Exosomal membrane proteins analysis using a silicon nanowire field effect transistor biosensor.

Exosomes are of great significance in clinical diagnosis, due to their high homology with parental generation, which can reflect the pathophysiological status. However, the quantitative and classification detection of exosomes is still faced with the challenges of low sensitivity and complex operation. In this study, we develop an electrical and label-free method to directly detect exosomes with high sensitivity based on a Silicon nanowire field effect transistor biosensor (Si-NW Bio-FET). First, the impact of Debye length on Si-NW Bio-FET detection was investigated through simulation. The simulation results demonstrated that as the Debye length increased, the electrical response to Si-NW produced by charged particle at a certain distance from the surface of Si-NW was greater. A Si-NW Bio-FET modified with specific antibody CD81 on the nanowire was fabricated then used for detection of cell line-derived exosomes, which achieved a low limit of detection (LOD) of 1078 particles/mL in 0.01 × PBS. Furthermore, the Si-NW Bio-FETs modified with specific antibody CD9, CD81 and CD63 respectively, were employed to distinguish exosomes derived from human promyelocytic leukemia (HL-60) cell line in three different states (control group, lipopolysaccharide (LPS) inflammation group, and LPS + Romidepsin (FK228) drug treatment group), which was consistent with nano-flow cytometry. This study provides a highly sensitive method of directly quantifying exosomes without labeling, indicating its potential as a tool for disease surveillance and medication instruction.

Additive antitumor effect of arsenic trioxide with exposure to ionizing radiation to human acute promyelocytic leukemia HL­60 cells.

Arsenic trioxide (ATO) is expected to be a chemical drug with antitumor activity against acute promyelocytic leukemia (APL), a type of acute myeloid leukemia. In Japan, its antitumor effects were confirmed in clinical trials for APL, and it has been approved in various countries around the world. However, there have been no reports on ATO's antitumor effects on radioresistant leukemia cells, which can be developed during radiotherapy and in combination with therapeutic radiation beams. The present study sought to clarify the antitumor effect of ATO on APL cells with radiation resistance and determine its efficacy when combined with ionizing radiation (IR). The radiation­resistant HL60 (Res­HL60) cell line was generated by subjecting the native cells to 4­Gy irradiation every week for 4 weeks. The half­maximal inhibitory concentration (IC50) for cell proliferation by ATO on native cell was 0.87 µM (R2=0.67), while the IC50 for cell proliferation by ATO on Res­HL60 was 2.24 µM (R2=0.91). IR exposure increased the sub­G1 and G2/M phase ratios in both cell lines. The addition of ATO resulted in a higher population of G2/M after 24 h rather than 48 h. When the rate of change in the sub­G1 phase was examined in greater detail, the sub­G1 phase in both control cells without ATO significantly increased by exposure to IR at 24 h, but only under the condition of 2 Gy irradiation, it had continued to increase at 48 h. Res­HL60 supplemented with ATO showed a higher rate of sub­G1 change at 24 h; however, 2 Gy irradiation resulted in a decrease compared with the control. There was a significant increase in the ratio of the G2/M phase in cells after incubation with ATO for 24 h, and exposure to 2 Gy irradiation caused an even greater increase. To determine whether the inhibition of cell proliferation and cell cycle disruptions is related to reactive oxygen species (ROS) activity, intracellular ROS levels were measured with a flow cytometric assay. Although the ROS levels of Res­HL60 were higher than those of native cells in the absence of irradiation, they did not change after 0.5 or 2 Gy irradiation. Furthermore, adding ATO to Res­HL60 reduced intracellular ROS levels. These findings provide important information that radioresistant leukemia cells respond differently to the antitumor effect of ATO and the combined effect of IR.

Evaluating the combined and individual cytotoxic effect of beauvericin, enniatin B and ochratoxin a on breast cancer cells, leukemia cells, and fresh peripheral blood mononuclear cells.

