Form Of Non-apoptotic Cell Death Research Articles

MCM4 potentiates evasion of hepatocellular carcinoma from sorafenib-induced ferroptosis through Nrf2 signaling pathway

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It poses an enormous socioeconomic burden and is a serious public health threat globally due to its poor prognosis. Ferroptosis is a newly identified non-apoptotic form of cell death characterized by lipid peroxidation, iron accumulation, and reactive oxygen species (ROS) generation. However, tumor cells have evolved diverse mechanisms to evade ferroptosis, conferring resistance to drugs. Sorafenib, a first-line therapy for advanced HCC, triggers ferroptosis by selectively targeting solute carrier family 7 member 11 (SLC7A11) to deplete glutathione and inhibit glutathione peroxidase 4 (GPX4), thereby effectively eliminating tumor cells. However, sorafenib resistance has been widely reported, and the precise mechanisms underlying sorafenib drug resistance remain unclear. The minichromosome maintenance (MCM) protein family contains 10 members with vital roles in DNA replication and cell cycle progression. MCM4, a member of the MCM protein family, might be a potential biomarker in pan-cancer analysis. The present study found that MCM4 was upregulated in liver cancer using bioinformatics analysis and sorafenib-treated HCC cells. Moreover, MCM4 might be regarded as a prognostic biomarker for HCC. Further experiments revealed that MCM4-inhibition enhanced the efficacy of sorafenib through elevation of ferroptosis both in vitro and in vivo. Mechanistically, MCM4 potentiates sorafenib-induced ferroptosis evasion in HCC by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) signaling activation. However, no direct interactions were found between Nrf2 and MCM4. Overall, these findings suggest a potential therapeutic strategy for HCC by targeting MCM4 inhibition.

CRISPR screening identifies PRMT1 as a key pro-ferroptotic gene via a two-layer regulatory mechanism

Ferroptosis is a form of nonapoptotic cell death characterized by iron-dependent peroxidation of polyunsaturated phospholipids. However, much remains unknown about the regulators of ferroptosis. Here, using CRISPR-Cas9-mediated genetic screening, we identify protein arginine methyltransferase 1 (PRMT1) as a crucial promoter of ferroptosis. We find that PRMT1 decreases the expression of solute carrier family 7 member 11 (SLC7A11) to limit the abundance of intracellular glutathione (GSH). Moreover, we show that PRMT1 interacts with ferroptosis suppressor protein 1 (FSP1), a GSH-independent ferroptosis suppressor, to inhibit the membrane localization and enzymatic activity of FSP1 through arginine dimethylation at R316, thus reducing CoQ10H2 content and inducing ferroptosis sensitivity. Importantly, genetic depletion or pharmacological inhibition of PRMT1 in mice prevents ferroptotic events in the liver and improves the overall survival under concanavalin A (ConA) exposure. Hence, our findings suggest that PRMT1 is a key regulator of ferroptosis and a potential target for antiferroptosis therapeutics.

Sulfiredoxin-1 accelerates erastin-induced ferroptosis in HT-22 hippocampal neurons by driving heme Oxygenase-1 activation

Ferroptosis, a recently identified non-apoptotic form of cell death, is strongly associated with neurological diseases and has emerged as a potential therapeutic target. Nevertheless, the fundamental mechanisms are still predominantly unidentified. In the current investigation, sulfiredoxin-1 (SRXN1) has been identified as a crucial regulator that enhances the susceptibility to ferroptosis in HT-22 mouse hippocampal cells treated with erastin. Utilizing TMT-based proteomics, a significant increase in SRXN1 expression was observed in erastin-exposed HT-22 cells. Efficient amelioration of erastin-induced ferroptosis was achieved via the knockdown of SRXN1, which resulted in the reduction of intracellular Fe2+ levels and reactive oxygen species (ROS) in HT-22 cells. Notably, the activation of Heme Oxygenase-1 (HO-1) was found to be crucial for inducing SRXN1 expression in HT-22 cells upon treatment with erastin. SRXN1 increased intracellular ROS and Fe2+ levels by activating HO-1 expression, which promoted erastin-induced ferroptosis in HT-22 cells. Inhibiting SRXN1 or HO-1 alleviated erastin-induced autophagy in HT-22 cells. Additionally, upregulation of SRXN1 or HO-1 increased the susceptibility of HT-22 cells to ferroptosis, a process that was counteracted by the autophagy inhibitor 3-Methyladenine (3-MA). These results indicate that SRXN1 is a key regulator of ferroptosis, activating the HO-1 protein through cellular redox regulation, ferrous iron accumulation, and autophagy in HT-22 cells. These findings elucidate a novel molecular mechanism of erastin-induced ferroptosis sensitivity and suggest that SRXN1-HO-1-autophagy-dependent ferroptosis serves as a promising treatment approach for neurodegenerative diseases.

