Necroptosis Pathway Research Articles

Identification and Characterization of Necroptosis-Related Differentially Expressed Genes in Acute Myocardial Infarction: Insights into Immune-Related Pathways and Protein-Protein Interactions.

Acute myocardial infarction (AMI) is a serious cardiovascular medical emergency that can lead to death. Necroptosis, a programmed cell death pathway, has been implicated in the development and progression of AMI. The aim of our study was to identify necroptosis-related differentially expressed genes (NRDEGs) in AMI and investigate their interactions and functions. The GSE66360 dataset was screened for NRDEGs using the 'limma' R package, with a threshold of p 0.05. A set of 159 necroptosis-related genes (NRGs) was retrieved from the KEGG database. The protein-protein interactions (PPI) network was constructed using the STRING data resource. Molecular Complex Detection (MCODE) and cytohHubba plugin was applied to find the major modules and genes. Gene ontology (GO) and KEGG pathway analyses were performed using the R 'clusterProfiler' package. The enrichment scores for immune cell types and associated biological pathways or functions were gained using the ssGSEA method. Our study identified 5 down-regulated and 16 up-regulated NRDEGs in AMI. The PPI network analysis revealed several important modules and hub genes, including TNF, IL1B, TLR4, STAT3, NLRP3, TNFAIP3, CYBB, IFNGR1, FADD, and IL33. GO analysis revealed that NRDEGs were enriched in multiple biological processes, cellular components, and molecular functions, including those related to cytokine production, response to cytokine stimulus, and necroptotic process. NRDEGs were found to be particularly abundant in a number of non-disease pathways, such as necroptosis and immune-related pathways like cytokine-cytokine receptor interaction and TNF signaling pathway, according to KEGG pathway analysis. The ssGSEA analysis revealed a correlation between immune cells and NRDEGs in AMI. The study identified NRDEGs and their interactions in AMI, providing insights into the potential function of necroptosis in the pathological process of AMI. The results imply that immune-related pathways and cytokines may be crucial in the initiation and development of AMI. The study provides a foundation for further research on the underlying mechanisms of necroptosis in AMI and the potential for developing novel therapies.

Synthesis of a bisindole enyne with anticancer properties

We herein synthesize a bisindole enyne using the iron-catalyzed environmentally benign dimerization method. The enyne kills cancer cells by arresting the cell cycle at the G2/M phase via the necroptosis pathway, a non-apoptotic form of cell death that is also considered a promising and attractive pathway to induce cancer cell death. The inherent fluorescent property has been used to study its localization in cells and its binding interactions with genomic DNA. This work demonstrates bisindole enyne motifs could be a new pharmacophore for developing anticancer agents.

Arsenic trioxide-induced cytotoxicity in A549 cells: The role of necroptosis.

Lung cancer is one of the deadliest cancers globally. Arsenic trioxide (ATO) is still present as a highly effective drug in treating acute promyelocytic leukemia (APL). Chemotherapy resistance is one of the major problems in cancer therapy. Necroptosis, can overcomes resistance to apoptosis, and can promote cancer treatment. This study examines the necroptosis pathway in A549 cancer cells exposed to ATO. We used the MTT test to determine the ATO effects on the viability of A549 cells at three different time intervals. Also, the reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were performed in three-time intervals. The effect of ATO on apoptosis was evaluated by Annexin V / PI staining and, the RIPK1 and MLKL gene expression were measured by Real-Time PCR. The ATO has dose and time-dependent cytotoxic effects, so at 24, 48, and 72 h, the IC50 doses were 33.81 '11.44 '2.535 µM respectively. A 50 μM ATO is the most appropriate to increase the MMP loss significantly at all three times. At 24 and 48 h after exposure of cells to ATO, the ROS levels increased. The RIPK1 gene expression increased significantly compared to the control group at concentrations of 50 and 100 μM; however, MLKL gene expression decreased. The A549 cells, after 48 h exposure to ATO at 50 and 100 μM, induces apoptosis and necroptosis. Due to the reduced expression of MLKL, it can be concluded that ATO is probably effective in the metastatic stage of cancer cells.

Variation in CD8 T cell IFNγ differentiation to strains of Toxoplasma gondii is characterized by small effect QTLs with contribution from ROP16.

Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.