Beauvericin (BEA), Enniatin B (ENN B), and Ochratoxin A (OTA) are mycotoxins produced by fungi species. Their main effect on several organs and systems is associated with chronic exposure going from immunotoxicity, estrogenic disorders, and renal failure to cancer (in animals and humans). OTA belongs to Group 1 according to the International Agency for Research in Cancer (IARC) and it has legislated limited values; not happening for BEA nor ENN B. Exposure to mixtures of mycotoxins occurs through food intake in daily consumption. The aim of this study was to evaluate the implication of BEA, ENN B, and OTA individually and combined in producing cytotoxicity in cells for immunological studies and cancer cell lines (human leukemia cells (HL-60), fresh human peripheral blood mononuclear cells (PBMCs), and human breast cancer (MDA-MB-231) cells). Cells were treated for 4 h and 24 h at different concentrations of BEA, ENN B, and OTA, respectively. Viability assays were carried out by flow cytometry using DAPI (4',6-diamindino-2-phenylindole, dihydrochloride) as a viability dye and the potential effects of synergism, addition, and antagonism were assessed through the Chou and Talalay method. Individual OTA treatment exerted the greatest cytotoxicity for PBMC cells (IC50 0.5 µM) while ENN B for HL-60 (IC50 0.25 µM) and MDA-MB-231 (IC50 0.15 µM). In binary combination [ENN B + OTA] resulted in exerting the greatest cytotoxicity for HL-60 and MDA-MB-231 cells; while [BEA + OTA] in PBMC cells. The triple combination resulted in being highly cytotoxic for PBMC cells compared to HL-60 and MDA-MB-231 cells. In summary, PBMC cells were the most sensible cells for all three mycotoxins and the presence of OTA in any of the combinations had the greatest toxicity causing synergism as the most common cytotoxic effect.

Molecular and biochemical evaluation of oxidative effects of cord blood CD34+ MPs on hematopoietic cells.

A graft source for allogeneic hematopoietic stem cell transplantation is umbilical cord blood, which contains umbilical cord blood mononuclear cells (MNCs and mesenchymal stem cells, both an excellent source of extracellular microparticles (MPs). MPs act as cell communication mediators, which are implicated in reactive oxygen species formation or detoxification depending on their origin. Oxidative stress plays a crucial role in both the development of cancer and its treatment by triggering apoptotic mechanisms, in which CD34+ cells are implicated. The aim of this work is to investigate the oxidative stress status and the apoptosis of HL-60 and mononuclear cells isolated from umbilical cord blood (UCB) following a 24- and 48-hour exposure to CD34 + microparticles (CD34 + MPs). The activity of superoxide dismutase, glutathione reductase, and glutathione S-transferase, as well as lipid peroxidation in the cells, were employed as oxidative stress markers. A 24- and 48-hour exposure of leukemic and mononuclear cells to CD34 + -MPs resulted in a statistically significant increase in the antioxidant activity and lipid peroxidation in both cells types. Moreover, CD34 + MPs affect the expression of BCL2 and FAS and related proteins and downregulate the hematopoietic differentiation program in both HL-60 and mononuclear cells. Our results indicate that MPs through activation of antioxidant enzymes in both homozygous and nonhomozygous cells might serve as a means for graft optimization and enhancement.

In vitro antiproliferative effects of Vatairea macrocarpa (Benth.) Ducke lectin on human tumor cell lines and in vivo evaluation of its toxicity in Drosophila melanogaster.