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response

Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

GPX4 allosteric activators inhibit ferroptosis and exert myocardial protection in doxorubicin-induced myocardial injury mouse model

Ferroptosis is a unique type of non-apoptotic form of cell death characterized by increased lipid hydroperoxide levels. It has relevance for a number of pathological conditions including multiple organ injuries and degenerative diseases. GPX4 plays an important role in ferroptosis by repairing lipid hydroperoxides. Based on the reported allosteric sites, we obtained the GPX4 allosteric activator hit compound A9 through virtual screening. A9 can bind to GPX4 and prevent RSL3-induced lipid peroxidation production in HT-1080 cells. In addition, A9 can specifically rescue erastin-induced cell death. Further chemical modification and structure-activity relationship studies afforded the optimized compound C3. C3 showed the activity of alleviating myocardial injury in the doxorubicin-induced myocardial injury mouse model. This study demonstrated that inhibiting ferroptosis by activating GPX4 is expected to be a potential solution to treat myocardial injury.

Assessment of the Ferroptosis Regulators: Glutathione Peroxidase 4, Acyl-Coenzyme A Synthetase Long-Chain Family Member 4, and Transferrin Receptor 1 in Patient-Derived Endometriosis Tissue.

Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case-control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = -2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2-13.5) g/dL versus 12.5 (12.2-13.4) g/dL in the control group; t (54) = -0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations.

Forms of Non-Apoptotic Cell Death and Their Role in Gliomas-Presentation of the Current State of Knowledge.

In addition to necrosis and apoptosis, the two forms of cell death that have been known for many decades, other non-apoptotic forms of cell death have been discovered, many of which also play a role in tumors. Starting with the description of autophagy more than 60 years ago, newer forms of cell death have become important for the biology of tumors, such as ferroptosis, pyroptosis, necroptosis, and paraptosis. In this review, all non-apoptotic and oncologically relevant forms of programmed cell death are presented, starting with their first descriptions, their molecular characteristics, and their role and their interactions in cell physiology and pathophysiology. Based on these descriptions, the current state of knowledge about their alterations and their role in gliomas will be presented. In addition, current efforts to therapeutically influence the molecular components of these forms of cell death will be discussed. Although research into their exact role in gliomas is still at a rather early stage, our review clarifies that all these non-apoptotic forms of cell death show significant alterations in gliomas and that important insight into understanding them has already been gained.

Uncovering the ferroptosis related mechanism of laduviglusib in the cell-type-specific targets of the striatum in Huntington’s disease

