in cells sensitive to PT-112 compared to cells with an intact OXPHOS pathway, suggesting potential clinical applications.

Nanoencapsulation of Docetaxel Induces Concurrent Apoptosis and Necroptosis in Human Oral Cancer Cells (SCC-9) via TNF-α/RIP1/RIP3 Pathway.

Human oral squamous cell carcinoma is the sixth most frequent malignant cancer, with an unacceptably high death rate that affects people's health. Albeit, there are several clinical approaches for diagnosing and treating oral cancer they are still far from ideal. We previously synthesised and characterised the docetaxel nanoformulation (PLGA-Dtx) and discovered that docetaxel nanoencapsulation may suppress oral cancer cells. The goal of this study was to figure out the mechanism involved in the suppression of oral cancer cell proliferation. We discovered that PLGA-Dtx inhibited SCC-9 cell growth considerably as compared to free docetaxel (Dtx), and that the viability of SCC-9 cells treated with PLGA-Dtx was decreased dose-dependently. MTT assay showed that PLGA-Dtx selectively inhibited the growth of PBMCs from oral cancer patients while sparing PBMCs from normal healthy controls. Further, flow cytometry analysis showed that PLGA-Dtx induced apoptosis and necroptosis in SCC-9 cells. G2/M cell cycle arrest has been confirmed on exposure of PLGA-Dtx for 24h in SCC-9 cells. Interestingly, western blot investigation found that PLGA-Dtx increased the amounts of necroptic proteins and apoptosis-related proteins more efficiently than Dtx. Furthermore, PLGA-Dtx was more effective in terms of ROS generation, and mitochondrial membrane potential depletion. Pretreatment with necroptosis inhibitor Nec-1 efficiently reversed the ROS production and further recover MMP caused by PLGA-Dtx. Overall, this study revealed a mechanistic model of therapeutic response for PLGA-Dtx in SCC-9 cells and proposed its potency by inducing cell death via activation of concurrent apoptosis and necroptosis in SCC-9 cells via TNF-α/RIP1/RIP3 and caspase-dependent pathway.

Discovery, optimization and evaluation of isothiazolo[5,4-b]pyridine derivatives as RIPK1 inhibitors with potent in vivo anti-SIRS activity

Receptor-interacting protein kinase-1 (RIPK1) is involved in the necroptosis pathway, which regulates inflammatory signaling and cell death in a variety of diseases, including inflammatory and neurodegenerative disorders. We identified a novel hit compound 36 by a cell-based screening assay (anti-necroptosis EC50 = 58 nM). Starting from compound 36, we designed a series of scaffolds to improve anti-necroptosis activity, physicochemical properties and metabolic stability. The isothiazolo[5,4-b]pyridine backbone proved to be a promising scaffold which provided a number of potent necroptosis inhibitors. Compound 56, for example, effectively blocked necroptosis in both human and mouse cells (EC50 = 1–5 nM). A binding assay showed that compound 56 potently binds to RIPK1 (Kd = 13 nM), but not RIPK3 (Kd > 10,000 nM). Kinase functional assay (ADP-Glo) confirmed that compound 56 inhibits RIPK1 phosphorylation with an IC50 at 5.8 nM. Importantly, compound 56 displayed excellent cross-species liver microsomal metabolic stability (t1/2 > 90 min). Furthermore, compound 56 exhibited favorable in vitro safety profiles in hERG and CYP assays. Finally, pre-treatment with 56 significantly reduced hypothermia and lethal shock in the systemic inflammatory response syndrome mice model. Taken together, compound 56 represented a promising prototype for the development of therapeutic agent to treat inflammation-related diseases.

Suppression of the necroptotic cell death pathways improves survival in Smn 2B/- mice.

Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by low levels of the Survival Motor Neuron (SMN) protein. Motor neuron degeneration is the central hallmark of the disease. However, the SMN protein is ubiquitously expressed and depletion of the protein in peripheral tissues results in intrinsic disease manifestations, including muscle defects, independent of neurodegeneration. The approved SMN-restoring therapies have led to remarkable clinical improvements in SMA patients. Yet, the presence of a significant number of non-responders stresses the need for complementary therapeutic strategies targeting processes which do not rely solely on restoring SMN. Dysregulated cell death pathways are candidates for SMN-independent pathomechanisms in SMA. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 have been widely recognized as critical therapeutic targets of necroptosis, an important form of programmed cell death. In addition, Caspase-1 plays a fundamental role in inflammation and cell death. In this study, we evaluate the role of necroptosis, particularly RIPK3 and Caspase-1, in the Smn2B/− mouse model of SMA. We have generated a triple mutant (TKO), the Smn2B/−; Ripk3−/−; Casp1−/− mouse. TKO mice displayed a robust increase in survival and improved motor function compared to Smn2B/− mice. While there was no protection against motor neuron loss or neuromuscular junction pathology, larger muscle fibers were observed in TKO mice compared to Smn2B/− mice. Our study shows that necroptosis modulates survival, motor behavior and muscle fiber size independent of SMN levels and independent of neurodegeneration. Thus, small-molecule inhibitors of necroptosis as a combinatorial approach together with SMN-restoring drugs could be a future strategy for the treatment of SMA.

Social isolation reinforces aging-related behavioral inflexibility by promoting neuronal necroptosis in basolateral amygdala.

Aging is characterized with a progressive decline in many cognitive functions, including behavioral flexibility, an important ability to respond appropriately to changing environmental contingencies. However, the underlying mechanisms of impaired behavioral flexibility in aging are not clear. In this study, we reported that necroptosis-induced reduction of neuronal activity in the basolateral amygdala (BLA) plays an important role in behavioral inflexibility in 5-month-old mice of the senescence-accelerated mice prone-8 (SAMP8) line, a well-established model with age-related phenotypes. Application of Nec-1s, a specific inhibitor of necroptosis, reversed the impairment of behavioral flexibility in SAMP8 mice. We further observed that the loss of glycogen synthase kinase 3α (GSK-3α) was strongly correlated with necroptosis in the BLA of aged mice and the amygdala of aged cynomolgus monkeys (Macaca fascicularis). Moreover, genetic deletion or knockdown of GSK-3α led to the activation of necroptosis and impaired behavioral flexibility in wild-type mice, while the restoration of GSK-3α expression in the BLA arrested necroptosis and behavioral inflexibility in aged mice. We further observed that GSK-3α loss resulted in the activation of mTORC1 signaling to promote RIPK3-dependent necroptosis. Importantly, we discovered that social isolation, a prevalent phenomenon in aged people, facilitated necroptosis and behavioral inflexibility in 4-month-old SAMP8 mice. Overall, our study not only revealed the molecular mechanisms of the dysfunction of behavioral flexibility in aged people but also identified a critical lifestyle risk factor and a possible intervention strategy.

Homoharringtonine combined with cladribine and aclarubicin (HCA) in acute myeloid leukemia: A new regimen of conventional drugs and its mechanism.

Objective The prognosis of children with refractory acute myeloid leukemia (AML) is poor. Complete remission (CR) is not always achieved with current salvage chemotherapy regimens before transplantation, and some patients have no chance of transplantation. Here, we aimed to describe a new regimen of conventional chemotherapy drugs (homoharringtonine, cladribine , and aclarubicin (HCA)) for refractory AML and its mechanism in vitro. Methods We retrospectively collected the clinical data of 5 children with primary refractory AML using HCA as reinduction chemotherapy, and CR rates, adverse reactions, and disease-free survival (DFS) were analyzed. The effects of homoharringtonine, cladribine, and aclarubicin alone or in combination on the proliferation of HL60 and THP1 cells were analyzed by CCK-8 assay. Furthermore, CCK-8 was used to determine the effects of HCA, alone or in combination with apoptosis inhibitors, necroptosis inhibitors, ferroptosis inhibitors, or autophagy inhibitors, on the proliferation of HL60 and THP1 cells and to screen for possible HCA-mediated death pathways in AML cells. The pathway of HCA-mediated AML cell death was further verified by Hoechst/PI staining, flow cytometry, and Western blotting. Results After 2 cycles of conventional chemotherapy, none of the 5 children with AML achieved CR and were then treated with the HCA regimen for two cycles, 4 of 5 achieved CR, and another child achieved CR with incomplete hematological recovery (CRi). After CR, 3 children underwent hematopoietic stem cell transplantation (HSCT), and only 2 of them received consolidation therapy. As of the last follow-up, all 5 patients had been in DFS for a range of 23 to 28 months. The inhibition rate of homoharringtonine, cladribine, and aclarubicin in combination on HL60 and THP1 cells was significantly greater than that of a single drug or a combination of two drugs. We found that inhibitors of apoptosis and necroptosis were able to inhibit HCA-mediated cell death but not ferroptosis or autophagy inhibitors. Compared with the control group, the number of apoptotic cells in the HCA group was significantly increased and could be reduced by an apoptosis inhibitor. Western blot results showed that PARP, caspase-3, and caspase-8 proteins were activated and cleaved in the HCA group, the expression of Bax was upregulated and that of Bcl-2 was downregulated. The expression of apoptosis-related proteins could be reversed by apoptosis inhibition. Compared with the control group, the expression levels of the necroptosis-related proteins RIP1, RIP3, and MLKL were downregulated in the HCA group but were not phosphorylated. The necroptosis inhibitor increased the expression of RIP1 but caused no significant changes in RIP3 and MLKL, and none were phosphorylated. Conclusions HCA, as a new regimen of conventional drugs, was a safe and efficacious reinduction salvage strategy in children with refractory AML before HSCT. HCA exhibits the synergistic growth inhibition of AML cells and induces cell death mainly through apoptosis.