Nyctanthes arbor-tristis alkaloids activates p53 independent cell death receptor and necroptosis pathways in HepG2 cells.

Nyctanthes arbor-tristis is a traditional medicinal plant with potential anti-cancer properties. In this study, crude and alkaloid extracts were prepared from different parts of the plant, and their cytotoxicity was evaluated on four different cancer cell lines. The alkaloid extracts from the leaf and fruit showed promising results, with the HepG2 cell line exhibiting significant cytotoxicity. The promising extracts were further studied for their apoptotic potential using various methods, including DNA fragmentation, TUNEL, Caspase-3 activity, Giemsa, and Hoechst staining. Our results indicated that the fruit extract had the highest apoptotic potential, with clear nuclear condensation, fragmentation, and apoptotic bodies observed. We also investigated the alteration of the Bax/Bcl-2 ratio both at the mRNA and protein levels. Our results showed a significant upregulation of the Bax gene and downregulation of the Bcl-2 gene for the fruit alkaloid extract. This indicates that the phenomenon of cell death expression might be following a p53-independent extrinsic pathway and Bax-activated caspase-independent AIF-mediated necroptosis in the HepG2 cancer cell line. Overall, our findings suggest that Nyctanthes arbor-tristis has potential as a therapeutic option for cancer treatment. The alkaloid extracts from the leaf and fruit may hold promise as a source of bioactive compounds for further development into anti-cancer agents. Further studies are needed to explore the underlying mechanisms of their cytotoxic and apoptotic effects and to evaluate their safety and efficacy in animal models and clinical trials.

Comprehensive prediction of immune microenvironment and hot and cold tumor differentiation in cutaneous melanoma based on necroptosis-related lncRNA

As per research, causing cancer cells to necroptosis might be used as a therapy to combat cancer drug susceptibility. Long non-coding RNA (lncRNA) modulates the necroptosis process in Skin Cutaneous Melanoma (SKCM), even though the precise mechanism by which it does so has yet been unknown. RNA sequencing and clinical evidence of SKCM patients were accessed from The Cancer Genome Atlas database, and normal skin tissue sequencing data was available from the Genotype-Tissue Expression database. Person correlation analysis, differential screening, and univariate Cox regression were successively utilized to identify necroptosis-related hub lncRNAs. Following this, we adopt the least absolute shrinkage and selection operator regression analysis to construct a risk model. The model was evaluated on various clinical characteristics using many integrated approaches to ensure it generated accurate predictions. Through risk score comparisons and consistent cluster analysis, SKCM patients were sorted either high-risk or low-risk subgroups as well as distinct clusters. Finally, the effect of immune microenvironment, m7G methylation, and viable anti-cancer drugs in risk groups and potential clusters was evaluated in further detail. Included USP30-AS1, LINC01711, LINC00520, NRIR, BASP1-AS1, and LINC02178, the 6 necroptosis-related hub lncRNAs were utilized to construct a novel prediction model with excellent accuracy and sensitivity, which was not influenced by confounding clinical factors. Immune-related, necroptosis, and apoptosis pathways were enhanced in the model structure, as shown by Gene Set Enrichment Analysis findings. TME score, immune factors, immune checkpoint-related genes, m7G methylation-related genes, and anti-cancer drug sensitivity differed significantly between the high-risk and low-risk groups. Cluster 2 was identified as a hot tumor with a better immune response and therapeutic effect. Our study may provide potential biomarkers for predicting prognosis in SKCM and provide personalized clinical therapy for patients based on hot and cold tumor classification.

Clinicopathological and prognosis significance of RIPK1 in patients with cervical squamous cell carcinoma: a retrospective cohort study.

