Mixed Lineage Kinase Domain-like Research Articles

Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries.

Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway. Type 2 diabetes models were established using db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein-coupled receptor-targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies. Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase-mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes Rip1, Rip3, and Mlkl. Under hyperglycemic conditions, high glucose induced CB2R internalization in a β-arrestin 2-dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of Bach2 diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries. CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.

Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains

Necroptosis is a form of programmed necrotic cell death in which a signaling cascade induces oligomerization of mixed lineage kinase domain-like (MLKL) protein, leading to plasma membrane rupture. Necroptotic cell death is recognized as important for protection against viral infection and has roles in a variety of diseases, including cancer and diabetes. Despite its relevance to health and disease states, many questions remain about the precise mechanism of necroptotic cell death, cellular factors that can protect cells from necroptosis, and the role of necroptosis in disease models. In this study, we engineered a light-activated version of MLKL that rapidly oligomerizes and is recruited to the plasma membrane in cells exposed to light, inducing rapid cell death. We demonstrate this tool can be controlled spatially and temporally, used in a chemical genetic screen to identify chemicals and pathways that protect cells from MLKL-induced cell death, and used to study signaling responses of non-dying bystander cells. In additional studies, we re-engineered MLKL to block its cell-killing capacity but retain light-mediated membrane recruitment, developing a new single-component optogenetic tool that allows modulation of protein function at the plasma membrane.

Z-DNA/RNA Binding Protein 1 Senses Mitochondrial DNA to Induce Receptor-Interacting Protein Kinase-3/Mixed Lineage Kinase Domain-Like-Driven Necroptosis in Developmental Sevoflurane Neurotoxicity

Developmental sevoflurane exposure leads to widespread neuronal cell death known as sevoflurane-induced neurotoxicity (SIN). Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL)-driven necroptosis plays an important role in cell fate. Previous research has shown that inhibition of RIPK1 activity alone did not attenuate SIN. Since RIPK3/MLKL signaling could also be activated by Z-DNA/RNA binding protein 1 (ZBP1), the present study was designed to investigate whether ZBP1-mediated and RIPK3/MLKL-driven necroptosis is involved in SIN through in vitro and in vivo experiments. We found that sevoflurane priming triggers neuronal cell death and LDH release in a time-dependent manner. The expression levels of RIPK1, RIPK3, ZBP1 and membrane phosphorylated MLKL were also dramatically enhanced in SIN. Intriguingly, knockdown of RIPK3, but not RIPK1, abolished MLKL-mediated neuronal necroptosis in SIN. Additionally, inhibition of RIPK3-mediated necroptosis with GSK’872, rather than inhibition of apoptosis with zVAD, significantly ameliorated SIN. Further investigation showed that sevoflurane treatment causes mitochondrial DNA (mtDNA) release into the cytosol. Accordingly, ZBP1 senses cytosolic mtDNA and consequently activates RIPK3/MLKL signaling. This conclusion was reinforced by the evidence that knockdown of ZBP1 or depleting mtDNA with ethidium bromide remarkably improved SIN. Finally, the administration of the RIPK3 inhibitor GSK'872 relieved sevoflurane-induced spatial and emotional disorders without influence on locomotor activity. Altogether, these results illustrate that ZBP1 senses cytosolic mtDNA to induce RIPK3/MLKL-driven necroptosis in SIN. Elucidating the role of necroptosis in SIN will provide new insights into understanding the mechanism of anesthetic exposure in the developing brain.

