Necroptosis Of Cells Research Articles

Abstract 1321: CarboCell provides new opportunities for image-guided high-precision intratumoral combinatorial immunotherapy

Abstract Intratumoral immune activating therapy is biologically supported by the cancer immune response being generated in the tumor microenvironment (TME) and draining lymph nodes. Successful intratumoral immune activation is however challenging as the cells in the TME are plastic and reactive, and therefore requires continuous stimulation. This is problematic as drugs are rapidly washed out of tumors, and frequent intratumoral injections limit manageable locations, patient compliance and provides risks for cancer cell dissemination. The CarboCell technology overcomes this by forming a positionally stable drug release depot at the injection site with tailored release profiles of single or multiple drugs. Here we demonstrate the therapeutic performance of a CarboCell formulation releasing a TLR7/8 agonist and a TGFb inhibitor (CarboCell TLR:TGFb). CarboCell provides intratumoral drug retention up to 90% at day 3 and 50% at day 7 after injection in mouse tumors and beyond 14 days in canines. By control of the release kinetics, more than five times the systemically tolerated drug dose was tolerated for intratumoral CarboCell TLR:TGFb even at repeated dosing. CarboCell TLR:TGFb combinatorial therapy provided complete tumor rejection in 71%, 67% and 89% of mice carrying large MC38, EMT-6 and CT26 tumors. The dual drug CarboCell TLR:TGFb formulation was therapeutically superior to single drug CarboCell formulations, which underlines the synergistic value the combinatorial therapy. CarboCell TLR:TGFb was further demonstrated to inhibit lung metastasis in highly metastatic cancer models. Mechanistically, CarboCell TLR:TGFb activated the innate and adaptive immune system. Inflammatory cytokines were rapidly induced and accompanied by cancer cells necroptosis. This was followed by an increased number and activation of central DCs in the tumor draining lymph node and anti-cancer specific T cells in the tumor. CarboCell TLR:TGFb demonstrate potent immune activating properties and overcomes the multifactorial immunosuppressive barriers in the TME. Clinically attractive features of CarboCell further includes intrinsic MR and US visibility and radiographic contrast. This, in combination with full compatibility with high precision thin needle injection technologies, makes image guided injection, therapeutic planning, interpretation and validation of clinical performance possible. Combined, the unprecedented drug release and imaging properties of CarboCell makes the technology highly attractive for clinical advancement. Citation Format: Sophie B. Jensen, Elizabeth S. Chavez, Hólmfridur R. Haldórsdóttir, Fredrik Melander, Marouschka J. Scheeper, Linda M. Bruun, Jonas R. Henriksen, Anders Elias Hansen, Thomas L. Andresen. CarboCell provides new opportunities for image-guided high-precision intratumoral combinatorial immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1321.

Structure‐Based Drug Design: Multi‐Arm PEG/Peptidomimetic Conjugate Inhibitors of DR6/APP Interaction Block Hematogenous Tumor Cell Extravasation (Adv. Sci. 11/2021)

In article number 2003558 by Hongze Liao, Houwen Lin, Yourong Duan, and co‐workers, first class of APP/DR6 inhibitor, polymer–peptidomimetic PEG‐tAHP‐DRI, are developed by utilizing three rationaldesigned screening strategies, which demonstrate promising anti‐hematogenous metastatic activity by inhibiting the binding of amyloid precursor protein (APP) and death receptor 6 (DR6) as well as blocking the necroptosis of vascular endothelial cells induced by tumors cells.

