Membrane Phosphatidylserine Exposure Research Articles

Perillaldehyde Functions as a Potential Antifungal Agent by Triggering Metacaspase-Independent Apoptosis in Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold, is an important plant pathogen causing preharvest and postharvest diseases. Due to the extensive use of commercial fungicides, fungicide-resistant strains have emerged. Natural compounds with antifungal properties are widely present in various kinds of organisms. Perillaldehyde (PA), derived from the plant species Perilla frutescens, is generally recognized as a potent antimicrobial substance and to be safe to humans and the environment. In this study, we demonstrated that PA could significantly inhibit the mycelial growth of B. cinerea and reduced its pathogenicity on tomato leaves. We also found that PA had a significant protective effect on tomato, grape, and strawberry. The antifungal mechanism of PA was investigated by measuring the reactive oxygen species (ROS) accumulation, the intracellular Ca2+ level, the mitochondrial membrane potential, DNA fragmentation, and phosphatidylserine exposure. Further analyses revealed that PA promoted protein ubiquitination and induced autophagic activities and then triggered protein degradation. When the two metacaspase genes, BcMca1 and BcMca2, were knocked out from B. cinerea, all mutants did not exhibit reduced sensitivity to PA. These findings demonstrated that PA could induce metacaspase-independent apoptosis in B. cinerea. Based on our results, we proposed that PA could be used as an effective control agent for gray mold management. IMPORTANCE Botrytis cinerea causes gray mold disease, is considered one of the most important dangerous pathogens worldwide, and leads to severe economic losses worldwide. Due to the lack of resistant varieties of B. cinerea, gray mold control has mainly relied on application of synthetic fungicides. However, long-term and extensive use of synthetic fungicides has increased fungicide resistance in B. cinerea and is harmful to humans and the environment. In this study, we found that perillaldehyde has a significant protective effect on tomato, grape, and strawberry. We further characterized the antifungal mechanism of PA on B. cinerea. Our results indicated that PA induced apoptosis that was independent of metacaspase function.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Water-Soluble Tomato Concentrate, a Potential Antioxidant Supplement, Can Attenuate Platelet Apoptosis and Oxidative Stress in Healthy Middle-Aged and Elderly Adults: A Randomized, Double-Blinded, Crossover Clinical Trial.

Increased oxidative stress and platelet apoptotic in middle-aged and elderly adults are important risk factors for atherosclerotic cardiovascular disease (ASCVD). Therefore, it is of great significance to control the oxidative stress and platelet apoptosis in middle-aged and elderly adults. Previous acute clinical trials have shown that water-soluble tomato concentrate (WSTC) from fresh tomatoes could exert antiplatelet benefits after 3 h or 7 h, but its effects on platelet apoptosis and oxidative stress are still unknown, especially in healthy middle-aged and elderly adults. This current study aimed to examine the efficacies of WSTC on platelet apoptosis and oxidative stress in healthy middle-aged and elderly adults via a randomized double-blinded placebo-controlled crossover clinical trial (10 weeks in total). A total of 52 healthy middle-aged and elderly adults completed this trial. The results showed that WSTC could increase the serum total antioxidant capacity levels (p < 0.05) and decrease the serum malondialdehyde levels (p < 0.05) after a 4-week WSTC supplementation in healthy middle-aged and elderly adults. Platelet endogenous reactive oxygen species generation (p < 0.05), mitochondrial membrane potential dissipation (p < 0.05) and phosphatidylserine exposure (p < 0.05) were attenuated. In addition, our present study also found that WSTC could inhibit platelet aggregation and activation induced by collagen or ADP after intervention (p < 0.05), while having no effects on adverse events (p > 0.05). The results suggest that WSTC can inhibit oxidative stress and its related platelet apoptosis, which may provide a basis for the primary prevention of WSTC in ASCVD.

P106: EARLY PREVENTIVE DIAGNOSIS OF HEMOLYTIC ANEMIA IN SICKLE CELL PATIENTS BY DETECTING THE TRIGGERING OF ERYPTOSIS.