A novel gut-restricted RIPK1 inhibitor, SZ-15, ameliorates DSS-induced ulcerative colitis

As a key mediator of cell death and inflammation, receptor-interacting protein kinase 1 (RIPK1) responds to a broad set of inflammatory and pro-death stimuli in human diseases. Inhibitors targeting RIPK1 are being investigated for the treatment of a wide range of human diseases, including ulcerative colitis. In the present study, we designed, synthesized, and investigated the anti-necroptosis and RIPK1-inhibition effects of SZ-15—a symmetrical high-molecular-weight (>500 Da) compound. SZ-15 effectively inhibited necroptosis in U937 and HT-29 cells at concentrations of 1 nM and 10 nM, respectively, and SZ-15 at a concentration of 10 nM almost completely blocked RIPK1, RIPK3, and mixed-lineage kinase domain-like (MLKL) protein phosphorylation induced by necrosis inducers. SZ-15 suppressed the pro-necroptosis function of RIPK1 by downregulating the mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. The activities of SZ-15 were effectively restricted to the gut: The percent recovery of the parent form of SZ-15 in mouse feces was 85.75%. Nevertheless, SZ-15 was effectively absorbed and detected in colon tissues after 1 h at a concentration of 3335 ± 868 ng/g, indicating that membrane permeability was maintained. SZ-15 alleviated dextran sulfate sodium (DSS)-induced ulcerative colitis in vivo by decreasing TNF-α, IL-1β, IL-22, and IL-6 mRNA expression in colonic tissues. Our preclinical study describes a novel gut-restricted RIPK1 inhibitor that shows great potential for use in the clinical treatment of ulcerative colitis.

Necroptosis: A Pathogenic Negotiator in Human Diseases.

Over the past few decades, mechanisms of programmed cell death have attracted the scientific community because they are involved in diverse human diseases. Initially, apoptosis was considered as a crucial mechanistic pathway for programmed cell death; recently, an alternative regulated mode of cell death was identified, mimicking the features of both apoptosis and necrosis. Several lines of evidence have revealed that dysregulation of necroptosis leads to pathological diseases such as cancer, cardiovascular, lung, renal, hepatic, neurodegenerative, and inflammatory diseases. Regulated forms of necrosis are executed by death receptor ligands through the activation of receptor-interacting protein kinase (RIPK)-1/3 and mixed-lineage kinase domain-like (MLKL), resulting in the formation of a necrosome complex. Many papers based on genetic and pharmacological studies have shown that RIPKs and MLKL are the key regulatory effectors during the progression of multiple pathological diseases. This review focused on illuminating the mechanisms underlying necroptosis, the functions of necroptosis-associated proteins, and their influences on disease progression. We also discuss numerous natural and chemical compounds and novel targeted therapies that elicit beneficial roles of necroptotic cell death in malignant cells to bypass apoptosis and drug resistance and to provide suggestions for further research in this field.

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

A novel necroptosis-related lncRNA signature predicts the prognosis and immune microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the malignant tumors with high mortality and a worse prognosis globally. Necroptosis is a programmed death mediated by receptor-interacting Protein 1 (RIP1), receptor-interacting Protein 1 (RIP3), and Mixed Lineage Kinase Domain-Like (MLKL). Our study aimed to create a new Necroptosis-related lncRNAs (NRlncRNAs) risk model that can predict survival and tumor immunity in HCC patients. The RNA expression and clinical data originated from the TCGA database. Pearson correlation analysis was applied to identify the NRlncRNAs. The LASSO-Cox regression analysis was employed to build the risk model. Next, the ROC curve and the area under the Kaplan-Meier curve were utilized to evaluate the accuracy of the risk model. In addition, based on the two groups of risk model, we performed the following analysis: clinical correlation, differential expression, PCA, TMB, GSEA analysis, immune cells infiltration, and clinical drug prediction analysis. Plus, qRT-PCR was applied to test the expression of genes in the risk model. Finally, a prognosis model covering six necroptosis-related lncRNAs was constructed to predict the survival of HCC patients. The ROC curve results showed that the risk model possesses better accuracy. The 1, 3, and 5-years AUC values were 0.746, 0.712, and 0.670, respectively. Of course, we also observed that significant differences exist in the following analysis, such as functional signaling pathways, immunological state, mutation profiles, and medication sensitivity between high-risk and low-risk groups of HCC patients. The result of qRT-PCR confirmed that three NRlncRNAs were more highly expressed in HCC cell lines than in the normal cell line. In conclusion, based on the bioinformatics analysis, we constructed an NRlncRNAs associated risk model, which predicts the prognosis of HCC patients. Although our study has some limitations, it may greatly contribute to the treatment of HCC and medical progression.