Indole-3-carbinol ameliorates necroptosis and inflammation of intestinal epithelial cells in mice with ulcerative colitis by activating aryl hydrocarbon receptor

Ulcerative colitis (UC) is a disease characterized by inflammation and disruption of the intestinal epithelial barrier. Necroptosis plays a critical role in disease progression. Indole-3-carbinol (I3C), a natural dietary agonist of aryl hydrocarbon receptor (AHR), has shown alleviating effects on UC. However, its mechanisms of action have not been comprehensively elucidated. Therefore, we aimed at investigating the protective role of I3C in DSS-induced colitis mice models. I3C significantly ameliorated body weight loss, colon length shortening and colonic pathological damage in colitis mice, reduced disease activity index (DAI) and histological (HI) scores, as well as alleviated colonic necroptosis and inflammation. In vitro, I3C attenuated necroptosis and inflammation of colonoids and NCM460 cells. AHR, activated by I3C, inhibits activation of receptor-interacting protein kinase 1 (RIPK1) and the subsequent assembly of necrosome in a time-dependent manner, as well as suppressing NF-κB activation and decreasing TNF-α, IL-1β, IL-6 and IL-8 expression. Silencing of AHR aggravated necroptosis and inflammation of NCM460 cells, and did not be ameliorated by I3C. Furthermore, AHR activation induces the expression of inhibitor of apoptosis proteins (IAPs) and the ubiquitination of RIPK1. In conclusion, I3C exerts a protective effect in DSS-induced colitis mice models by alleviating the necroptosis and inflammation of IECs through activating AHR.

Non-muscle myosin heavy chain 9 maintains intestinal homeostasis by preventing epithelium necroptosis and colitis adenoma formation.

SummaryNon-muscle myosin IIA plays an important role in cell adhesion, cell migration, and tissue architecture. We previously showed that low activity of the heavy chain of non-muscle myosin II Myh9 is beneficial to LGR5+ intestinal stem cell maintenance. However, the function of Myh9 in adult mouse intestinal epithelium is largely unclear. In this study, we used the inducible Villin-creERT2 knockout approach to delete Myh9 in adult mouse intestinal epithelium and observed that homozygous deletion of Myh9 causes colitis-like morphologic changes in intestine, leads to a high sensitivity to dextran sulfate sodium and promotes colitis-related adenoma formation in the colon. Myh9 deletion disturbs cell junctions and impairs intestinal lumen barrier integrity, promoting the necroptosis of epithelial cells. Consistently, these changes can be partially rescued by Ripk3 knockout. Our results indicate that Myh9 is required for the maintenance of intestinal epithelium integrity and the prevention of cell necroptosis.

ABT‑737, a Bcl‑2 family inhibitor, has a synergistic effect with apoptosis by inducing urothelial carcinoma cell necroptosis.

ABT-737 is a recently reported inhibitor of members of the Bcl-2 family of apoptosis regulators. However, to the best of our knowledge, its necroptosis-inducing function in bladder cancer has not yet been researched. Thus, the present study aimed to investigate whether this Bcl-2 family inhibitor can induce both apoptosis and necroptosis of urothelial carcinoma cells. The proliferation and survival of urothelial carcinoma cell lines treated with a combination of both Z-VAD-FMK as a pan-caspase inhibitor and ABT-737 were assessed in vitro. Z-DNA binding protein 1 (ZBP1), receptor-interacting protein (RIP)1 and RIP3 were knocked down using small interfering RNA in urothelial carcinoma cell lines. The protein expression levels of ZBP1, RIP1 and RIP3 following cell transfection were measured via western blot analysis. Cell viability was determined using an MTT assay. Cell invasion was examined using cell invasion assays. The expression levels of necroptosis-related proteins, high mobility group box 1, ZBP1, mixed-lineage kinase domain-like protein (MLKL) and RIP3, were measured via western blotting. It was found that ABT-737 inhibited the proliferation and invasion of bladder cancer cells by inducing cell necrosis. The data demonstrated that ZBP1 and RIP3 have main roles in the cell necrosis induced by ABT-737. In addition, RIP3 and ZBP1, without interacting with RIP1, directly induced MLKL-mediated programmed cell necrosis. Thus, understanding how urothelial carcinoma cells react to Bcl-2 family inhibitors may accelerate the discovery of drugs to treat bladder cancer.

Necroptosis in Macrophage Foam Cells Promotes Fat Graft Fibrosis in Mice.

