Phosphatidylserine Externalization Research Articles

Temozolomide and Lomustine Induce Tissue Factor Expression and Procoagulant Activity in Glioblastoma Cells In Vitro

Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.

Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II.

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13-AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13-AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13-AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13-AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.

The magnetic cell separation method reveals protective effect of melatonin on human spermatozoa from peroxide-induced apoptosis

BackgroundThe selection of non-apoptotic sperm is related to successful fertilization. This study investigated the protective effects of melatonin and the role of the MACS (magnetically activated cell sorting) method to prevent oxidative damage in vitro and improve sperm quality parameters such as viability and DNA integrity.Materials and methodsSemen analysis was performed on 20 different eligible donors participating in the study. Sperm viability and concentration were checked at time of 0 (0 h). In order to conduct more studies after 24 h (24 h), the remaining sperm suspension was divided into a control group and six experimental groups. DNA fragmentation was assessed using the TUNEL assay.ResultsThe treatment of human spermatozoa with 100 µM hydrogen peroxide for 24 h induced a significant increase in phosphatidylserine externalization and significantly increases apoptotic sperm (p ≤ 0.001). TUNEL analysis of human sperm pretreated with 100 µM hydrogen peroxide for 24 h showed that the percentage of sperm with fragmented DNA was significantly reduced after sorting by MACS (P ≤ 0.001). However, pretreated human sperm with 1 μM melatonin for 24 h could effectively maintain sperm motility and progressive motility.ConclusionsPretreated human spermatozoa with 1 µM melatonin for 24 h could be effective for maintenance of sperm motility and progressive motility. Although 100 µM hydrogen peroxide-treated sperm were used, MACS was used to retain the appropriate sperm and select high-quality sperm.

Silica nanoparticles trigger phosphatidylserine exposure in red blood cells and induce thrombosis risk

Silica nanoparticles (SiNPs) have attracted increasing attention for their health effects due to the increased risk of exposure to human bodies via diverse routes. Considering that SiNPs enter the circulatory system and inevitably encounter red blood cells (RBCs), it is necessary to investigate their risk of causing erythrocytotoxicity. In this study, three sizes of SiNPs (SiNP-60, SiNP-120, and SiNP-200) were tested for their effects on mouse RBCs. The results showed that SiNPs could induce hemolysis, morphological changes, and phosphatidylserine (PS) exposure in RBCs in a particulate size-related manner. Further investigations on the underlying mechanism indicated that SiNP-60 exposure increased intracellular reactive oxidative species (ROS) generation and subsequently caused the phosphorylation of p38 and ERK1/2 in RBCs. The addition of antioxidants or inhibitors of mitogen-activated protein kinase (MAPK) signaling significantly attenuated PS exposure in RBCs and ameliorated SiNP-induced erythrocytotoxicity. Moreover, ex vivo assays using platelet-rich plasma (PRP) showed that SiNP-60-induced PS exposure in RBCs could trigger thrombin-dependent platelet activation. The contrary evidence from the assays of PS blockage and thrombin inhibition further confirmed that SiNP-60-induced platelet activation was dependent on PS externalization in RBCs, concomitantly with thrombin formation. These findings revealed the procoagulant and prothrombotic effects of SiNPs through the regulation of PS externalization in RBCs, and may be of great help in bridging the knowledge gap on the potential cardiovascular hazards of particulate silica from both artificial and naturally occurring origins.

Microparticle Phosphatidylserine Mediates Coagulation: Involvement in Tumor Progression and Metastasis

Tumor progression and cancer metastasis has been linked to the release of microparticles (MPs), which are shed upon cell activation or apoptosis and display parental cell antigens, phospholipids such as phosphatidylserine (PS), and nucleic acids on their external surfaces. In this review, we highlight the biogenesis of MPs as well as the pathophysiological processes of PS externalization and its involvement in coagulation activation. We review the available evidence, suggesting that coagulation factors (mainly tissue factor, thrombin, and fibrin) assist in multiple steps of tumor dissemination, including epithelial-mesenchymal transition, extracellular matrix remodeling, immune escape, and tumor angiogenesis to support the formation of the pre-metastatic niche. Platelets are not just bystander cells in circulation but are functional players in primary tumor growth and metastasis. Tumor-induced platelet aggregation protects circulating tumor cells (CTCs) from the blood flow shear forces and immune cell attack while also promoting the binding of CTCs to endothelial cells and extravasation, which activates tumor invasion and sustains metastasis. Finally, in terms of therapy, lactadherin can inhibit coagulation by competing effectively with coagulation factors for PS binding sites and may similarly delay tumor progression. Furthermore, we also investigate the therapeutic potential of coagulation factor inhibitors within the context of cancer treatment. The development of multiple therapies targeting platelet activation and platelet-tumor cell interactions may not only reduce the lethal consequences of thrombosis but also impede tumor growth and spread.