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder featured by abnormal movements, arising from the extensive neuronal loss and glial dysfunction in the striatum. Although the causes and pathogenetic mechanisms of HD are well established, the development of disease-modifying pharmacological therapies for HD remains a formidable challenge. Laduviglusib has demonstrated neuroprotective effects through the enhancement of mitochondrial function in the striatum of HD animal models. Ferroptosis is a nonapoptotic form of cell death that occurs as a consequence of lethal iron-dependent lipid peroxidation and mitochondrial dysfunction. However, the ferroptosis-related mechanisms underlying the neuroprotective effects of laduviglusib in the striatum of HD patients remain largely uncharted. In this study, we leveraged single-nucleus RNA sequencing data obtained from the striatum of HD patients in stages 2–4 to identify differentially expressed genes within distinct cell-type. We subsequently integrated these differentially expressed genes of HD, laduviglusib target genes and ferroptosis-related genes to predict the ferroptosis-related mechanisms underpinning the neuroprotective effects of laduviglusib in HD patients. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses unveiled that the effects of laduviglusib on direct pathway striatal projection neurons (dSPNs) is mainly associated with Th17 cell differentiation pathways. Conversely, its impact on indirect pathway striatal projection neurons (iSPNs) extends to the Neurotrophin signaling pathway, FoxO signaling pathway, and reactive oxygen species pathway. In microglia, laduviglusib appears to contribute to HD pathology via mechanisms related to Th17 cell differentiation and the FoxO signaling pathway. Further, molecular docking results indicated favorable binding of laduviglusib with PARP1 (associated with dSPNs and iSPNs), SCD (associated with astrocytes), ALOX5 (associated with microglia), and HIF1A (associated with dSPNs, iSPNs, and microglia). In addition, the KEGG results suggest that laduviglusib may enhance mitochondrial function and protect against neuronal loss by targeting ferroptosis-related signaling pathways, particularly mediated by ALOX5 in microglia. These findings provide valuable insights into the potential mechanisms through which laduviglusib exerts its effects on distinct cell-types within the HD striatum.

Tumor suppressor Par-4 activates autophagy-dependent ferroptosis

Ferroptosis is a unique iron-dependent form of non-apoptotic cell death characterized by devastating lipid peroxidation. Whilst growing evidence suggests that ferroptosis is a type of autophagy-dependent cell death, the underlying molecular mechanisms regulating ferroptosis are largely unknown. In this study, through an unbiased RNA-sequencing screening, we demonstrate the activation of a multi-faceted tumor-suppressor protein Par-4/PAWR during ferroptosis. Functional studies reveal that genetic depletion of Par-4 effectively blocks ferroptosis, whereas Par-4 overexpression sensitizes cells to undergo ferroptosis. More importantly, we have determined that Par-4-triggered ferroptosis is mechanistically driven by the autophagic machinery. Upregulation of Par-4 promotes activation of ferritinophagy (autophagic degradation of ferritin) via the nuclear receptor co-activator 4 (NCOA4), resulting in excessive release of free labile iron and, hence, enhanced lipid peroxidation and ferroptosis. Inhibition of Par-4 dramatically suppresses the NCOA4-mediated ferritinophagy signaling axis. Our results also establish that Par-4 activation positively correlates with reactive oxygen species (ROS) production, which is critical for ferritinophagy-mediated ferroptosis. Furthermore, Par-4 knockdown effectively blocked ferroptosis-mediated tumor suppression in the mouse xenograft models. Collectively, these findings reveal that Par-4 has a crucial role in ferroptosis, which could be further exploited for cancer therapy.

A homologous membrane-camouflaged self-assembled nanodrug for synergistic antitumor therapy

Limited success has been achieved in ferroptosis-induced cancer treatment due to the challenges related to low production of toxic reactive oxygen species (ROS) and inherent ROS resistance in cancer cells. To address this issue, a self-assembled nanodrug have been investigated that enhances ferroptosis therapy by increasing ROS production and reducing ROS inhibition. The nanodrug is constructed by allowing doxorubicin (DOX) to interact with Fe2+ through coordination interactions, forming a stable DOX-Fe2+ chelate, and this chelate further interacts with sorafenib (SRF), resulting in a stable and uniform nanoparticle. In tumor cells, overexpressed glutathione (GSH) triggers the disassembly of nanodrug, thereby activating the drug release. Interestingly, the released DOX not only activates nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) to produce abundant H2O2 production for enhanced ROS production, but also acts as a chemotherapeutics agent, synergizing with ferroptosis. To enhance tumor selectivity and improve the blood clearance, the nanodrug is coated with a related cancer cell membrane, which enhances the selective inhibition of tumor growth and metastasis in a B16F10 mice model. Our findings provide valuable insights into the rational design of self-assembled nanodrug for enhanced ferroptosis therapy in cancer treatment. Statement of significanceFerroptosis is a non-apoptotic form of cell death induced by the iron-regulated lipid peroxides (LPOs), offering a promising potential for effective and safe anti-cancer treatment. However, two significant challenges hinder its clinical application: 1) The easily oxidized nature of Fe2+ and the low concentration of H2O2 leads to a low efficiency of intracellular Fenton reaction, resulting in poor therapeutic efficacy; 2) The instinctive ROS resistance of cancer cells induce drug resistance. Therefore, we developed a simple and high-efficiency nanodrug composed of self-assembling by Fe2+ sources, H2O2 inducer and ROS resistance inhibitors. This nanodrug can effectively deliver the Fe2+ sources into tumor tissue, enhance intracellular concentration of H2O2, and reduce ROS resistance, achieving a high-efficiency, precise and safe ferroptosis therapy.