Cervical cancer is one of the most common types of carcinoma in women and has high morbidity and mortality rates worldwide. Recurrent and metastatic disease remains difficult to treat. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in mediating apoptosis, necroptosis, and inflammatory pathways downstream of death receptors and pattern recognition receptors. This study sought to explore the clinicopathological and prognosis significance of RIPK1 expression in cervical squamous cell carcinoma (CSCC). The data of 100 CSCC patients who underwent curative surgery from 2019 to 2020 were retrospectively included in this study. We collected the clinicopathological information of the patients and detected RIPK1 protein expression using immunohistochemistry. The Chi-square test and a 1-way analysis of variance were used to make comparisons between groups stratified by RIPK1 expression. A Pearson linear correlation analysis was used to evaluate the association between RIPK1 expression and the clinicopathological characteristics of the patients. A Cox regression analysis and Kaplan-Meier curves were used to analyze overall survival (OS) and progression-free survival (PFS). A multivariable regression analysis was conducted to identify the risk factors for an impaired prognosis in CSCC. RIPK1 was found to be overexpressed in the CSCC tissues. RIPK1 expression was significantly associated with age, the preoperative serum squamous cell carcinoma antigen (SCC-Ag) level, lymph node metastasis, invasion depth, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor size, PFS, and OS (P<0.05). The PFS and OS differed significantly among patients with RIPK1 expression (P<0.05). The multivariate analysis showed that RIPK1 was not independent risk factors for PFS and OS in CSCC patients (P>0.05). The expression of RIPK1 was significantly upregulated in CSCC and was associated with the clinicopathological features of CSCC. RIPK1 might serve as a novel marker that can be used to predict the prognosis of CSCC patients and as a biological target for the treatment of CSCC.

Lyso-globotriaosylsphingosine induces endothelial dysfunction via autophagy-dependent regulation of necroptosis

Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient α-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease.

Luteolin ameliorates necroptosis in Glucocorticoid-induced osteonecrosis of the femoral head via RIPK1/RIPK3/MLKL pathway based on network pharmacology analysis

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is deeply relevant to damage and dysfunction of bone microvascular endothelial cells (BMECs). Recently, necroptosis, a newly programmed cell death with necrotic appearance, has garnered increasing attention. Luteolin, a flavonoid compound derived from Rhizoma Drynariae, has numerous pharmacological properties. However, the effect of Luteolin on BMECs in GIONFH through the necroptosis pathway has not been extensively investigated. Based on network pharmacology analysis, 23 genes were identified as potential targets for the therapeutic effect of Luteolin in GIONFH via the necroptosis pathway, with RIPK1, RIPK3, and MLKL being the hub genes. Immunofluorescence staining results revealed high expression of vWF and CD31 in BMECs. In vitro experiments showed that incubation with dexamethasone led to reduced proliferation, migration, angiogenesis ability, and increased necroptosis of BMECs. However, pretreatment with Luteolin attenuated this effect. Based on molecular docking analysis, Luteolin exhibited strong binding affinity with MLKL, RIPK1, and RIPK3. Western blotting was utilized to detect the expression of p-MLKL, MLKL, p-RIPK3, RIPK3, p-RIPK1, and RIPK1. Intervention with dexamethasone resulted in a significant increase in the p-RIPK1/RIPK1 ratio, but the effects of dexamethasone were effectively counteracted by Luteolin. Similar findings were observed for the p-RIPK3/RIPK3 ratio and the p-MLKL/MLKL ratio, as anticipated. Therefore, this study demonstrates that Luteolin can reduce dexamethasone-induced necroptosis in BMECs via the RIPK1/RIPK3/MLKL pathway. These findings provide new insights into the mechanisms underlying the therapeutic effects of Luteolin in GIONFH treatment. Additionally, inhibiting necroptosis could be a promising novel approach for GIONFH therapy.

Abstract 2525: Analyzing the role of MDMX for regulating cell death in prostate cancer cells