Background: Fibrosis is a major grafting-related complication that leads to fat tissue dysfunction. Macrophage-induced inflammation is related to the development of fat tissue fibrosis. Necroptosis is a recently discovered pathway of programmed cell necrosis that results in severe inflammation and subsequent tissue fibrosis. Thus, in this study, we investigated the role of macrophage necroptosis in fat graft fibrosis and the underlying mechanisms.Methods: Fibrosis and necroptosis were investigated in mouse fat tissue before and after grafting. An in vitro “crown-like” structure (CLS) cell culture model was developed by co-culturing RAW 264.7 macrophages with apoptotic adipocytes to reproduce in vivo CLS macrophage-adipocyte interactions. Lipid uptake and necroptosis in CLS macrophages were analyzed using Oil-Red-O staining, western blotting, and immunofluorescence. RAW264.7 macrophages were cultured alone or with apoptotic adipocytes and treated with a necroptosis inhibitor (Nec-1 or GSK872) to explore the paracrine effect of necroptotic CLS macrophages on collagen synthesis in fibroblasts in vitro. Mice were treated with Nec-1 to analyze the effect of blocking necroptosis on fat graft fibrosis.Results: Fibrosis was increased after grafting in fat grafts of mice. Macrophages clustered around apoptotic adipocytes or large oil droplets to form a typical CLS in fibrotic depots. This was accompanied by formation and necroptosis of macrophage foam cells (MFCs) in CLSs. RAW 264.7 macrophages co-cultured with apoptotic adipocytes induced CLS formation in vitro, and lipid accumulation in CLS macrophages resulted in the formation and necroptosis of MFCs. Necroptosis of MFCs altered the expression of collagen I and VI in fibroblasts via a paracrine mechanism involving inflammatory cytokines/chemokines, which was reversed by GSK872 or Nec-1 treatment. Furthermore, treatment with Nec-1 ameliorated fat graft fibrosis in mice.Conclusion: Apoptotic adipocytes induced necroptosis of MFCs, and necroptosis of these cells activated collagen synthesis in fibroblasts via a paracrine mechanism. Inhibition of necroptosis in macrophages is a potential approach to prevent fibrosis in fat grafts.

Involvement of necroptosis in contrast-induced nephropathy in a rat CKD model.

The risk of contrast-induced nephropathy (CIN) is high in patients with chronic kidney disease (CKD). However, the mechanism of CIN in CKD is not fully understood. Here, we prepared a clinically relevant model of CIN and examined the role of necroptosis, which potentially cross-talks with autophagy, in CIN. In Sprague-Dawley rats, CKD was induced by subtotal nephrectomy (SNx, 5/6 nephrectomy) 4weeks before induction of CIN. CIN was induced by administration of a contrast medium (CM), iohexol, following administration of indomethacin and N-omega-Nitro-L-arginine methyl ester. Renal function and tissue injuries were assessed 48h after CM injection. Serum creatinine (s-Cre) and BUN were increased from 0.28 ± 0.01 to 0.52 ± 0.02mg/dl and from 15.1 ± 0.7 to 29.2 ± 1.2mg/dl, respectively, after SNx alone. CM further increased s-Cre and BUN to 0.69 ± 0.03 and 37.2 ± 2.1, respectively. In the renal tissue after CM injection, protein levels of receptor-interacting serine/threonine-protein kinase (RIP) 1, RIP3, cleaved caspase 3, and caspase 8 were increased by 64 ~ 212%, while there was reduction in LC3-II and accumulation of p62. Necrostatin-1, an RIP1 inhibitor, administered before and 24h after CM injection significantly suppressed elevation of s-Cre, BUN and urinary albumin levels, kidney injury molecule-1 expression and infiltration of CD68-positive macrophages in renal tissues after CM injection. The results suggest that necroptosis of proximal tubular cells contributes to CIN in CKD and that suppression of protective autophagy by pro-necroptotic signaling may also be involved.

TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

Angiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

Heparanase induces necroptosis of microvascular endothelial cells to promote the metastasis of hepatocellular carcinoma

Heparanase (HPSE) is a kind of multifunctional extracellular hydrolase, and related to metastasis of hepatocellular carcinoma (HCC). Endothelial necroptosis promotes the metastasis of cancer cells. It is not clear whether HPSE could mediate necroptosis of microvascular endothelial cells (MVECs) to promote HCC metastasis. Here we found HPSE expression was up-regulated in HCC tissues and its over-expression was correlated with multiple tumor foci, microvascular invasion, and poor outcome of HCC patients. Non-contact co-culture experiments showed high-expressed HPSE in HCC cells mediated the necroptosis of human umbilical vein endothelial cells (HUVECs) and elevated the expression levels of syndecan-1 (SDC-1) and tumor necrosis factor-α (TNF-α) in vitro. As a result of necroptosis, trans-endothelial migration (TEM) of HCC cells was increased. Conversely, both HPSE and SDC-1 knockdowns reversed necroptosis and decreased TNF-α expression level, while HPSE over-expression increased SDC-1 and TNF-α expression and aggravated necroptosis. Animal experiments found that the nude mice, intraperitoneally injected with HPSE high expressing HCC cells, had obvious necroptosis of MVECs and high intrahepatic metastasis rate, which could be relieved by inhibitor of necroptosis. Morever, HPSE elevated the expression levels of p38 mitogen-activated protein kinase (p38 MAPK) rather than nuclear factor kappa B in vitro. Our data suggest that HPSE induces necroptosis of MVECs to promote the metastasis of HCC by activating HPSE/SDC-1/TNF-α axis and p38 MAPK pathway.

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Meet the First Authors.

Osthole induces necroptosis via ROS overproduction in glioma cells.

Glioma is a common primary malignant tumor that has a poor prognosis and often develops drug resistance. The coumarin derivative osthole has previously been reported to induce cancer cell apoptosis. Recently, we found that it could also trigger glioma cell necroptosis, a type of cell death that is usually accompanied with reactive oxygen species (ROS) production. However, the relationship between ROS production and necroptosis induced by osthole has not been fully elucidated. In this study, we found that osthole could induce necroptosis of glioma cell lines U87 and C6; such cell death was distinct from apoptosis induced by MG‐132. Expression of necroptosis inhibitor caspase‐8 was decreased, and levels of necroptosis proteins receptor‐interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain‐like protein were increased in U87 and C6 cells after treatment with osthole, whereas levels of apoptosis‐related proteins caspase‐3, caspase‐7, and caspase‐9 were not increased. Lactate dehydrogenase release and flow cytometry assays confirmed that cell death induced by osthole was primarily necrosis. In addition, necroptosis induced by osthole was accompanied by excessive production of ROS, as observed for other necroptosis‐inducing reagents. Pretreatment with the RIP1 inhibitor necrostatin‐1 attenuated both osthole‐induced necroptosis and the production of ROS in U87 cells. Furthermore, the ROS inhibitor N‐acetylcysteine decreased osthole‐induced necroptosis and growth inhibition. Overall, these findings suggest that osthole induces necroptosis of glioma cells via ROS production and thus may have potential for development into a therapeutic drug for glioma therapy.

Carbon ion triggered immunogenic necroptosis of nasopharyngeal carcinoma cells involving necroptotic inhibitor BCL-x.

