Cell Line MEG-01 Research Articles

Compound K Promotes Megakaryocytic Differentiation by NLRP3 Inflammasome Activation.

Platelets are essential blood components that maintain hemostasis, prevent excessive bleeding, and facilitate wound healing. Reduced platelet counts are implicated in various diseases, including leukemia, hepatitis, cancer, and Alzheimer's disease. Enhancing megakaryocytic differentiation is a promising strategy to increase platelet production. Compound K (CK), a major bioactive metabolite of ginsenosides from Panax ginseng, has demonstrated anti-cancer and neuroprotective properties. In this study, we investigated the effects of CK on megakaryocytic differentiation and apoptosis in chronic myeloid leukemia (CML) cell lines K562 and Meg-01. CK treatment significantly upregulated the mRNA expression of key megakaryocytic differentiation markers, including CD61, CD41, and CD42a, and promoted the formation of large, multinucleated cells in K562 cells. Additionally, flow cytometry analysis revealed that CK at 5 µM induced apoptosis, a critical process in thrombocytopoiesis, in both K562 and Meg-01 cells. RT2 Profiler PCR array analysis further identified a marked increase in the expression of genes associated with the activation of the NLRP3 inflammasome in CK-treated K562 and Meg-01 cells. This study is the first to demonstrate that CK promotes megakaryocytic differentiation and apoptosis through the activation of the ERK/EGR1 and NLRP3 inflammasome pathways. These findings suggest that CK may enhance platelet production, indicating its potential as a therapeutic candidate for platelet-related disorders and other associated diseases.

Elevated levels of pro-thrombotic eNOS-negative platelets in COVID-19 patients

BackgroundPlatelet-rich microvascular thrombi are common in severe COVID-19. Endogenous nitric oxide (NO)-signaling limits thrombus formation and previously we identified platelet subpopulations with a differential ability to produce NO based on the presence or absence of endothelial nitric oxide synthase (eNOS). eNOS expression is counter-regulated by cytokines, and COVID-19-associated immune/inflammatory responses may affect the transcriptome profile of megakaryocytes and their platelet progeny. ObjectivesWe investigated whether the percentage of eNOS-negative to eNOS-positive platelets increases in COVID-19 patients and whether this change may be due to the actions of pro-inflammatory cytokines on megakaryocytes. MethodsPlatelets were isolated from hospitalized COVID-19 patients and COVID-19-negative controls. Platelet eNOS was measured by flow cytometry and plasma inflammatory cytokines by ELISA. Megakaryocytes from eNOS-GFP transgenic mice and the Meg-01 cell line were characterized to identify an appropriate model to study eNOS-based platelet subpopulation formation in response to inflammatory cytokines. ResultsCOVID-19 patients demonstrated a significant increase in eNOS-negative and a concomitant decrease in eNOS-positive platelets compared to controls, and this change was associated with disease severity as assessed by ICU admission. A higher eNOS-negative to –positive platelet percentage was associated with enhanced platelet activation as measured by surface CD62P. Accordingly, COVID-19 patients demonstrated higher TNF-α, IL-6, and IL-1β plasma concentrations than controls. Inflammatory cytokines associated with COVID-19 promoted eNOS-negative Meg-01 formation and enhanced subsequent eNOS-negative platelet-like particle formation. ConclusionsCOVID-19 patients have a higher percentage of eNOS-negative to –positive platelets, likely as a result of inflammatory response reducing megakaryocyte eNOS expression, which predisposes to thrombosis.

HIV-1 inhibits IFITM3 expression to promote the infection of megakaryocytes.