Abstract Prostate cancer (PCa) is the most prominent cancer type and the second leading cause of cancer death among men in the USA. The five-year survival rate for localized PCa is 98% but drops to 31% when PCa spreads to different parts of the body. Various treatment strategies that can induce cell death through different mechanisms are used while treating cancers. In recent years, necroptosis has been established as one of the effective mechanisms of inducing cell death in cancers. Necroptosis can be identified as a regulated form of cell death that can occur independently of executioner caspases. Despite numerous studies on the effects of necroptosis in cancer, it’s role on PCa has not been fully elucidated. In this study, we have evaluated the role of MDMX, a proto-oncogene involved in the negative regulation of p53, in causing cell death through p53-independent mechanisms. We have observed that inhibition of MDMX decreased the cell viability of LNCaP cells in response to 20 µM of NSC (NSC-207895), a MDMX specific inhibitor treatment, in a concentration-dependent manner. MDMX inhibition appears to induce necroptosis in LNCaP prostate cancer cells by elevating the expression of receptor-interacting protein kinase 1 (RIP1), which is a key element of the necroptotic pathway. In general, the necroptosis pathway can trigger two opposing mechanisms in cancer where it can promote tumor migration and metastasis on one hand and can also act as a defensive mechanism on the other hand when apoptosis is disrupted. To further assess the effect of MDMX on cell migration, the LNCaP cells were treated with 20 µM of NSC, which effectively blocked the cancer cell migration for up to 48 h, which was confirmed using the scratch assay. Interestingly, NSC treatment also reduced the expression of Vascular Endothelial Growth Factor A (VEGFA), which is one of the main markers for angiogenesis. Our results show that the p53-independent mechanisms that are activated following MDMX inhibition can regulate both cancer cell migration and angiogenesis. MDMX also seemed to be involved in regulating PCa cell survival through necroptosis. The outcomes of our experiments provide additional knowledge and strategies for making an accurate prognosis and choosing the most suitable therapeutic strategies for offering effective treatments for MDMX-positive PCa. (This research was supported by the Royal Dames of Cancer Research Inc. Ft. Lauderdale, Florida) Citation Format: Ahmed Alsarrani, Umamaheswari Natarajan, Samia Alsubhi, Amal Alzahrani, Appu Rathinavelu. Analyzing the role of MDMX for regulating cell death in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2525.

Abstract 6143: Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies

Abstract Background: The recent discovery that multiple chemo- and immuno-therapies activate non-apoptotic cell death pathways such as necroptosis, pyroptosis, and ferroptosis in cancer cells has necessitated development of pharmacodynamic biomarker assays to study their novel mechanisms of action. We describe the development, analytical validation, and proof-of-concept demonstration of assays for potential biomarkers of ferroptosis, pyroptosis and necroptosis pathways to support anti-cancer drug development. Methods: We developed multiplex sandwich immunoassays on the Luminex platform to quantify transferrin receptor (TfR), glutathione peroxidase 4 (GPx4), active caspase-3, cleaved and full-length gasdermins D and E (GSDMD, GSDME), MLKL oligomers, and MLKL heterodimers with RIPK1 and RIPK3. Novel antibodies to gasdermin cleavage neoepitopes were generated and their specificity validated with knockout (GSDMD) or non-expressing (GSDME) cell lines. We utilized well-established in vitro cancer cell line models (NCI-H522, THP-1, MV411, MB231, HT29) and inducers/inhibitors of pyroptosis, necroptosis and ferroptosis to test for expected directional changes of biomarker modulation. Results: Analytical validity of the measurements is demonstrated with acceptable reproducibility, precision, dilutional linearity, dilutional recovery, spike recovery, sample freeze-thaw and temperature stability. The fitness-for-purpose of the assays is demonstrated by increased cleavage of GSDME, loss of full-length GSDME, and activation of caspase-3 in NCI-H522 cells treated with doxorubicin; similarly, cleavage of GSDMD and loss of full-length GSDMD is shown in THP-1 cells primed with LPS and treated with nigericin, and in MV-4-11 cells treated with val-boroPro. HT29 cells treated with ZVAD + birinapant + TNFα were used to demonstrate increased MLKL oligomerization or heterodimer formation with RIPK1/RIPK3. Modulation of ferroptosis biomarkers TfR and GPx4 (SU-DHL-5, MDA-MB-231) in response to imidazole ketone erastin (IKE) or RSL3 was cell line-dependent. Studies are underway to validate modulation of select ferroptosis biomarkers using orthogonal LC-MS methods. Conclusions: We describe development and translational readiness of novel multiplex assays to interrogate drugs inducing ferroptosis, pyroptosis and necroptosis cell death pathways. Our data demonstrate utility of biomarkers of pyroptosis and necroptosis, whereas biomarkers of ferroptosis require further appraisal. In vivo pharmacodynamic studies of pyroptosis, ferroptosis and necroptosis inducers are ongoing to verify assay fitness-for-purpose with tumor biopsy specimens. This project was funded with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: William G. Herrick, Jeevan Govindharajulu, Ralph E. Parchment, James H. Doroshow, Apurva K. Srivastava. Novel multiplexed assays of ferroptosis, pyroptosis and necroptosis biomarkers for translational studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6143.

Unwinding the modalities of necrosome activation and necroptosis machinery in neurological diseases.

