ABSTRACT
Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.
Subject(s)
Biomarkers, Tumor/genetics , Cell Proliferation , Knowledge Bases , Neoplasms/pathology , Software , Spheroids, Cellular/pathology , Tumor Microenvironment , Cell Culture Techniques/methods , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/classification , Neoplasms/metabolism , RNA-Seq , Reproducibility of Results , Spheroids, Cellular/immunology , Spheroids, Cellular/metabolism , Tumor Cells, CulturedABSTRACT
Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.
Subject(s)
Necrosis , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Systemic Inflammatory Response Syndrome/enzymology , Animals , Apoptosis/drug effects , Caspases/metabolism , Cecal Diseases/genetics , Cecal Diseases/pathology , Gene Deletion , Imidazoles/administration & dosage , Imidazoles/pharmacology , Indoles/administration & dosage , Indoles/pharmacology , Intestinal Mucosa/metabolism , Intestines/drug effects , Intestines/pathology , Kaplan-Meier Estimate , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Systemic Inflammatory Response Syndrome/genetics , Systemic Inflammatory Response Syndrome/mortality , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.
Subject(s)
Apoptosis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelium/metabolism , Homeostasis , Intestinal Mucosa/metabolism , Intestines/cytology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Apoptosis/drug effects , Caspase 8/genetics , Caspase 8/metabolism , Cell Survival/drug effects , Epithelial Cells/drug effects , Epithelial Cells/pathology , Epithelium/drug effects , Epithelium/pathology , Female , Gene Deletion , Homeostasis/drug effects , Inflammation/metabolism , Inflammation/pathology , Intestines/drug effects , Intestines/pathology , Male , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/deficiency , NF-kappa B/metabolism , Necrosis , Organoids/cytology , Organoids/drug effects , Organoids/enzymology , Organoids/metabolism , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/deficiency , Survival Analysis , Tumor Necrosis Factors/pharmacologyABSTRACT
Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2(-)) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2(-) production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2(-) levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Apoptosis/drug effects , Boranes/pharmacology , Carbonates/pharmacology , Cycloheximide/toxicity , Epithelial Cells/drug effects , Intestinal Mucosa/drug effects , Oxidative Stress/drug effects , Stilbenes/pharmacology , Tumor Necrosis Factor-alpha/toxicity , Animals , Cell Line , Cytoprotection , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/drug effects , Mitochondria/metabolism , NADPH Oxidases/metabolism , Oxygen Consumption/drug effects , Resveratrol , Superoxides/metabolismABSTRACT
Cell death research during the last decades has revealed many molecular signaling cascades, often leading to distinct cell death modalities followed by immune responses. For historical reasons, the prototypic and best characterized cell death modes are apoptosis and necrosis (dubbed necroptosis, to indicate that it is regulated). There is mounting evidence for the interplay between cell death modalities and their redundant action when one of them is interfered with. This increase in cell death research points to the need for characterizing cell death pathways by different approaches at the biochemical, cellular and if possible, physiological level. In this review we present a selection of techniques to detect cell death and to distinguish necrosis from apoptosis. The distinction should be based on pharmacologic and transgenic approaches in combination with several biochemical and morphological criteria. A particular problem in defining necrosis is that in the absence of phagocytosis, apoptotic cells become secondary necrotic and develop morphologic and biochemical features of primary necrosis.
Subject(s)
Apoptosis/genetics , Cell Membrane/metabolism , Fibroblasts/metabolism , Macrophages/metabolism , Animals , Caspases/genetics , Caspases/metabolism , Cell Line , Cell Membrane/ultrastructure , DNA Fragmentation , Enzyme Activation , Fibroblasts/ultrastructure , Flow Cytometry , Macrophages/ultrastructure , Mice , Microscopy , Necrosis/genetics , Necrosis/pathology , Phagocytosis , Time-Lapse ImagingABSTRACT
Generation of cardiomyocytes from human pluripotent stem cells (hPSCs) is of high interest for disease modelling and regenerative medicine. hPSCs can provide an unlimited source of patient-specific cardiomyocytes that are otherwise difficult to obtain from individuals. Moreover, the low proliferation rate of adult cardiomyocytes and low viability ex vivo limits the quantity of study material. Most protocols for the differentiation of cardiomyocytes from hPSCs are based on the temporal modulation of the Wnt pathway. However, during the initial stage of GSK-3 inhibition, a substantial number of cells are lost due to detachment. In this study, we aimed to increase the efficiency of generating cardiomyocytes from hPSCs. We identified cell death as a detrimental factor during this initial stage of in vitro cardiomyocyte differentiation. Through pharmacological targeting of different types of cell death, we discovered that ferroptosis was the main cell death type during the first 48 h of the in vitro differentiation procedure. Inhibiting ferroptosis using ferrostatin-1 during cardiomyocyte differentiation resulted in increased robustness and cell yield.