Exploration of Antiproliferative Activity and Apoptosis Induction of New Nickel(II) Complexes Encompassing Carbazole Ligands.

To attest the effectiveness of nickel complexes as anticancer drug candidates with minimum side effects, the present investigation describes the facile synthesis and anticancer activities of nickel(II) complexes enriched with three derivatives of carbazolone-based benzhydrazone ligands(L) having a [Ni(L)2] composition. Analytical and spectral techniques were used to characterize the synthesized Ni(II) complexes. The single-crystal X-ray diffraction performed for complex 4 confirmed the square planar geometry with a [Ni(κ2-N,O-L)2] arrangement. The MTT assay was carried out for the complexes to determine in vitro cytotoxicity against cancerous human-cervical carcinoma, human-colon carcinoma, and non-cancerous L929 (fibroblast) cells. All three complexes exhibited good toxicity against the cancer cells with a low IC50 concentration. Complex 4, containing -OCH3 fragment, exhibits high lipophilicity and revealed exceptional cytotoxicity against cancer cells. AO-EB fluorescent staining indicated apoptosis-associated cell morphological changes after exposure to complex 4. The apoptosis induction was further confirmed by a HOECHST-33342 fluorescent staining technique via chromosomal condensation and nuclear fragmentation. Further, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) mechanistic studies revealed that complex 4 can raise ROS levels and reduce MMP and promote mitochondrial dysfunction-mediated apoptotic cell death. Further, stimulation of late apoptosis by complex 4 in cervical cancer cells was quantitatively differentiated through the staining of phosphatidylserine externalization by flow cytometry. Furthermore, the ELISA analysis confirmed that complex 4 induced apoptosis through caspase activation.

Platelet activation and ferroptosis mediated NETosis drives heme induced pulmonary thrombosis

Cell-free heme (CFH) is a product of hemoglobin, myoglobin and hemoprotein degradation, which is a hallmark of pathologies associated with extensive hemolysis and tissue damage. CHF and iron collectively induce cytokine storm, lung injury, respiratory distress and infection susceptibility in the lungs suggesting their key role in the progression of lung disease pathology. We have previously demonstrated that heme-mediated reactive oxygen species (ROS) induces platelet activation and ferroptosis. However, interaction of ferroptotic platelets and neutrophils, the mechanism of action and associated complications remain unclear. In this study, we demonstrate that heme-induced P-selectin expression and Phosphatidylserine (PS) externalization in platelets via ASK-1-inflammasome axis increases platelet-neutrophil aggregates in circulation, resulting in Neutrophil extracellular traps (NET) formation in vitro and in vivo. Further, heme-induced platelet activation in mice increased platelet-neutrophil aggregates and accumulation of NETs in the lungs causing pulmonary damage. Thus, connecting CFH-mediated platelet activation to NETosis and pulmonary thrombosis. As lung infections induce acute respiratory stress, thrombosis and NETosis, we propose that heme -mediated platelet activation and ferroptosis might be crucial in such clinical manifestations. Further, considering the ability of redox modulators and ferroptosis inhibitors like FS-1, Lpx-1 and DFO to inhibit heme-induced ferroptotic platelets-mediated NETosis and pulmonary thrombosis. They could be potential adjuvant therapy to regulate respiratory distress-associated clinical complications.

Phosphatidylserine-Exposing Annexin A1-Positive Extracellular Vesicles: Potential Cancer Biomarkers.

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an "eat-me" signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1+) derived from cancer cells compared to m/lEVs (annexin A1+) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1+) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes.

Myricetin-induced suicidal erythrocyte death.

Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

The survival of Amblyomma sculptum ticks upon blood-feeding depends on the expression of an inhibitor of apoptosis protein