Necroptosis, a regulated form of cell death, is involved in the genesis and development of various life-threatening diseases, including cancer, neurological disorders, cardiac myopathy, and diabetes. Necroptosis initiates with the formation and activation of a necrosome complex, which consists of RIPK1, RIPK2, RIPK3, and MLKL. Emerging studies has demonstrated the regulation of the necroptosis cell death pathway through the implication of numerous post-translational modifications, namely ubiquitination, acetylation, methylation, SUMOylation, hydroxylation, and others. In addition, the negative regulation of the necroptosis pathway has been shown to interfere with brain homeostasis through the regulation of axonal degeneration, mitochondrial dynamics, lysosomal defects, and inflammatory response. Necroptosis is controlled by the activity and expression of signaling molecules, namely VEGF/VEGFR, PI3K/Akt/GSK-3β, c-Jun N-terminal kinases (JNK), ERK/MAPK, and Wnt/β-catenin. Herein, we briefly discussed the implication and potential of necrosome activation in the pathogenesis and progression of neurological manifestations, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, traumatic brain injury, and others. Further, we present a detailed picture of natural compounds, micro-RNAs, and chemical compounds as therapeutic agents for treating neurological manifestations.

Targeting Receptor-Interacting Protein Kinase 1 by Novel Benzothiazole Derivatives: Treatment of Acute Lung Injury through the Necroptosis Pathway.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are serious and devastating pulmonary manifestations of acute systemic inflammation with high morbidity and mortality worldwide. Currently, there are no specific effective treatments for ALI/ARDS. RIPK1, which contributes to necroptosis and inflammation, is confirmed to be a promising strategy for the treatment of ALI. Herein, 23 benzothiazole derivatives were designed to specifically target RIPK1, and SZM-1209 showed high anti-necroptotic activity (EC50 = 22.4 nM) and kinase selectivity on RIPK1 over RIPK3 (Kd,RIPK1 = 85 nM, Kd,RIPK3 > 10,000 nM). In a mTNF-α-induced systemic inflammatory response syndrome (SIRS) model, SZM-1209 could completely reverse mouse deaths with significant anti-inflammatory effects. Furthermore, in a NNK short-term intratracheal exposure-induced ALI model, SZM-1209 significantly alleviated ALI by reducing pulmonary edema and pathological damage. Collectively, activities of SZM-1209 against RIPK1, necroptosis, SIRS, and ALI warranted further investigation of optimized benzothiazoles as promising lead structures against ALI-related diseases.

Transposable elements activation triggers necroptosis in mouse embryonic stem cells

Deficiency of the histone H3K9 methyltransferase SETDB1 induces RIPK3-dependent necroptosis in mouse embryonic stem cells (mESCs). However, how necroptosis pathway is activated in this process remains elusive. Here we report that the reactivation of transposable elements (TEs) upon SETDB1 knockout is responsible for the RIPK3 regulation through both cis and trans mechanisms. IAPLTR2_Mm and MMERVK10c-int, both of which are suppressed by SETDB1-dependent H3K9me3, act as enhancer-like cis-regulatory elements and their RIPK3 nearby members enhance RIPK3 expression when SETDB1 is knockout. Moreover, reactivated endogenous retroviruses generate excessive viral mimicry, which promotes necroptosis mainly through Z-DNA-binding protein 1 (ZBP1). These results indicate TEs play an important role in regulating necroptosis.

The Evaluation of Androctonus crassicauda Antivenom against the Effects of Aegaeobuthus nigrocinctus Scorpion Venom on Autophagy, Apoptosis and Necroptosis.

In this study aimed to show the role of autophagy acting as a seesaw between apoptosis and necroptosis in certain vital organs under the effects of the Aegaeobuthus nigricinctus venom and different dosages of the Androctonus crassicauda antivenom administration in mice. In the venom group (VG), mice (n= 6) were inoculated with 2LD50 A. nigrocinctus venom. In the antivenom administered groups (AVG), the effects of the potency of the A. crassicauda antivenom were evaluated to have a neutralization effect against 20LD50 of the A. nigrocinctus venom. After histopathological examination, expressions of mammalian target of rapamycin (mTOR) as an autophagy activator, receptor-interacting serine/threonine-protein kinase 3 (RIPK3) as a necroptosis activator, and caspase-3, caspase-9 as the markers of apoptotic cell death signals were evaluated by the immunoperoxidase method in addition to DNA in-situ fragmentations by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Only in VG, caspases and TUNEL expressions were found to be higher after the envenomation process in contrast to the elevated RIPK3 expressions. mTOR expressions remained almost stable in the organs. In AG, mTOR expressions were further increased in the 30LD50 and 40LD50 groups. There were an increased mTOR expression and stabilized caspases and TUNEL expression in these subgroups, the RIPK3 expressions were found to be low when compared with all of the antivenom administration groups. Increasing doses of the antivenom drifts more the cells to autophagy while cell fate in organs under envenomation getting rid of apoptosis and necroptosis pathways.