Characterizing the prognostic and therapeutic value of necroptosis in sarcoma based on necroptosis subtypes.

Necroptosis, a type of necrotic cell death independent of caspase regulation, is mainly mediated by receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). Necroptosis plays an essential role in many tumors. However, the potential roles of necroptosis in tumor microenvironment (TME) of sarcoma (SARC) remain unknown. This study analyzed the expression, prognosis, genetic alterations of necroptosis genes in SARC. We identified two subtypes (cluster A and B) by performing unsupervised consensus clustering. Cluster A and B greatly differed in prognosis and immune infiltration, with cluster A showing more favorable prognosis, higher immune infiltration and higher expression levels of necroptosis genes than cluster B. Based on the differentially expressed genes (DEGs) between two clusters, a necroptosis scoring system was developed for predicting overall survival of SARC patients. Patients with high necroptosis score had worse survival status, with a decreased infiltration level of most immune cells. Our findings demonstrated the potential role of necroptosis in regulating tumor microenvironment and the prognostic value of necroptosis-related genes for SARC patients.

The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes

SummaryReceptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/β-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.

The Expression Pattern of Non-apoptotic Cell Death Pathway in Osteosarcoma: Necroptosis and Autophagy as Backup Mechanisms for Therapeutics Strategy

Background: Among the primary bone tumors, osteosarcoma accounts for a malignant tumor with a high rate of progression and poor prognosis. Despite the achievement of combined therapy regimens in improving patients’ overall survival, patients with osteosarcoma confront the chemoresistance obstacle. Objectives: This study aimed at determining the expression pattern of autophagy and necroptosis pathways mediators in osteosarcoma tumors. Methods: The expression level of autophagy main mediators such as autophagy-associated protein 5 (ATG5), Beclin 1 (BECN1), and microtubule-associated protein 1A/1B-light chain 3 (LC3), necroptosis biomarkers such as receptor-interacting protein kinases (RIPK1 and RIPK3), and mixed lineage kinase domain-like (MLKL) were evaluated in 80 bone tissues including 60 bone tumors (40 malignant tumors and 20 benign tumors) and 20 margin tissues, using real-time PCR. The correlations of gene expression levels with the patient’s clinical and pathological features were considered. Results: Based on our data, ATG5, BECN1 and LC3 expression were down-regulated in osteosarcoma tumors compared to margin tissues. Also, malignant osteosarcoma tumors showed a significant decrease in the expression level of RIPK1 and MLKL as necroptosis regulators, which revealed a correlation with tumor malignancy. In addition, the higher expression levels of BECN1, LC3, RIPK1, and MLKL were observed in tumor tissues of patients under the chemotherapy regimen, indicating the relevance of autophagy and necroptosis pathways with the patient’s response to therapy. Conclusions: Reduction in the expression level of autophagy and necroptosis mediators in high-grade osteosarcoma tumors indicates the possible impact of these pathways on the rate of proliferation and growth of osteosarcoma tumor cells and can emphasize the importance of cell death alternative pathways for treatment when apoptosis machinery is mutated and cause chemoresistance.

Necroptosis of neuronal cells is related to the neuropathology of tick-borne encephalitis

Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes tick-borne encephalitis (TBE) in humans. Infections of Sapporo-17-Io1 (Sapporo) and Oshima 5-10 (Oshima) TBEV strains showed different pathogenic effects in mice. However, the differences between the two strains are unknown. In this study, we examined neuronal degeneration and death, and activation of glial cells in mice inoculated with each strain to investigate the pathogenesis of TBE. Viral growth was similar between Sapporo and Oshima, but neuronal degeneration and death, and activation of glial cells, was more prominent with Oshima. In human neuroblastoma cells, apoptosis and pyroptosis were not observed after TBEV infection. However, the expression of the necroptosis marker, mixed lineage kinase domain-like (MLKL) protein, was upregulated by TBEV infection, and this upregulation was more pronounced in Oshima than Sapporo infections. As necroptosis is a pro-inflammatory type of cell death, differences in necroptosis induction might be involved in the differences in neuropathogenicity of TBE.

Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector—mixed lineage kinase domain like (MLKL)—was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry‐based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase‐3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis‐associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3‐mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation‐mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune‐complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Necroptosis at a glance.

Necroptosis, or programmed necrosis, is an inflammatory form of cell death with important functions in host defense against pathogens and tissue homeostasis. The four cytosolic receptor-interacting protein kinase homotypic interaction motif (RHIM)-containing adaptor proteins RIPK1, RIPK3, TRIF (also known as TICAM1) and ZBP1 mediate necroptosis induction in response to infection and cytokine or innate immune receptor activation. Activation of the RHIM adaptors leads to phosphorylation, oligomerization and membrane targeting of the necroptosis effector protein mixed lineage kinase domain-like (MLKL). Active MLKL induces lesions on the plasma membrane, leading to the release of pro-inflammatory damage-associated molecular patterns (DAMPs). Thus, activities of the RHIM adaptors and MLKL are tightly regulated by posttranslational modifications to prevent inadvertent release of immunogenic contents. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of the regulatory mechanisms of necroptosis and its biological functions in tissue homeostasis, pathogen infection and other inflammatory diseases.

The role of RHIM in necroptosis.

The RIP homotypic interaction motif (RHIM) is a conserved protein domain that is approximately 18–22 amino acids in length. In humans, four proteins carrying RHIM domains have been identified: receptor-interacting serine/threonine protein kinase (RIPK) 1, RIPK3, Z-DNA-binding protein 1 (ZBP1), and TIR domain-containing adapter-inducing IFN-β (TRIF), which are all major players in necroptosis, a distinct form of regulated cell death. Necroptosis is mostly presumed to be a fail-safe form of cell death, occurring in cells in which apoptosis is compromised. Upon activation, RIPK1, ZBP1, and TRIF each hetero-oligomerize with RIPK3 and induce the assembly of an amyloid-like structure of RIPK3 homo-oligomers. These act as docking stations for the recruitment of the pseudokinase mixed-lineage kinase domain like (MLKL), the pore-forming executioner of necroptosis. As RHIM domain interactions are a vital component of the signaling cascade and can also be involved in apoptosis and pyroptosis activation, it is unsurprising that viral and bacterial pathogens have developed means of disrupting RHIM-mediated signaling to ensure survival. Moreover, as these mechanisms play an essential part of regulated cell death signaling, they have received much attention in recent years. Herein, we present the latest insights into the supramolecular structure of interacting RHIM proteins and their distinct signaling cascades in inflammation and infection. Their uncovering will ultimately contribute to the development of new therapeutic strategies in the regulation of lytic cell death.

RIPK3-MLKL signaling activates mitochondrial CaMKII and drives intrarenal extracellular matrix production during CKD