Background/Aims: Sickle cell disease (SCD) also known as sickle cell anemia is one of the most worldwide-disseminated hereditary hemoglobinopathies. It is caused by a single amino acid substitution at the sixth residue of β globin gene (Glu6Val), which results in an abnormal hemoglobin called hemoglobin S (HbS). The acceleration of HbS polymerization induces rigid and dysfunctional erythrocytes that play a central role in acute and chronic clinical manifestations of SCD(Conran et al.,2009). Vaso-occlusion and hemolytic anemia are the hallmarks of SCD. We aim to explore the cellular environment of red blood cells to explain the physiopathology of SCD. In fact, the life span of circulating erythrocytes in healthy individuals vary from 100 to 120 days. In SCD, red blood cells undergo a form of cell death, namely, eryptosis before they reach their full life span. Eryptosis is triggered by a wide variety of factors as hyperosmolarity, oxidative stress and energy depletion. It is characterized by the presence of membrane blebbing, cell shrinkage, and phosphatidylserine (PS) exposure (Lang et al.,2012). In this study, we will explore the mechanism of triggering of eryptosis in Sickle cell disease. Methods: Following clinical diagnosis, 50 homozygous SCD patients and 30 healthy donors were identified for hematological and cellular assays. Flow cytometry was performed in order to determine the viability parameters of erythrocytes. The morphology of red blood cells and the externalization of phosphatidylserine was detected by labeling red blood cells with Annexine V. Moreover, we had identified intracellular calcium concentration and ceramide level by labeling erythrocytes with Fluo3-am and anti-ceramide antibodies. Finally, we had quantified reactive oxygen species (ROS) by labeling red blood cells with CM-H2DCFDA. Results and Discussion: Eryptosis in sickle cell patients is accelerated. In fact, PS (+) red blood cells are more present in patients than in healthy subjects. Therefore, eryptosis is triggered by oxidative stress, which stimulates the increase of calcium activity and subsequent externalization of PS and red blood cells shrinkage in sickle cell patients. However, the pathway of ceramide can also be considered a potential stimulating factor of eryptosis in SCD. Eryptosis ensures healthy erythrocyte quantity in circulation, whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.

Serotonin type-3 receptor antagonists selectively kill melanoma cells through classical apoptosis, microtubule depolymerisation, ERK activation, and NF-κB downregulation.

Malignant melanoma is a highly metastatic tumour, resistant to treatment. Serotonin type-3 (5-HT3) receptor antagonists, such as tropisetron and ondansetron, are well-tolerated antiemetic drugs commonly used to prevent nausea caused by chemotherapy or radiotherapy. We investigated the anticancer effects of these drugs on melanoma cancer cell lines WM-266-4 and B16F10 with or without paclitaxel. We constructed IC50 curves and performed Chou-Talalay analysis, using data obtained with the MTT assay. Flow cytometry and fluorescent microscopy were used to examine characteristics of the cell cycle, cell death and cytoskeleton changes. Protein levels and activation were analysed by western blotting and molecular docking studies carried out. Data were analysed by one way ANOVA and post hoc testing. Ondansetron and tropisetron showed selective concentration-dependent cytotoxicity in melanoma cell lines WM-266-4 and B16F10. The effect in combination with paclitaxel was synergistic. The drugs did not cause cell cycle arrest but did promote characteristics of classical apoptosis, including accumulation of subG1 DNA, cleaved caspase-3, mitochondrial membrane permeability and phosphatidylserine exposure. As well, the cytosolic calcium level in the melanoma cells was enhanced, phosphorylated ERK1/2 induced and NF-κB inhibited. Finally, the formation of microtubules was shown to be impaired in melanoma cells treated with ondansetron or tropisetron. Docking studies were used to predict that these drugs could bind to the colchicine binding site on the tubulin molecule. Antiemetic drugs, already given in combination with chemotherapy, may enhance the cytotoxic effect of chemotherapy, following successful delivery to the tumour site.

The Role of RBC Oxidative Stress in Sickle Cell Disease: From the Molecular Basis to Pathologic Implications.