Standard of care drugs do not modulate activity of senescent primary human lung fibroblasts

Cellular senescence is crucial in the progression of idiopathic pulmonary fibrosis (IPF), but it is not evident whether the standard-of-care (SOC) drugs, nintedanib and pirfenidone, have senolytic properties. To address this question, we performed colorimetric and fluorimetric assays, qRT-PCR, and western blotting to evaluate the effect of SOC drugs and D + Q on senescent normal and IPF lung fibroblasts. In this study, we found that SOC drugs did not provoke apoptosis in the absence of death ligand in normal or IPF senescent lung fibroblasts. Nintedanib increased caspase-3 activity in the presence of Fas Ligand in normal but not in IPF senescent fibroblasts. Conversely, nintedanib enhanced B cell lymphoma 2 expression in senescent IPF lung fibroblasts. Moreover, in senescent IPF cells, pirfenidone induced mixed lineage kinase domain-like pseudokinase phosphorylation, provoking necroptosis. Furthermore, pirfenidone increased transcript levels of FN1 and COL1A1 in senescent IPF fibroblasts. Lastly, D + Q augmented growth differentiation factor 15 (GDF15) transcript and protein levels in both normal and IPF senescent fibroblasts. Taken together, these results establish that SOC drugs failed to trigger apoptosis in senescent primary human lung fibroblasts, possibly due to enhanced Bcl-2 levels by nintedanib and the activation of the necroptosis pathway by pirfenidone. Together, these data revealed the inefficacy of SOC drugs to target senescent cells in IPF.

Innate immune inflammatory cell death: PANoptosis and PANoptosomes in host defense and disease.

Regulated cell death (RCD) triggered by innate immune activation is an important strategy for host survival during pathogen invasion and perturbations of cellular homeostasis. There are two main categories of RCD, including nonlytic and lytic pathways. Apoptosis is the most well-characterized nonlytic RCD, and the inflammatory pyroptosis and necroptosis pathways are among the best known lytic forms. While these were historically viewed as independent RCD pathways, extensive evidence of cross-talk among their molecular components created a knowledge gap in our mechanistic understanding of RCD and innate immune pathway components, which led to the identification of PANoptosis. PANoptosis is a unique innate immune inflammatory RCD pathway that is regulated by PANoptosome complexes upon sensing pathogens, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs) or the cytokines produced downstream. Cytosolic innate immune sensors and regulators, such as ZBP1, AIM2 and RIPK1, promote the assembly of PANoptosomes to drive PANoptosis. In this review, we discuss the molecular components of the known PANoptosomes and highlight the mechanisms of PANoptosome assembly, activation and regulation identified to date. We also discuss how PANoptosomes and mutations in PANoptosome components are linked to diseases. Given the impact of RCD, and PANoptosis specifically, across the disease spectrum, improved understanding of PANoptosomes and their regulation will be critical for identifying new therapeutic targets and strategies.

Identification of a necroptosis-related gene signature as a novel prognostic biomarker of cholangiocarcinoma.