BackgroundThe tick Amblyomma sculptum is the major vector of Rickettsia rickettsii, the causative agent of the highly lethal Brazilian spotted fever. It has been shown that R. rickettsii inhibits apoptosis in both human endothelial cells and tick cells. Apoptosis is regulated by different factors, among which inhibitors of apoptosis proteins (IAPs) play a central role. In the study reported here, we selected an IAP of A. sculptum that has not yet been characterized to assess its role in cell death and to determine the effects of its gene silencing on tick fitness and R. rickettsii infection.MethodsAn A. sculptum cell line (IBU/ASE-16) was treated with specific double-stranded RNA (dsRNA) for either IAP (dsIAP) or green fluorescent protein (dsGFP; as a control). The activity of caspase-3 and the exposure of phosphatidylserine were determined in both groups. In addition, unfed adult ticks, infected or not infected with R. rickettsii, were treated with either dsIAP or dsGFP and allowed to feed on noninfected rabbits. In parallel, noninfected ticks were allowed to feed on an R. rickettsii-infected rabbit. Ticks (infected or not with R. rickettsii) that remained unfed were used as a control.ResultsCaspase-3 activity and the externalization of phosphatidylserine were significantly higher in IBU/ASE-16 cells treated with dsIAP than in those treated with dsGFP. The mortality rates of ticks in the dsIAP group were much higher than those in the dsGFP group when they were allowed to feed on rabbits, independent of the presence of R. rickettsii. Conversely, lower mortality rates were recorded in unfed ticks.ConclusionsOur results show that IAP negatively regulates apoptosis in A. sculptum cells. Moreover, IAP-silenced ticks experienced higher mortality rates following the acquisition of a blood meal, suggesting that feeding may trigger the activation of apoptosis in the absence of this physiological regulator. These findings indicate that IAP is a potential antigen for an anti-tick vaccine.Graphical

Effect of liquiritigenin on chloroquine accumulation in digestive vacuole leading to apoptosis-like death of chloroquine-resistant P. falciparum

BackgroundMalaria remains one of the major health concerns, especially in tropical countries. Although drugs such as artemisinin-based combinations are efficient for treating Plasmodium falciparum, the growing threat from multi-drug resistance has become a major challenge. Thus, there is a constant need to identify and validate new combinations to sustain current disease control strategies to overcome the challenge of drug resistance in the malaria parasites. To meet this demand, liquiritigenin (LTG) has been found to positively interact in combination with the existing clinically used drug chloroquine (CQ), which has become unfunctional due to acquired drug resistance. PurposeTo evaluate the best interaction between LTG and CQ against CQ- resistant strain of P. falciparum. Furthermore, the in vivo antimalarial efficacy and possible mechanism of action of the best combination was also assessed. MethodsThe in vitro anti-plasmodial potential of LTG against CQ- resistant strain K1 of P. falciparum was tested using Giemsa staining method. The behaviour of the combinations was evaluated using the fix ratio method and evaluated the interaction of LTG and CQ by calculating the fractional inhibitory concentration index (FICI). Oral toxicity study was carried out in a mice model. In vivo antimalarial efficacy of LTG alone and in combination with CQ was evaluated using a four-day suppression test in a mouse model. The effect of LTG on CQ accumulation was measured using HPLC and the rate of alkalinization of the digestive vacuole. Cytosolic Ca2+ level, mitochondrial membrane potential, caspase-like activity, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and Annexin V Apoptosis assay to assess anti-plasmodial potential. Proteomics analysis was evaluated by LC-MS/MS analysis. ResultsLTG possesses anti-plasmodial activity on its own and it showed to be an adjuvant of CQ. In in vitro studies, LTG showed synergy with CQ only in the ratio (CQ: LTG-1:4) against CQ-resistant strain (K1) of P. falciparum. Interestingly, in vivo studies, LTG in combination with CQ showed higher chemo-suppression and enhanced mean survival time at much lower concentrations compared to individual doses of LTG and CQ against CQ- resistant strain (N67) of Plasmodium yoelli nigeriensis. LTG was found to increase the CQ accumulation into digestive vacuole, reducing the rate of alkalinization, in turn increasing cytosolic Ca2+ level, loss of mitochondrial potential, caspase-3 activity, DNA damage and externalization of phosphatidylserine of the membrane (in vitro). These observations indicate the involvement of apoptosis-like death of P. falciparum that might be due to the accumulation of CQ. ConclusionLTG showed synergy with CQ in the ratio LTG: CQ, 4:1) in vitro and was able to curtail the IC50 of CQ and LTG. Interestingly, in vivo in combination with CQ, LTG showed higher chemo-suppression as well as enhanced mean survival time at a much lower concentrations of both the partners as compared to an individual dose of CQ and LTG. Thus, synergistic drug combination offers the possibility to enhance CQ efficacy in chemotherapy.

