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Frontiers in Immunology 2023T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in...
INTRODUCTION
T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in acute myeloid leukemia and master cell survival and proliferation. In this study, we aimed to explore the effect of TIM-3 interaction with its ligand galectin-9 (Gal-9) on glucose and lipid metabolism in AML cell lines.
METHODS
HL-60 and THP-1 cell lines, representing M3 and M5 AML subtypes, respectively, were cultured under appropriate conditions. The expression of TIM-3 on the cell surface was ascertained by flow cytometric assay. We used real-time PCR to examine the mRNA expression of GLUT-1, HK-2, PFKFB-3, G6PD, ACC-1, ATGL, and CPT-1A; colorimetric assays to measure the concentration of glucose, lactate, GSH, and the enzymatic activity of G6PD; MTT assay to determine cellular proliferation; and gas chromatography-mass spectrometry (GC-MS) to designate FFAs.
RESULTS
We observed the significant upregulated expression of , , , , , and and the enzymatic activity of G6PD in a time-dependent manner in the presence of Gal-9 compared to the PMA and control groups in both HL-60 and THP-1 cell lines ( > 0.05). Moreover, the elevation of extracellular free fatty acids, glucose consumption, lactate release, the concentration of cellular glutathione (GSH) and cell proliferation were significantly higher in the presence of Gal-9 compared to the PMA and control groups in both cell lines (p < 0.05).
CONCLUSION
TIM-3/Gal-9 ligation on AML cell lines results in aerobic glycolysis and altered lipid metabolism and also protects cells from oxidative stress, all in favor of leukemic cell survival and proliferation.
Topics: Humans; Galectins; Hepatitis A Virus Cellular Receptor 2; HL-60 Cells; Lactates; Leukemia, Myeloid, Acute; Lipid Metabolism
PubMed: 38022614
DOI: 10.3389/fimmu.2023.1267578 -
International Journal of Molecular... Oct 2022The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin ()-1364A/C polymorphism was shown...
The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin ()-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele-leading to lower AQP5 expression-has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that promoter methylation differs between genotypes in specific types of immune cells.: promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of -1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils ( = 0.0055) and a four-fold increase in monocytes ( = 0.0005) and lymphocytes ( = 0.0184) in septic patients and healthy controls as well. In addition, a decreased promoter methylation was accompanied by an increased AQP5 expression in HL-60 ( = 0.0102) and REH cells ( = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the promoter. Hence, promoter methylation could depict a key mechanism in genotype-dependent expression.
Topics: Aquaporin 5; DNA Methylation; HL-60 Cells; Humans; Promoter Regions, Genetic; Sepsis
PubMed: 36233114
DOI: 10.3390/ijms231911813 -
Cytometry. Part B, Clinical Cytometry Sep 2021Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its...
Exploring blast composition in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms: CD45RA and CD371 improve diagnostic value of flow cytometry through assessment of myeloblast heterogeneity and stem cell aberrancy.
BACKGROUND
Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its application is challenging due to difficulties in standardization, complexity of antibody panels and subjective interpretation of data. Since blasts are invariably affected in these disorders, we developed a FCIP approach for detailed and objective analysis of the blast population.
METHODS
FCIP using a one-tube 10-color (13-marker) antibody panel was performed on bone marrow samples from 23 MDS and 8 MDS/MPN patients, 21 cytopenic patients non-diagnostic for MDS (Non-MDS), and 16 Control samples.
RESULTS
MDS and MDS/MPN cases demonstrated one to several immunophenotypic abnormalities including: increased myeloblasts, decreased stage-1 hematogones, aberrant stem cells, abnormal myeloblast heterogeneity/divergence from normal, increased or decreased CD45 intensity, increased CD117 or CD123 intensity, decreased CD38 intensity, and aberrant expression of lineage markers (CD5, CD19, CD56). A Blast score was developed that showed sensitivity of 80.6% and specificity of 90.5% for immunophenotypic diagnosis of MDS and MDS/MPN. Expression levels of CD45RA and CD371 were used to evaluate abnormal myeloblast heterogeneity and stem cell aberrancy. Both these features were, for the first time, incorporated into a scoring system and resulted in 19% increase in the sensitivity of the assay for lower-risk MDS.