Tumor cells may develop alterations in glycosylation patterns during the initial phase of carcinogenesis. These alterations may be important therapeutic targets for lectins with antitumor action. This work aimed to evaluate the in vitro cytotoxicity of VML on tumor and non-tumor cells (concentration of 25 µg/mL and then microdiluted) and evaluate its in vivo toxicity at different concentrations (1.8, 3.5 and 7.0 µg/mL), using Drosophila melanogaster. Toxicity in D. melanogaster evaluated mortality rate, as well as oxidative stress markers (TBARS, iron levels, nitric oxide levels, protein and non-protein thiols). The cytotoxicity assay showed that VML had cytotoxic effect on leukemic lines HL-60 (IC50 = 3.5 µg/mL), KG1 (IC50 = 18.6 µg/mL) and K562 (102.0 µg/mL). In the toxicity assay, VML showed no reduction in survival at concentrations of 3.5 and 7.0 µg/mL and did not alter oxidative stress markers at any concentrations tested. Cytotoxicity of VML from HL-60, KG1 and K562 cells could arise from the interaction between the lectin and specific carbohydrates of tumor cells. In contrast, effective concentrations of VML against no-tumor cells human keratinocyte - HaCat and in the D. melanogaster model did not show toxicity, suggesting that VML is a promising molecule in vivo studies involving leukemic cells.

13C-metabolic flux analysis reveals metabolic rewiring in HL-60 neutrophil-like cells through differentiation and immune stimulation.

Neutrophils are innate immune cells and the first line of defense for the maintenance of homeostasis. However, our knowledge of the metabolic rewiring associated with their differentiation and immune stimulation is limited. Here, quantitative 13C-metabolic flux analysis was performed using HL-60 cells as the neutrophil model. A metabolic model for 13C-metabolic flux analysis of neutrophils was developed based on the accumulation of 13C in intracellular metabolites derived from 13C-labeled extracellular carbon sources and intracellular macromolecules. Aspartate and glutamate in the medium were identified as carbon sources that enter central carbon metabolism. Furthermore, the breakdown of macromolecules, estimated to be fatty acids and nucleic acids, was observed. Based on these results, a modified metabolic model was used for 13C-metabolic flux analysis of undifferentiated, differentiated, and lipopolysaccharide (LPS)-activated HL-60 cells. The glucose uptake rate and glycolytic flux decreased with differentiation, whereas the tricarboxylic acid (TCA) cycle flux remained constant. The addition of LPS to differentiated HL-60 cells activated the glucose uptake rate and pentose phosphate pathway (PPP) flux levels, resulting in an increased rate of total NADPH regeneration, which could be used to generate reactive oxygen species. The flux levels of fatty acid degradation and synthesis were also increased in LPS-activated HL-60 cells. Overall, this study highlights the quantitative metabolic alterations in multiple pathways via the differentiation and activation of HL-60 cells using 13C-metabolic flux analysis.

Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90-100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

Invasive and Non-invasive Clinical Haemophilus influenzae Type A Isolates Activate Differentiated HL-60 Cells In Vitro.

Background: The effective elimination of encapsulated bacteria like Haemophilus influenzae type a (Hia) relies on immune mechanisms such as complement-mediated opsonophagocytosis by neutrophils in coordination with opsonization by anti-capsular antibodies. This study evaluated if Hia could activate the immune response through neutrophils and if these responses differed between encapsulated versus unencapsulated or invasive versus non-invasive strains. Methods: HL-60-derived neutrophil-like cells (dHL-60), differentiated with 1.25% dimethyl sulfoxide over 9 days, were used in an opsonophagocytosis assay and in vitro infection model to measure Hia's susceptibility to killing and dHL-60 surface molecule expression, respectively. The impact of strain-specific features on the immune response was investigated using clinical isolates of a dominant North American sequence type (ST)-23, including Hia 11-139 (encapsulated, invasive), 14-61 (encapsulated, non-invasive), 13-0074 (unencapsulated, invasive), as well as a representative ST-4 isolate (Hia 13-240, encapsulated, invasive), and a nontypeable strain (NTHi 375, unencapsulated, non-invasive). Results: Unencapsulated and non-invasive Hi strains were more susceptible to killing by the innate immune response while the ST-23 invasive strain, Hia 11-139 required serum antibodies for destruction. Flow cytometry analysis showed increased expression of co-stimulatory molecule ICAM-1 and Fc receptors (CD89, CD64) but decreased expression of the Fc receptor CD16, revealing potential mechanisms of neutrophil-mediated defense against Hia that extend to both non-invasive and invasive strains. Conclusions: Hia clinical isolates with diverse pathogenicity illustrated contrasting susceptibility to killing by immune mechanisms while maintaining the same capacity to activate neutrophil-like cells, further underscoring the need for additional studies on Hia's pathogenesis.

Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

Expression and secretion of galectin-12 in the context of neutrophilic differentiation of human promyeloblastic HL-60 cells.

Galectin-12 is a tissue-specific galectin that has been largely defined by its role in the regulation of adipocyte differentiation and lipogenesis. This study aimed to evaluate the role of galectin-12 in the differentiation and polarization of neutrophils within a model of acute myeloid leukemia HL-60 cells. All-trans retinoic acid and dimethyl sulfoxide were used to induce differentiation of HL-60 cells which led to the generation of two phenotypes of neutrophil-like cells with opposite changes in galectin-12 gene (LGALS12) expression and different functional responses to N-formyl- l-methionyl- l-leucyl- l-phenylalanine. These phenotypes showed significant differences of differentially expressed genes on a global scale based on bioinformatics analysis of available Gene Expression Omnibus (GEO) data sets. We also demonstrated that HL-60 cells could secrete and accumulate galectin-12 in cell culture medium under normal growth conditions. This secretion was found to be entirely inhibited upon neutrophilic differentiation and was accompanied by an increase in intracellular lipid droplet content and significant enrichment of 22 lipid gene ontology terms related to lipid metabolism in differentiated cells. These findings suggest that galectin-12 could serve as a marker of neutrophilic plasticity or polarization into different phenotypes and that galectin-12 secretion may be influenced by lipid droplet biogenesis.

Intracellular Accumulation and Secretion of YKL-40 (CHI3L1) in the Course of DMSO-Induced HL-60 Cell Differentiation.

YKL-40 (CHI3L1) is a matrix glycoprotein stored in human neutrophil-specific granules and released upon activation. While it is implicated in inflammation, cancer progression, and cell differentiation, its exact physiological role remains unclear. This study investigated the intracellular expression and secretion of YKL-40 by untreated and DMSO-treated HL-60 cells in association with surface expression of CD11b and CD66b throughout the differentiation process (up to 120 h). Secreted YKL-40 protein and mRNA levels of YKL-40, CD66b, and CD11b were measured by ELISA and quantitative RT-PCR, respectively. The intracellular YKL-40 and surface CD11b and CD66b expression were assessed by flow cytometry. A significant increase in CD11b expression confirmed DMSO-induced differentiation of HL-60 cells. Upon DMSO stimulation, YKL-40 mRNA expression increased in a time-dependent manner, unlike CD66b. The lack of CD66b (a granulocyte maturation and activation marker) on the surface of HL-60 cells might suggest that DMSO treatment did not induce full maturation or activation. The intracellular YKL-40 protein expression was increasing up to 96 h of DMSO treatment and then declined. YKL-40 secretion into the culture medium was detectable only at later time points (96 and 120 h), which was correlated with a decreased proliferation of DMSO-treated HL-60 cells. These findings suggest sequential changes in YKL-40 production and secretion during DMSO-induced differentiation of HL-60 cells and might contribute to a better understanding of YKL-40's involvement in both physiological processes and disease development, including multiple sclerosis.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity.

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 µM for normoxia, and 0.001 µM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 µM and Abl inhibitory activity with IC500.08 µM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 µM, VEGFR-2 IC500.68 µM, B-raf IC500.33 µM, ERK IC501.41 µM, CK1 IC500.29 µM and p38-MAPK IC500.38 µM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Granulocyte pro-myeloperoxidase is redundantly processed by proprotein convertase furin and PC7 in HL-60 cells.

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.