Intrarenal extracellular matrix production or kidney fibrosis is a prevalent feature of all forms of chronic kidney disease (CKD). The transforming growth factor-beta (TGFβ) is believed to be a major driver of extracellular matrix production. Nevertheless, anti-TGFβ therapies have consistently failed to reduce extracellular matrix production in CKD patients indicating the need for novel therapeutic strategies. We have previously shown that necroinflammation contributes to acute kidney injury. Here, we show that chronic/persistent necroinflammation drives intrarenal extracellular matrix production during CKD. We found that renal expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) increases with the production of intrarenal extracellular matrix and declined kidney function in both humans and mice. Furthermore, we found that TGFβ exposure induces the translocation of RIPK3 and MLKL to mitochondria resulting in mitochondrial dysfunction and ROS production. Mitochondrial ROS activates the serine-threonine kinase calcium/calmodulin-dependent protein kinases-II (CaMKII) that increases phosphorylation of Smad2/3 and subsequent production of alpha-smooth muscle actin (αSMA), collagen (Col) 1α1, etc. in response to TGFβ during the intrarenal extracellular matrix production. Consistent with this, deficiency or knockdown of RIPK3 or MLKL as well as pharmacological inhibition of RIPK1, RIPK3, and CaMKII prevents the intrarenal extracellular matrix production in oxalate-induced CKD and unilateral ureteral obstruction (UUO). Together, RIPK1, RIPK3, MLKL, CaMKII, and Smad2/3 are molecular targets to inhibit intrarenal extracellular matrix production and preserve kidney function during CKD.

Protective effect of inhibiting necroptosis on gentamicin-induced nephrotoxicity.

Necroptosis is defined as a novel programmed cell necrosis that is mediated by receptor interacting serine-threonine protein kinase 1 (RIPK1) and other related signals. Necrosis, apoptosis and inflammation are commonly considered as the leading mechanism in acute kidney injury (AKI) induced by gentamicin (GEN), which is a useful antibiotic for treating the infection of Gram-negative bacterial. However, the necroptosis in the pathogenesis of GEN-induced AKI is unknown. In this study, to investigate the process and function of necroptosis in GEN-induced AKI, NRK-52E and HK-2 cells and SD rats were used as the models. The necroptosis-related proteins, including RIPK1, RIPK3, mixed lineage kinase domain-like (MLKL) and phosphorylated MLKL (p-MLKL), were all increasing time-dependently when GEN was continuously given. By using the RIPK1 inhibitor necrostatin-1 (NEC-1) and RIPK3 inhibitor (CPD42), the GEN-induced toxicity of tubular cells was alleviated. Moreover, it was validated that GEN-induced cell apoptosis and inflammation were attenuated after treating with NEC-1 or CPD42, both in vivo and in vitro. When MLKL was knocked down by siRNA, NEC-1 and CPD42 can not further protect the damage of tubular cells by GEN. Although the using of pan-caspase inhibitor Z-VAD significantly decreased GEN-induced apoptosis, it enhanced necroptosis and slightly promoted the decreased cell viability in GEN-treated cells, with the protective effects weaker than NEC-1 or CPD42. Finally, in vitro minimum inhibitory concentration (MIC) tests and bacteriostatic ring studies showed that NEC-1 did not interfere with the antibiotic effects of GEN. Thus, suppressing necroptosis can serve as a promising strategy for the prevention of GEN-induced nephrotoxicity.

Development and validation of a robust necroptosis related classifier for colon adenocarcinoma.

Background: Necroptosis, a novel form of apoptosis, plays a crucial function in the progression of colon adenocarcinoma (COAD) and is expected to be triggered in cancer therapy for enhancing anti-tumor immunity. However, the function of necroptosis in tumors and its relationship with the tumor microenvironment (TME) remains largely unclear. Methods: Necroptosis-related genes (NRGs) were collected from high-quality literature. Using The Cancer Genome Atlas (TCGA) (https://cancergenome.nih.gov) and the Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/geo) meta-cohorts, a robust risk model was constructed to systematically examine the clinical value, functional status, the role of TME based on the risk model, as also the genomic variations. Results: A risk model containing nine NRGs, including TNF receptor-associated factor (TRAF2), TNF receptor 1 associated via death domain (TRADD), ubiquitin carboxyl-terminal hydrolase 21 (USP21), TNF receptor superfamily, member 6 (FAS), tumor necrosis factor receptor superfamily 10B (TNFRSF10B), mitogen-activated protein kinase 8 (MAPK8), mixed lineage kinase domain-like (MLKL), TNF receptor-associated factor 5 (TRAF5), and recombinant receptor-interacting serine-threonine kinase 3 (RIPK3), was constructed. The risk model’s stability and accuracy were demonstrated in training, as also the validation cohorts; it was verified as an independent prognostic model for COAD. High-risk group patients developed “cold” tumors having active tumor proliferation and immunosuppression, while those in the low-risk group developed “hot” tumors with active immune and cell killing functions. Moreover, a higher number of copy number variations in the genome and fewer somatic mutations were found in high-risk group patients. Furthermore, higher sensitivity towards immunotherapy and chemotherapy was seen in patients of the low-risk group. Conclusion: A reliable risk model based on NRGs to assess patient prognosis and guide clinical decision-making was constructed and validated. Our findings may contribute to the understanding of necroptosis and are expected to aid clinical management and guide precision treatment for patients with COAD.