CONCLUSION
Deep immunophenotypic analysis of the blast population is valuable for diagnosis of MDS and MDS/MPN and can potentially provide sensitivity and specificity figures comparable to those previously described using more comprehensive panels that assess maturing myelomonocytic and erythroid elements in addition to progenitor cells.
Topics: Aged; Aged, 80 and over; Female; Flow Cytometry; Granulocyte Precursor Cells; Humans; Lectins, C-Type; Leukocyte Common Antigens; Male; Middle Aged; Myelodysplastic Syndromes; Myelodysplastic-Myeloproliferative Diseases; Receptors, Mitogen; Stem Cells
PubMed: 33369070
DOI: 10.1002/cyto.b.21983 -
Leukemia & Lymphoma Dec 2021All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present...
All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib-ATRA (tra-ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra-ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra-ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.
Topics: Cell Differentiation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidinones; Tretinoin
PubMed: 34355652
DOI: 10.1080/10428194.2021.1961231 -
Experimental Parasitology Dec 2022The naturally occurring polyether ionophore salinomycin was previously found to display promising anti-proliferative activity against bloodstream forms of Trypanosoma...
The naturally occurring polyether ionophore salinomycin was previously found to display promising anti-proliferative activity against bloodstream forms of Trypanosoma brucei. Here, we report the evaluation of 20-deoxysalinomycin, a naturally occurring homolog to salinomycin, for trypanocidal and cell swelling activity. The concentration of 20-deoxysalinomycin required to reduce the growth rate of bloodstream-form trypanosomes by 50% was determined to be 0.12 μM and found to be 8 times more trypanocidal than that of salinomycin. Moreover, 20-deoxysalinomycin and salinomycin displayed similar cytotoxic activity against human HL-60 cells. Measured as the ratio of cytotoxic to trypanocidal activity, 20-deoxysalinomycin thus exhibits a four-fold higher selectivity compared to salinomycin. The stronger trypanocidal activity of 20-deoxysalinomycin is attributed to an enhanced ability to induce cell swelling in trypanosomes. The findings support 20-deoxysalinomycin as a useful lead in the rational development of new and improved anti-trypanosomal drugs.
Topics: Humans; Trypanocidal Agents; Trypanosoma brucei brucei; HL-60 Cells
PubMed: 36273616
DOI: 10.1016/j.exppara.2022.108414 -
Molecules (Basel, Switzerland) Jun 2022Quinolinones have been known for a long time as broad-spectrum synthetic antibiotics. More recently, the anticancer potential of this group of compounds has been...
Quinolinones have been known for a long time as broad-spectrum synthetic antibiotics. More recently, the anticancer potential of this group of compounds has been investigated. Following this direction, we obtained a small library of 3-methylidene-1-sulfonyl-2,3-dihydroquinolin-4(1)-ones with various substituents at positions 1, 2, 6, and 7 of the quinolinone ring system. The cytotoxic activity of the synthesized analogs was tested in the MTT assay on two cancer cell lines in order to determine the structure-activity relationship. All compounds produced high cytotoxic effects in MCF-7, and even higher in HL-60 cells. 2-Ethyl-3-methylidene-1-phenylsulfonyl-2,3-dihydroquinolin-4(1)-one, which was over 5-fold more cytotoxic for HL-60 than for normal HUVEC cells, was selected for further tests. This analog was shown to inhibit proliferation and induce DNA damage and apoptosis in HL-60 cells.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Molecular Structure; Quinolones; Structure-Activity Relationship
PubMed: 35684532
DOI: 10.3390/molecules27113597 -
Journal of Periodontal Research Jan 2021Neutrophils are emerging as a key player in periodontal pathogenesis. The surface expression of cellular markers enables functional phenotyping of neutrophils which have...
BACKGROUND AND OBJECTIVES
Neutrophils are emerging as a key player in periodontal pathogenesis. The surface expression of cellular markers enables functional phenotyping of neutrophils which have distinct roles in disease states. This study aimed to evaluate the effect of periodontal management on neutrophil phenotypes in peripheral blood in periodontitis patients over one year.
MATERIALS AND METHODS
Peripheral blood and the periodontal parameters, mean probing depth and percentage of sites with bleeding on probing (%BOP), were collected from 40 healthy controls and 54 periodontitis patients at baseline and 3-, 6- and 12- months post-treatment. Flow cytometry was used to identify CD11b , CD16b , CD62L and CD66b expression on neutrophils, neutrophil maturation stages as promyelocytes (CD11b CD16b ), metamyelocytes (CD11b CD16b ) and mature neutrophils (CD11b CD16b ), and suppressive neutrophil phenotype as bands (CD16 CD62L ), normal neutrophils (CD16 CD62L ) and suppressive neutrophils (CD16 CD62L ).
