Non-apoptotic Cell Death Pathways Research Articles

Parallel Lipid Peroxide Accumulation Strategy Based on Bimetal-Organic Frameworks for Enhanced Ferrotherapy.

Ferroptosis, a nonapoptotic cell-death pathway, is commonly regulated by ether lipid peroxide generation or glutathione consumption. In this work, a parallel lipid peroxide accumulation strategy was designed based on catalytic metal-organic frameworks (MOFs) for enhanced ferrotherapy. The bimetallic MOF was synthesized with iron porphyrin as a linker and cupric ion as a metal node, and erastin, a ferroptosis inducer, was sandwiched between the MOF layers with 4,4'-dipyridyl disulfide as spacers. In a tumor microenvironment, erastin was released from the layered MOFs through glutathione-responsive cleavage. The exfoliated MOFs served as a dual Fenton reaction inducer to generate numerous hydroxyl radicals for the accumulation of lipid peroxide, while erastin-aggravated glutathione depletion down-regulated glutathione peroxidase 4; this then inhibited the consumption of lipid peroxide. Therefore, a parallel lipid peroxide accumulation strategy was established for enhanced ferrotherapy that effectively inhibited tumor growth in live mice, opening up new opportunities to treat apoptosis-insensitive tumors.

Optogenetic Control of Non-Apoptotic Cell Death

We introduce optogenetic tools (designated LiPOP) that enable photoswitchable necroptosis and pyroptosis in live cells with varying kinetics. The LiPOP tools allow us to reconstruct the key molecular steps involved in these two non-apoptotic cell death pathways by harnessing the power of light. We further demonstrate the use of LiPOP, by itself or coupled with bioluminescence, to achieve optochemical killing of bacteria and cancer cells in vivo. LiPOPs set the stage for triggering necroptotic and pyroptotic cell death both in vitro and in vivo and will likely find use in studying the role of nonapoptotic cell death pathways within the tumor microenvironment.

Breast Cancer Stem Cell Potency of Nickel(II)-Polypyridyl Complexes Containing Non-steroidal Anti-inflammatory Drugs.

We report the breast cancer stem cell (CSC) potency of two nickel(II)‐3,4,7,8‐tetramethyl‐1,10‐phenanthroline complexes, 1 and 3, containing the non‐steroidal anti‐inflammatory drugs (NSAIDs), naproxen and indomethacin, respectively. The nickel(II) complexes, 1 and 3 kill breast CSCs and bulk breast cancer cells in the micromolar range. Notably, 1 and 3 display comparable or better potency towards breast CSCs than salinomycin, an established CSC‐active agent. The complexes, 1 and 3 also display significantly lower toxicity towards non‐cancerous epithelial breast cells than breast CSCs or bulk breast cancer cells (up to 4.6‐fold). Mechanistic studies suggest that 1 and 3 downregulate cyclooxygenase‐2 (COX‐2) in breast CSCs and kill breast CSCs in a COX‐2 dependent manner. Furthermore, the potency of 1 and 3 towards breast CSCs decreased upon co‐treatment with necroptosis inhibitors (necrostatin‐1 and dabrafenib), implying that 1 and 3 induce necroptosis, an ordered form of necrosis, in breast CSCs. As apoptosis resistance is a hallmark of CSCs, compounds like 1 and 3, which potentially provide access to alternative (non‐apoptotic) cell death pathways could hold the key to overcoming hard‐to‐kill CSCs. To the best of our knowledge, 1 and 3 are the first compounds to be associated to COX‐2 inhibition and necroptosis induction in CSCs.

Mechanisms of Photoreceptor Death in Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the most common cause of inherited blindness and is characterised by the progressive loss of retinal photoreceptors. However, RP is a highly heterogeneous disease and, while much progress has been made in developing gene replacement and gene editing treatments for RP, it is also necessary to develop treatments that are applicable to all causative mutations. Further understanding of the mechanisms leading to photoreceptor death is essential for the development of these treatments. Recent work has therefore focused on the role of apoptotic and non-apoptotic cell death pathways in RP and the various mechanisms that trigger these pathways in degenerating photoreceptors. In particular, several recent studies have begun to elucidate the role of microglia and innate immune response in the progression of RP. Here, we discuss some of the recent progress in understanding mechanisms of rod and cone photoreceptor death in RP and summarise recent clinical trials targeting these pathways.