Iron chelator deferasirox inhibits NF-κB activity in hepatoma cells and changes sorafenib-induced programmed cell deaths

ObjectiveThe improvements of antitumor effects and tolerability on chemotherapy for advanced hepatocellular carcinoma (HCC) are warranted. Here, we aimed to elucidate the mechanism of the combining effect of tyrosine kinase inhibitor sorafenib (SOR) and iron chelator deferasirox (DFX) in human hepatoma cell lines, HepG2 and Huh-7. MethodsThe types of programmed cell deaths (PCDs); necrosis/necroptosis and apoptosis, were evaluated by flow cytometry and fluorescent microscopy. Human cleaved caspase-3 was analyzed by ELISA for apoptosis. GSH assay was used for ferroptosis. PCDs inhibition was analyzed by adding apoptosis inhibitor Z-VAD-FMK, ferroptosis inhibitor ferrostatin-1, necroptosis inhibitor necrosulfonamide, respectively. The expression of NF-κB was quantified by Western blotting. ResultsIn SOR monotherapy, cleaved caspase-3 expression was increased in all concentrations, confirming the result that SOR induces apoptosis. In SOR monotherapy, GSH/GSSG ratio was decreased on concentration-dependent, showing that SOR also induced ferroptosis. Lipid Peroxidation caused by SOR, corresponding to ferroptosis, was suppressed by DFX. In fluorescence microscopy of SOR monotherapy, apoptosis was observed at a constant rate on all concentrations, while necroptosis and ferroptosis were increased on high concentration. In sorafenib and deferasirox combinations, sub G1 phase increased additively. In SOR and DFX combinations, the cytotoxic effects were not suppressed by ferrostatin-1, but suppressed by Z-VAD-FMK and necrosulfonamide. In each monotherapy, and SOR + DFX combinations, the expression of NF-κB in nucleus was suppressed. Regarding PCD by SOR and DFX combination, ferroptosis was suppressed and both apoptosis and necroptosis became dominant. ConclusionSuppression of NF-κB is possibly involved in the effect of DFX. As a result, SOR and DFX combination showed additive antitumor effects for HCC through the mechanism of programed cell deaths and NF-kB signal modification.

Free docosahexaenoic acid promotes ferroptotic cell death via lipoxygenase dependent and independent pathways in cancer cells.

