Staurosporine-induced Apoptosis Research Articles

Contribution of TRPC channels in human and experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is due to progressive distal pulmonary artery (PA) obstruction leading to right ventricular hypertrophy and failure. Exacerbated store-operated Ca2+ entry (SOCE) contributes to PAH pathogenesis, mediating human PA smooth muscle cells (hPASMCs) abnormalities. The transient receptor potential canonical channels (TRPC family) are Ca2+-permeable channels contributing to SOCE in different cell types, including PASMCs. However, the properties, signaling pathways, and contribution to Ca2+ signaling of each TRPC isoform are unclear in human PAH. We studied in vitro the impact of TRPC knockdown on control and PAH-hPASMCs function. In vivo, we analyzed the consequences of pharmacological TRPC inhibition using the experimental model of pulmonary hypertension (PH) induced by monocrotaline (MCT)-exposure. Compared to control-hPASMCs cells, in PAH-hPASMCs, we found a decreased TRPC4 expression, overexpression of TRPC3 and TRPC6, and unchanged TRPC1 expression. Using the siRNA strategy, we found that the knockdown of TRPC1-C3-C4-C6 reduced the SOCE and the proliferation rate of PAH-hPASMCs. Only TRPC1 knockdown decreased the migration capacity of PAH-hPASMCs. After PAH-hPASMCs exposure to the apoptosis inducer staurosporine, TRPC1-C3-C4-C6 knockdown increased the percentage of apoptotic cells, suggesting that these channels promote apoptosis resistance. Only TRPC3 function contributed to exacerbated calcineurin activity. In the MCT-PH rats' model, only TRPC3 protein expression was increased in lungs compared to control rats, and in vivo "curative" administration of a TRPC3 inhibitor attenuated PH development in rats. These results suggest that TRPC channels contribute to PAH-hPASMCs dysfunctions, including SOCE, proliferation, migration, apoptosis resistance, and could be considered as therapeutic targets in PAH.

Fiber density and matrix stiffness modulate distinct cell migration modes in a 3D stroma mimetic composite hydrogel.

The peritumoral stroma is a complex 3D tissue that provides cells with myriad biophysical and biochemical cues. Histologic observations suggest that during metastatic spread of carcinomas, these cues influence transformed epithelial cells, prompting a diversity of migration modes spanning single cell and multicellular phenotypes. Purported consequences of these variations in tumor escape strategies include differential metastatic capability and therapy resistance. Therefore, understanding how cues from the peritumoral stromal microenvironment regulate migration mode has both prognostic and therapeutic value. Here, we utilize a synthetic stromal mimetic in which matrix fiber density and bulk hydrogel mechanics can be orthogonally tuned to investigate the contribution of these two key matrix attributes on MCF10A migration mode phenotypes, epithelial-mesenchymal transition (EMT), and invasive potential. We develop an automated computational image analysis framework to extract migratory phenotypes from fluorescent images and determine 3D migration metrics relevant to metastatic spread. Using this analysis, we find that matrix fiber density and bulk hydrogel mechanics distinctly contribute to a variety of MCF10A migration modes including amoeboid, single mesenchymal, clusters, and strands. We identify combinations of physical and soluble cues that induce a variety of migration modes originating from the same MCF10A spheroid and use these settings to examine a functional consequence of migration mode -resistance to apoptosis. We find that cells migrating as strands are more resistant to staurosporine-induced apoptosis than either disconnected clusters or individual invading cells. Improved models of the peritumoral stromal microenvironment and understanding of the relationships between matrix attributes and cell migration mode can aid ongoing efforts to identify effective cancer therapeutics that address cell plasticity-based therapy resistances. STATEMENT OF SIGNIFICANCE: Stromal extracellular matrix structure dictates both cell homeostasis and activation towards migratory phenotypes. However decoupling the effects of myriad biophysical cues has been difficult to achieve. Here, we encapsulate electrospun fiber segments within an amorphous hydrogel to create a fiber-reinforced hydrogel composite in which fiber density and hydrogel stiffness can be orthogonally tuned. Quantification of 3D cell migration reveal these two parameters uniquely contribute to a diversity of migration phenotypes spanning amoeboid, single mesenchymal, multicellular cluster, and collective strand. By tuning biophysical and biochemical cues to elicit heterogeneous migration phenotypes, we find that collective strands best resist apoptosis. This work establishes a composite approach to modulate fibrous topography and bulk hydrogel mechanics and identified biomaterial parameters to direct distinct 3D cell migration phenotypes.