Transcriptomic analysis of lipoteichoic acid­treated undifferentiated and neutrophil­like differentiated HL­60 cells.

Toll-like receptor 2 (TLR2) is an important sensor for innate immune cells, including neutrophils, for the recognition of pathogen infection. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is a TLR2 ligand. LTA-induced TLR2 signaling pathways are well established in neutrophils. However, experimental studies regarding transcriptional regulation and the molecular mechanisms in primary human neutrophils are limited due to their short lifespan. The promyelocytic leukemia cell line, HL-60, can differentiate into a neutrophil-like phenotype following treatment with dimethyl sulfoxide. The aim of the present study was to investigate whether differentiated HL-60 (dHL-60) cells induced a similar gene expression profile upon LTA treatment as that previously determined for primary human neutrophils. After 4 or 24 h of Staphylococcus aureus LTA treatment, undifferentiated HL-60 (uHL-60) and dHL-60 cells were collected for RNA sequencing. The results demonstrated that hundreds of identical differentially expressed genes (DEGs) were observed in 1 and 10 µg/ml LTA-treated dHL-60 cells following 4 and 24 h of incubation, while almost no DEGs between LTA-treated HL-60 and dHL-60 cells were observed. Using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses (KEGG), it was noted that the pathways of shared DEGs between the 1 and 10 µg/ml LTA-treated dHL-60 cells at both time points were significantly enriched in immune and inflammatory response-related pathways, such as cellular response to tumor necrosis factor, interleukin-1, interferon γ, neutrophil chemotaxis, the NF-κB signaling pathway and the Toll-like receptor signaling pathway. In addition, when comparing the effect of 1 and 10 µg/ml LTA treatment on dHL60 cells, it was found that all enriched GO and KEGG pathways were associated with the TLR signaling pathways of neutrophils. The results of the present study provided important information for the implementation of mRNA profiling in LTA-treated dHL-60 cells and may indicate the feasibility of using dHL-60 cells as a research model for TLR2 signaling in human neutrophils.

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells.

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1ß, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1ß, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

Prediction of miRNA­mRNA network regulating the migration ability of cytarabine­resistant HL60 cells.

Cytarabine is an important medicine for acute myeloid leukemia (AML) treatment, however, drug resistance hinders the treatment of AML. Although microRNA (miRNA or miR) alteration is one of the well-recognized mechanisms underlying drug resistance in AML, few studies have investigated the role and function of miRNAs in the development of cytarabine resistance. In the present study, total RNA was isolated from parental HL60 and cytarabine-resistant HL60 (R-HL60) cells. Subsequently, miRNAs and mRNAs were detected using small RNA sequencing and gene expression array, respectively. Differentially expressed mRNAs (DEMs) and differentially expressed genes (DEGs) with more than two-fold changes between HL60 and R-HL60 cells were screened out. Negatively associated miRNA-mRNA pairs were selected as candidate miRNA-mRNA target pairs according to the miRDB, Targetscan or miRTar databases. Functional enrichment analysis of DEGs included in the candidate miRNA-mRNA pairs was performed. The results indicated that 10 DEGs (CCL2, SOX9, SLC8A1, ICAM1, CXCL10, SIPR2, FGFR1, OVOL2, MITF and CARD10) were simultaneously involved in seven Gene Ontology pathways related to the regulation of migration ability, namely the 'regulation of cell migration', 'regulation of locomotion', 'regulation of cellular component movement', 'cell migration', 'locomotion', 'cell motility', and 'localization of cell'. DEMs predicted to negatively regulate the aforementioned 10 DEGs were paired with DEGs into 16 candidate miRNA-mRNA pairs related to the regulation of migration ability. In addition, migration assays revealed that the migration ability of R-HL60 cells was greater than that of HL60 cells. These findings provide a new perspective for the treatment of cytarabine-resistant AML and advance our understanding of altered migration ability underlying cytarabine resistance development, specifically related to miRNAs.