Fenton reaction-independent ferroptosis therapy via glutathione and iron redox couple sequentially triggered lipid peroxide generator

Ferroptosis, a newfound non-apoptotic cell death pathway, results from the accumulation of iron-dependent lipid peroxide (LPO). Recently, emerging iron-based nanomaterials have been extensively developed to induce Fenton reaction-dependent ferroptosis for cancer therapy. However, insufficient amount of H2O2 and limited acidity of tumor could not satisfy the optimal conditions for Fenton reaction, which extremely limited the efficacy of ferroptosis therapy. Herein, we report a novel glutathione (GSH) and iron redox couple sequentially triggered LPO generator (LPOgener) which can directly supply the Fenton reaction-independent downstream executioner of ferroptosis for cancer therapy. By harnessing GSH-mediated Fe3+ reduction and the well-established iron redox couple-mediated lipid peroxidation, LPOgener was constructed by complete ferric ammonium citrate (FAC) and unsaturated lipids-rich phosphatidylcholine, and formed as FAC loaded liposome. The Fe3+ encapsulated in LPOgener could be efficiently reduced to Fe2+ under high GSH level in tumor cells. Subsequently, the formed iron redox couple could trigger overwhelming lipid peroxidation for Fenton reaction-independent ferroptosis. Superior anticancer therapeutic effect with little systemic toxicity demonstrated that LPOgener was a potent ferroptosis-inducing agent for cancer therapy. Therefore, to directly supply the druglike, easily prepared, GSH and iron redox couple sequentially triggered LPOgener would provide a new direction in designing strategies for ferroptosis therapy.

Delineation of cell death mechanisms induced by synergistic effects of statins and erlotinib in non-small cell lung cancer cell (NSCLC) lines.

Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) have been shown to overcome tyrosine kinase inhibitor (TKI) resistance in epithelial growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC) cells in vivo and in vitro. However, little is known about the putative induction of non-apoptotic cell death pathways by statins. We investigated the effects of pitavastatin and fluvastatin alone or in combination with erlotinib in three NSCLC cell lines and examined the activation of different cell death pathways. We assessed apoptosis via fluorometric caspase assay and poly (ADP-ribose) polymerase 1 (PARP) cleavage. Furthermore, annexinV/propidium iodide (PI) flow cytometry was performed. Small molecule inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD), necrostatin 1 (Nec1), ferrostatin 1 (Fer1), Ac-Lys-Lys-Norleucinal (Calp1) were used to characterise cell death pathway(s) putatively (co-)activated by pitavastatin/erlotinib co-treatment. Synergism was calculated by additivity and isobolographic analyses. Pitavastatin and fluvastatin induced cell death in EGFR TKI resistant NSCLC cells lines A549, Calu6 and H1993 as shown by caspase 3 activation and PARP cleavage. Co-treatment of cells with pitavastatin and the EGFR TKI erlotinib resulted in synergistically enhanced cytotoxicity compared to pitavastatin monotherapy. Flow cytometry indicated the induction of alternative regulated cell death pathways. However, only co-treatment with mevalonic acid (Mev) or the pan-caspase inhibitor zVAD could restore cell viability. The results show that cytotoxicity mediated by statin/erlotinib co-treatment is synergistic and can overcome erlotinib resistance in K-ras mutated NSCLC and relies only on apoptosis.

The DNA-damage response and nuclear events as regulators of nonapoptotic forms of cell death.

The maintenance of genome stability is essential for the cell as the integrity of genomic information guaranties reproduction of a whole organism. DNA damage occurring in response to different natural and nonnatural stimuli (errors in DNA replication, UV radiation, chemical agents, etc.) is normally detected by special cellular machinery that induces DNA repair. However, further accumulation of genetic lesions drives the activation of cell death to eliminate cells with defective genome. This particular feature is used for targeting fast-proliferating tumor cells during chemo-, radio-, and immunotherapy. Among different cell death modalities induced by DNA damage, apoptosis is the best studied. Nevertheless, nonapoptotic cell death and adaptive stress responses are also activated following genotoxic stress and play a crucial role in the outcome of anticancer therapy. Here, we provide an overview of nonapoptotic cell death pathways induced by DNA damage and discuss their interplay with cellular senescence, mitotic catastrophe, and autophagy.