Influences of edaravone on necroptosis-related proteins and oxidative stress in rats with lower extremity ischemia/reperfusion injury

The study aimed to investigate the influences of edaravone on necroptosis-related proteins and oxidative stress in rats with lower extremity ischemia/reperfusion (I/R) injury. The normal group (n=10), model group (lower extremity I/R injury model, n=10), treatment group (treatment with edaravone, n=10) and intervention group [lower extremity I/R injury model intervened with necrostatin-1 (Nec-1), n=10] were set. A conventional biochemical test was adopted to detect hepatic function indexes, and an enzyme-linked immunosorbent assay was performed to measure the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO). The apoptosis level in rat tissues was determined via terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay. The expression levels of genes and proteins were measured via quantitative polymerase chain reaction (qPCR) and Western blotting assay. The content of serum alkaline phosphatase (ALP), glutamic-pyruvic transaminase (GPT) and creatine kinase isoenzyme (CK-MB) was remarkably higher in the model group than that in the normal group. The levels of TNF-α, IL-6 and IL-1 were increased markedly in the model group, and the content of MDA in anterior tibial muscle tissues was also raised. The SOD content was elevated in the treatment group and intervention group. The number of apoptotic cells was larger than that in other groups (p<0.05). The gene expression levels of receptor-interacting protein kinase 1 (RIPK1), RIPK3, mixed lineage kinase domain-like (MLKL) and Caspase-3 were prominently higher in the model group than those in the treatment group and intervention group (p<0.05). The expression level of SOD in the treatment group and intervention group increased remarkably compared with that in the model group (p<0.05). RIPK1 and MLKL were raised evidently in the model group (p<0.05). Edaravone may regulate necroptosis-related proteins and oxidative stress in rats with lower extremity I/R injury by inhibiting the RIPK1-MLKL signaling pathway.

Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death.

Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.

Alteration of intestinal microecology by oral antibiotics promotes oral squamous cell carcinoma development

Oral antibiotics can influence cancers and immunotherapy by interfering with the intestinal microbiota. However, the association between oral antibiotics and oral squamous cell carcinoma (OSCC) as well as the mechanisms underlying the effects of oral antibiotics on OSCC remain unclear. Here, we found that oral antibiotics cocktail (4Abx) promoted the tumor development and shifted the microbiota, decreasing the abundance of probiotic bacteria, and altered microbial metabolism in the gut of OSCC mice, increasing tyrosine and decreasing glutamate levels. In vitro experiments showed that tyrosine upregulated the PD-1 expression in T cells, SCC7 cell proliferation, and necroptosis expression. IL-10 expression level in CD11c+ cells was reduced by glutamate. Furthermore, the expression of the necroptosis-related proteins, including receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL), was higher in the OSCC mice treated with 4Abx. Supplementation with glutamate or healthy mouse feces by gavage alleviated the tumor-promoting effect of 4Abx with restored balance of microbial metabolism. Overall, we identified the detrimental role of oral antibiotics in promoting OSCC development through altered intestinal microbiota, microbial metabolism, and immune dysbiosis, implying the need for antibiotic stewardship in OSCC treatment.