Unidirectional fluxes of monovalent ions in human erythrocytes compared with lymphoid U937 cells: Transient processes after stopping the sodium pump and in response to osmotic challenge.

Recently, we have developed software that allows, using a minimum of required experimental data, to find the characteristics of ion homeostasis and a list of all unidirectional fluxes of monovalent ions through the main pathways in the cell membrane both in a balanced state and during the transient processes. Our approach has been successfully validated in human proliferating lymphoid U937 cells during transient processes after stopping the Na/K pump by ouabain and for staurosporine-induced apoptosis. In present study, we used this approach to find the characteristics of ion homeostasis and the monovalent ion fluxes through the cell membrane of human erythrocytes in a resting state and during the transient processes after stopping the Na/K pump with ouabain and in response to osmotic challenge. Due to their physiological significance, erythrocytes remain the object of numerous studies, both experimental and computational methods. Calculations showed that, under physiological conditions, the K+ fluxes through electrodiffusion channels in the entire erythrocyte ion balance is small compared to the fluxes through the Na/K pump and cation-chloride cotransporters. The proposed computer program well predicts the dynamics of the erythrocyte ion balance disorders after stopping the Na/K pump with ouabain. In full accordance with predictions, transient processes in human erythrocytes are much slower than in proliferating cells such as lymphoid U937 cells. Comparison of real changes in the distribution of monovalent ions under osmotic challenge with the calculated ones indicates a change in the parameters of the ion transport pathways through the plasma membrane of erythrocytes in this case. The proposed approach may be useful in studying the mechanisms of various erythrocyte dysfunctions.

A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection

Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped β-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.

Expression Ratios of the Antiapoptotic BCL2 Family Members Dictate the Selective Addiction of Kaposi's Sarcoma-Associated Herpesvirus-Transformed Primary Effusion Lymphoma Cell Lines to MCL1.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several malignancies in people living with HIV, including primary effusion lymphoma (PEL). PEL cell lines exhibit oncogene addictions to both viral and cellular genes. Using CRISPR screens, we previously identified cellular oncogene addictions in PEL cell lines, including MCL1. MCL1 is a member of the BCL2 family, which functions to prevent intrinsic apoptosis and has been implicated in several cancers. Despite the overlapping functions of the BCL2 family members, PEL cells are dependent only on MCL1, suggesting that MCL1 may have nonredundant functions. To investigate why PEL cells exhibit selective addiction to MCL1, we inactivated the intrinsic apoptosis pathway by engineering BAX/BAK1 double knockout cells. In this context, PEL cells become resistant to MCL1 knockdown or MCL1 inactivation by the MCL1 inhibitor S63845, indicating that the main function of MCL1 in PEL cells is to prevent BAX/BAK1-mediated apoptosis. The selective requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Ectopic expression of several BCL2 family proteins, as well as the KSHV BCL2 homolog, significantly decreased basal caspase 3/7 activity and buffered against staurosporine-induced apoptosis. Finally, overexpressed BCL2 family members can functionally substitute for MCL1, when it is inhibited by S63845. Together, our data indicate that the expression levels of the BCL2 family likely explain why PEL tumor cells are highly addicted to MCL1. Importantly, our results suggest that caution should be taken when considering MCL1 inhibitors as a monotherapy regimen for PEL because resistance can develop easily. IMPORTANCE Primary effusion lymphoma (PEL) is caused by Kaposi's sarcoma-associated herpesvirus. We showed previously that PEL cell lines require the antiapoptotic protein MCL1 for survival but not the other BCL2 family proteins. This selective dependence on MCL1 is unexpected as the BCL2 family functions similarly in preventing intrinsic apoptosis. Recently, new roles for MCL1 not shared with the BCL2 family have emerged. Here, we show that noncanonical functions of MCL1 are unlikely essential. Instead, MCL1 functions mainly to prevent apoptosis. The specific requirement to MCL1 is due to MCL1 being expressed in excess over the BCL2 family. Consistent with this model, shifting these expression ratios changes the requirement away from MCL1 and toward the dominant BCL2 family gene. Together, our results indicate that although MCL1 is an attractive chemotherapeutic target to treat PEL, careful consideration must be taken, as resistance to MCL1-specific inhibitors easily develops through BCL2 family overexpression.

Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.

Minimizing Molecular Misidentification in Imaging Low-Abundance Protein Interactions Using Spectroscopic Single-Molecule Localization Microscopy.

Super-resolution microscopy can capture spatiotemporal organizations of protein interactions with resolution down to 10 nm; however, the analyses of more than two proteins involving low-abundance protein are challenging because spectral crosstalk and heterogeneities of individual fluorescent labels result in molecular misidentification. Here we developed a deep learning-based imaging analysis method for spectroscopic single-molecule localization microscopy to minimize molecular misidentification in three-color super-resolution imaging. We characterized the 3-fold reduction of molecular misidentification in the new imaging method using pure samples of different photoswitchable fluorophores and visualized three distinct subcellular proteins in U2-OS cell lines. We further validated the protein counts and interactions of TOMM20, DRP1, and SUMO1 in a well-studied biological process, Staurosporine-induced apoptosis, by comparing the imaging results with Western-blot analyses of different subcellular portions.

Hcmv-miR-UL148D regulates the staurosporine-induced apoptosis by targeting the Endoplasmic Reticulum to Nucleus signaling 1(ERN1).

The propensity of viruses to co-opt host cellular machinery by reprogramming the host’s RNA-interference machinery has been a major focus of research, however, regulation of host defense mechanisms by virus-encoded miRNA, is an additional regulatory realm gaining momentum in the arena of host-viral interactions. The Human Cytomegalovirus (HCMV) miRNAs, regulate many cellular pathways alone or in concordance with HCMV proteins, thereby paving a conducive environment for successful infection in the human host. We show that HCMV miRNA, hcmv-miR-UL148D inhibits staurosporine-induced apoptosis in HEK293T cells. We establish that ERN1 mRNA is a bonafide target of hcmv-miR-UL148D and its encoded protein IRE1α is translationally repressed by the overexpression of hcmv-miR-UL148D resulting in the attenuation of apoptosis. Unlike the host microRNA seed sequence (6–8 nucleotides), hcmv-miR-UL148D has long complementarity to 3’ UTR of ERN1 mRNA resulting in mRNA degradation. The repression of IRE1α by the hcmv-miR-UL148D further downregulates Xbp1 splicing and c-Jun N-terminal kinase phosphorylation thus regulating ER-stress and ER-stress induced apoptotic pathways. Strikingly, depletion of ERN1 attenuates staurosporine-induced apoptosis which further suggests that hcmv-miR-UL148D functions through regulation of its target ERN1. These results uncover a role for hcmv-miR-UL148D and its target ERN1 in regulating ER stress-induced apoptosis.

The Phosphofurin Acidic Cluster Sorting Protein 2 (PACS-2) E209K Mutation Responsible for PACS-2 Syndrome Increases Susceptibility to Apoptosis.