Sickle cell disease (SCD) is an inherited monogenic disorder and the most common severe hemoglobinopathy in the world. SCD is characterized by a point mutation in the β-globin gene, which results in hemoglobin (Hb) S production, leading to a variety of mechanistic and phenotypic changes within the sickle red blood cell (RBC). In SCD, the sickle RBCs are the root cause of the disease and they are a primary source of oxidative stress since sickle RBC redox state is compromised due to an imbalance between prooxidants and antioxidants. This imbalance in redox state is a result of a continuous production of reactive oxygen species (ROS) within the sickle RBC caused by the constant endogenous Hb autoxidation and NADPH oxidase activation, as well as by a deficiency in the antioxidant defense system. Accumulation of non-neutralized ROS within the sickle RBCs affects RBC membrane structure and function, leading to membrane integrity deficiency, low deformability, phosphatidylserine exposure, and release of micro-vesicles. These oxidative stress-associated RBC phenotypic modifications consequently evoke a myriad of physiological changes involved in multi-system manifestations. Thus, RBC oxidative stress in SCD can ultimately instigate major processes involved in organ damage. The critical role of the sickle RBC ROS production and its regulation in SCD pathophysiology are discussed here.

The binding of autotaxin to integrins mediates hyperhomocysteinemia-potentiated platelet activation and thrombosis in mice and humans

AbstractHyperhomocysteinemia (HHcy) is associated with an exaggerated platelet thrombotic response at sites of vascular injury. In this study, human medical examination showed that elevated human plasma Hcy levels correlated positively with enhanced blood coagulation and platelet activity, suggesting that humans with HHcy are more prone to thrombus formation at the sites of vascular injury. Accordingly, we observed accelerated platelet activation, primary hemostasis, and thrombus formation in apolipoprotein E-deficient (ApoE−/−) mice with acute or chronic HHcy. Upon homocysteine (Hcy) administration in C57BL/6J mice, platelet aggregation, spreading and clot retraction were markedly induced. More important, Hcy increased the affinity of platelet integrin αIIbβ3 with ligands and enhanced integrin outside-in signaling by promoting membrane phosphatidylserine exposure in vitro. Mechanistically, lipidomics analysis showed that lysophosphatidylcholines were the primary metabolites leading to clustering of HHcy-stimulated platelets. Cytosolic phospholipase A2 (cPLA2) activity and autotaxin (ATX, a secreted lysophospholipase D) secretion were upregulated by Hcy, leading to membrane phospholipid hydrolysis and PS exposure. Moreover, secreted ATX directly interacted with integrin β3. Inhibitors of cPLA2 and ATX activity blocked integrin αIIbβ3 outside-in signaling and thrombosis in HHcy ApoE−/− mice. In this study, we identified a novel mechanism by which HHcy promotes platelet membrane phospholipid catabolism and extracellular ATX secretion to activate integrin outside-in signaling, consequently exacerbating thrombosis and the results revealed an innovative approach to treating HHcy-related thrombotic diseases.

Analogs of a Natural Peptaibol Exert Anticancer Activity in Both Cisplatin- and Doxorubicin-Resistant Cells and in Multicellular Tumor Spheroids.

Peptaibols, by disturbing the permeability of phospholipid membranes, can overcome anticancer drug resistance, but their natural hydrophobicity hampers their administration. By a green peptide synthesis protocol, we produced two water-soluble analogs of the peptaibol trichogin GA IV, termed K6-Lol and K6-NH2. To reduce production costs, we successfully explored the possibility of changing the naturally occurring 1,2-aminoalcohol leucinol to a C-terminal amide. Peptaibol activity was evaluated in ovarian cancer (OvCa) and Hodgkin lymphoma (HL) cell lines. Peptaibols exerted comparable cytotoxic effects in cancer cell lines that were sensitive—and had acquired resistance—to cisplatin and doxorubicin, as well as in the extrinsic-drug-resistant OvCa 3-dimensional spheroids. Peptaibols, rapidly taken up by tumor cells, deeply penetrated and killed OvCa-spheroids. They led to cell membrane permeabilization and phosphatidylserine exposure and were taken up faster by cancer cells than normal cells. They were resistant to proteolysis and maintained a stable helical structure in the presence of cancer cells. In conclusion, these promising results strongly point out the need for further preclinical evaluation of our peptaibols as new anticancer agents.

CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood–Brain Barrier Models

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood–brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood–brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells

AbstractAcquisition of CCR7 expression is an important phenotype change during dendritic cell (DC) maturation that endows these cells with the capability to migrate to lymph nodes. We have analyzed the possible role of CCR7 on the regulation of the survival of DCs. Stimulation with CCR7 ligands CCL19 and CCL21 inhibits apoptotic hallmarks of serum-deprived DCs, including membrane phosphatidylserine exposure, loss of mitochondria membrane potential, increased membrane blebs, and nuclear changes. Both chemokines induced a rapid activation of phosphatidylinositol 3′-kinase/Akt1 (PI3K/Akt1), with a prolonged and persistent activation of Akt1. Interference with PI3K, Gi, or G protein βγ subunits abrogated the effects of the chemokines on Akt1 activation and on survival. In contrast, inhibition of extracellular signal-related kinase 1/2 (Erk1/2), p38, or c-Jun N-terminal kinase (JNK) was ineffective. Nuclear factor–κB (NFκB) was involved in the antiapoptotic effects of chemokines because inhibition of NFκB blunted the effects of CCL19 and CCL21 on survival. Furthermore, chemokines induced down-regulation of the NFκB inhibitor IκB, an increase of NFκB DNA-binding capability, and translocation of the NFκB subunit p65 to the nucleus. In summary, in addition to its well-established role in chemotaxis, we show that CCR7 also induces antiapoptotic signaling in mature DCs.

Abstract 608: The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML)

Abstract Background: AML is a genetically heterogenous disease with poor prognosis and new treatments are needed. MPS1 is the main kinase of the spindle assembly checkpoint (SAC), critical for segregation of chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. MPS1 inhibition has been investigated in several solid tumors but not yet in AML. Here, we demonstrate the efficacy of the small molecule MPS1 inhibitor (MPS1i) S81694 in AML cell lines, primary cells and mouse models. Materials and Methods: Cell viability, cell stress and apoptosis were assessed by MTS assays, DiOC2(3) or annexin-V modifications and PI incorporation after exposure to S81694. Cell-cycle and polyploidy were determined by cytofluorometry and FISH. Phosphorylation of MPS1 was detected in synchronized cells by immunofluorescence detecting phosphorylated Thr33/Ser37 residues. Protein modifications were studied by WB. Time-lapse microscopy was used to determine mitosis duration using cells stained with the SiR-DNA live cell probe. Bone marrow cells from patients were obtained after informed consent and incubated on a MSC feeder or in methylcellulose for colony formation assays. All animal studies were performed in accordance with the animal ethics committee's guidelines. NSG mice were sub-lethally-irradiated and injected with AML-NS8 or AML-PS patient derived xenografts. Mice were treated intravenously with S81694 or with vehicle. Results: IC50 for viability after S81694 exposure alone was &amp;lt;1000nM in a panel of 8 AML cell lines. Three cell lines had IC50 &amp;gt;1000nM: the NPM1cmut cell line OCI-AML3, OCI-AML3-TP53mut and K562. In the most sensitive cell lines, including OCI-AML2, S81694 led to inhibition of MPS1 auto-phosphorylation, induced significant cell stress and apoptosis as detected by mitochondrial membrane potential loss, phosphatidylserine exposure and PI incorporation. In these cell lines, the cell cycle was strongly affected by treatment with S81694 showing aberrant 2n/4n ploidy distribution due to SAC abrogation as well as polyploidy (8n and 16n). S81694 exposure triggered mitotic exit as shown by cyclin B1 downregulation and significantly accelerated mitosis. For treated OCI-AML2 and OCI-AML3 cells we observed induction of gamma-H2AX (ser139), p53 upregulation and downstream caspase-3 and PARP cleavage indicating that S81694 induces p53 dependent apoptosis. Furthermore, MPS1i induced downregulation of anti-apoptotic proteins MCL-1 and BCLXL. Finally, for AML patient derived blast cells cells we observed apoptosis after exposure to S81694 and significant reduction of colony number. In two AML PDX models, intravenous administration of S81694 was shown to improve significantly median survival time, as compared to control, by 28 to 41 days and by 33 to 46 days respectively. Conclusion: The MPS1i S81694 shows significant preclinical activity in vitro and in vivo in AML. Combinations with different drugs active in AML are ongoing. Citation Format: Anna Kaci, Emilie Adicéam, Etienne De Braekeleer, Marine Garrido, Jeannig Berrou, Mélanie Dupont, Hanane Djamai, Virginie Eclache, Baruchel André, Claude Gardin, Hervé Dombret, Mike Burbridge, Thorsten Braun. The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 608.