RESULTS
CD62L expression decreased with treatment. No differences were observed in neutrophil maturation stages in health or disease upon treatment. Suppressive and normal neutrophils showed a reciprocal relationship, where suppressive neutrophils decreased with treatment and normal neutrophils increased with treatment. In addition, %BOP was associated with suppressive neutrophils.
CONCLUSION
This study demonstrates that management of periodontitis significantly modifies distinct neutrophil phenotypes in peripheral blood. Suppressive neutrophils may play a role in the pathogenesis of periodontitis. However, their exact role is unclear and requires further investigation.
Topics: Flow Cytometry; Humans; Neutrophils; Periodontitis; Phenotype
PubMed: 32803891
DOI: 10.1111/jre.12793 -
Scientific Reports Apr 2022Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear...
Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear leukocyte (PMN), are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. In our study, we analyzed temperature-dependent neutrophil migration using differentiated HL-60 cells. The migration speed of differentiated HL-60 cells was found to correlate positively with temperature from 30 to 42 °C, with higher temperatures inducing a concomitant increase in cell detachment. The migration persistence time of differentiated HL-60 cells was higher at lower temperatures (30-33 °C), while the migration persistence length stayed constant throughout the temperature range. Coupled with the increased speed observed at high temperatures, this suggests that neutrophils are primed to migrate more effectively at the elevated temperatures characteristic of inflammation. Temperature gradients exist on both cell and tissue scales. Taking this into consideration, we also investigated the ability of differentiated HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis. Using a two-dimensional temperature gradient chamber with a range of 27-43 °C, we observed a migration bias parallel to the gradient, resulting in both positive and negative thermotaxis. To better mimic the extracellular matrix (ECM) environment in vivo, a three-dimensional collagen temperature gradient chamber was constructed, allowing observation of biased neutrophil-like differentiated HL-60 migration toward the heat source.
Topics: Cell Movement; HL-60 Cells; Humans; Inflammation; Neutrophils; Temperature
PubMed: 35488042
DOI: 10.1038/s41598-022-10858-w -
Molecules (Basel, Switzerland) Aug 2019Acute myeloid leukemia (AML) is a neoplastic disorder resulting from clonal proliferation of poorly differentiated immature myeloid cells. Distinct genetic and... (Review)
Review
Acute myeloid leukemia (AML) is a neoplastic disorder resulting from clonal proliferation of poorly differentiated immature myeloid cells. Distinct genetic and epigenetic aberrations are key features of AML that account for its variable response to standard therapy. Irrespective of their oncogenic mutations, AML cells produce elevated levels of reactive oxygen species (ROS). They also alter expression and activity of antioxidant enzymes to promote cell proliferation and survival. Subsequently, selective targeting of redox homeostasis in a molecularly heterogeneous disease, such as AML, has been an appealing approach in the development of novel anti-leukemic chemotherapeutics. Naphthoquinones are able to undergo redox cycling and generate ROS in cancer cells, which have made them excellent candidates for testing against AML cells. In addition to inducing oxidative imbalance in AML cells, depending on their structure, naphthoquinones negatively affect other cellular apparatus causing neoplastic cell death. Here we provide an overview of the anti-AML activities of naphthoquinone derivatives, as well as analysis of their mechanism of action, including induction of reduction-oxidation imbalance, alteration in mitochondrial transmembrane potential, Bcl-2 modulation, initiation of DNA damage, and modulation of MAPK and STAT3 activity, alterations in the unfolded protein response and translocation of FOX-related transcription factors to the nucleus.
Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Naphthoquinones; Reactive Oxygen Species
PubMed: 31466259
DOI: 10.3390/molecules24173121 -
Nature Jun 2020Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and...
Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state.
Topics: Animals; Candida albicans; Cell Lineage; DNA-Binding Proteins; Disease Models, Animal; Female; Granulocyte Precursor Cells; Humans; Immunity, Innate; Male; Mice; Mice, Transgenic; Mutation; Neutropenia; Neutrophils; Transcription Factors
PubMed: 32494068
DOI: 10.1038/s41586-020-2227-7