Programmed necroptosis is upregulated in low-grade myelodysplastic syndromes and may play a role in the pathogenesis.

Myelodysplastic syndrome (MDS) is characterized by persistent cytopenias and evidence of morphologic dysplasia in the bone marrow (BM). Excessive hematopoietic programmed cell death (PCD) and inflammation have been observed in the bone marrow of patients with MDS, and are thought to play a significant role in the pathogenesis of the disease. Necroptosis is a major pathway of PCD that incites inflammation; however, the role of necroptosis in human MDS has not been extensively investigated. To assess PCD status in newly diagnosed MDS, we performed immunofluorescence staining with computational image analysis of formalin-fixed, paraffin-embedded BM core biopsies using cleaved caspase-3 (apoptosis marker) and necroptosis markers (receptor-interacting serine/threonine-protein kinase 1 [RIPK1], phospho-mixed lineage kinase domain-like protein [pMLKL]). Patients with MDS, but not controls without MDS or patients with de novo acute myeloid leukemia, had significantly increased expression of RIPK1 and pMLKL but not cleaved caspase-3, which was most evident in morphologically low-grade MDS (<5% BM blasts) and in MDS with low International Prognostic Scoring System risk score. RIPK1 expression highly correlated with the distribution of CD71+ erythroid precursors but not with CD34+ blast cells. We found that necroptosis is upregulated in early/low-grade MDS relative to control participants, warranting further study to define the role of necroptosis in the pathogenesis of MDS and as a potential biomarker for the diagnosis of low-grade MDS.

The Trend of ripk1/ripk3 and mlkl Mediated Necroptosis Pathway in Patients with Different Stages of Prostate Cancer as Promising Progression Biomarkers.

BACKGROUND: Programmed cell death is critical to maintain tissue homeostasis. Necroptosis, as well as apoptosis, has been considered as another form of regulated cell death which can be used as an effective way to overcome apoptosis-resistant tumor tissue growth. The aim of present study was to test whether or not ripk1, ripk3, or mlkl expression levels, as the key necroptotic modulators in different stages of prostate tumor growth. METHODS: Sixty-seven prostate tissues representing histologically confirmed cancer were selected. The cancer samples were categorized into 4 different stages based on cellular differentiation, tumor growth rate, and extra tissue expansion to regional lymph nodes, average PSA levels, and tumor volume. RNA extraction, cDNA synthesis and quantitative real time PCR were done based on standard guidelines. RESULTS: No statistically significant changes in ripk1 expression showed in all three stages (stage II to IV). The expression pattern of ripk3 represented a remarkable elevation in early stage, while, predominantly repressed in final cancer stage (IV). Also, there has been a significant negative correlation between ripk3 gene expression and tumor size and PSA levels. CONCLUSIONS: We cannot exclude the importance of the key regulator proteins in development and progression of prevalent lethal disease like prostate cancer. The ripk1/ripk3 mediated necroptosis pathway is more activated in early stages of prostate cancer via induced ripk3 expression, while repressed during prostate cancer final stages. Also, the repression of ripk3 is related to elevation of both PSA levels and tumor volume which represented the tumor progression in final stages.

RIPK3 mediates renal tubular epithelial cell apoptosis in endotoxin­induced acute kidney injury.