P-5 - Screening for novel inhibitors of glutathione peroxidase

Cancer cells have higher basal levels of reactive oxygen species (ROS) production, and altered expression of antioxidant enzymes, when compared to normal cells. At high levels, ROS can cause substantial cellular damage, often leading to cell death, and many cancer therapeutics function through targeting these imbalanced redox systems. Glutathione peroxidases (GPx) protect cells from increased levels of ROS and oxidative stress by coupling the reduction of hydrogen and organic peroxides with the oxidization of glutathione (GSH) to glutathione disulfide (GSSG) [1]. Because of the role of glutathione peroxidases in controlling ROS levels, reducing oxidative stress, and regulating cell death pathways, we are interested in identifying inhibitors of GPx1 and GPx4. Moreover, GPx4 has recently been identified as a unique and important regulator of non-apoptotic cell death pathway, ferroptosis [2]. Using a novel method of recombinant GPx expression [3] we are developing an assay amenable to high-throughput screening of small molecules targeting GPx, and will characterize the identified inhibitors as potential chemotherapeutics for treating Glioblastoma Multiforme.

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies.

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.

Protein synthesis inhibition enhances paraptotic death induced by inhibition of cyclophilins in glioblastoma cells

Treatment of cancer is frequently unsuccessful related to the loss of apoptotic signaling in malignant cells. This is a particular problem for high-grade gliomas, such as Glioblastoma Multiforme (GBM), which are almost universally fatal within a year or so of diagnosis. Novel therapies that capitalize on non-apoptotic cell death pathways may yield more effective outcomes, if their underlying mechanisms can be more completely deciphered. In a recent publication (ref 10), the mechanisms by which cellular cyclophilins support GBM cell survival have been identified. Inhibition of cyclophilins activated paraptosis, which relied on a combination of endoplasmic reticulum (ER) stress and transient activation of autophagy. An important aspect of this effect was the relative rates of cap-dependent versus cap-independent protein synthesis, which were differentially modulated by protein synthesis inhibitors or mTOR inhibition. Although cycloheximide has previously been characterized as an inhibitor of paraptosis, in the case of cyclophilin inhibition, it appears to significantly enhance stress-related paraptosis and cell death. This work reveals an important role for cap-independent protein translation and autophagy in the ability of GBM cells to resist non-apoptotic death, and adds to our understanding of the events that underlie paraptosis.

Eosinophil ETosis and DNA Traps: a New Look at Eosinophilic Inflammation.

The traditional paradigm of eosinophils as end-stage damaging cells has mainly relied on their release of cytotoxic proteins. Cytokine-induced cell survival and secretion of granular contents from tissue-dwelling eosinophil are thought to be important mechanisms for eosinophilic inflammatory disorders, although the occurrence of cytolysis and its products (i.e., free extracellular granules) has been observed in affected lesions. Recent evidence indicates that activated eosinophils can exhibit a non-apoptotic cell death pathway, namely extracellular trap cell death (ETosis) that mediates the eosinophil cytolytic degranulation. Here, we discuss the current concept of eosinophil ETosis which provides a new look at eosinophilic inflammation. Lessons from eosinophilic chronic rhinosinusitis revealed that ETosis-derived DNA traps, composed of stable web-like chromatin, contribute to the properties of highly viscous eosinophilic mucin and impairments in its clearance. Intact granules entrapped in DNA traps are causing long-lasting inflammation but also might have immunoregulatory roles. Eosinophils possess a way to have post-postmortem impacts on innate immunity, local immune response, sterile inflammation, and tissue damage.

Establishment and characterization of a skin epidermal cell line from mud loach, Misgurnus anguillicaudatus, (MASE) and its interaction with three bacterial pathogens.

A continuous skin epidermal cell line from mud Loach (Misgurnus anguillicaudatus) (MASE cell line) was established with its application in bacteria infection demonstrated in this study. Primary MASE cell culture was initiated at 26°C in Dulbecco's modified Eagle medium/F12 medium (1:1; pH7.2) supplemented with 20% fetal bovine serum (FBS). The primary MASE cells in spindle morphology proliferated into a confluent monolayer within 2 weeks, and were continuously subcultured even in 10% FBS- DMEM/F12 after 10 passages. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 26°C. The MASE cells have been subcultured steadily over Passage 90 with a population doubling time of 53.3hat Passage 60. Chromosome analysis revealed that 60.5% of MASE cells at Passage 60 maintained the normal diploid chromosome number (50) with a normal karyotype of 10m+4sm+36t. Bacteria from the three species (Aeromonas veronii, Vibrio parahaemolyticus and Escherichia coli) were used to investigate the interactions between bacteria and cellular hosts. The three strains could be attached to the MASE cells and replicate at different levels. A.veronii could induce apoptosis in the MASE cells, with highest adherence rate among the three strains, whereas V.parahaemolyticus could cause highest cell death rate through a non-apoptotic cell death pathway, with high level of replication. The results revealed that different bacteria could interact with the MASE cells in different manners, and divergent pathways might lie in mediating cell death when cellular hosts confronted with pathogen infection. Therefore, the MASE cell line may serve as a useful tool for studying the interaction between skin bacteria and fish cells.