Steroidal glycosides from Ornithogalum thyrsoides bulbs and their cytotoxicity toward HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells.

Ornithogalum thyrsoides Jacq belongs to the Asparagaceae family and is cultivated for ornamental purposes. The authors have previously reported several cholestane- and spirostan-type steroidal glycosides from O. thyrsoides. Conventional TLC analysis of the methanolic bulb extract of O. thyrsoides suggested the presence of unprecedented compounds; therefore, a detailed phytochemical investigation of the extract was performed and 35 steroidal glycosides (1-35), including 21 previously undescribed ones (1-21) were collected. The structures of 1-21 were determined mainly by analyses of their 1H and 13C NMR spectra with the aid of two-dimensional NMR spectroscopy. The isolated compounds were classified into three distinct groups: furostan-type (1, 2, 8-12, and 22), spirostan-type (3-7 and 23-26), and cholestane-type (13-21 and 27-35). Although the C/D-ring junction of the steroidal skeleton is typically trans-oriented, except for some cardiotonic and pregnane-type steroidal derivatives, 7 possess a cis C/D-ring junction. This is the first reported instance of such a configuration in spirostan-type steroidal derivatives, marking it as a finding of significant interest. Compounds 1-35 were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells. Compounds 3-6, 9, 17-21, 23-25, and 30-35 demonstrated cytotoxicity in a dose-dependent manner with IC50 values ranging from 0.000086 to 18 µM and from 0.00014 to 37 µM toward HL-60 and SBC-3 cells, respectively. Compound 19, which is obtained in a good yield and shows relatively potent cytotoxicity among the undescribed compounds, induces apoptosis in HL-60 cells, accompanied by arresting the cell cycle of HL-60 cells at the G2/M phase. In contrast, 19 causes oxidative stress-associated necrosis in SBC-3 cells. The cytotoxic mechanism of 19 is different between HL-60 and SBC-3 cells.

Dengue virus infection in mice induces bone marrow myeloid cell differentiation and generates Ly6Glow immature neutrophils with modulated functions.

While neutrophil activation during dengue virus infection is known, the effect of dengue virus infection on neutrophil biogenesis has not been studied. We demonstrate that dengue virus serotype 2 induces the differentiation of mice progenitor cells ex vivo toward the CD11b+Ly6C+Ly6G+ granulocyte population. We further observed an expansion of CD11b+Ly6CintLy6Glow myeloid cells in the bone marrow of dengue virus serotype 2-infected AG129 mice with low CXCR2 expression, implying an immature population. Additionally, dengue virus serotype 2 alone could induce the differentiation of promyelocyte cell line HL-60 into neutrophil-like cells, as evidenced by increased expression of CD10, CD66b, CD16, CD11b, and CD62L, corroborating the preferential shift toward neutrophil differentiation by dengue virus serotype 2 in the mouse model of dengue infection. The functional analysis showed that dengue virus serotype 2-induced neutrophil-like cells exhibited reduced phagocytic activity and enhanced NETosis, as evidenced by the increased production of myeloperoxidase, citrullinated histones, extracellular DNA, and superoxide. These neutrophil-like cells lose their ability to proliferate irreversibly and undergo arrest in the G0 to G1 phase of the cell cycle. Further studies show that myeloperoxidase-mediated signaling operating through the reactive oxygen species axis may be involved in dengue virus serotype 2-induced proliferation and differentiation of bone marrow cells as ABAH, a myeloperoxidase inhibitor, limits cell proliferation in vitro and ex vivo, affects the cell cycle, and reduces reactive oxygen species production. Additionally, myeloperoxidase inhibitor reduced NETosis and vascular leakage in dengue virus serotype 2-infected AG129 mice. Our study thus provides evidence that dengue virus serotype 2 can accelerate the differentiation of bone marrow progenitor cells into neutrophils through myeloperoxidase and modulate their functions.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites.

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.