Renal tubular epithelial cell apoptosis is an important pathological mechanism of septic acute kidney injury (AKI). Endotoxin, also known as lipopolysaccharide (LPS), has a key role in septic AKI and can directly induce tubular epithelial cell apoptosis. The upregulation of receptor­interacting protein kinase 3 (RIPK3) in tubular epithelial cells has been reported in septic AKI, with RIPK3 mediating apoptosis in several cell types. In the present study, the effect of RIPK3 on endotoxin­induced AKI was investigated in mouse tubular epithelial cell apoptosis in vitro and in vivo. It was found that the expression of RIPK3 was markedly increased in endotoxin­induced AKI. Endotoxin­induced AKI and tubular epithelial cell apoptosis could be attenuated by GSK'872, a RIPK3 inhibitor. LPS stimulation also upregulated RIPK3 expression in tubular epithelial cells in a time­dependent manner. Both RIPK3 inhibitor and small interfering RNA (siRNA) targeting RIPK3 reduced LPS­induced tubular epithelial cell apoptosis in vitro. The expression of the proapoptotic protein Bax was induced by LPS and reversed by GSK'872 or RIPK3­siRNA. The present study revealed that RIPK3 mediated renal tubular cell apoptosis in endotoxin­induced AKI. RIPK3 may be a potential target for the prevention of renal tubular cell apoptosis in endotoxin­induced AKI.

Plasma receptor interacting protein kinase-3 levels are associated with acute respiratory distress syndrome in sepsis and trauma: a cohort study.

BACKGROUND: Necroptosis, a form of programmed cell death mediated by receptor interacting serine/threonine-protein kinase-3 (RIPK3), is implicated in murine models of acute respiratory distress syndrome (ARDS). We hypothesized that plasma RIPK3 concentrations in sepsis and trauma would be associated with ARDS development and that plasma RIPK3 would reflect changes in lung tissue RIPK3 in a murine model of systemic inflammation. METHODS: We utilized prospective cohort studies of critically ill sepsis (n = 120) and trauma (n = 180) patients and measured plasma RIPK3 at presentation and 48 h. Patients were followed for 6 days for ARDS by the Berlin definition. We used multivariable logistic regression to determine the association of plasma RIPK3 with ARDS in each cohort, adjusting for confounders. In mice, we determined whether plasma and lung tissue RIPK3 levels rise concomitantly 4 h after injection with lipopolysaccharide and ZVAD-FMK, an apoptosis inhibitor. RESULTS: The change in plasma RIPK3 from presentation to 48 h (ΔRIPK3) was associated with ARDS in sepsis (OR 1.30, 95% CI 1.03-1.63, per ½ standard deviation) and trauma (OR 1.79, 95% CI 1.33-2.40). This association was not evident for presentation RIPK3 levels. Secondary analyses showed similar findings for the association of ΔRIPK3 with acute kidney injury and 30-day mortality. Mice injected with lipopolysaccharide and ZVAD-FMK had significantly higher plasma (p < 0.001) and lung (p = 0.005) RIPK3 than control mice. CONCLUSIONS: The change in plasma RIPK3 from presentation to 48 h in both sepsis and trauma patients is independently associated with ARDS, and plasma RIPK3 may reflect RIPK3 activity in lung tissue.

RIP3 inhibition protects locomotion function through ameliorating mitochondrial antioxidative capacity after spinal cord injury.

A novel type of programmed necrosis called necroptosis has been identified in the field of cell death, thereby offering an opportunity for re-examining necrosis after spinal cord injury (SCI). Several recent studies have suggested receptor-interacting protein kinase 3 (RIP3) plays an important role in necrosis in many cell types. However, it is still unclear what downstream events that lead to cell death are triggered by RIP3 activation. Hence, link between RIP3 inhibition and induction of neuronal cell death via mitochondrial function and antioxidative capacity after SCI was studied in our work. We examined the protective effects of RIP3 inhibition in SCI-mice. Furthermore, mimicking the pathological conditions of SCI in vitro, spinal cord neurons were subjected to oxygen-glucose deprivation. Notably, we found GSK872 and Nec-1 ameliorated the locomotor function and spinal cord edema, and conferred reverse of SCI-induced loss of mitochondrial integrity, ATP, glutathione and superoxide dismutase and elevation of reactive oxygen species and malonyldialdehyde in SCI-mice. Moreover, GSK872 alleviated OGD-inducted mitochondrial dysfunction, decreased antioxidative capacity and cell death in spinal cord neurons, through inhibiting RIP3 activity. The data suggest improving antioxidative capacity as a potential multifunctional treatment after SCI and the broader possibility of targeting RIP3 activity as a therapeutic window for spinal neuroprotective intervention.