Induction of Erythrocyte Shrinkage by Omeprazole.

Omeprazole, a proton pump inhibitor blocks the H+/K+-ATPase channels of gastric parietal cells. It is used for the treatment of peptic ulcer. Prolonged use of omeprazole may involve in inducing anemia. The key marker of eryptosis includes membrane blebbing, cell shrinkage and phosphatidylserine (PS) exposure at the cell surface. In current study, the eryptotic, oxidative as well as hemolytic effects of therapeutical doses (0.5, 1 and 1.5 µM) of omeprazole were investigated after exposing erythrocytes for 48 hours. Investigation of eryptosis was done by cell size measurement, PS exposure determination and calcium channel inhibition. As a possible mechanism of omeprazole induced eryptosis, oxidative stress was investigated by determining the catalase, glutathione peroxidase and superoxide dismutase activities. Similarly, necrotic effect of omeprazole on erythrocytes was also evaluated through hemolysis measurement. Results of our study illustrated that 1.5 µM of omeprazole may induce significant decrease in superoxide dismutase, glutathione peroxidase and catalase activities as well as triggered the erythrocytes shrinkage, PS exposure and hemolysis. Role of calcium was also confirmed in inducing erythrocyte shrinkage. It is concluded that the exposure of erythrocytes with 1.5 µM omeprazole may enhance the rate of eryptosis and hemolysis by inducing oxidative stress.

Combined Fluorimetric Caspase-3/7 Assay and Bradford Protein Determination for Assessment of Polycation-Mediated Cytotoxicity.

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell-death related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of caspase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro.Methods:In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention.Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion.Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. DisclosuresNo relevant conflicts of interest to declare.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Caspase-independence and characterization of bisnaphthalimidopropyl spermidine induced cytotoxicity in HL60 cells

Bisnaphthalimides are DNA intercalators of potential use as chemotherapeutics but for which the range of mechanism of action is only gradually being elucidated. Using human promyelocytic HL-60 cells, we extend characterization of the cytotoxicity of bisnaphthalimidopropylspermidine (BNIPSpd) and examine the relationship with caspase-activity. Within 4 h exposure, BNIPSpd (1–10 μM) induced significant DNA strand breakage. Evidence of apoptosis was progressive through the experimental period. Within 6 h, BNIPSpd increased the proportion of cells exhibiting plasma membrane phosphatidylserine exposure. Within 12 h, active caspase expression increased and was sustained with 5 and 10 μM BNIPSpd. Flow cytometric analysis revealed caspase activity in cells with and without damaged membranes. By 24 h, 5 and 10 μM BNIPSpd increased hypodiploid DNA content and internucleosomal DNA fragmentation (DNA ladders) typical of the later stages of apoptosis. 1 μM BNIPSpd exposure also increased hypodiploid DNA content by 48 h. Polyamine levels decreased by 24 h BNIPSpd exposure. The pan-caspase inhibitor, z-VAD-fmk, significantly decreased DNA degradation (hypodiploid DNA and DNA ladders) and cytotoxicity. Despite this, cell growth and viability remained significantly impaired. We propose that BNIPSpd cytotoxicity arises through DNA damage and not polyamine depletion and that cytotoxicity is dominated by but not dependent upon caspase driven apoptosis.

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure.

Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis. Using flow cytometry and/or fluorescence microscopy, we analyzed desialylation patterns and eryptotic alterations in erythrocytes exposed to Clostridium perfringens-derived neuraminidase. Exogenous bacterial neuraminidase significantly augmented membrane PS exposure and cytosolic Ca2+ levels in a dose- and time-dependent manner. Neuraminidase treatment significantly reduced fluorescence-tagged agglutinin binding, an effect temporally preceding the increase in PS externalization. Neuraminidase-induced PS exposure was significantly curtailed by pretreatment with the pan-sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. Neuraminidase treatment further induced hemolysis but did not significantly impact erythrocyte volume, ceramide abundance, or the generation of reactive oxygen species. Collectively, our data reveal that alteration of erythrocyte sialylation status by bacterial neuraminidase favors eryptotic cell death, an effect potentially contributing to reduced erythrocyte lifespan and anemia in sepsis.