To explore the potential and mechanisms of necroptosis, a form of immunogenic cell death, induced by carbon ion as compared to photon beams in established photon resistant- (PR-) and sensitive nasopharyngeal carcinoma (NPC) cells. MLKL is considered a central executor of necroptosis and phosphorylation of MLKL (p-MLKL) was a critical event of necroptosis. The clonogenic survival and DNA microarray demonstrated that after repeated photon irradiation, radiosensitive NPC cells became apoptosis-resistant but could be effectively inhibited by carbon ion irradiation. The relative biologic effectiveness (RBE) at D10 and D37 were 2.15 and 2.78 for PR-NPC cells. Carbon ion induced delayed DNA damage repair, cell cycle arrest, cytogenetic damage, morphological change and cell necrosis, indicating the possibility of necroptosis in both PR- and sensitive NPC cell types. The lower expression of necroptotic inhibitors (caspase-8 and Bcl-x) and higher level of MLKL in PR-NPC cells showed it was relatively more predisposed to necroptosis compared to the sensitive cells. Subsequent experiments demonstrated the significant upregulation of p-MLKL in the PR-NPC cells treated by carbon ion (4 Gy) compared with photon irradiation at both physical (4 Gy) and RBE (10 Gy) doses (P≤0.0001). Moreover, carbon ion induced a robust (up to 28 folds) p-MLKL in the PR-NPC cells as well as sensitive cells (up to 6-fold) coupled with a lower level of BCL-x expression and increased GM-CSF implicated in resculputure of immune system. These results suggested that carbon ion could induce necroptosis of NPC cells, especially in PR-NPC cells, and its mechanisms involve BCL-x.

Self-assembled polymeric nanocarrier-mediated co-delivery of metformin and doxorubicin for melanoma therapy

Malignant melanoma is a life-threatening form of skin cancer with a low response rate to single-agent chemotherapy. Although combined therapies of metformin (MET) and doxorubicin (DOX) are effective in treating a variety of cancers, including breast cancer, their different physicochemical properties and administration routines reduce the effective co-accumulation of both drugs in tumors. Nanoparticles (NPs) have been demonstrated to potentially improve drug delivery efficiency in cancer therapy of, for example, liver and lung cancers. Hence, in this study, we prepared pH-sensitive, biocompatible, tumor-targeting NPs based on the conjugation of biomaterials, including sodium alginate, cholesterol, and folic acid (FCA). As expected, since cholesterol and folic acid are two essentials, but insufficient, substrates for melanoma growth, we observed that the FCA NPs specifically and highly effectively accumulated in xenograft melanoma tumors. Taking advantage of the FCA NP system, we successfully co-delivered a combination of MET and DOX into melanoma tumors to trigger pyroptosis, apoptosis, and necroptosis (PANoptosis) of the melanoma cells, thus blocking melanoma progression. Combined, the establishment of such an FCA NP system provides a promising vector for effective drug delivery into melanoma and increases the possibility and efficiency of drug combinations for cancer treatment.

Necroptosis specific inhibitor-1 inhibits necroptosis in rat brain and improves brain injury after cardiac arrest

To explore the effects of necroptosis specific inhibitor-1 (Nec-1) on brain injury in rats after cardiac arrest and its mechanism. A total of 24 Sprague-Dawley (SD) rats were divided into Sham group, model group and Nec-1 group (n = 8 per group) according to random number table method. In the Sham group, only general surgical procedures were underdone without inducing cardiac arrest. In the model group, the rats were subjected to asphyxial cardiac arrest followed by cardiopulmonary resuscitation (CPR) at 6 minutes after cardiac arrest. In the Nec-1 group, Nec-1 of 1 mg/kg was administered after cardiac arrest, and CPR was performed at 6 minutes after cardiac arrest. At 72 hours after CPR, neurological deficit scores (NDS) were assessed, serum S100B levels were measured by enzyme linked immunosorbent assay (ELISA), receptor-interacting protein 3 (RIP3) expression in cerebral cortex and hippocampus was observed under immunofluorescence and positive rate was calculated, and the levels of RIP3 protein expression in brain were analyzed by Western blotting. At 72 hours after CPR, the rats in the model group showed obvious necroptosis and injury in brain. Compared with the Sham group, the NDS scores in the model group were significantly decreased [57.0 (52.7, 60.0) vs. 80.0 (80.0, 80.0), P < 0.05], the serum S100B was significantly increased (ng/L: 44.9±4.5 vs. 18.6±1.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly elevated [cerebral cortex: (31.7±4.8)% vs. (11.6±3.2)%, hippocampus: (28.4±0.8)% vs. (10.9±0.6)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly increased [RIP3 protein (RIP3/GAPDH): 0.708 (0.642, 0.722) vs. 0.408 (0.253, 0.504), P < 0.05]. After Nec-1 intervention, necroptosis and injury in brain were obviously improved. Compared with the model group, the NDS scores at 72 hours after CPR in the Nec-1 group were significantly increased [70.5 (68.5, 71.7) vs. 57.0 (52.7, 60.0), P < 0.05), the serum S100B was significantly decreased (ng/L: 31.9±2.7 vs. 44.9±4.5, P < 0.05), the percentages of RIP3 positive cells in cerebral cortex and hippocampus were significantly lowered [cerebral cortex: (23.7±4.1)% vs. (31.7±4.8)%,hippocampus: (20.4±0.4)% vs. (28.4±0.8)%, both P < 0.05], and the levels of RIP3 protein expression in brain were significantly declined [RIP3 protein (RIP3/GAPDH): 0.437 (0.379, 0.507) vs. 0.708 (0.642, 0.722), P < 0.05]. Nec-1 attenuated necroptosis of brain cells by inhibiting the expression of RIP3 protein, so as to reduce brain injury after cardiac arrest in rats.