Ferroptosis is a form of regulated cell death that has the potential to be targeted as a cancer therapeutic strategy. But cancer cells have a wide range of sensitivities to ferroptosis, which limits its therapeutic potential. Accumulation of lipid peroxides determines the occurrence of ferroptosis. However, the type of lipid involved in peroxidation and the mechanism of lipid peroxide accumulation are less studied. The effects of fatty acids (10μM) with different carbon chain length and unsaturation on ferroptosis were evaluated by MTT and LDH release assay in cell lines derived from prostate cancer (PC3, 22RV1, DU145 and LNCaP), colorectal cancer (HT-29), cervical cancer (HeLa) and liver cancer (HepG2). Inhibitors of apoptosis, necroptosis, autophagy and ferroptosis were used to determinethe type of cell death. Then the regulation of reactive oxygen species (ROS) and lipid peroxidation by docosahexaenoic acid (DHA) wasmeasured by HPLC-MS and flow cytometry. The avtive form of DHA was determined by siRNA mediated gene silencing. The role of lipoxygenases was checked by inhibitors and gene silencing.Finally, the effect of DHA on ferroptosis-mediated tumor killing was verified in xenografts. The sensitivity of ferroptosis was positively correlated with the unsaturation of exogenouslyadded fatty acid. DHA (22:6 n-3) sensitized cancer cells to ferroptosis-inducing reagents (FINs) at the highest level in vitro and in vivo. In this process, DHA increasedROS accumulation, lipid peroxidation and protein oxidation independent of its membrane receptor, GPR120. Inhibition of long chain fatty acid-CoA ligases and lysophosphatidylcholine acyltransferases didn't affect the role of DHA.DHA-involved ferroptosis can be induced in both arachidonate lipoxygenase 5 (ALOX5) negative and positive cells. Down regulation of ALOX5 inhibited ferroptosis, while overexpression of ALOX5 promoted ferroptosis. DHA can effectively promote ferroptosis-mediated tumor killing by increasing intracellular lipid peroxidation. Both ALOX5 dependent and independent pathways are involved in DHA-FIN induced ferroptosis. And during this process, free DHA plays an important role.

Necroptosis-Mediated eCIRP Release in Sepsis.

IntroductionExtracellular cold-inducible RNA-binding protein (eCIRP) is an endogenous pro-inflammatory mediator that exacerbates injury in inflammation and sepsis. The mechanisms in which eCIRP is released have yet to be fully explored. Necroptosis is a programmed cell death that is dependent on the activation of mixed lineage kinase domain-like pseudo kinase (MLKL) which causes the release of damage-associated molecular patterns. We hypothesize that eCIRP is released through necroptosis and intensifies inflammation in sepsis.MethodsRAW264.7 cells were treated with pan-caspase inhibitor z-VAD (15 μM) 1 h before stimulation with LPS (1 μg/mL). Necroptosis inhibitor, Necrostatin-1 (Nec-1) (10 μM) was added to the cells with LPS simultaneously. After 24 h of LPS stimulation, cytotoxicity was determined by LDH assay. eCIRP levels in the culture supernatants and phospho-MLKL (p-MLKL) from cell lysates were assessed by Western blot. p-MLKL interaction with the cell membrane was visualized by immunofluorescence. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Mice were treated with Nec-1 (1 mg/kg) or DMSO. 20 h post-surgery, serum and peritoneal fluid levels of eCIRP, TNF-α and IL-6 were determined by ELISA. H&E staining of lung tissue sections was performed.ResultsWe found that in RAW264.7 cells, LPS+z-VAD induces necroptosis as evidenced by an increase in p-MLKL levels and causes eCIRP release. Nec-1 reduces both p-MLKL activation and eCIRP release in LPS+z-VAD-treated RAW264.7 cells. Nec-1 also inhibits the release of eCIRP, TNF-α and IL-6 in the serum and peritoneal fluid in CLP-induced septic mice. We predicted a transient interaction between eCIRP and MLKL using a computational model, suggesting that eCIRP may exit the cell via the pores formed by p-MLKL.ConclusionNecroptosis is a novel mechanism of eCIRP release in sepsis. Targeting necroptosis may ameliorate inflammation and injury in sepsis by inhibiting eCIRP release.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

Discovery of a chalcone derivative as potent necroptosis inhibitor for the treatment of acute kidney injury.

Necroptosis, a form of inflammation-related programmed cell death, is a major mechanism of proximal tubular cell injury in acute kidney injury (AKI). Blockade of necroptosis signalling represents a promising strategy for clinical therapy of AKI. Previously, we identified a small molecular receptor-interacting protein kinases (RIPK)1 inhibitor Cpd-71 with nephroprotective activities. To discover more nephroprotective agents, in this study, 20 chalcone derivatives were synthesized and evaluated for their anti-necroptosis and nephroprotective activities. Among the chalcone derivatives, Cpd-2 exhibited the most potent anti-necroptosis activity (IC50 =1.08 μM) and protective activity (EC50 =1.49 μM) through directly binding to RIPK1 and blocking RIPK1-RIPK3-mixed-lineage kinase domain-like protein (MLKL) signalling pathway. Furthermore, Cpd-2 effectively attenuated cisplatin or hypoxia/reoxygenation (H/R)-induced injury and necroptotic inflammation in renal cell models. Moreover, in cisplatin- or ischemia/reperfusion (I/R) induced AKI mouse model, detection of creatinine and urea nitrogen in blood showed that Cpd-2 improved kidney function. Periodic acid-Schiff (PAS) staining and immunofluorescence analysis indicated that Cpd-2 also reduced pathological damage and inhibited inflammatory development in kidney tissues. In summary, although some chalcone derivatives have been reported to prevent kidney injury previously, our present study not only discovered a promising leading compound Cpd-2, but also provided a novel and successful practice for the development of necroptosis inhibitors from natural products derivatives as AKI therapeutic agents.