Phosphofurin acidic cluster sorting protein 2 (PACS-2) is a multifunctional cytosolic membrane trafficking protein with distinct roles in maintaining cellular homeostasis. Recent clinical reports have described 28 individuals possessing a de novo PACS-2 E209K mutation that present with epileptic seizures and cerebellar dysgenesis. As the PACS-2 E209K missense mutation has become a marker for neurodevelopmental disorders, we sought to characterize its biochemical properties. Accordingly, we observed that the PACS-2 E209K protein exhibited a slower turnover rate relative to PACS-2 wild type (WT) upon cycloheximide treatment in 293T cells. The longer half-life of PACS-2 E209K suggests a disruption in its proteostasis, with the potential for altered protein–protein interactions. Indeed, a regulatory protein in neurodevelopment known as 14-3-3ε was identified as having an increased association with PACS-2 E209K. Subsequently, when comparing the effect of PACS-2 WT and E209K expression on the staurosporine-induced apoptosis response, we found that PACS-2 E209K increased susceptibility to staurosporine-induced apoptosis in HCT 116 cells. Overall, our findings suggest PACS-2 E209K alters PACS-2 proteostasis and favors complex formation with 14-3-3ε, leading to increased cell death in the presence of environmental stressors.

Pro-angiogenic activity of isofuran.

Isofurans (IsoFs) are a series of novel discovered lipid peroxidation products. This study focused on the investigation of the angiogenic property of IsoF. MTT stain assay indicated that 1 µm IsoF had the most bioactivity in rat brain endothelial cells (RBECs). IsoF significantly promoted cellular proliferation and migration and remarkably decreased staurosporine-induced apoptosis by TUNEL assay in the RBECs. It successfully up-regulated rat aortic vascularization and choroid explant sprouting, extracellular regulated protein kinases (ERK)1/2, and triggered calcium release. RT-PCR examination indicated that IsoF up-regulated tumor necrosis factor (TNF)α, angiopoietin-1 receptor (Tie2), and vascular endothelial growth factor (VEGF)-A, but did not interfere with caspase 2 and VEGF-C in the RBECs. IsoF has pro-angiogenic activity. Calcium release and ERK1/2 phosphorylation may be involved in the signaling of the IsoF-induced up-regulation of TNFα, Tie2, and VEGF-A, which could be the molecular mechanism of the pro-angiogenic activity of the IsoF.

Direct cleavage of caspase-8 by herpes simplex virus 1 tegument protein US11

The HSV-1 tegument protein Us11 counteracts the antiviral defense mechanisms by precluding the host protein shutoff. Previous works demonstrated that Us11 prevents heat-and staurosporine-induced apoptosis and inhibits autophagy. Therefore, in the present study, we investigated the hypothesis that HSV-1, through Us11, could recruit caspase-8, a key enzyme regulating programmed cell death. We first show that HSV-1 promotes the accumulation of caspase-8-p18 active fragments in both semi permissive THP-1 cells and fully permissive HEp-2 cells to HSV-1 replication. Using a recombinant virus R3630 (ΔUs11/ΔUs12) and a plasmid encoding Us11-recombinant protein we have proven that Us11 promotes p18 accumulation, which does not trigger the apoptotic signaling. Additional, in an in vitro model, we demonstrated that Us11-recombinant protein induces caspase-8-p18 cleavage by physically interacting with the caspase-8 recombinant protein. Finally, we found that, during HSV-1 replication, activated-caspase-8 cleaves Atg3 protein to potentially block autophagy and support its replication.

Modulatory Impact of Lamiaceae Metabolites on Apoptosis of Human Leukemia Cells.