Ferroptosis inhibitor improves outcome after early and delayed treatment in mild spinal cord injury

We show that redox active iron can induce a regulated form of non-apoptotic cell death and tissue damage called ferroptosis that can contribute to secondary damage and functional loss in the acute and chronic periods after spinal cord injury (SCI) in young, adult, female mice. Phagocytosis of red blood cells at sites of hemorrhage is the main source of iron derived from hemoglobin after SCI. Expression of hemeoxygenase-1 that induces release of iron from heme, is increased in spinal cord macrophages 7 days after injury. While iron is stored safely in ferritin in the injured spinal cord, it can, however, be released by NCOA4-mediated shuttling of ferritin to autophagosomes for degradation (ferritinophagy). This leads to the release of redox active iron that can cause free radical damage. Expression of NCOA4 is increased after SCI, mainly in macrophages. Increase in the ratio of redox active ferrous (Fe2+) to ferric iron (Fe3+) is also detected after SCI by capillary electrophoresis inductively coupled mass spectrometry. These changes are accompanied by other hallmarks of ferroptosis, i.e., deficiency in various elements of the antioxidant glutathione (GSH) pathway. We also detect increases in enzymes that repair membrane lipids (ACSL4 and LPCAT3) and thus promote on-going ferroptosis. These changes are associated with increased levels of 4-hydroxynonenal (4-HNE), a toxic lipid peroxidation product. Mice with mild SCI (30 kdyne force) treated with the ferroptosis inhibitor (UAMC-3203-HCL) either early or delayed times after injury showed improvement in locomotor recovery and secondary damage. Cerebrospinal fluid and serum samples from human SCI cases show evidence of increased iron storage (ferritin), and other iron related molecules, and reduction in GSH. Collectively, these data suggest that ferroptosis contributes to secondary damage after SCI and highlights the possible use of ferroptosis inhibitors to treat SCI.

Mutant p53 reactivators protect breast cancer cells from ferroptosis.

Ferroptosis is a novel nonapoptotic form of cell death characterized by iron-dependent reactive oxygen species-mediated lipid peroxidation. In several different cell systems, the tumor suppressor p53 can enhance sensitivity to ferroptotic inducers. At least half of all human cancers show loss of function of p53. Furthermore, many of those tumors express mutant forms of p53 that has lost its wild-type function. Several groups have designed small molecules that can reactivate the wild-type function of these missense p53 mutants. We reasoned that p53 reactivators may also enhance sensitivity of certain cancer cells to ferroptosis stimuli. To test this idea we combined a number of different p53 reactivators with small molecule inducers of ferroptosis. In contrast, we observed that several p53 reactivators protected cells from cell death induced by ferroptotic inducers. Surprisingly, this protection still occurred in p53-null cell lines. We observed that these reactivators were neither free radical scavengers nor ion chelators. One of these p53 reactivator molecules, NSC 59984, reduced expression of GPX4, which is unlikely to explain its ability to reduce sensitivity to ferroptosis. We suggest that these p53 reactivators function via an unknown, p53-independent manner to suppress ferroptosis.