Sinoporphyrin Sodium-Mediated Sonodynamic Therapy Inhibits RIP3 Expression and Induces Apoptosis in the H446 Small Cell Lung Cancer Cell Line.

BACKGROUND/AIMS: Sonodynamic therapy (SDT) is expected to be a new method to solve the clinical problems caused by advanced metastasis in patients with lung cancer. The use of ultrasound has the advantage of being noninvasive, with deep-penetration properties. This study explored the anti-tumor effect of SDT with a new sonosensitizer, sinoporphyrin sodium (DVDMS), on the human small cell lung cancer H446 cell line in vitro and in vivo. METHODS: Absorption of DVDMS was detected by a fluorescence spectrophotometer, and DVDMS toxicity was determined using a Cell Counting Kit-8. Mitochondrial membrane potential (MMP) was assessed using the JC-1 fluorescent probe. Cell apoptosis was measured by flow cytometry, and apoptosis-related proteins were detected by western blotting. The expression of cytokines was measured using an enzyme-linked immunosorbent assay and quantitative real-time PCR. To verify the in vitro results, we detected tumor volumes and weight changes in a xenograft nude mouse model after DVDMS-SDT. Hematoxylin and eosin staining was used to observe changes to the tumor, heart, liver, spleen, lung, and kidney of the mice, and immunohistochemistry was used to examine changes in the expression of tumor CD34 and receptor-interacting protein kinase-3 (RIP3), while terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to observe apoptosis in tumor tissues. RESULTS: DVDMS-SDT-treated H446 cells increased the rate of cellular apoptosis and the levels of reactive oxygen species (ROS), cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, and caspase-10, and decreased the levels of MMP, RIP3, B-cell lymphoma 2, vascular endothelial growth factor, and tumor necrosis factor-α. The sonotoxic effect was mediated by ROS and was reduced by a ROS scavenger (N-acetyl-L-cysteine). In the in vivo mouse xenograft model, DVDMS-SDT showed efficient anti-cancer effects with no visible side effects. CONCLUSION: DVDMS-SDT induced apoptosis in H446 cells, in part by targeting mitochondria through the mitochondria-mediated apoptosis signaling pathway, and the extrinsic apoptosis pathway was also shown to be involved. Both apoptosis and changes in RIP3 expression were closely related to the generation of ROS. DVDMS-SDT will be advantageous for the management of small cell lung cancer due to its noninvasive characteristics.

A Role for Receptor-Interacting Protein Kinase-1 in Neutrophil Extracellular Trap Formation in Patients with Systemic Lupus Erythematosus: a Preliminary Study.

BACKGROUND/AIMS: Neutrophil extracellular traps (NETs) are known to play an important role in systemic lupus erythematosus (SLE) by triggering innate and adaptive immune responses. The molecular mechanisms responsible for their formation in SLE are still unclear. In this study, we aim to characterize the role of the receptor-interacting protein kinase-1 (RIPK1), a homologous serine/threonine kinase previously implicated in the regulation of necroptosis and tissue injury, in decreasing neutrophil death and formation of NETs, and to investigate the clinical implications of RIPK1 in SLE. METHODS: Patients with SLE (n = 50) and healthy donors (n = 35) were enrolled in in vitro studies. Management of SLE patients was evaluated using the SLE disease activity index 2000 (SLEDAI-2K) score and laboratory variables. The mRNA level of RIPKs was measured by quantitative polymerase chain reaction (qPCR). Intracellular RIPK1 and RIPK3 production by peripheral blood leukocytes was detected by four-color flow cytometry and confirmed by automatic western blotting. TNF-α, IFN-γ, IL-1ß, IL-2, IL-8, IL-18, and RIPK1 were measured by enzyme-linked immunosorbent assay. Cell death was assayed by Sytox green dye from peripheral neutrophils stimulated by RIPK-1-stabilizer necrostatin-1 (nec-1) and phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining and confocal microscopy were used to detect NET formation ex vivo. Quantification of NETs was determined by fluorescence spectrometry. RESULTS: IFN-γ, IL-1ß, IL-8, and IL-18 levels in serum were increased in SLE patients compared to controls. However, the expression of TNF-α, IL-2, and RIPK1 were decreased. In addition, we observed significant differences in the expression of RIPK1 in peripheral blood leukocytes. Of all the leukocytes, RIPK1 expression was significantly lower in neutrophils. Furthermore, we studied NETs formation in neutrophils of SLE with decreased RIPK1 expression, and these show increased susceptibility to NETosis, when stimulated with PMA and/or nec-1. Importantly, RIPK1 expression in neutrophils negatively correlated with ESR, CRP, 24-hour urine total protein, and the disease activity index in SLE. CONCLUSION: These data represent the first report of decreased RIPK1 expression in neutrophils of SLE patients and imply that RIPK1 may be involved in neutrophil death and NET formation. We suggest that RIPK1 is a potential biomarker to predict disease activity.