Despite an undetectable plasma viral load as a result of antiretroviral therapy, HIV-1-infected individuals with poor immune reconstitution harbor infectious HIV-1 within their platelets. Megakaryocytes, as platelet precursors, are the likely cellular origin of these HIV-1-containing platelets. To investigate the mechanisms that allow megakaryocytes to support HIV-1 infection, we established in vitro models of viral infection using hematopoietic stem cell-derived megakaryocytes and the megakaryocytic MEG-01 cell line. We observed HIV-1 DNA provirus integration into the megakaryocyte cell genome, self-limiting virus production, and HIV-1 protein and RNA compartmentalization, which are hallmarks of HIV-1 infection in myeloid cells. In addition, following HIV-1 infection of megakaryocyte precursors, the expression of interferon-induced transmembrane protein 3 (IFITM3), an antiviral factor constitutively expressed in megakaryocytes, was inhibited in terminally differentiated HIV-1-infected megakaryocytes. IFITM3 knockdown in MEG-01 cells prior to infection led to enhanced HIV-1 infection, indicating that IFITM3 acts as an HIV-1 restriction factor in megakaryocytes. Together, these findings indicate that megakaryocyte precursors are susceptible to HIV-1 infection, leading to terminally differentiated megakaryocytes harboring virus in a process regulated by IFITM3. Megakaryocytes may thus constitute a neglected HIV-1 reservoir that warrants further study in order to develop improved antiretroviral therapies and to facilitate HIV-1 eradication.

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

Gestodene, a novel positive allosteric modulator of PAR1, enhances PAR1-mediated human platelet aggregation.

Background: Protease-activated receptor 1 (PAR1) is expressed in human platelets and can be activated by low concentrations of thrombin. Vorapaxar, a selective antagonist of PAR1, inhibits thrombin-induced calcium mobilization in human platelet, which is associated with an increased risk of bleeding. Conversely, the administration of a positive allosteric modulator (PAM) of PAR1 may pose a substantial risk of thrombosis due to inducing excessive platelet activation. In this study, we discovered a novel PAM of PAR1 and investigated the effect of enhanced PAR1 activation by PAM of PAR1 on platelet activation. Methods: To find PAMs of PAR1, a cell-based screen was performed in HT29 cells, and finally, gestodene, an oral contraceptive drug (OC), was identified as a novel PAM of PAR1. The mechanism of action of gestodene and its effects on platelet activation were investigated in human megakaryocytic leukemia cell line MEG-01 cells and human platelet. Results: Gestodene enhanced both thrombin- and PAR1-activating peptide (AP)-induced intracellular calcium levels in a dose-dependent manner without altering PAR2 and PAR4 activity. Gestodene significantly increased PAR1-AP-induced internalization of PAR1 and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP induced morphological changes in MEG-01 cells. Remarkably, in human blood, gestodene exerted a robust augmentation of PAR1-AP-induced platelet aggregation, and vorapaxar effectively attenuated the gestodene-induced enhancement of platelet aggregation mediated by PAR1. Conclusion: Gestodene is a selective PAM of PAR1 and suggest one possible mechanism for the increased risk of venous thromboembolism associated with OCs containing gestodene.

TRPM8 Contributes to Early Temperature-Induced Platelet Activation

Background: Like sensory neurons, platelets are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Platelet activation by temperature could contribute to numerous clinical observations, most importantly to the well-known reduced lifespan of ex vivo-stored platelets for transfusion. In this study, we sought to dissect the molecular basis of the cold-induced platelet activation response and specifically, evaluate the role of the thermosensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in platelets and their precursor cells. Methods: We tested for the presence of TRPM8 on the megakaryocytic cell line MEG-01 and human platelets by immunoblotting, confocal microscopy, and imaging flow cytometry. The effect of TRPM8 on platelets was evaluated using the commercially available TRPM8 inhibitor PF-05105679. In addition, we used TRPM8 agonists such as menthol, WS-12, and icilin. Platelet shape change and αIIbβ3 integrin activation were assessed by imaging flow cytometry and conventional flow cytometry. We tested for platelet aggregation using light transmission aggregometry and for intracellular calcium by reading fluorescence on a plate reader after loading platelets with Fura-2-AM. Calcium release in response to temperature decrease was assessed after loading platelets or TRPM8-transfected HEK293T/17 and reading fluorescence in a Real-Time PCR Detection System. Results: We detected TRPM8 mRNA and protein in MEG-01 cells and platelets but there was a remarkable variability among donors. Further evidence for TRPM8's presence in platelets came from the fact that inhibitors of TRPM8 prevented early temperature-induced platelet activation events, such as αIIbβ3 integrin activation and shape change. Unlike temperature changes, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. Platelets exposed to below-normal body temperature displayed a cytosolic calcium increase, but this was independent of TRPM8 and entirely dependent on the calcium release from the endoplasmic reticulum. Conclusion: We found evidence for TRPM8 expression in platelets but the expression varies greatly between individuals and is of unclear physiologic significance. Our study suggests that the cold response of platelets is complex, and TRPM8 appears to play a calcium-independent role in the early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies.