Targeting SOX13 inhibits assembly of respiratory chain supercomplexes to overcome ferroptosis resistance in gastric cancer

Therapeutic resistance represents a bottleneck to treatment in advanced gastric cancer (GC). Ferroptosis is an iron-dependent form of non-apoptotic cell death and is associated with anti-cancer therapeutic efficacy. Further investigations are required to clarify the underlying mechanisms. Ferroptosis-resistant GC cell lines are constructed. Dysregulated mRNAs between ferroptosis-resistant and parental cell lines are identified. The expression of SOX13/SCAF1 is manipulated in GC cell lines where relevant biological and molecular analyses are performed. Molecular docking and computational screening are performed to screen potential inhibitors of SOX13. We show that SOX13 boosts protein remodeling of electron transport chain (ETC) complexes by directly transactivating SCAF1. This leads to increased supercomplexes (SCs) assembly, mitochondrial respiration, mitochondrial energetics and chemo- and immune-resistance. Zanamivir, reverts the ferroptosis-resistant phenotype via directly targeting SOX13 and promoting TRIM25-mediated ubiquitination and degradation of SOX13. Here we show, SOX13/SCAF1 are important in ferroptosis-resistance, and targeting SOX13 with zanamivir has therapeutic potential.

LPCAT1-mediated membrane phospholipid remodelling promotes ferroptosis evasion and tumour growth.

The mechanisms underlying the dynamic remodelling of cellular membrane phospholipids to prevent phospholipid peroxidation-induced membrane damage and evade ferroptosis, a non-apoptotic form of cell death driven by iron-dependent lipid peroxidation, remain poorly understood. Here we show that lysophosphatidylcholine acyltransferase 1 (LPCAT1) plays a critical role in ferroptosis resistance by increasing membrane phospholipid saturation via the Lands cycle, thereby reducing membrane levels of polyunsaturated fatty acids, protecting cells from phospholipid peroxidation-induced membrane damage and inhibiting ferroptosis. Furthermore, the enhanced in vivo tumour-forming capability of tumour cells is closely associated with the upregulation of LPCAT1 and emergence of a ferroptosis-resistant state. Combining LPCAT1 inhibition with a ferroptosis inducer synergistically triggers ferroptosis and suppresses tumour growth. Therefore, our results unveil a plausible role for LPCAT1 in evading ferroptosis and suggest it as a promising target for clinical intervention in human cancer.

Abstract LB164: Inducing immunogenic cell death in difficult-to-treat cancers with an orally-bioavailable GPX4 inhibitor

Abstract Recent advancements in immunotherapy have transformed cancer treatment, resulting in extended survival times for some patients. However, despite these advancements, many patients do not benefit from immunotherapy because of an immunosuppressive tumor microenvironment. Consequently, there is a need for new strategies to improve the effectiveness of immunotherapy by stimulating inflammatory immunogenic cell death in tumors. Ferroptosis is a recently-described form of non-apoptotic cell death that induces a uniquely inflammatory immunogenic cell death. Glutathione Peroxidase 4 (GPX4) is the cell’s major defense system against ferroptosis as it is the only known enzyme capable of reducing toxic lipid hydroperoxides to inert lipid alcohols. Here we describe the discovery of a series of potent and selective small molecule GPX4 inhibitors that result in significant tumor growth inhibition in several aggressive tumor CDX models. We verified induction of ferroptosis in tumors using known biomarkers of ferroptosis such as HMOX1 mRNA induction as well as with 4-HNE staining. GPX4 target engagement and degradation was confirmed by mass spectrometry and western blot in tumors. GPX4 inhibition, when combined with PDL-1 blockade in a syngeneic sarcoma model, resulted in sustained significant tumor growth inhibition throughout the course of a 28-day study at well-tolerated doses when compared to either monotherapy alone. Current studies are underway to more carefully define the unique immunogenic cell death imparted by GPX4 inhibition and resulting ferroptosis. Citation Format: Timothy J. Read, Richard Steel, Ardeshir Golialei, Jenna Rimel, Leah Damon, Joey Azofeifa. Inducing immunogenic cell death in difficult-to-treat cancers with an orally-bioavailable GPX4 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB164.