Length of intact plasma membrane determines the diffusion properties of cellular water.

Molecular diffusion in a boundary-free medium depends only on the molecular size, the temperature, and medium viscosity. However, the critical determinant of the molecular diffusion property in inhomogeneous biological tissues has not been identified. Here, using an in vitro system and a high-resolution MR imaging technique, we show that the length of the intact plasma membrane is a major determinant of water diffusion in a controlled cellular environment and that the cell perimeter length (CPL) is sufficient to estimate the apparent diffusion coefficient (ADC) of water in any cellular environment in our experimental system (ADC = −0.21 × CPL + 1.10). We used this finding to further explain the different diffusion kinetics of cells that are dying via apoptotic or non-apoptotic cell death pathways exhibiting characteristic changes in size, nuclear and cytoplasmic architectures, and membrane integrity. These results suggest that the ADC value can be used as a potential biomarker for cell death.

FLIP the Switch: Regulation of Apoptosis and Necroptosis by cFLIP.

cFLIP (cellular FLICE-like inhibitory protein) is structurally related to caspase-8 but lacks proteolytic activity due to multiple amino acid substitutions of catalytically important residues. cFLIP protein is evolutionarily conserved and expressed as three functionally different isoforms in humans (cFLIPL, cFLIPS, and cFLIPR). cFLIP controls not only the classical death receptor-mediated extrinsic apoptosis pathway, but also the non-conventional pattern recognition receptor-dependent apoptotic pathway. In addition, cFLIP regulates the formation of the death receptor-independent apoptotic platform named the ripoptosome. Moreover, recent studies have revealed that cFLIP is also involved in a non-apoptotic cell death pathway known as programmed necrosis or necroptosis. These functions of cFLIP are strictly controlled in an isoform-, concentration- and tissue-specific manner, and the ubiquitin-proteasome system plays an important role in regulating the stability of cFLIP. In this review, we summarize the current scientific findings from biochemical analyses, cell biological studies, mathematical modeling, and gene-manipulated mice models to illustrate the critical role of cFLIP as a switch to determine the destiny of cells among survival, apoptosis, and necroptosis.

Mlkl Pores Release Neutrophil Extracellular Traps in Necroptotic Neutrophils

Neutrophil extracellular traps (NETs) are networks of extracellular nuclear DNA and microbicidal proteins released from neutrophils in response to tissue damage and infection. Despite evidence of pathogenic roles for NETs in systemic lupus erythematosus, rheumatoid arthritis, diabetes, artherosclerosis and Alzheimer's disease, the major biochemical pathways controlling their formation remains poorly understood. Apoptosis does not contribute to NET formation but the role of regulated non-apoptotic cell death pathways such as necroptosis is not known. We have investigated the role of positive and negative regulators of necroptosis including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase domain-like (MLKL), receptor-interacting protein kinase-1 (RIPK1) and Caspase-8. Using immunogold electron microscopy, flow cytometry, imaging flow cytometry and fluorescence microscopy, we demonstrate that necroptosis can drive NET formation via MLKL pore formation at the cell surface. This process is caspase-independent but reactive oxygen species-dependent. Genetically-modified mouse peripheral blood and bone marrow neutrophils were used to show that Caspase-8 and RIPK1 negatively regulate NET formation driven by RIPK3 and MLKL. Mice that lack MLKL are deficient in necroptosis and NET formation, and were sensitive to methicillin-resistant Staphylococcus aureus (MRSA). Neutrophil-specific Caspase-8-deficiency also leads to increased susceptibility to MRSA due to increased rates of necroptotic neutrophil death. Killing of MRSA by necroptotic neutrophils is sensitive to DNase, and is dependent on MLKL, suggesting that necroptosis-driven NET formation contributes to the bactericidal activity of neutrophils. Human peripheral blood neutrophils also generate NETs that are sensitive to pharmacological inhibitors of necroptosis, suggesting that targeting necroptosis in general may help combat autoimmune responses to DNA. This study provides a framework to investigate the role of extracellular DNA release and cell death in the setting of infection, autoimmunity and autoinflammatory disease. DisclosuresNo relevant conflicts of interest to declare.

Synergistic effect of apoptosis and necroptosis inhibitors in cisplatin-induced nephrotoxicity.