Isobavachalcone induces cell death through multiple pathways in human breast cancer MCF-7 cells

To explore the effects of isobavachalcone (IBC) on cell death of human breast cancer MCF-7 cells and explore the possible mechanism. MCF-7 cells were treated with different concentrations of IBC, and the changes in cell proliferation were assessed using MTT assay. Apoptosis of MCF-7 cells following treatment with 10, 20, and 40 μmol/L IBC was analyzed using flow cytometry with annexin V-FITC/PI double staining and fluorescence microscopy, and the expressions of apoptosis- and autophagy-related proteins (Bax, Bcl-2, Akt, p-Akt, p62, and LC3) were detected with Western blotting. Electron microscopy was used to observe the changes in submicrostructure of the cells following treatment with 40 μmol/L IBC. JC-1 assay kit, ATP assay kit, and reactive oxygen species (ROS) kit were used to determine the effect of IBC on mitochondrial function of the cells. MTT assay showed that IBC significantly inhibited the proliferation of MCF-7 cells in a concentration- and time-dependent manner, with IC50 values of 38.46, 31.31, and 28.26 μmol/L at 24, 48, and 72 h, respectively. IBC also concentration-dependently induced apoptosis of MCF-7 cells. IBC-induced cell death was inhibited by z-VAD-fmk, a caspase inhibitor (P < 0.05), but not by the necroptosis inhibitor necrostatin-1 (Nec-1). Western blotting showed that IBC-induced MCF-7 cell apoptosis by increasing Bax expression and down-regulating the expressions of Bcl-2, Akt and p-Akt-473 (all P < 0.05). With the increase of IBC concentration, the expression of autophagy-related protein p62 and the LC3-II/I ratio increased progressively. Electron microscopy revealed the presence of autophagic bodies in IBC-treated MCF-7 cells. IBC treatment also resulted in decreased mitochondrial membrane potential and intracellular ATP level and increased ROS accumulation in MCF-7 cells (P < 0.05). IBC is capable of inducing both apoptosis and autophagy in MCF-7 cells, suggesting the potential value of IBC as a lead compound in the development of anti-breast cancer agents.

Enterococcus faecalis-Induced Macrophage Necroptosis Promotes Refractory Apical Periodontitis.

ABSTRACTThe persistence of residual bacteria, particularly Enterococcus faecalis, contributes to refractory periapical periodontitis, which still lacks effective therapy. The role of receptor-interacting protein kinase 3 (RIPK3)- and mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis, a highly proinflammatory form of regulated cell death, has recently drawn much attention. However, the role of necroptosis in the pathogenesis of refractory periapical periodontitis remains unclear. We investigated whether the RIPK3/MLKL signaling pathway was activated in periapical lesion specimens obtained from patients diagnosed with refractory periapical periodontitis. RIPK3-deficient mice were then used to determine the role of necroptosis under this condition in vivo. We found that the phosphorylation levels of RIPK3 and MLKL were elevated in periapical lesion specimens of patients with refractory periapical periodontitis. In addition, necroptosis was induced in an E. faecalis-infected refractory periapical periodontitis mouse model, in which inhibition of necroptosis by RIPK3 deficiency could markedly alleviate inflammation and bone destruction. Moreover, double-labeling immunofluorescence suggested that macrophage necroptosis may be involved in the development of refractory periapical periodontitis. Then, we established an in vitro macrophage infection model with E. faecalis. E. faecalis infection was found to induce necroptotic cell death in macrophages through the RIPK3/MLKL signaling pathway, which was markedly alleviated by the RIPK3- or MLKL-specific inhibitor. Our study revealed that RIPK3/MLKL-mediated macrophage necroptosis contributes to the development of refractory periapical periodontitis and suggests that inhibitors or treatments targeting necroptosis represent a plausible strategy for the management of refractory periapical periodontitis.IMPORTANCE Oral infectious diseases represent a major neglected global population health challenge, imposing an increasing burden on public health and economy. Refractory apical periodontitis (RAP), mainly caused by Enterococcus faecalis, is a representative oral infectious disease with considerable therapeutic challenges. The interplay between E. faecalis and the host often leads to the activation of programmed cell death. This study identifies an important role of macrophage necroptosis induced by E. faecalis in the pathogenesis of RAP. Manipulating RIPK3/MLKL-mediated necroptosis may represent novel therapeutic targets, not only for RAP but also for other E. faecalis-associated infectious diseases.