Abstract LB299: Dihydrouridine synthase 2 sustains levels of tRNACys and prevents ferroptosis in lung cancer

Abstract Dihydrouridine is a universally conserved tRNA modification installed by enzymes that are important for human health. High expression of dihydrouridine synthase 2 (DUS2) predicts poor patient outcomes in lung adenocarcinoma (LUAD) for reasons that are not yet clear. Here, we show in human cells and mouse xenografts that DUS2 suppresses ferroptosis, a metal-dependent non-apoptotic form of cell death that is emerging as a therapeutic target in lung cancer. Elevated DUS2 correlates with resistance to ferroptosis inducers and loss of DUS2 causes increased sensitivity with concomitant accumulation of toxic lipid peroxides. Mechanistically, DUS2 is required to maintain tRNA CysGCA levels, supporting translation of cysteine-rich proteins including metallothioneins, key regulators of metal and redox homeostasis Notably, DUS2 KO cells cannot sustain normal levels of metallothioneins, a class of very cysteine rich proteins that serve as key regulators of both metal and redox homeostasis. Accordingly, DUS2 knockout cells are more susceptible to zinc intoxication and have lower levels of reduced glutathione, which partially explains their sensitivity to ferroptosis. Our findings demonstrate that DUS2 is required to support tRNACys levels and fend off ferroptosis in lung cancer cells. This highlights that individual tRNA substrates can play an outsized role in the biological functions of tRNA modifying enzymes, and demonstrates the therapeutic potential of targeting DUS2 in cancer. Citation Format: Austin Draycott, Matthew C. Wang, Diana Martínez Saucedo, Luisa Escobar-Hoyos, Wendy Gilbert. Dihydrouridine synthase 2 sustains levels of tRNACys and prevents ferroptosis in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB299.

Juglone induces ferroptosis in glioblastoma cells by inhibiting the Nrf2-GPX4 axis through the phosphorylation of p38MAPK

BackgroundFerroptosis, a non-apoptotic form of cell death induced by accumulation of free iron ions and lipid peroxidation, its importance for cancer treatment is gradually being recognized. Research on the anti-cancer mechanism of juglone is accumulating. However, the specific mechanism by which it directs glioblastoma (GBM) to death is unknown.MethodsWe used in vitro and in vivo experiments to explore the anti-GBM effect generated by juglone through the ferroptosis pathway.ResultsJuglone mainly causes cell death by inducing ferroptosis. Mechanistically, juglone can significantly activate the phosphorylation of p38MAPK. According to transcriptome sequencing and protein interaction analysis, the Nrf2-GPX4 signaling pathway is identified as the primary pathway through which juglone mediates ferroptosis. In vitro and in vivo experiments further verified that juglone induces the ferroptosis of GBM by activating the phosphorylation of p38MAPK and negatively regulating the Nrf2-GPX4 signaling pathway.ConclusionJuglone induces ferroptosis and inhibits the growth of GBM by targeting the Nrf2/Gpx4 signaling pathway and thus holds promise as a novel ferroptosis inducer or anti-GBM drug.

Arnicolide D induces endoplasmic reticulum stress-mediated oncosis via ATF4 and CHOP in hepatocellular carcinoma cells

Endoplasmic reticulum (ER) stress can trigger various cell death mechanisms beyond apoptosis, providing promise in cancer treatment. Oncosis, characterized by cellular swelling and increased membrane permeability, represents a non-apoptotic form of cell death. In our study, we discovered that Arnicolide D (AD), a natural sesquiterpene lactone compound, induces ER stress-mediated oncosis in hepatocellular carcinoma (HCC) cells, and this process is reactive oxygen species (ROS)-dependent. Furthermore, we identified the activation of the PERK-eIF2α-ATF4-CHOP pathway during ER stress as a pivotal factor in AD-induced oncosis. Notably, the protein synthesis inhibitor cycloheximide (CHX) was found to effectively reverse AD-induced oncosis, suggesting ATF4 and CHOP may hold crucial roles in the induction of oncosis by AD. These proteins play a vital part in promoting protein synthesis during ER stress, ultimately leading to cell death. Subsequent studies, in where we individually or simultaneously knocked down ATF4 and CHOP in HCC cells, provided further confirmation of their indispensable roles in AD-induced oncosis. Moreover, additional animal experiments not only substantiated AD’s ability to inhibit HCC tumor growth but also solidified the essential role of ER stress-mediated and ROS-dependent oncosis in AD’s therapeutic potential. In summary, our research findings strongly indicate that AD holds promise as a therapeutic agent for HCC by its ability to induce oncosis.