The Expression of Dectin-1, Irak1 and Rip2 During the Host Response to Aspergillus fumigatus.

BACKGROUND: C-type lectin receptors (CLRs), Toll-like receptors (TLRs), and Nod-like receptors (NLRs) have the ability to recognize Aspergillus fumigatus (A. fumigates) and induce innate immune response. Dectin-1 is a well-described CLR, while interleukin-1 receptor-associated kinase 1 (Irak1) and receptor-interacting protein 2 (Rip2) are pivotal adaptor proteins of TLRs and NLRs signaling pathways, respectively. OBJECTIVES: Our primary aim is to elucidate whether Dectin-1 regulates the expression of Irak1 and Rip2, and confirm that CLRs, TLRs, and NLRs pathways act synergistically in response to A. fumigatus infection. METHODS: Pulmonary infection mouse models were established. Myeloid cells were differentiated in cell culture and examined by inverted microscopy, flow cytometry, and scanning electron microscopy. The relative mRNA levels were determined by qRT-PCR. The protein expression levels were determined by immunohistochemistry and Western blot. RESULTS: The expression of Dectin-1, Irak1, Rip2, and phosphorylation level of nuclear factor (NF)-κB p65 were induced by conidia in immunocompetent mice, while their expression and phosphorylation level were inhibited in immunocompromised mice after the administration of conidia. Conidia increased the expression of Dectin-1, Irak1, and Rip2 in myeloid cells, while Dectin-1 silencing significantly reduced their expression. CONCLUSION: Our findings demonstrate that Dectin-1, Irak1, and Rip2 are involved in response to A. fumigatus infection. Dectin-1 modulates the expression of Irak1 and Rip2. Additionally, these three signaling pathways are interconnected, and CLRs pathway plays a dominant role against A. fumigatus invasion.

RIP2 deficiency attenuates cardiac hypertrophy, inflammation and fibrosis in pressure overload induced mice.

Although the pathological cardiac hypertrophy presents a leading cause of morbidity and mortality worldwide, our knowledge of the molecular mechanisms underlying the disease is still poor. Here, we reported that receptor-interacting serine/threonine-protein kinase 2 (RIP2), promoting pro-inflammatory gene expression, enhanced the pathological cardiac hypertrophy in animals. The effects of RIP2 on the cardiac hypertrophy triggered by pathological stimuli have not been fully investigated. In our study, mice were subjected to aortic banding (AB) surgery to explore the pathological, echocardiographic and molecular mechanisms. RIP2 expressed highly in cardiomyocytes after AB operation in wild type (WT) mice. RIP2-knockout (KO) attenuated cardiac hypertrophy, inflammation and fibrosis in mice 4 weeks after AB-surgery. First, RIP2 knockout down-regulated hypertrophic markers of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and ß-myosin heavy chain (ß-MHC) in the heart of AB-operated mice.in addition, RIP2-deficiency reduced toll-like receptor 4/myeloid differentiation factor 88/nuclear factor kappa B (TLR4/MyD88/NF-κB) activation, mitogen-activated protein kinases (MAPKs) phosphorylation and transforming growth factor-ß1 (TGF-ß1)/SMADs expressions, contributing to the suppression of inflammatory response and fibrosis, as further evidenced by down-regulated pro-inflammatory cytokines, including Tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and IL-18, as well as fibrosis markers of Collagen I, Collagen III and α-smooth muscle actin (α-SMA). Taken together, our data indicated that RIP2-deficience ameliorated cardiac hypertrophy, inflammation and fibrosis through modulating multiple signaling pathways.