ABSTRACT
Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release.
Subject(s)
Acyltransferases , Hepatic Stellate Cells , Hepatic Stellate Cells/metabolism , Acyltransferases/metabolism , Acyltransferases/genetics , Animals , Retinyl Esters/metabolism , Cells, Cultured , Diterpenes/metabolism , Diterpenes/pharmacology , Rats , MiceABSTRACT
Tauopathies encompass a group of neurodegenerative disorders characterised by diverse tau amyloid fibril structures. The persistence of polymorphism across tauopathies suggests that distinct pathological conditions dictate the adopted polymorph for each disease. However, the extent to which intrinsic structural tendencies of tau amyloid cores contribute to fibril polymorphism remains uncertain. Using a combination of experimental approaches, we here identify a new amyloidogenic motif, PAM4 (Polymorphic Amyloid Motif of Repeat 4), as a significant contributor to tau polymorphism. Calculation of per-residue contributions to the stability of the fibril cores of different pathologic tau structures suggests that PAM4 plays a central role in preserving structural integrity across amyloid polymorphs. Consistent with this, cryo-EM structural analysis of fibrils formed from a synthetic PAM4 peptide shows that the sequence adopts alternative structures that closely correspond to distinct disease-associated tau strains. Furthermore, in-cell experiments revealed that PAM4 deletion hampers the cellular seeding efficiency of tau aggregates extracted from Alzheimer's disease, corticobasal degeneration, and progressive supranuclear palsy patients, underscoring PAM4's pivotal role in these tauopathies. Together, our results highlight the importance of the intrinsic structural propensity of amyloid core segments to determine the structure of tau in cells, and in propagating amyloid structures in disease.
Subject(s)
Alzheimer Disease , Supranuclear Palsy, Progressive , Tauopathies , Humans , Alzheimer Disease/genetics , Amyloid/chemistry , Amyloidogenic Proteins , Supranuclear Palsy, Progressive/pathology , tau Proteins/genetics , tau Proteins/chemistry , Tauopathies/genetics , Tauopathies/pathologyABSTRACT
BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.
Subject(s)
CRISPR-Cas Systems , Zea mays , Zea mays/genetics , Triticum/genetics , Gene Editing/methods , MutagenesisABSTRACT
RIPK3 partially protects against disease caused by influenza A virus (IAV) infection in the mouse model. Here, we compared the immune protection of active vaccination with a universal influenza A vaccine candidate based on the matrix protein 2 ectodomain (M2e) and of passive immunization with anti-M2e IgG antibodies in wild type and Ripk3-/- mice. We observed that the protection against IAV after active vaccination with M2e viral antigen is lost in Ripk3-/- mice. Interestingly, M2e-specific serum IgG levels induced by M2e vaccination were not significantly different between wild type and Ripk3-/- vaccinated mice demonstrating that the at least the humoral immune response was not affected by the absence of RIPK3 during active vaccination. Moreover, following IAV challenge, lungs of M2e vaccinated Ripk3-/- mice revealed a decreased number of immune cell infiltrates and an increased accumulation of dead cells, suggesting that phagocytosis could be reduced in Ripk3-/- mice. However, neither efferocytosis nor antibody-dependent phagocytosis were affected in macrophages isolated from Ripk3-/- mice. Likewise following IAV infection of Ripk3-/- mice, active vaccination and infection resulted in decreased presence of CD8+ T-cells in the lung. However, it is unclear whether this reflects a deficiency in vaccination or an inability following infection. Finally, passively transferred anti-M2e monoclonal antibodies at higher dose than littermate wild type mice completely protected Ripk3-/- mice against an otherwise lethal IAV infection, demonstrating that the increased sensitivity of Ripk3-/- mice could be overcome by increased antibodies. Therefore we conclude that passive immunization strategies with monoclonal antibody could be useful for individuals with reduced IAV vaccine efficacy or increased IAV sensitivity, such as may be expected in patients treated with future anti-inflammatory therapeutics for chronic inflammatory diseases such as RIPK inhibitors.