Lamiaceae species are rich sources of biologically active compounds which have been applied in medicine since ancient times. Especially their antineoplastic properties have been thoroughly studied with respect to their putative application in chemoprevention and adjuvant therapy of cancer. However, the most known biological effects of Lamiaceae have been ascribed to their essential oil fractions, whereas their (poly)phenolic metabolites being also abundant in these plants, are much less recognized, nevertheless contributing to their beneficial properties, such as anti-cancer actions. The aim of this study was to evaluate the impact of dried aqueous extracts from common thyme (Thymus vulgaris L.) (ExTv), wild thyme (Thymus serpyllum L.) (ExTs), sweet marjoram (Origanum majorana L.) (ExOm), and peppermint (Mentha × piperita L.) (ExMp), as well as (poly)phenolic compounds: caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), luteolin-7-O-β-glucuronide (Lgr), luteolin-7-O-rutinoside (Lr), eriodictyol-7-O-rutinoside (Er), and arbutin (Ab), on unstimulated Jurkat cells, in comparison with their effect on staurosporine-stimulated Jurkat cells. Jurkat T cells were incubated with different concentrations of ExTv, ExTs, ExOm, ExMp, Lgr, LA, Er, Lr, RA, CA, or Ab. Subsequently, staurosporine was added to half of the samples and flow cytometry combined with fluorescence-activated cell sorting analysis was conducted, which allowed for the selection of early and late apoptotic cells. Both ExTs and ExOm stimulated apoptosis of Jurkat cells and enhanced the proapoptotic effect of staurosporine. Conversely, ExTv and ExMp demonstrated no clear effect on apoptosis. CA and RA raised the staurosporine-induced apoptotic effect. The impact of Er and Lgr on Jurkat cells showed fluctuations depending on the compound concentration. Neither Er nor Ab altered staurosporine-induced apoptosis in Jurkat cells, whereas Lgr seemed to weaken the proapoptotic action of staurosporine. The most evident observation in this study was the pro-apoptotic action of ExTs and ExOm observed both in staurosporine-unstimulated and stimulated Jurkat cells. Additionally, an enhancement of staurosporine-induced apoptosis by caffeic and rosmarinic acids was reported. Therefore, it might be concluded that these are the mixtures of biologically active polyphenols which often exert more pronounced beneficial effects than purified molecules.

Purine nucleoside phosphorylase deficiency induces p53-mediated intrinsic apoptosis in human induced pluripotent stem cell-derived neurons

Purine nucleoside phosphorylase (PNP) is an important enzyme in the purine degradation and salvage pathway. PNP deficiency results in marked T lineage lymphopenia and severe immunodeficiency. Additionally, PNP-deficient patients and mice suffer from diverse non-infectious neurological abnormalities of unknown etiology. To further investigate the cause for these neurologic abnormalities, induced pluripotent stem cells (iPSC) from two PNP-deficient patients were differentiated into neurons. The iPSC-derived PNP-deficient neurons had significantly reduced soma and nuclei volumes. The PNP-deficient neurons demonstrated increased spontaneous and staurosporine-induced apoptosis, measured by cleaved caspase-3 expression, together with decreased mitochondrial membrane potential and increased cleaved caspase-9 expression, indicative of enhanced intrinsic apoptosis. Greater expression of tumor protein p53 was also observed in these neurons, and inhibition of p53 using pifithrin-α prevented the apoptosis. Importantly, treatment of the iPSC-derived PNP-deficient neurons with exogenous PNP enzyme alleviated the apoptosis. Inhibition of ribonucleotide reductase (RNR) in iPSC derived from PNP-proficient neurons with hydroxyurea or with nicotinamide and trichostatin A increased the intrinsic neuronal apoptosis, implicating RNR dysfunction as the potential mechanism for the damage caused by PNP deficiency. The findings presented here establish a potential mechanism for the neurological defects observed in PNP-deficient patients and reinforce the critical role that PNP has for neuronal viability.