Unexpected Noremestrin with a Sulfur-Bearing 15-Membered Macrocyclic Lactone from Emericella sp. 1454.

Two previous unreported epipolythiodioxopiperazines of the emestrin family, namely, noremestrin A (1) and secoemestrin E (2), were successfully isolated from the fungal source Emericella sp. 1454. Employing comprehensive spectroscopic techniques, such as high-resolution electrospray ionization mass spectrometry, infrared, and nuclear magnetic resonance (NMR), along with NMR and electronic circular dichroism calculations, the chemical structures of compounds 1 and 2 were elucidated. Particularly noteworthy is the distinctive nature of noremestrin A, representing the inaugural instance of a noremestrin variant incorporating a sulfur-bearing 15-membered macrocyclic lactone moiety. Compounds 1 and 2 exhibited weak cytotoxic activities against the human chronic myelocytic leukemia cell lines MEG-01 and K562.

Inflammation Mediated By TNFα Disrupts the Autophagic Flux and Oxidative Phosphorylation of Megakaryocytes and Platelets through the Downregulation of STX17

Background. JAK2 V617F-positive polycythemia vera (PV) is the most common Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Elevated levels of TNFα, and a significantly higher risk of thrombosis and bleeding (thrombohemorrhage) are hallmarks of JAK2 V617F PV. Platelets from patients with PV are characterized by hyperreactivity (fast activation of the αIIbβ3 integrin), higher P-selectin exposure (thromboinflammation), and phosphatidylserine (procoagulant potential). We have also found that in addition to these phenotypes, platelets from PV patients have dysfunctional autophagy promoting mitochondrial dysfunction, accumulation of dysmorphic mitochondria, and decreased clot retraction. Therefore, we hypothesized that inflammation mediated by TNFα (like in some cancers) dysregulates autophagy in JAK2 V617F PV by reprogramming (immunometabolism) metabolism of MKs and platelets. Methods. Platelets from sex and age-matched healthy subjects and patients with PV were analyzed. Platelets were treated with chloroquine (CQ) to analyze the autophagic flux by western blot and electron microscopy. Platelet bioenergetics was analyzed using the Seahorse extracellular flux analyzer. Semi-quantitative metabolomics was performed using the Vanquish UHPLC system. Thrombus formation was measured by microfluidics using T-TAS and ATLAS equipment. Clot retraction was assessed by measuring the weight of serum extruded from the clot. Platelets for clot retraction were treated with CQ or EACC. The MEG-01 cell line was treated with TNFα to analyze its impact on the autophagic flux, mitochondrial metabolism, and transcription (RNAseq). Murine studies using platelets from wild type (WT), DARE (transgenic mice with constitutive expression of TNFα or a neutralizing antibody against TNFα were performed. Results. Platelets from patients with PV have significantly impaired autophagic flux (p<0.01), mitochondrial dysfunction (p<0.002), and accumulation of dysmorphic mitochondria (p<0.007). Platelets from PV patients have significantly lower levels of STX17 (p<0.008), a key regulatory molecule of autophagosome and lysosome fusion (Figure 1). The inhibition of STX17 from healthy donor platelets decreased clot stability (p<0.004). Moreover, platelets from chronically (DARE) and acutely inflamed mice (WT mice treated with TNFα) exhibited the same autophagic defect (p<0.03 and 0.02, respectively) secondary to the downregulation of STX17 (p<0.01 and 0.02, respectively). In vitro experiments showed that megakaryocytes downregulate the transcription and translation STX17 (p<0.006 and p<0.03, respectively) in response to TNFα. Finally, in vivo treatment of inflamed mice with a neutralizing anti-TNFa antibody restored the expression of STX17 (p<0.0001) and the autophagic flux (p<0.002). Conclusion. Using several in vitro and in vivo approaches, we demonstrated for the first time the immunometabolic effect of TNFα in platelets and megakaryocytes. TNFα is a key regulator of the autophagic flux in platelets inducing platelet hyperreactivity and decreasing clot stability. Our findings may explain why patients with JAK2 V617F PV are at increased risk for both thrombo-hemorrhagic events and suggest that targeting autophagy might lower this risk.