Synergistic Utilization of Necrostatin-1 and Z-VAD-FMK Efficiently Promotes the Survival of Compression-Induced Nucleus Pulposus Cells via Alleviating Mitochondrial Dysfunction.

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function.

ROS-Mediated Necroptosis Is Involved in Iron Overload-Induced Osteoblastic Cell Death.

Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.

Intestinal vitamin D receptor signaling ameliorates dextran sulfate sodium-induced colitis by suppressing necroptosis of intestinal epithelial cells.

Vitamin D status is closely related to inflammatory bowel disease (IBD), but the mechanism has not been fully elucidated. This study explored the effect of intestinal vitamin D signaling on necroptosis and the underlying mechanism in colitis. Serum 25(OH)D levels and the expression of necroptotic proteins were examined in patients with IBD. Colitis was induced in an intestinal-specific hVDR transgenic model, and the gross manifestation, histological integrity, and intestinal barrier function were tested. The findings were further confirmed in vitro. Immunoprecipitation and colocalization were performed to investigate the association between the vitamin D receptor and necroptotic proteins. We found that serum 25(OH)D decreased in patients with IBD, while the expression of necroptotic proteins increased. The intestinal hVDR transgenic model could largely ameliorate the structural destruction, restore barrier dysfunction, and suppress necroptosis caused by DSS. This was probably achieved by binding to RIPK1/3 necrosomes, as we observed decreased RIPK1/3 necrosome formation and increased VDR expression in the cytosol. This study demonstrated an inhibitory effect of the intestinal vitamin D signaling pathway on necroptosis in DSS-induced colitis. The vitamin D receptor shifts from the nucleus to the cytosol to impede the formation of RIPK1/3. Our findings may offer some theoretical basis for a novel treatment of IBD in clinical practice.

MiR-141-3p ameliorates RIPK1-mediated necroptosis of intestinal epithelial cells in necrotizing enterocolitis.

Aim: To explore the effects of miR-141-3p on intestinal epithelial cells in necrotizing enterocolitis and the underlying mechanism.Results: The expression of miR-141-3p was significantly downregulated in serum samples of patients with NEC and LPS-treated Caco-2 cells. The in vitro assays showed that miR-141-3p mimics inhibited expression of IL-6 and TNF-α and reduced PI positive rate of the LPS-treated Caco-2 cells. Next, receptor interacting protein kinase 1 (RIPK1) was identified as the downstream molecule of miR-141-3p, and RIPK1 overexpression aggravated LPS-induced Caco-2 cell injury, which was ameliorated by miR-141-3p mimics. Finally, we found miR-141-3p mimics inhibited upregulation of necroptosis-related molecules and interaction of RIPK1 and RIPK3 in LPS-treated Caco-2 cells.Conclusion: Our research indicated that miR-141-3p protected intestinal epithelial cells from LPS damage by suppressing RIPK1-mediated inflammation and necroptosis, providing an alternative perspective to explore the pathogenesis of NEC.Methods: Quantitative real time-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-141-3p in serum samples of participants and lipopolysaccharide (LPS)-treated Caco-2 cells. Cell Counting Kit-8 (CCK-8) assay, Propidium Iodide (PI) staining and detection of inflammatory cytokines were performed to evaluate the role of miR-141-3p in LPS-treated Caco-2 cells. TargetScanHuman database and luciferase reporter gene assay were utilized to confirm the direct downstream molecule of miR-141-3p. Western blot analysis was used to explore the mechanism.