Toll-like receptor 3 activation promotes joint degeneration in osteoarthritis

Osteoarthritis (OA) is characterized by cartilage degradation that is induced by inflammation. Sterile inflammation can be caused by damage-associated molecular patterns that are released by chondrocytes and activate pattern recognition receptors. We evaluate the role of toll-like receptor-3-activating RNA in the pathogenesis of OA. Toll-like receptor 3 (TLR3) was detected by semiquantitative reverse transcriptase PCR, western blotting and microscopy. Rhodamine-labelled poly(I:C) was used to image uptake in chondrocytes and full-thickness cartilage. The production of IFNβ in chondrocytes after stimulation with poly(I:C) as well as in the synovial fluid of OA patients was measured using ELISA. Chondrocyte apoptosis was chemically induced using staurosporine. Immunohistochemistry was performed to examine TLR3 expression and apoptosis in human and murine OA cartilage. RNA in synovial fluid was quantified by RiboGreen assay. Destabilisation of the medial meniscus was performed in TLR3−/− and wildtype mice. OA was assessed after eight weeks using OARSI score. TLR3 expression was confirmed by western blot and RT-PCR. Poly(I:C) was internalised by chondrocytes as well as cartilage and caused an increase of IFNβ production in murine (11.46 ± 11.63 (wo) to 108.7 ± 25.53 pg/ml; N = 6) and human chondrocytes (1.88 ± 0.32 (wo) to 737.6 ± 130.5 pg/ml; N = 3; p < 0.001). OA cartilage showed significantly more TLR3-positive (KL0 = 0.22 ± 0.24; KL4 = 6.02 ± 6.75; N ≥ 15) and apoptotic chondrocytes (KL0 = 0.6 ± 1.02; KL4 = 9.78 ± 7.79; N ≥ 12) than healthy cartilage (p < 0.001). Staurosporine-induced chondrocyte apoptosis causes a dose-dependent RNA release (0 ng/ml = 1090 ± 39.1 ng/ml; 1000 ng/ml=2014 ± 160 ng/ml; N = 4; p < 0.001). Human OA synovial fluid contained increased concentrations of RNA (KL0-2 = 3408 ± 1129 ng/ml; KL4 = 4870 ± 1612ng/ml; N ≥ 7; p < 0.05) and IFNβ (KL0-2 = 41.95 ± 92.94 ng/ml; KL3 = 1181 ± 1865ng/ml; N ≥ 8; p < 0.05). TLR3−/− mice showed reduced cartilage degradation eight weeks after OA induction (OARSI WT = 5.5 ± 0.04; TLR3−/− = 3.75 ± 1.04; N ≥ 6) which was accompanied by gradually decreasing levels of TUNEL-positive cells (WT = 34.87 ± 24.10; TLR3−/ = 19.64 ± 7.89) resulting in decreased IFNβ expression (WT = 12.57 ± 5.43; TLR3−/− = 6.09 ± 2.07) in cartilage (p < 0.05). The release of RNA by apoptotic chondrocytes thus activating TLR3 signalling is one possible way of perpetuating inflammatory cartilage changes. The inhibition of TLR3 could be a possible therapeutic target for OA treatment.

PINK1 Protects against Staurosporine-Induced Apoptosis by Interacting with Beclin1 and Impairing Its Pro-Apoptotic Cleavage.

PINK1 is a causative gene for Parkinson’s disease and the corresponding protein has been identified as a master regulator of mitophagy—the autophagic degradation of damaged mitochondria. It interacts with Beclin1 to regulate autophagy and initiate autophagosome formation, even outside the context of mitophagy. Several other pro-survival functions of this protein have been described and indicate that it might play a role in other disorders, such as cancer and proliferative diseases. In this study, we investigated a novel anti-apoptotic function of PINK1. To do so, we used SH-SY5Y neuroblastoma cells, a neuronal model used in Parkinson’s disease and cancer studies, to characterize the pro-survival functions of PINK1 in response to the apoptosis inducer staurosporine. In this setting, we found that staurosporine induces apoptosis but not mitophagy, and we demonstrated that PINK1 protects against staurosporine-induced apoptosis by impairing the pro-apoptotic cleavage of Beclin1. Our data also show that staurosporine-induced apoptosis is preceded by a phase of enhanced autophagy, and that PINK1 in this context regulates the switch from autophagy to apoptosis. PINK1 protein levels progressively decrease after treatment, inducing this switch. The PINK1–Beclin1 interaction is crucial in exerting this function, as mutants that are unable to interact do not show the anti-apoptotic effect. We characterized a new anti-apoptotic function of PINK1 that could provide options for treatment in proliferative or neurodegenerative diseases.

Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes.