Multiomic Characterization of Myelodysplastic Neoplasms (MDS) with Micromegakaryocytes Highlights the Role of EZH2-RUNX1 deregulation in Disease Physiopathology and Response to Targeted Therapies

Micromegakaryocytes (micro-MKs), an unequivocal myeloid dysplastic feature in adults, are present in 15-20% of myelodysplastic neoplasms (MDS). We recently associated the detection of these small megakaryocytes with a mono- or bi-lobated nucleus in bone marrow (BM) smears of MDS patients with an unfavorable prognosis. The aim of the present study was to elucidate the molecular bases of micro-MK formation and its impact on MDS response to targeted therapies. We selected retrospectively a cohort of 40 MDS patient samples subjected to NGS myeloid panel and RNA sequencing and carried out a morphological re-evaluation that included micro-MKs recounting. Cases with isolated del(5q) or SF3B1 mutation were excluded. MDS cases were selected according to the following morphological findings: no dismegakaryopoiesis, dismegakaryopoiesis without micro-MK, low rate of micro-MK (<10%; micro-MK low) and high rate of micro-MK (>40%; micro-MK high). No patient harbored an intermediate (10-40%) rate of micro-MK. A comparative characterization of 8 representative patients was carried out subjecting diagnosis BM samples to Tapestri and Chromium single cell proteogenomic and transcriptomic platforms, gene set enrichment analysis, and deep cytogenetic characterization by optical genome mapping. Functional validation studies were performed using the megakaryoblastic cell line MEG01 differentiated to a mature MK phenotype by exposure to phorbol myristate acetate (PMA), as well as using the CRISPR-Cas9 tool for gene editing. Finally, activity of investigational drugs including 5-azacytidine, lenalidomide, venetoclax, rigosertib and pevonedistat, was evaluated in MEG01-activated cells and in vivo using a chicken embryo chorioallantoic membrane (CAM) model of MDS patient-derived xenograft (PDX). The most frequently mutated genes across all cohorts were ASXL1 (58%), SRSF2 (38%), RUNX1 (30%), TET2 (30%), STAG2 (20%), EZH2 (18%), DDX41 (10%), U2AF1 (10%), and ZRSR2 (10%) . Interestingly, EZH2 and RUNX1 mutations were enriched in the micro-MK high group ( EZH2: 0%, 10%, 14.3% vs. 66.7%, p 0.006; RUNX1: 0%, 20%, 35.7% vs. 83.3%, p 0.003), and both mutations co-occurred in 85.7% (6/7) of the EZH2 mutated cases. Single cell analyses revealed an enrichment of RUNX1 alterations in three populations within micro-MK high BM samples, including hematopoietic stem and progenitor cells, non-classical monocytes and immature erythroid cells. These samples were also characterized by a 5-fold increase in MK progenitor abundance and by the upregulation of a set of 10 EZH2-repressed genes in neutrophil-like cells and myeloid progenitors, with 7q loss presumably affecting the EZH2 locus in 50% of the cases with micro-MK. Using the in vitro MK differentiation model, we further demonstrated that EZH2 downregulation hampered the acquisition of MK surface markers CD41 and CD61, and that venetoclax was the sole agent retaining notable cytotoxic activity in differentiated cells when compared to undifferentiated megakaryoblasts. Accordingly, in a first-in-kind in vivo CAM-MDS transplant model, venetoclax was able to impair BM infiltration by human cells in a PDX model of micro-MK+ MDS. In summary, supporting the notion that EZH2 and RUNX1 exert a crucial role in the regulation of megakaryopoiesis, our present findings suggest that deregulation of EZH2-RUNX1 activity by loss-of-function mutation and/or genetic deletion affecting myeloid precursors, impairs a proper megakaryocytic differentiation process in MDS. Thus, this phenomenon may be considered as a characteristic feature of MDS cases with an elevated proportion of micro-MK. Of note, preliminary in vitro and in vivo results support the use of venetoclax for the treatment of this subgroup of patients with dismal outcome.