Subject(s)
Influenza A virus , Influenza Vaccines , Influenza, Human , Orthomyxoviridae Infections , Animals , Antibodies, Viral , Humans , Immunization, Passive , Immunoglobulin G , Mice , Mice, Inbred BALB C , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Vaccination , Viral Matrix ProteinsABSTRACT
Heterotypic amyloid interactions between related protein sequences have been observed in functional and disease amyloids. While sequence homology seems to favour heterotypic amyloid interactions, we have no systematic understanding of the structural rules determining such interactions nor whether they inhibit or facilitate amyloid assembly. Using structure-based thermodynamic calculations and extensive experimental validation, we performed a comprehensive exploration of the defining role of sequence promiscuity in amyloid interactions. Using tau as a model system we demonstrate that proteins with local sequence homology to tau amyloid nucleating regions can modify fibril nucleation, morphology, assembly and spreading of aggregates in cultured cells. Depending on the type of mutation such interactions inhibit or promote aggregation in a manner that can be predicted from structure. We find that these heterotypic amyloid interactions can result in the subcellular mis-localisation of these proteins. Moreover, equilibrium studies indicate that the critical concentration of aggregation is altered by heterotypic interactions. Our findings suggest a structural mechanism by which the proteomic background can modulate the aggregation propensity of amyloidogenic proteins and we discuss how such sequence-specific proteostatic perturbations could contribute to the selective cellular susceptibility of amyloid disease progression.
Subject(s)
Amyloidosis , Proteomics , Amino Acid Sequence , Amyloid/metabolism , Amyloidogenic Proteins/metabolism , HumansABSTRACT
Radiotherapy is commonly used as a cytotoxic treatment of a wide variety of tumors. Interestingly, few case reports underlined its potential to induce immune-mediated abscopal effects, resulting in regression of metastases, distant from the irradiated site. These observations are rare, and apparently depend on the dose used, suggesting that dose-related cellular responses may be involved in the distant immunogenic responses. Ionizing radiation (IR) has been reported to elicit immunogenic apoptosis, necroptosis, mitotic catastrophe, and senescence. In order to link a cellular outcome with a particular dose of irradiation, we performed a systematic study in a panel of cell lines on the cellular responses at different doses of X-rays. Remarkably, we observed that all cell lines tested responded in a similar fashion to IR with characteristics of mitotic catastrophe, senescence, lipid peroxidation, and caspase activity. Iron chelators (but not Ferrostatin-1 or vitamin E) could prevent the formation of lipid peroxides and cell death induced by IR, suggesting a crucial role of iron-dependent cell death during high-dose irradiation. We also show that in K-Ras-mutated cells, IR can induce morphological features reminiscent of methuosis, a cell death modality that has been recently described following H-Ras or K-Ras mutation overexpression.
Subject(s)
Cell Death/drug effects , Cellular Senescence/drug effects , Radiation, Ionizing , Animals , Humans , MiceABSTRACT
The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.
Subject(s)
Antibodies, Monoclonal/immunology , Hepacivirus/immunology , Hepatitis C Antibodies/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Cell Line , Epitope Mapping , Hepatitis C Antibodies/isolation & purification , Humans , Mice , Neutralization Tests , Pan troglodytes , Viral Envelope Proteins/immunologyABSTRACT
Livers exposed to prolonged warm ischemia (WI), such as those from non-heart-beating donors (NHBDs), are at higher risk of primary graft nonfunction (PNF). In a pig model of liver transplantation (LTx) from NHBDs, hepatocellular vacuolation, focal hepatocyte dropout, congestion, and sinusoidal dilatation appeared on biopsies taken after exposure to WI. In functioning grafts, vacuolation and sinusoidal dilatation were reversible after LTx, in contrast to PNF grafts. We studied whether the extent of these morphological signs and particularly vacuolation, present on pre-LTx biopsies, was associated with WI length and able to predict PNF, hepatocellular damage, and survival. Pre-LTx biopsies from pig livers exposed to incremental periods of WI were reviewed retrospectively. The extent of vacuolation was quantified blindly by a pathologist's semiquantitative score, validated by stereological point counting and digital image analysis, and then used to predict PNF and hepatocellular damage. On biopsies taken after WI, stereological point counting and digital analysis scoring contributed significantly in predicting PNF (P = 0.027 and P = 0.043, respectively) versus the pathologist's semiquantitative score (P = 0.058). Stereological point counting and digital image analysis predicted the extent of hepatocellular damage (P < 0.0001 and P = 0.001) versus the pathologist's semiquantitative score (P = 0.085). In conclusion, the extent of parenchymal vacuolation present on WI liver grafts reflects the severity of hepatocellular damage and predicts pig liver graft viability before LTx. Further studies are now warranted to evaluate whether these anoxic changes that are associated with liver graft viability in pigs also apply to human NHBD liver biopsies.