In a variety of physiological and pathophysiological conditions, cells are exposed to acidic environments. Severe synovial fluid acidification also occurs in a progressive state of osteoarthritis (OA) affecting articular chondrocytes. In prior studies extracellular acidification has been shown to protect cells from apoptosis but the underlying mechanisms remain elusive. In the present study, we demonstrate that the inhibition of Cl− currents plays a significant role in the antiapoptotic effect of acidification in human articular chondrocytes. Drug-induced apoptosis was analyzed after exposure to staurosporine by caspase 3/7 activity and by annexin-V/7-actinomycin D (7-AAD) staining, followed by flow cytometry. Cell viability was assessed by resazurin, CellTiter-Glo and CellTiter-Fluor assays. Cl− currents and the mean cell volume were determined using the whole cell patch clamp technique and the Coulter method, respectively. The results reveal that in C28/I2 cells extracellular acidification decreases caspase 3/7 activity, enhances cell viability following staurosporine treatment and gradually deactivates the volume-sensitive outwardly rectifying (VSOR) Cl− current. Furthermore, the regulatory volume decrease (RVD) as well as the apoptotic volume decrease (ADV), which represents an early event during apoptosis, were absent under acidic conditions after hypotonicity-induced cell swelling and staurosporine-induced apoptosis, respectively. Like acidosis, the VSOR Cl− current inhibitor DIDS rescued chondrocytes from apoptotic cell death and suppressed AVD after induction of apoptosis with staurosporine. Similar to acidosis and DIDS, the VSOR channel blockers NPPB, niflumic acid (NFA) and DCPIB attenuated the staurosporine-induced AVD. NPPB and NFA also suppressed staurosporine-induced caspase 3/7 activation, while DCPIB and Tamoxifen showed cytotoxic effects per se. From these data, we conclude that the deactivation of VSOR Cl− currents impairs cell volume regulation under acidic conditions, which is likely to play an important role in the survivability of human articular chondrocytes.

Plasma-derived extracellular vesicles from myocardial infarction patients inhibits tumor necrosis factor-alpha induced cardiac cell death

RationaleExtracellular vesicles (EVs) derived exogenously from pluripotent stem cells or endogenously from healthy human serum exert cardioprotective effects after injury. However role of endogenous EVs from myocardial infarction (MI) patients not well understood in this settings. Methods and resultsThe EVs from plasma of MI patients with preserved or reduced left ventricular ejection fraction (LVEF) and healthy controls (HC) were purified and characterized by flow cytometry, mass spectrometry (MS) and transmission electron microscopy (TEM). HCM and human cardiac microvascular endothelial cells (hCMVECs), under individual culture or co-culture, were used to study functional effects of EVs upon TNFα stimulation. These effects of EVs on HCM and hCMVECs were observed using cell death assays, western blots and confocal microscopy. Higher concentrations of platelet-, leukocyte-, endothelial- and erythrocyte-derived EVs were found in MI patients, both with preserved and reduced LVEF, compared to HC, and MS data on MI EVs proteome displayed alteration in several proteins. MI EVs protected HCM and hCMVECs against staurosporine-induced apoptosis. Furthermore, MI EVs were observed to abrogate TNFα-triggered HCM and hCMVECs death under both individually cultured and co-cultured conditions. MI EVs failed to inhibit TNFα induced hCMVECs and HCM activation when cultured individually, however co-cultured hCMVECs with HCM supported MI EVs capacity to attenuate TNFα induced cells activation. MI CD41+ EVs but not HC EVs were found to be internalized by HCM directly or migrated through hCMVECs to HCM. MI EVs indirectly restores TNFα mediated drop in mitochondrial membrane potential. ConclusionsEndogenous EVs from MI patients, regardless of severity of the MI exert cardioprotective potential upon TNFα-induced cell death. Patient-derived EVs needs to be further explored to elucidate their potential cardioprotective role during MI.

ORAI1 regulates sustained cytosolic free calcium fluctuations during breast cancer cell apoptosis and apoptotic resistance via a STIM1 independent pathway.