Suppression of CXCL-1 Could Restore Necroptotic Pathway in Chronic Lymphocytic Leukemia.

PurposeTo clarify the role of different cytokines and selenite in the defective necroptotic pathway of chronic lymphocytic leukemia (CLL).Patients and MethodsWe randomly collected the peripheral blood samples of 11 untreated CLL patients and 10 healthy volunteers, and then separated B lymphocytes from peripheral blood. Then, real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blot were performed to detect the expression of different cytokines, including CXC-motif chemokine ligand 1 (CXCL-1). Finally, we used flow cytometry to analyze the percentage of surviving cells to figure out whether CLL cells or normal B lymphocytes underwent necroptosis.Results1) The high expression of CXCL-1 was seen in CLL cells compared with normal B lymphocytes (p = 0.0001, adjusted p =0.0012); 2) The downregulation of CXCL-1 was shown in normal B lymphocytes after induction by TNF-α and z-VAD; 3) CLL cells could restore necroptosis induced by TNF-α and z-VAD after knockdown of CXCL-1; 4) The transcriptional and translational expression of LEF-1 were downregulated after the knockdown of CXCL-1 in CLL cells; 5. 3.2μM selenite could help CLL cells restore necroptosis (p = 0.0102) and inhibit the transcriptional and translational expression of CXCL-1.ConclusionCXCL-1 played an important role in the defective necroptosis of CLL cells and regulated the expression of LEF-1. Selenite could inhibit the expression of CXCL-1 and help CLL cells restore necroptosis together with TNF-α and z-VAD. Selenite might be the potential medication of CLL in the future.

Enterococcus faecalis induces necroptosis in human osteoblastic MG63 cells through the RIPK3 / MLKL signalling pathway.

To explore the activation of necroptosis triggered by Enterococcus faecalis in human osteoblastic MG63 cells and provide new insights into the pathogenesis of refractory apical periodontitis. The viability of MG63 cells exposed to live E.faecalis was investigated using the cell counting kit-8 assay. The relative expressions of specific markers for necroptosis, namely p-RIPK3 and p-MLKL, were determined by western blotting. Cells pretreated with necrosulfonamide and GSK'872, which are specific inhibitors for MLKL and RIPK3, respectively, were then subjected to lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry analysis and Hoechst 33342/PI double fluorescence staining. Lentiviral-delivered short hairpin RNA (shRNA) targeting MLKL was employed to further confirm the activation of necroptosis in MG63 cells infected with E.faecalis. Transmission electron microscopy was additionally used to observe the morphological characteristics. Statistical analysis was conducted using Student's t-tests or one-way ANOVA followed by the Student-Newman-Keuls test. The infection with E.faecalis significantly inhibited the viability of MG63 cells in a multiplicity of infection- and infection time-dependent manners (P<0.05). In line with this, the expression levels of necroptosis-related markers, p-RIPK3 and p-MLKL, were significantly increased postinfection (P<0.05). Significant reductions in death rate were detected in the case of E.faecalis-infected MG63 cells following pretreatment with the inhibitors of RIPK3 and MLKL (P<0.01). Furthermore, silencing of MLKL by shRNA significantly decreased LDH release (P<0.01) and resulted in less mitochondrial swelling and vacuole-like changes, as well as reduced endoplasmic reticulum expansion. Enterococcus faecalis infection-induced necroptosis of MG63 cells via the RIPK3/MLKL signalling pathway, which may exert a negative influence on the healing process of refractory apical periodontitis. This study may offer novel insights into the pathogenesis and potential therapeutic targets of refractory apical periodontitis.