A novel receptor-interacting protein-1 (RIP1) inhibitor (547) protects human cardiac cells from ischemia/reperfusion-triggered necroptotic cell death

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): M.M.C.P. is supported by a Netherlands Cardiovascular Research Initiative (CVON) grant (REMAIN 2014B27) and J.P.G.S is supported by Horizon2020 ERC-2016-COG-EVICARE [725229] and BRAVE (grant number 874827. Purpose Restoring perfusion, either by coronary angioplasty or bypass anastomosis, is performed around 2 million times per year throughout Europe to treat patients after myocardial infarction. Although this treatment is highly efficient to resolve myocardial ischemia, reperfusion is accompanied by damage through the release of reactive oxygen species (ROS). This ischemia/reperfusion (I/R) injury is manifested by cardiomyocyte death, caused to a certain extent by necroptosis. As a potential means of cardioprotection, we applied the small molecule compound 547 inhibiting RIP1-kinase, a central mediator of the necroptosis pathway, in an in vitro model of human cardiac I/R injury. Methods Human foetal cardiomyocyte progenitor cells (hfCPCs) were cultured and characterized for cell type defining markers before being subjected to simulated I/R damage using hydrogen peroxide (H2O2). Cardioprotective effects of 547 were assessed 24 hours after induction of damage and application of the compound. Cell viability and necroptosis pathway activation were assessed by flow cytometry, immunocytochemistry, and western blotting. Furthermore, mitochondrial damage was determined by JC-1 staining. Results hfCPCs expressed indicative markers SCA1, GATA4, PECAM-1, VEGF and C-KIT. Treatment with compound 547 significantly protected hfCPCs from H2O2 induced damage (viable cells: 84,3 ± 4,3% vs 1,6 ± 0,31%; P&amp;lt;0,0001). 547 decreased the necrotic cell population (DAPI+ AnnV-) (3,0 ± 1,1% vs 10,1 ± 1,5%, P&amp;lt;0,0001) and reduced phosphorylation of necrosome components RIP1 (1,3 ± 0,35 fold vs 3,7 ± 0,78 fold; P&amp;lt; 0,01), RIP3 (1,0 ± 0,46 fold vs 4,3 ± 1,5 fold; P&amp;lt; 0,05), and MLKL (1,1 ± 0,15 fold vs 5,7 ± 1,7 fold; P&amp;lt; 0,01) without decreasing unphosphorylated protein or mRNA levels. Interestingly, 547 treatment prevented oxidative stress induced loss of nuclear RIP1 and RIP3 as measured by immunocytochemistry and western blotting of cell fractions (nuclear/cytoplasmic ratio RIP1: 0,80 ± 0,0006 [Control] vs 1,5 ± 0,17 fold [547]; P&amp;lt; 0,01; nuclear/cytoplasmic ratio RIP3: 0,76 ± 0,10 [Control] vs 1,6 ± 0,18 fold [547]; P&amp;lt; 0,01). Remarkably, compound 547 also decreased H2O2–induced mitochondrial depolarization (JC-1 staining/membrane depolarization index: 4,5 ± 0,85% vs 1,2 ± 0,38%; P&amp;lt; 0,05)) and expression and activation of CAMKII (0,46 ± 0,13 fold; P&amp;lt;0,01), a protein mediating mitochondria-dependent cell death. Conclusions Administration of compound 547 led to inhibition of necroptosis, increased cell viability and reduced mitochondrial damage in an in vitro human cardiac I/R injury model, emphasizing the potential of necroptosis inhibition for cardioprotection. Assessing clinical applicability will require further studies in relevant in vivo models.