Subject(s)
Graft Survival , Liver Transplantation/methods , Animals , Biopsy , Cold Temperature , Cytoplasm/metabolism , Hepatocytes/metabolism , Ischemia , Liver/pathology , Microscopy, Electron , Reperfusion Injury , Retrospective Studies , Risk , Swine , Vacuoles/pathologyABSTRACT
Phenotypic screening and subsequent target identification approaches are very valuable to identify chemical probes that can be used to explore the connection between phenotypes and biological pathways. However, assessing a phenotypic effect in plants in a high-throughput fashion is a challenging task and often requires expensive readout devices. In this chapter, we describe a cost-effective multi-parametric screening procedure that is compatible with liquid-handling systems and that enables the assessment of phenotypes in Arabidopsis thaliana seedlings in an automated way.
Subject(s)
Arabidopsis/physiology , Seedlings/physiology , Biomarkers , Germination , High-Throughput Screening Assays , Phenotype , Plants, Genetically Modified , SeedsABSTRACT
The Aurora kinase family (Aurora A, B and C) are crucial regulators of several mitotic events, including cytokinesis. Increased expression of these kinases is associated with tumorigenesis and several compounds targeting Aurora kinase are under evaluation in clinical trials (a.o. AT9283, AZD1152, Danusertib, MLN8054). Here, we demonstrate that the pan-Aurora kinase inhibitor Tozasertib (VX-680 and MK-0457) not only causes cytokinesis defects through Aurora kinase inhibition, but is also a potent inhibitor of necroptosis, a cell death process regulated and executed by the RIPK1, RIPK3 and MLKL signalling axis. Tozasertib's potency to inhibit RIPK1-dependent necroptosis and to block cytokinesis in cells is in the same concentration range, with an IC50 of 1.06 µM and 0.554 µM, respectively. A structure activity relationship (SAR) analysis of 67 Tozasertib analogues, modified at 4 different positions, allowed the identification of analogues that showed increased specificity for either cytokinesis inhibition or for necroptosis inhibition, reflecting more specific inhibition of Aurora kinase or RIPK1, respectively. These results also suggested that RIPK1 and Aurora kinases are functionally non-interacting targets of Tozasertib and its analogues. Indeed, more specific Aurora kinase inhibitors did not show any effect in necroptosis and Necrostatin-1s treatment did not result in cytokinesis defects, demonstrating that both cellular processes are not interrelated. Finally, Tozasertib inhibited recombinant human RIPK1, human Aurora A and human Aurora B kinase activity, but not RIPK3. The potency ranking of the newly derived Tozasertib analogues and their specificity profile, as observed in cellular assays, coincide with ADP-Glo recombinant kinase activity assays. Overall, we show that Tozasertib not only targets Aurora kinases but also RIPK1 independently, and that we could generate analogues with increased selectivity to RIPK1 or Aurora kinases, respectively.
Subject(s)
Apoptosis/drug effects , Aurora Kinases/antagonists & inhibitors , Piperazines/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , Apoptosis/genetics , Aurora Kinases/genetics , Aurora Kinases/metabolism , Cell Line , Humans , Mice , Receptor-Interacting Protein Serine-Threonine Kinases/geneticsABSTRACT
Receptor interacting protein kinase 1 (RIPK1) plays a crucial role in tumor necrosis factor (TNF)-induced necroptosis, suggesting that this pathway might be druggable. Most inhibitors of RIPK1 are classified as either type II or type III kinase inhibitors. This opened up some interesting perspectives for the discovery of novel inhibitors that target the active site of RIPK1. Tozasertib, a type I pan-aurora kinase (AurK) inhibitor, was found to show a very high affinity for RIPK1. Because tozasertib presents the typical structural elements of a type I kinase inhibitor, the development of structural analogues of tozasertib is a good starting point for identifying novel type I RIPK1 inhibitors. In this paper, we identified interesting inhibitors of mTNF-induced necroptosis with no significant effect on AurK A and B, resulting in no nuclear abnormalities as is the case for tozasertib. Compounds 71 and 72 outperformed tozasertib in an in vivo TNF-induced systemic inflammatory response syndrome (SIRS) mouse model.