SARS-CoV-2 Lysate Stimulation Impairs the Release of Platelet-like Particles and Megakaryopoiesis in the MEG-01 Cell Line.

SARS-CoV-2 infection causes a considerable inflammatory response coupled with impaired platelet reactivity, which can lead to platelet disorders recognized as negative prognostic factors in COVID-19 patients. The virus may cause thrombocytopenia or thrombocytosis during the different disease stages by destroying or activating platelets and influencing platelet production. While it is known that several viruses can impair megakaryopoiesis by generating an improper production and activation of platelets, the potential involvement of SARS-CoV-2 in affecting megakaryopoiesis is poorly understood. To this purpose, we explored, in vitro, the impact of SARS-CoV-2 stimulation in the MEG-01 cell line, a human megakaryoblastic leukemia cell line, considering its spontaneous capacity of releasing platelet-like particles (PLPs). We interrogated the effect of heat-inactivated SARS-CoV-2 lysate in the release of PLPs and activation from MEG-01, the signaling pathway influenced by SARS-CoV-2, and the functional effect on macrophagic skewing. The results highlight the potential influence of SARS-CoV-2 in the early stages of megakaryopoiesis by enhancing the production and activation of platelets, very likely due to the impairment of STATs signaling and AMPK activity. Overall, these findings provide new insight into the role of SARS-CoV-2 in affecting megakaryocyte-platelet compartment, possibly unlocking another avenue by which SARS-CoV-2 moves.

AMPK inhibition induces MCL1 mRNA destabilization via the p38 MAPK/miR-22/HuR axis in chronic myeloid leukemia cells

The oncogenic and tumor-suppressive roles of AMPK in chronic myeloid leukemia (CML) are controvertible. This study aimed to investigate the cytotoxic effects of the AMPK inhibitor Compound C in the CML cell lines K562, KU812, and MEG-01. Compared to K562 cells, KU812 and MEG-01 cells were more sensitive to Compound C-mediated cytotoxicity. Moreover, Compound C induced SIRT3 upregulation in K562 cells but not in KU812 or MEG-01 cells. SIRT3 silencing increased the sensitivity of K562 cells to Compound C. Additionally; Compound C-induced autophagy attenuated its induced apoptosis in KU812 and MEG-01 cells. Compound C-induced ROS-mediated AMPKα inactivation resulted in the downregulation of apoptotic regulator MCL1 in KU812 and MEG-01 cells. Mechanistically, AMPK inhibition activated p38 MAPK-mediated miR-22 expression, which in turn inhibited HuR expression, thereby reducing MCL1 mRNA stability. Overexpression of constitutively active AMPKα1 and abolishment of the activation of p38 MAPK inhibited Compound C-induced cell death and MCL1 downregulation. Furthermore, Compound C synergistically enhanced the cytotoxicity of BCR-ABL inhibitors and the BCL2 inhibitor ABT-199. Collectively, this study indicates that Compound C induces MCL1 downregulation through the AMPK/p38 MAPK/miR-22/HuR pathway, thereby inducing apoptosis of KU812 and MEG-01 cells. Furthermore, our findings suggest that AMPK inhibition is a promising strategy for improving CML therapy.