Tandem mass tag-based proteomics technology provides insights into multi-targeted mechanism of peptide MOp2 from Moringa oleifera seeds against Staphylococcus aureus

Our previous study proved that the peptide MOp2 from Moringa oleifera seeds exhibited membrane-damaging effects on Staphylococcus aureus. In this study, TMT-based proteomics technology was mainly used to investigate the antimicrobial mechanism of MOp2 against S. aureus. A total of 541 differentially expressed proteins (DEPs) were identified in S. aureus treated with MOp2, among which 256 and 285 DEPs were upregulated and downregulated, respectively. They were mainly involved in inositol phosphate metabolism, S. aureus infection, citrate cycle (TCA cycle), and phosphotransferase system (PTS) and acted as ABC transporters and ribosomes. Moreover, the decreasing AKP activity indicated that MOp2 affected cell wall biosynthesis, and excessive ROS accumulation caused apoptosis, resulting in DNA fragmentation, nuclear morphological changes, and phosphatidylserine externalization. Additionally, molecular docking showed that MOp2 could interact with eight DEPs, including ProC, QoxB, SOD2, DnaK, GroEL, RplY, AcpS, and FabG, indicating that MOp2 might act on these molecular targets, leading to increased cell permeability, oxidative damage, impaired protein synthesis, cell wall synthesis obstruction, and energy metabolism disorder. Overall, these findings provided new insights into the multi-targeted mechanism of MOp2 against S. aureus, and could provide a theoretical basis for its application as a novel food preservative and antibiotic substitute.

The Prognostic Value of Eryptosis Parameters in the Cerebrospinal Fluid for Cerebral Vasospasm and Delayed Cerebral Ischemia Formation

Delayed cerebral ischemia (DCI) and cerebral vasospasm (VS.) contribute to poor outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH). The pathophysiology of DCI is not fully understood, and this has hindered the adoption of a uniform definition. Reliable diagnostic tests and effective evidence-based treatment are lacking. This study explored the possibility of using eryptosis parameters in the cerebrospinal fluid (CSF) as a marker for early detection of VS and DCI. Twenty-one SAH patients were recruited and treated at Kharkiv Regional Hospital. The occurrences of DCI and VS were also recorded. Flow cytometry was used to assess eryptosis indices in the CSF by analyzing phosphatidylserine externalization in erythrocytes using annexin V staining and evaluating reactive oxygen species generation using 2,7-dichlorodihydrofluorescein (DCF) diacetate staining. The percentage of annexin-positive red blood cells (RBCs) in the VS group was significantly higher than that in the non-VS group (P=0.0017). Furthermore, higher values of this index were significantly associated with DCI formation (P<0.0001). Patients with VS had higher mean fluorescence intensity values of DCF in RBCs compared to patients without VS (P=0.0258). Patients with DCI also had higher mean fluorescence intensity values of DCF in RBCs (P=0.0282). A higher percentage of annexin-positive RBCs following 3days of aSAH was correlated with poor 3-month neurological outcomes (r=0.7). Our findings indicate a strong correlation between eryptosis level and DCI in a sizable series of patients with aSAH. Correlations between eryptosis indicators in the CSF and clinical and radiological manifestations suggest that eryptosis parameters are promising diagnostic biomarkers for DCI.

Dysregulation of ADAM10 shedding activity in naked mole-rat fibroblasts is due to deficient phosphatidylserine externalization.

The naked mole-rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age-associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblastsionomycin induced ADAM10-dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+ -dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10-dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

Therapeutic potential of Indian medicinal plants against Leishmania donovani: a review

A prevalent medical condition in the tropical and sub-tropical regions of the world, Leishmaniasis; due to the unavailability of effective drugs, caters the need for its eradication and therapeutic control. Modern-day pentavalent antimonials such as Sodium Stibogluconate, Miltefosine used for disease treatment are costly and have low efficacy, apart from triggering severe side effects after prolonged treatment. Plants offer active natural components that have miraculous medicinal properties such as antimicrobial, antimalarial, and even antileishmanial. In this review, research articles on Indian medicinal plants effective against visceral leishmaniasis causal organism, Leishmania donovani, from several renowned domains from 1900s to 20th century were thoroughly studied and the results discussed. Among the 16 plant families from over 50 articles, Piperaceae has been found to treasure plant species with potent leishmanicidal chemical constituents, followed by Meliaceae. The review highlights the plant derived biologically active extracts, essential oils, and isolated compounds effective against the parasite both in vitro and in vivo. While Plumbago zeylanica was found to be the most effective in vitro, momordicatin from Momordica charantia lead to 100% parasite clearance at 10 mg/kg body weight dosage in infected hamsters. The course of study revealed that apart from directly inhibiting parasite growth, plant extracts also led to phosphatidylserine externalization, DNA fragmentation, cell cycle arrest and a significant increase in ROS and pro- inflammatory cytokines expression as well. This article can be further utilized for the selection of naturally derived drugs for clinical trials and their successful use as effective anti-leishmanial medicines.