Subject(s)
Necrosis/prevention & control , Piperazines/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Aurora Kinase A/drug effects , Aurora Kinase B/drug effects , Cell Death/drug effects , Mice , Piperazines/adverse effects , Protein Kinase Inhibitors/chemistry , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Systemic Inflammatory Response Syndrome/chemically induced , Systemic Inflammatory Response Syndrome/drug therapy , Tumor Necrosis Factor-alpha/adverse effectsABSTRACT
Necroptosis contributes to the pathophysiology of several inflammatory, infectious and degenerative disorders. TNF-induced necroptosis involves activation of the receptor-interacting protein kinases 1 and 3 (RIPK1/3) in a necrosome complex, eventually leading to the phosphorylation and relocation of mixed lineage kinase domain like protein (MLKL). Using a high-content screening of small compounds and FDA-approved drug libraries, we identified the anti-cancer drug Sorafenib tosylate as a potent inhibitor of TNF-dependent necroptosis. Interestingly, Sorafenib has a dual activity spectrum depending on its concentration. In murine and human cell lines it induces cell death, while at lower concentrations it inhibits necroptosis, without affecting NF-κB activation. Pull down experiments with biotinylated Sorafenib show that it binds independently RIPK1, RIPK3 and MLKL. Moreover, it inhibits RIPK1 and RIPK3 kinase activity. In vivo Sorafenib protects against TNF-induced systemic inflammatory response syndrome (SIRS) and renal ischemia-reperfusion injury (IRI). Altogether, we show that Sorafenib can, next to the reported Braf/Mek/Erk and VEGFR pathways, also target the necroptotic pathway and that it can protect in an acute inflammatory RIPK1/3-mediated pathology.
Subject(s)
Inflammation/drug therapy , Necrosis/genetics , Protein Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Death/drug effects , Disease Models, Animal , Humans , Inflammation/genetics , Inflammation/pathology , Mice , Necrosis/pathology , Niacinamide/administration & dosage , Niacinamide/analogs & derivatives , Phenylurea Compounds/administration & dosage , Phosphorylation/genetics , Reperfusion Injury/chemically induced , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Sorafenib , Tumor Necrosis Factor-alpha/adverse effects , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Several cell death assays have been developed based on a single biochemical parameter such as caspase activation or plasma membrane permeabilization. Our fluorescent apoptosis/necrosis (FAN) assay directly measures cell death and distinguishes between caspase-dependent apoptosis and caspase-independent necrosis of cells grown in any multiwell plate. Cell death is monitored in standard growth medium as an increase in fluorescence intensity of a cell-impermeable dye (SYTOX Green) after plasma membrane disintegration, whereas apoptosis is detected through caspase-mediated release of a fluorophore from its quencher (DEVD-amc). The assay determines the normalized percentage of dead cells and caspase activation per condition as an end-point measurement or in real time (automated). The protocol can be applied to screen drugs, proteins or siRNAs for interference with cell death while simultaneously detecting cell death modality switching between apoptosis and necrosis. Initial preparation may take up to 5 d, but the typical hands-on time is â¼2 h.
Subject(s)
Cell Death , Fluorometry/methods , Animals , Cell Line , Humans , Mice , Staining and Labeling , Time FactorsABSTRACT
TNF-α/cycloheximide (CHX)-induced apoptosis of the mouse intestinal epithelial cell line MODE-K corresponds with the production of reactive oxygen species (ROS). The aim of the study is to investigate the sources of ROS production contributing to apoptotic cell death during TNF-α/CHX-induced oxidative stress in MODE-K cells. Total ROS or mitochondrial superoxide anion production was measured simultaneously with cell death in the absence or presence of pharmacological inhibitors of various ROS-producing systems, and of ROS scavengers/antioxidants. The influence of TNF-α/CHX on mitochondrial membrane potential (Ψ(m)) and cellular oxygen consumption was also studied. TNF-α/CHX time-dependently increased intracellular total ROS and mitochondrial superoxide anion production in MODE-K cells, starting from 2h. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) by a pan-NOX inhibitor (VAS-2870) and a specific inhibitor of Rac1 (NSC23766) significantly reduced TNF-α/CHX-induced total ROS and cell death levels. The mitochondrial electron transport chain inhibitors, amytal (IQ site of complex I) and TTFA (Qp site of complex II) showed a pronounced decrease in TNF-α/CHX-induced total ROS, mitochondrial superoxide anion and cell death levels. TNF-α/CHX treatment caused an immediate decrease in mitochondrial respiration, and a loss of Ψ(m) and increase in mitochondrial dysfunction from 1 h on. The results suggest that mitochondria and NOX are the two major sources of ROS overproduction during TNF-α/CHX-induced cell death in MODE-K cells, with superoxide anions being the major ROS species. Particularly, the quinone-binding sites of mitochondrial complex I (site I(Q)) and complex II (site Qp) seem to be the major sites of mitochondrial ROS production.