Dvl proteins regulate SMAD1, AHR, mTOR, BRD7 protein expression while differentially regulating canonical and non-canonical Wnt signaling pathways in CML cell lines

Dishevelled (Dvl) is a scaffold protein that transmits Wnt signals to downstream effector molecules via both canonical and non-canonical Wnt signaling pathways. Deregulated activation of Dvl proteins has been reported in various solid tumors. However, it is not clear which pathway and proteins are responsible for observed aberrant activities and their relevance in disease prognosis. In addition, there is relatively limited knowledge on the role Dvl proteins may have in hematologic malignancy etiopathogenesis. In this study, we demonstrated that Dvl genes are not expressed in normal bone marrow but are expressed at different levels in the bone marrow of patients with chronic myeloid leukemia. We showed SMAD1, AHR, mTOR, BRD7 protein expressions are significantly affected by Dvl silencing and overexpression in CML cell lines. Wnt/β-catenin and Wnt/PCP signaling pathway components are effectively repressed after Dvl silencing in K562 cells, while regulator of Wnt/Ca2+ signaling showed increase in both CML cell lines. Targeting Dvl proteins increases imatinib susceptibility of the K562 and MEG-01 cell lines. In light of our data, Dvl could be a potential therapeutic target in the treatment of CML.

Dengue and Zika virus differential infection of human megakaryoblast MEG-01 reveals unique cellular markers

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48–72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%–25%) and 5% (3%–8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-β, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections.

Simple Isolation of Cordycepin from Cordyceps militaris by Dual-Normal Phase Column Chromatography and Its Potential for Making Kombucha Functional Products

Cordycepin (3′-deoxyadenosine) is a potent bioactive metabolite of the medicinal fungus Cordyceps militaris, which has been increasingly exploited to treat various chronic diseases in humans. However, the current synthesis and purification procedures of cordycepin are principally laborious and complicated. This study provides a simple protocol approach to isolate and purify cordycepin from C. militaris by normal phase column chromatography at room temperature. Besides, this is the first to investigate the potential of cordycepin and cordycepin-included extracts from C. militaris for making Kombucha functional products. By a repeated column chromatography, an amount of 1.16 g of cordycepin is isolated from 2.8 kg of fruiting bodies of C. militaris, which obtained an efficiency of 83.26% compared to that estimated by high-performance liquid chromatography (HPLC). The purity of cordycepin is confirmed by thin-layer chromatography (TLC), HPLC, and proton nuclear magnetic resonance (1H NMR). In addition, kombucha-fermented extracts from cordycepin and cordycepin-included fractions show potential biological activities in terms of antioxidant, anti-diabetes via α-glucosidase inhibitory assay, and cytotoxicity via MTT assay on Meg-01 and HL-60 cell lines. Further studies on optimization of extraction protocol and verification of health benefits of kombucha products from cordycepin should be conducted prior to the official mass production.

Prenylemestrins A and B: Two Unexpected Epipolythiodioxopiperazines with a Thioethanothio Bridge from Emericella sp. Isolated by Genomic Analysis.

Prenylemestrins A and B (1 and 2, respectively), two unusual epipolythiodioxopiperazines featuring a thioethanothio bridge instead of a polysulfide bridge, were isolated from the fungus Emericella sp. CPCC 400858 guided by genomic analysis. Their structures were determined by extensive spectroscopic data, NMR and ECD calculations, and X-ray diffraction analysis. A plausible biosynthetic pathway for 1 and 2 was proposed on the basis of gene cluster analysis. Prenylemestrins A and B exhibited cytotoxicities against human chronic myelocytic leukemia cell lines K562 and MEG-01.

Apoptosis of Megakaryocytic Leukemia Cell Line Meg-01 Induced by TSP-1 Via CD36/Caspase-3

To investigate the effect of thrombospondin-1 (TSP-1) on apoptosis of human megakaryocytic leukemia cell line Meg-01 and its possible mechanism. The expression of CD36 antigen in Meg-01 cells was detected by flow cytometry and immunocytochemistry. Meg-01 cells were cultured for 48 hours with TSP-1 and CD36 antibody FA6-152 at different concentrations. The early apoptosis and activity of caspase-3 were detected by flow cytometry. The effect of TSP-1 on the growth and differentiation of megakaryocytes was investigated by cell counting and CFU-MK culture. The flow cytometry and immunocytochemistry showed that CD36 antigen was expressed on the surface of Meg-01 cells. TSP-1 (5 μg/ml) inhibited the growth of Meg-01 cells, but had unobvious effect on M-07e cells. After addition of CD36 antibody FA6-152 (5, 10, and 25 μg/ml), the inhibition effect of TSP-1 was significantly reduced. TSP-1 (2.5, 5, and 7.5 μg/ml) increased the positive expression of Annexin V (P<0.01) and caspase-3 activity (P<0.01), which indicated that TSP-1 had a significant effect on inducing apoptosis. After addition of CD36 antibody FA6-152 (25 μg/ml), the apoptosis induced by TSP-1 in Meg-01 cells was significantly reduced. TSP-1 (5, 10, and 25 μg/ml) could significantly inhibit the formation of CFU-MK in mouse bone marrow cells, while β-TG could not. CD36 antibody FA6-152 (25 μg/ml) could significantly reduce the inhibition of TSP-1 on CFU-MK. TSP-1 may induce apoptosis of megakaryocytic leukemia cell line Meg-01 cells via CD36/caspase-3, which provides a potential new drug development and treatment target for clinical treatment of megakaryocytic leukemia.