Acute generation of reactive oxygen species that induced by doxycycline pretreatment results in rapid cell death in polyphyllin G-treated osteosarcoma cell lines.

Polyphyllin G, a pennogenyl saponin extracted from Paris polyphylla, has been shown to possess antitumor effects. In this study, we demonstrated that doxycycline, an antibiotic medicine, could significantly enhance the sensitivities of osteosarcoma cell lines to polyphyllin G. As the cells were pretreated with doxycycline at non-toxic concentrations and then co-exposed to polyphyllin G, this combination could induce a rapid cell death distinct from apoptosis. The non-apoptotic cell death was characterized by a loss of integrity of plasma membrane without externalization of phosphatidyl serine. Furthermore, this combined treatment resulted in suppression of cell viability and colony-forming ability, and increased the level of γ-H2A.X, a critical marker for DNA damage, in osteosarcoma cell lines. When examining the underlying mechanism, it was revealed combination of polyphyllin G and doxycycline triggered an enhanced generation of reactive oxygen species (ROS), and up-regulated mitochondrial oxidative stress within 0.5h. Co-administration of the ROS inhibitor NAC reversed the suppressed cell viability and colony-forming ability, and abolished the increased level of γ-H2A.X in the cells with the combined treatment, indicating that the enhanced ROS was involved in the anti-proliferative effect of the combined treatment. Overall, the results demonstrated that doxycycline may function as chemosensitizers by inducing an acute and lethal ROS production to enhance cytotoxic of polyphyllin G in osteosarcoma cell lines, and the combined use of drugs may provide an alternative thinking for the development of new therapeutic agents.

Regulation of membrane homeostasis by TMC1 mechanoelectrical transduction channels.

The mechanoelectrical transduction (MET) channel in auditory hair cells converts sound into electrical signals, enabling hearing. Transmembrane-like channel 1 and 2 (TMC1 and TMC2) are implicated in forming the pore of the MET channel. Here, we demonstrate that inhibition of MET channels, breakage of the tip links required for MET, or buffering of intracellular Ca... induces pronounced phosphatidylserine externalization, membrane blebbing, and ectosome release at the hair cell sensory organelle, culminating in the loss of TMC1. Membrane homeostasis triggered by MET channel inhibition requires Tmc1 but not Tmc2, and three deafness-causing mutations in Tmc1 cause constitutive phosphatidylserine externalization that correlates with deafness phenotype. Our results suggest that, in addition to forming the pore of the MET channel, TMC1 is a critical regulator of membrane homeostasis in hair cells, and that Tmc1-related hearing loss may involve alterations in membrane homeostasis.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry

Cryopreservation of platelets, at − 80 °C with 5–6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1−/CD42b+/CD62P+) and a novel population (AnnV+/PAC1−/CD42b+/CD62P−) that did not align with the phenotype of aggregatory (AnnV−/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1−/CD42b−/CD62P−) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

A Mixture of Dietary Plant Sterols at Nutritional Relevant Serum Concentration Inhibits Extrinsic Pathway of Eryptosis Induced by Cigarette Smoke Extract.

Cell death program of red blood cells (RBCs), called eryptosis, is characterized by activation of caspases and scrambling of membrane phospholipids with externalization of phosphatidylserine (PS). Excessive eryptosis confers a procoagulant phenotype and is implicated in impairment of microcirculation and increased prothrombotic risk. It has recently been reported that cigarette smokers have high levels of circulating eryptotic erythrocytes, and a possible contribution of eryptosis to the vaso-occlusive complications associated to cigarette smoke has been postulated. In this study, we demonstrate how a mixture of plant sterols (MPtS) consisting of β-sitosterol, campesterol and stigmasterol, at serum concentration reached after ingestion of a drink enriched with plant sterols, inhibits eryptosis induced by cigarette smoke extract (CSE). Isolated RBCs were exposed for 4 h to CSE (10-20% v/v). When RBCs were co-treated with CSE in the presence of 22 µM MPtS, a significant reduction of the measured hallmarks of apoptotic death like assembly of the death-inducing signaling complex (DISC), PS outsourced, ceramide production, cleaved forms of caspase 8/caspase 3, and phosphorylated p38 MAPK, was evident. The new beneficial properties of plant sterols on CSE-induced eryptosis presented in this work open new perspectives to prevent the negative physio-pathological events caused by the eryptotic red blood cells circulating in smokers.

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity.

Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.