Giardipain-1, a protease secreted by Giardia duodenalis trophozoites, causes junctional, barrier and apoptotic damage in epithelial cell monolayers

The adhesion of Giardia duodenalis trophozoites to intestinal epithelial cells allows the onset and maintenance of giardiasis. During these interactions, epithelial cells can be committed to apoptosis by enzymes secreted by the parasites, including cysteine proteases that are increasingly identified as virulence factors in parasitic protozoa. In this work, a monoclonal antibody (mAb1G3) raised against G. duodenalis surface components was found to react with a 25 kDa protein expressed in the cell surface and flagella of G. duodenalis trophozoites. When trophozoites expressing this protein were cultured with IEC-6 intestinal epithelial cell monolayers, a dynamic release of this protein was observed with mAbIG3. Proteomic analysis identified the protein as a mature cathepsin B-like (gCatB) enzyme, whose proteolytic activity, detected in zymograms, was eliminated by CatB inhibitor E-64. This protein was named giardipain-1 due to its functional papain-like features and was purified by affinity chromatography using mAbIG3. Upon exposure to the purified, mature and secreted forms of giardipain-1, IEC-6 epithelial cell monolayers displayed membrane blebbing and phosphatidylserine exposure on the outer cell surface, indicating an apoptotic process. In Madin Darby Canine Kidney (MDCK) cell monolayers, giardipain-1 leads to the appearance of pore-like regions and of gaps along cell–cell junctions, to decreased transepithelial electrical resistance (TER), caspase-3 activation and poly-ADP-ribose polymerase (PARP) fragmentation. At early times during exposure, giardipain-1 co-localized at cell–cell junctions, associated with occludin and induced the delocalization and degradation of tight junction proteins occludin and claudin-1. The damage caused to epithelial monolayers by giardipain-1 was blocked by pre-incubation with the CatB B Inhibitor E-64. Furthermore, silencing the giardipain-1 gene in trophozoites lowered the proteolytic activity of giardipain-1 and reduced the damage in IEC-6 monolayers. The damage observed appears to be specific to giardipain activity since almost no damage was observed when IEC-6 monolayers were incubated with papain, a non-related cysteine protease. Hence this study suggests that giardipain-1 triggers, in epithelial cells, degradation of cell–cell junctional components and apoptotic damage, supporting the notion of giardiapain-1 as a virulence factor of Giardia.

Trypanosoma cruzi: death phenotypes induced by ortho-naphthoquinone substrates of the aldo-keto reductase (TcAKR). Role of this enzyme in the mechanism of action of β-lapachone.

Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as β-lapachone (β-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by β-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that β-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, β-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only β-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in β-Lap activation.

Combining cold physical plasma with pulsed electrical fields for cancer treatment

Pallation of end-stage melanoma and breast cancer patients is a challenge. Despite the generally successful use of electrochemotherapy (ECT) in these patients, response rates can still be improved. Previous studies demonstrated the anticancer potential of nano-second PEFs (nsPEFs), which are able to induce apoptosis [1] or nanopore formation when using different settings [2]. Likewise, the anticancer capacity of cold physical plasma has been demonstrated in various studies in cell lines and in xenograft tumors in mice [3-7]. The apoptotic effect of cold plasmas is mediated by a variety of reactive species being released onto the cells, where the species trigger redox signaling, and subsequent cell death in some instances [8, 9]. By contrast, PEFs act by a mechanism much less dependent on the newly generated reactive species. Our idea was to combine both treatments to improve palliative cancer treatment in the future. Malignant suspension cell lines were tested to investigate the proof of concept of additive or possible even synergistic cytotoxic effects. Plasma treatment time (kINPen) and PEF intensity as well as pulse length were varied to retrieve sublethal dosage regimens for each treatment. The sequence of combination (first plasma, then PEF or vice versa) was also modulated. To investigate the mode of action of both therapies, a number of cellular parameters were investigated. This included oxidation at cytosolic and membrane compartments, thiol content, mitochondrial depolarization, caspase activation and phosphatidylserine exposure, metabolic activity, cell membrane permeabilization, cell growth and morphology, and protection by antioxidants. Furthermore, we identified a synergistic effect of plasma and PEFs using tumorigenic adherent cells in preliminary tests.