Necroptosis is a nonapoptotic cell death pathway. We aim to study the effect of necrostatin-1 (a specific necroptosis inhibitor) in cisplatin-induced injury. We analyzed the effect of the combined use of inhibitors of apoptosis (z-vad) and necroptosis (necrostatin-1) in acute kidney injury by cisplatin in human proximal tubule cells. Our results showed moderate effectiveness in cytoprotection after treatment with z-vad. But the concomitant use of inhibitors (z-vad and necrostatin-1) presented synergistic and additive protection. The present study analyzed the caspase-3 activity and we observed a significant decrease in the group treated with z-vad and cisplatin. However we did not observe changes in the group treated with both inhibitors (z-vad and necrostatin-1) and cisplatin. Thus, demonstrating that necroptosis is a caspase-independent mechanism. We also analyzed the effect of necrostatin-1 in vivo model. C57BL/6 mice were treated with cisplatin and/or inhibitors. The concomitant use of inhibitors (z-vad and necrostatin-1) recovered renal function and decreased levels of urinary Ngal. Additionally, we analyzed the expression of RIP-1, a specific marker for necroptosis. In animals treated with cisplatin and z-VAD levels of RIP-1 were higher. This result reinforces that necroptosis occurs only in conditions where apoptosis was blocked. However, the use of both inhibitors (z-vad and necrostatin-1) provided additional protection. In conclusion, our study has a significant potential to show in vitro and in vivo protection obtained by necrostatin-1. Therefore, our results suggest that necroptosis may be an important mechanism of cell death after kidney injury.

Fight or flight: regulation of emergency hematopoiesis by pyroptosis and necroptosis.

A feature of the innate immune response that is conserved across kingdoms is the induction of cell death. In this review, we discuss the direct and indirect effects of increased inflammatory cell death, including pyroptosis - a caspase-1-dependent cell death - and necroptosis - a receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein-dependent, caspase-independent cell death - on emergency hematopoiesis. Activation of nonapoptotic cell death pathways during infection can trigger release of cytokines and/or damage-associated molecular patterns such as interleukin (IL)-1α, IL-1β, IL-18, IL-33, high-mobility group protein B1, and mitochondrial DNA to promote emergency hematopoiesis. During systemic infection, pyroptosis and necroptosis can directly kill hematopoietic stem and progenitor cells, which results in impaired hematopoiesis, cytopenia, and immunosuppression. Although originally described as discrete entities, there now appear to be more intimate connections between the nonapoptotic and death receptor signaling pathways. The choice to undergo pyroptotic and necroptotic cell death constitutes a rapid response system serving to eliminate infected cells, including hematopoietic stem and progenitor cells. This system has the potential to be detrimental to emergency hematopoiesis during severe infection. We discuss the potential of pharmacological intervention for the pyroptosis and necroptosis pathways that may be beneficial during periods of infection and emergency hematopoiesis.

Regulated necrosis: the expanding network of non-apoptotic cell death pathways

Cell death research was revitalized by the understanding that necrosis can occur in a highly regulated and genetically controlled manner. Although RIPK1 (receptor-interacting protein kinase 1)- and RIPK3-MLKL (mixed lineage kinase domain-like)-mediated necroptosis is the most understood form of regulated necrosis, other examples of this process are emerging, including cell death mechanisms known as parthanatos, oxytosis, ferroptosis, NETosis, pyronecrosis and pyroptosis. Elucidating how these pathways of regulated necrosis are interconnected at the molecular level should enable this process to be therapeutically targeted.

The dark side of TRAIL signaling

The dark side of TRAIL signaling

Identification of a small molecule that induces ATG5-and-cathepsin-l-dependent cell death and modulates polyglutamine toxicity

Non-apoptotic cell death mechanisms are largely uncharacterized despite their importance in physiology and disease [1]. Here we sought to systematically identify non-apoptotic cell death pathways in mammalian cells. We screened 69,612 compounds for those that induce non-canonical cell death by counter screening in the presence of inhibitors of apoptosis and necrosis. We further selected compounds that require active protein synthesis for inducing cell death. Using this tiered approach, we identified NID-1 (Novel Inducer of Death-1), a small molecule that induces an active, energy-dependent cell death in diverse mammalian cell lines. NID-1-induced death required components of the autophagic machinery, including ATG5, and the lysosomal hydrolase cathepsin L, but was distinct from classical macroautophagy. Since macroautophagy can prevent cell death in several contexts, we tested and found that NID-1 suppressed cell death in a cell-based model of Huntington's disease, suggesting that NID-1 activates a specific pathway. Thus the discovery of NID-1 identifies a previously unexplored cell death pathway, and modulating this pathway may have therapeutic applications. Furthermore, these findings provide a proof-of-principle for using chemical screening to identify novel cell death paradigms.