Cholangiocarcinoma (CHOL) is the most prevalent type of malignancy and the second most common form of primary liver cancer, resulting in high rates of morbidity and mortality. Necroptosis is a type of regulated cell death that appears to be involved in the regulation of several aspects of cancer biology, including tumorigenesis, metastasis, and cancer immunity. This study aimed to construct a necroptosis-related gene (NRG) signature to investigate the prognosis of CHOL patients using an integrated bioinformatics analysis. CHOL patient data were acquired from the Gene Expression Omnibus (GEO) (GSE89748, GSE107943) and The Cancer Genome Atlas (TCGA) databases, with NRGs data from the necroptosis pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Univariate and multivariate regression analyses were performed to establish the NRG signatures. Kaplan-Meier (KM) curves were used to evaluate the prognosis of patients with CHOL. Functional enrichment analysis was performed to identify key NRG-associated biological signaling pathways. We also applied integrative multi-omics analysis to the high- and low-risk score groups. Spearman's rank correlation was used to clarify the relationship between the NRG signature and immune infiltration. 65 differentially expressed (DE) NRGs were screened, five of which were selected to establish the prognostic signature of NRGS based on multivariate Cox regression analysis. We observed that low-risk patients survived significantly longer than high-risk patients. We found that patients with high-risk scores experienced higher immune cell infiltration, drug resistance, and more somatic mutations than patients with low-risk scores. We further found that sensitivities to GW843682X, mitomycin C, rapamycin, and S-trityl-L-cysteine were significantly higher in the low-risk group than in the high-risk group. Finally, we validated the expression of five NRGs in CHOL tissues using the TCGA database, HPA database and our clinical data. These findings demonstrate that the five-NRG prognostic signature for CHOL patients is reasonably accurate and valid, and it may prove to be of considerable value for the treatment and prognosis of CHOL patients in the future.

Limonin delays the progression of intervertebral disc degeneration in vivo and in vitro: the key role of the MAPK/NF-κB and necroptosis pathways.

Limonin has received significant attention due to its multiple biological effects, intervertebral disc degeneration (IDD) is also of interest due to the high prevalence of this disease. In this study, we determined the effects of limonin on IDD and the underlying mechanism of action to find novel ways to treat IDD. An IL-1β-induced cell inflammation model and a lumbar instability model inducing IDD were established to assess the progression of IDD with or without limonin treatment. We further evaluated MAPK/NF-κB and necroptosis pathways and alterations in the extracellular matrix specific within the disc. Limonin suppresses inflammation in the nucleus pulposus in vitro by reducing the production of pro-inflammatory markers such as iNOS and COX-2. Limonin reduced the activation of the MAPK/NF-κB signalling pathway and the RIP1/RIP3/MLKL necroptosis pathway in the NP cells. Moreover, limonin delays the IDD progression in the lumbar instability model. Limonin could potentially delay IDD by inhibiting NP cell necroptosis and modulating peripheral matrix proteins within the intervertebral disc and is a potential pharmacological research direction for the therapy in patients with IDD.

Construction and validation of a prognostic signature based on necroptosis-related genes in hepatocellular carcinoma.

Necroptosis is a necrotic programmed cell death with potent immunogenicity. Due to the dual effects of necroptosis on tumor growth, metastasis and immunosuppression, we evaluated the prognostic value of necroptosis-related genes (NRGs) in hepatocellular carcinoma (HCC). We first analyzed RNA sequencing and clinical HCC patient data obtained to develop an NRG prognostic signature based on the TCGA dataset. Differentially expressed NRGs were further evaluated by GO and KEGG pathway analyses. Next, we conducted univariate and multivariate Cox regression analyses to build a prognostic model. We also used the dataset obtained from the International Cancer Genome Consortium (ICGC) database to verify the signature. The Tumor Immune Dysfunction and Exclusion (TIDE) algorithm was used to investigate the immunotherapy response. Furthermore, we investigated the relationship between the prediction signature and chemotherapy treatment response in HCC. We first identified 36 differentially expressed genes out of 159 NRGs in hepatocellular carcinoma. Enrichment analysis showed that they were mainly enriched in the necroptosis pathway. Four NRGs were screened by Cox regression analysis to establish a prognostic model. The survival analysis revealed that the overall survival of patients with high-risk scores was significantly shorter than that of patients with low-risk scores. The nomogram demonstrated satisfactory discrimination and calibration. The calibration curves validated a fine concordance between the nomogram prediction and actual observation. The efficacy of the necroptosis-related signature was also validated by an independent dataset and immunohistochemistry experiments. TIDE analysis revealed that patients in the high-risk group were possibly more susceptible to immunotherapy. Furthermore, high-risk patients were found to be more sensitive to conventional chemotherapeutic medicines such as bleomycin, bortezomib, and imatinib. We identified 4 necroptosis-related genes and established a prognostic risk model that could potentially predict prognosis and response to chemotherapy and immunotherapy in HCC patients in the future.