A Three-Dimensional Lymphatic Endothelial Cell Tube Formation Assay to Identify Novel Kinases Involved in Lymphatic Vessel Remodeling.

The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes-ZAP70, IRAK4, RIPK1, and RIPK2-were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

The RIP1-RIP3 Complex Mediates Osteocyte Necroptosis after Ovariectomy in Rats.

Osteocyte apoptosis has been reported to play a central role in bone remodeling. In addition to apoptosis, other mechanisms may be involved in osteocyte loss. This study aimed to investigate the effect of necroptosis on osteocytes in ovariectomized (OVX) rats. Ninety-six female Sprague-Dawley rats were randomly divided into an OVX group and a sham group. At 0, 4, 8 and 12 weeks after surgery, specimens from each group (n = 12 each) were harvested. Bone mineral density (BMD) and body weight were measured. Transmission electron microscopy (TEM) and micro-CT were used to observe the changes in cellular morphology and bone microarchitecture induced by estrogen deficiency. Osteocyte apoptosis and necroptosis were evaluated via TUNEL and immunofluorescence staining for active caspase-3. At 8 weeks after ovariectomy, a greater number of osteocytes with typical necrotic morphological features were TUNEL positive but negative for active caspase-3. Western blotting, quantitative real-time PCR and immunofluorescence assessments demonstrated that the levels of receptor-interacting serine/threonine protein kinase 1 (RIP1) and RIP3 in osteocytes were significantly increased at 8 weeks after ovariectomy. These data are the first to suggest that necroptosis accelerates osteocyte loss under conditions of estrogen deficiency-induced osteoporosis in OVX rats. These findings provide evidence of a potential mechanism through which osteocyte necroptosis is associated with postmenopausal osteoporosis.

Ripoptosome Analysis by Caspase-8 Coimmunoprecipitation.

The biochemical signaling of cell death pathways is executed at a number of different intracellular and/or membrane-bound high-molecular mass complexes. It is crucial to be able to detect the formation, differences in assembly, and differential composition of such complexes to understand their contribution to the execution phase of apoptotic or necroptotic cell death. We describe here the use of caspase-8 coimmunoprecipitation in the spontaneously transformed keratinocyte cell line, HaCaT, to study the formation and composition of the Ripoptosome, a complex that is based on the serine-threonine kinase receptor-interacting protein 1 (RIPK1). However, the method can be adapted for use with other antibodies and cell lines. This protocol determines whether cells form the Ripoptosome complex, which is important for both apoptosis and necroptosis execution. Caspase-8 is an indispensible Ripoptosome component; therefore, caspase-8 antibodies are used to pull down the respective complex. However, the method cannot discriminate whether this complex triggers apoptosis (through the RIPK1 → FADD → caspase-8 activation pathway), necroptosis (through the RIPK1 → RIPK3 → MLKL activation pathway) or nondeath signaling. The actual signaling output (death or nondeath signaling) depends on the stoichiometry of the respective molecules as well as on the activity of FLIP, caspase-8, or other factors.

Protective effect of 2-deoxy-D-glucose on the cytotoxicity of cyclosporin A in vitro.