PIKfyve-Dependent Phosphoinositide Dynamics in Megakaryocyte/Platelet Granule Integrity and Platelet Functions.

Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P2 and the cation channel TRPML (transient receptor potential mucolipin) 1 and TPC (two pore segment channel) 2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P2 control granule integrity both in megakaryocytes and platelets.

Carboplatin-Induced Thrombocytopenia through JAK2 Downregulation, S-Phase Cell Cycle Arrest and Apoptosis in Megakaryocytes.

Chemotherapy-induced thrombocytopenia (CIT) is a common complication when treating malignancies with cytotoxic agents wherein carboplatin is one of the most typical agents causing CIT. Janus kinase 2 (JAK2) is one of the critical enzymes to megakaryocyte proliferation and differentiation. However, the role of the JAK2 in CIT remains unclear. In this study, we used both carboplatin-induced CIT mice and MEG-01 cell line to examine the expression of JAK2 and signal transducer and activator of transcription 3 (STAT3) pathway. Under CIT, the expression of JAK2 was significantly reduced in vivo and in vitro. More surprisingly, the JAK2/STAT3 pathway remained inactivated even when thrombopoietin (TPO) was administered. On the other hand, carboplatin could cause prominent S phase cell cycle arrest and markedly increased apoptosis in MEG-01 cells. These results showed that the thrombopoiesis might be interfered through the downregulation of JAK2/STAT3 pathway by carboplatin in CIT, and the fact that exogenous TPO supplement cannot reactivate this pathway.

PAR4 Primarily Mediates Thrombin‐induced Morphological Changes in MEG‐01 Cells through PI3K/Akt and RhoA/ROCK Signaling Pathways

Platelet activation is critical in hemostasis and thrombosis. Thrombin most potently activates platelets via two protease‐activated receptors (PARs): PAR1 and PAR4. While PAR1 activation results in a rapid and transient signal required for the initiation phase of platelet aggregation, PAR4 activation induces a sustained signal associated with later phases responsible for stable thrombus formation. The present study focused on the differential signaling pathways by PAR1 and PAR4 involved in thrombin‐induced intracellular events in human megakaryoblastic leukemia cell line, MEG‐01. Interestingly, we found that thrombin triggers not only Gq/PLC‐mediated calcium signaling, but also G12/13/RhoA/ROCK‐mediated morphological changes in MEG‐01 cells via PAR1 and PAR4, which showed close similarity to those observed in platelets. We developed a new image‐based assay to quantify the morphological changes in living cells and extensively investigated on the underlying mechanism for thrombin‐induced morphological changes in MEG‐01 cells. Selective blockade of PAR1 and PAR4 by vorapaxar and BMS‐986120, respectively, indicated that PAR4 activation primarily mediates thrombin‐induced morphological changes in MEG‐01 cells. Moreover, treatment of a set of kinase inhibitors showed that the PAR4‐mediated morphological changes were predominantly mediated via PI3K/Akt and RhoA/ROCK signaling pathways. As in the platelets, PAR4‐mediated signaling pathways are significant in thrombin‐induced morphological changes in MEG‐01 cells. Based on the close similarity to thrombin‐induced signaling in platelets, MEG‐01 cell line is a useful complementary in vitro model that can be used in research on platelets and thrombosis.