Accurate redox state indication by in situ derivatization with N-ethylmaleimide - Profiling of transsulfuration and glutathione pathway metabolites by UPLC-MS/MS

BackgroundReduced and oxidized glutathione play an important role for the intracellular detoxification of reactive oxygen species. The iron-dependent formation of such reactive oxygen species in conjunction with the inhibition of the redox-balancing enzyme glutathione peroxidase 4 underlie an imbalance in the cellular redox state, thereby resulting in a non-apoptotic form of cell death, defined as ferroptosis, which is relevant in several pathologies. MethodsHere we present a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) based method providing the accurate quantification of 12 glutathione pathway metabolites after in situ derivatization with N-Ethylmaleimide (NEM). The method was validated regards linearity, recovery and accuracy as well as precision. The assay includes glutathione and its oxidized form glutathione disulfide. Furthermore, the related precursors cysteine, cystine, glutamic acid, γ-glutamylcysteine and cysteinylglycine, biomarkers of protein crosslinking such as cystathionine and lanthionine, as well as metabolites of the transsulfuration pathway, methionine, homocysteine and serine are simultaneously determined. ResultsTwelve glutathione pathway metabolites were simultaneously analyzed in four different human cell line extracts within a total LC run time of 5.5 min. Interday coefficients of variation (1.7 % to 12.0 %), the mean observed accuracy (100.0 % ± 5.2 %), linear quantification ranges over three orders of magnitude for all analytes and sufficient metabolite stability after NEM-derivatization demonstrate method reliability. Immediate derivatization with NEM at cell harvesting prevents autooxidation of glutathione, ensures accurate results for the GSH/GSSG redox ratio and thereby allows interpretation of cellular redox state. ConclusionThe described UPLC-MS/MS method provides a sensitive and selective tool for a fast and simultaneous analysis of glutathione pathway metabolites, its direct precursors and related compounds. Assay performance characteristics demonstrate the suitability of the method for applications in different cell cultures. Therefore, by providing glutathione related functional metabolic readouts, the method enables investigations in mechanisms of ferroptosis and alterations in oxidative stress levels in several pathophysiologies.

Interplay of Ferroptosis and Cuproptosis in Cancer: Dissecting Metal-Driven Mechanisms for Therapeutic Potentials

Iron (Fe) and copper (Cu), essential transition metals, play pivotal roles in various cellular processes critical to cancer biology, including cell proliferation, mitochondrial respiration, distant metastases, and oxidative stress. The emergence of ferroptosis and cuproptosis as distinct forms of non-apoptotic cell death has heightened their significance, particularly in connection with these metal ions. While initially studied separately, recent evidence underscores the interdependence of ferroptosis and cuproptosis. Studies reveal a link between mitochondrial copper accumulation and ferroptosis induction. This interconnected relationship presents a promising strategy, especially for addressing refractory cancers marked by drug tolerance. Harnessing the toxicity of iron and copper in clinical settings becomes crucial. Simultaneous targeting of ferroptosis and cuproptosis, exemplified by the combination of sorafenib and elesclomol-Cu, represents an intriguing approach. Strategies targeting mitochondria further enhance the precision of these approaches, providing hope for improving treatment outcomes of drug-resistant cancers. Moreover, the combination of iron chelators and copper-lowering agents with established therapeutic modalities exhibits a synergy that holds promise for the augmentation of anti-tumor efficacy in various malignancies. This review elaborates on the complex interplay between ferroptosis and cuproptosis, including their underlying mechanisms, and explores their potential as druggable targets in both cancer research and clinical settings.