The present study aimed to investigate the mechanism underlying the protective effect of 2-deoxy-D-glucose (2-DG) on the cytotoxicity of cyclosporin A (CsA) in vitro using NRK-52E cells. Staining with Hoechst 33342/propidium iodide prior to flow cytometric analysis was performed to assess the rate of cellular apoptosis and necrosis induced by CsA. The expression levels of lactate dehydrogenase (LDH), caspase 3, receptor-interacting protein kinase 3 (RIP3), reactive oxygen species (ROS), glutathione (GSH) and malondialdehyde (MDA) were detected using colorimetry, ELISA, western blotting or flow cytometric analysis to determine the protective effects of 2-DG on CsA-induced cell death. The results demonstrated that 2-DG inhibited the release of LDH, the activation of caspase 3 and the generation of ROS induced by CsA, but had no effect on the expression of RIP3. Treatment with 2-DG increased the expression of GSH and decreased the expression of MDA in dose-dependent manner, and reduced the rate of the cellular apoptosis and necrosis induced by CsA. Therefore, 2-DG inhibited CsA-induced cellular apoptosis and necrosis, possibly by reducing the production of ROS. Inhibiting the activation of caspase 3 is one of the protective mechanisms of 2-DG, however, the expression of RIP3 remained unaltered following treatment with 2-DG. Whether 2-DG inhibits the CsA-induced necrosis and apoptosis by inhibiting the RIP3 signaling pathway remains to be elucidated.

Receptor-interacting protein (RIP) and Sirtuin-3 (SIRT3) are on opposite sides of anoikis and tumorigenesis.

BACKGROUND: Regulating cross-talk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like cancer. Previously, the authors demonstrated that anoikis activates a signaling pathway involving the CD95/Fas-mediated signaling pathway that is regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/focal adhesion kinase (FAK)-mediated survival pathways. Because it is known that sirtuin-3 (SIRT3), a nicotinamide adenine dinucleotide-dependent deacetylase, regulates cell survival, metabolism, and tumorigenesis, the authors hypothesized that SIRT3 may engage in cross-talk with Fas/RIP/integrin/FAK survival-death pathways in cancer cell systems. METHODS: Using immunohistochemical staining, immunoblotting, human tissue microarrays, and overexpression and suppression approaches in vitro and in vivo, the roles of RIP and SIRT3 were examined in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis. RESULTS: RIP and SIRT3 had opposite expression profiles in OSCC cells and tissues. Stable suppression of RIP enhanced SIRT3 levels, whereas stable suppression of SIRT3 did not impact RIP levels in OSCC cells. The authors observed that, as OSCC cells became anoikis-resistant, they formed multicellular aggregates or oraspheres in suspension conditions, and their expression of SIRT3 increased as their RIP expression decreased. Also, anoikis-resistant OSCC cells with higher SIRT3 and low RIP expression induced an increased tumor burden and incidence in mice, unlike their adherent OSCC cell counterparts. Furthermore, stable suppression of SIRT3 inhibited anoikis resistance and reduced tumor incidence. CONCLUSIONS: The current results indicted that RIP is a likely upstream, negative regulator of SIRT3 in anoikis resistance, and an anoikis-resistant orasphere phenotype defined by higher SIRT3 and low RIP expression contributes to a more aggressive phenotype in OSCC development.

Methods to analyze cellular necroptosis.

Necroptosis is a mechanism of necrotic cell death induced by external stimuli in the form of death domain receptor (DR) engagement by their respective ligands, TNF-alpha, Fas ligand (FasL) and TRAIL, under conditions when apoptotic cell death execution is prevented, e.g. by caspase inhibitors. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. RIP1 kinase activity is a key step in the necroptosis pathway. We have previously identified specific and potent small-molecule inhibitors of necroptosis, necrostatins, which efficiently prevent execution of this form of cell death. Herein, we describe the methods to analyze cellular necroptosis, and the methods to analyze the inhibitory effects of anti-necroptosis compounds (necrostatin-1).