Excessive rapid increases in cytosolic free Ca2+ have a clear association with the induction of cancer cell death. Whereas, characterizing the Ca2+ signaling events that occur during the progression of the apoptotic cascade over a period of hours or days, has not yet been possible. Now using genetically encoded Ca2+ indicators complemented with automated epifluorescence microscopy we have shown that staurosporine-induced apoptosis in MDA-MB-231 breast cancer cells was associated with delayed development of cytosolic free Ca2+ fluctuations, which were then maintained for 24 h. These cytosolic free Ca2+ fluctuations were dependent on the Ca2+ channel ORAI1. Silencing of ORAI1, but not its canonical activators STIM1 and STIM2, promoted apoptosis in this model. The pathway for this regulation implicates a mechanism previously associated with the migration of cancer cells involving ORAI1, the chaperone protein SigmaR1, and Ca2+ -activated K+ channels.

Rapid and automated quantification of TDP-43 and FUS mislocalisation for screening of frontotemporal dementia and amyotrophic lateral sclerosis gene variants.

Identified genetic mutations account for ∼50% of familial frontotemporal dementia (FTD) and ∼70% of familial amyotrophic lateral sclerosis (ALS) cases, however, for the remainder of patients the origin of the disease is uncertain. Post-mortem, affected neurons from 97% of ALS and ∼50% of FTD patients show cytoplasmic mislocalisation of the typically nuclear proteins, TDP-43 and/or FUS. We exploited this predominant neuropathological feature to develop a high-throughput, automated method for the quantification of cytoplasmic TDP-43 and FUS in human cell lines. Utilising fluorescently tagged cDNA constructs to identify cells of interest, fluorescence intensity of TDP-43 or FUS in the nucleus and cytoplasm of HEK293 and SH-SY5Y cells was measured from confocal microscope images using the freely available analysis software, CellProfiler. The assay was validated using known ALS-causative mutations in the genes encoding TDP-43 (TARDBP) and FUS, and pharmacological interventions known to cause TDP-43 and FUS mislocalisation. The ability of the assay to detect effects of a secondary gene on endogenous TDP-43 was tested using CYLD wild-type and mutant constructs. Expression of known TARDBP and FUS mutations showed significantly higher cytoplasmic to nuclear ratios when compared to wild-type protein. Treatment with apoptosis inducers MG132 and staurosporine induced a similar effect. Lastly, we recapitulated the effect of the CYLD FTD-ALS mutation p.M719V on TDP-43 mislocalisation, as observed previously by manual counting of primary mouse neurons [Dobson-Stone et al 2020, Brain 143:783-799]. The current study validates our methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used to rapidly and systematically assess the pathogenicity of predicted FTD/ALS gene variants. This methodology can be employed to aid genetic diagnosis of patients carrying novel variants in known FTD/ALS genes, and in the prioritisation of novel candidate genes for further functional analysis.

Phosphocatalytic Kinome Activity Profiling of Apoptotic and Ferroptotic Agents in Multiple Myeloma Cells.

Through phosphorylation of their substrate proteins, protein kinases are crucial for transducing cellular signals and orchestrating biological processes, including cell death and survival. Recent studies have revealed that kinases are involved in ferroptosis, an iron-dependent mode of cell death associated with toxic lipid peroxidation. Given that ferroptosis is being explored as an alternative strategy to eliminate apoptosis-resistant tumor cells, further characterization of ferroptosis-dependent kinase changes might aid in identifying novel druggable targets for protein kinase inhibitors in the context of cancer treatment. To this end, we performed a phosphopeptidome based kinase activity profiling of glucocorticoid-resistant multiple myeloma cells treated with either the apoptosis inducer staurosporine (STS) or ferroptosis inducer RSL3 and compared their kinome activity signatures. Our data demonstrate that both cell death mechanisms inhibit the activity of kinases classified into the CMGC and AGC families, with STS showing a broader spectrum of serine/threonine kinase inhibition. In contrast, RSL3 targets a significant number of tyrosine kinases, including key players of the B-cell receptor signaling pathway. Remarkably, additional kinase profiling of the anti-cancer agent withaferin A revealed considerable overlap with ferroptosis and apoptosis kinome activity, explaining why withaferin A can induce mixed ferroptotic and apoptotic cell death features. Altogether, we show that apoptotic and ferroptotic cell death induce different kinase signaling changes and that kinome profiling might become a valid approach to identify cell death chemosensitization modalities of novel anti-cancer agents.