Proliferation Of NB4 Cells Research Articles

Effects of imidazole derivatives on cellular proliferation and apoptosis in myeloid leukemia.

Acute promyelocytic leukemia (APL) is the sub-type of Acute myeloid leukemia (AML) which is described by differentiation block at promyelocytic stage and t(15; 17) translocation with All trans retinoic acid (ATRA) and arsenic trioxide (ATO) as standard treatments. Chronic myeloid leukemia (CML) translocation t (19; 22) causes a rise in granulocytes and their immature precursors in the blood. Different mutations cause resistance to first-line tyrosine kinase therapies in CML. Beside drug resistance, leukemia stem cells (LSC) are critical resources for relapse and resistance in APL and CML. The drug toxicity and resistant profile associated with LSC and current therapeutics of APL and CML necessitate the development of new therapies. Imidazoles are heterocyclic nitrogen compounds with diverse cellular actions. The purpose of this research was to assess the anti-leukemic properties of four novel imidazole derivatives including L-4, L-7, R-35, and R-NIM04. Pharmacological and biochemical approaches were used which showed that all four imidazole derivatives interfere with the NB4 cells proliferation, an APL cell line, while only L-7 exhibit anti-proliferative activity against K562 cells, a CML cell line. The anti-proliferative effect of imidazole derivatives was linked to apoptosis induction. Further real-time polymerase chain reaction (RT-PCR) analysis revealed downregulation of AXL-Receptor Tyrosine Kinase (AXL-RTK) and target genes of Wnt/beta-catenin pathway like c-Myc, Axin2 and EYA3. An additive effect was observed after combinatorial treatment of L-7 with standard drugs ATRA or Imatinib on the proliferation of NB4 and K562 cells respectively which was related to further downregulation of target genes of Wnt/beta catenin pathway. Imidazole derivatives significantly reduce proliferation of NB4 and K562 cells by inducing apoptosis, down regulating of AXL-RTK and Wnt/β-catenin target genes.

Multi-omics evaluation of the prognostic value and immune signature of FCN1 in pan-cancer and its relationship with proliferation and apoptosis in acute myeloid leukemia.

The FCN1 gene encodes the ficolin-1 protein, implicated in the pathogenesis of various diseases, though its precise role in tumorigenesis remains elusive. This study aims to elucidate the prognostic significance, immune signature, and treatment response associated with FCN1 across diverse cancer types. Employing multi-omics data, we conducted a comprehensive assessment, encompassing tissue-specific and single-cell-specific expression disparities, pan-cancer expression patterns, epigenetic modifications affecting FCN1 expression, and the immune microenvironment. Our investigation primarily focused on the clinical prognostic attributes, immune profiles, potential molecular mechanisms, and candidate therapeutic agents concerning FCN1 and acute myeloid leukemia (AML). Additionally, in vitro experiments were performed to scrutinize the impact of FCN1 knockdown on cell proliferation, apoptosis, and cell cycle dynamics within the AML cell line U937 and NB4. FCN1 expression exhibits widespread dysregulation across various cancers. Through both univariate and multivariate Cox regression analyses, FCN1 has been identified as an independent prognostic indicator for AML. Immunological investigations elucidate FCN1's involvement in modulating inflammatory responses within the tumor microenvironment and its correlation with treatment efficacy. Remarkably, the deletion of FCN1 influences the proliferation, apoptosis, and cell cycle dynamics of U937 cells and NB4 cells. These findings underscore FCN1 as a promising pan-cancer biomarker indicative of macrophage infiltration, intimately linked with the tumor microenvironment and treatment responsiveness, and pivotal for cellular mechanisms within AML cell lines.

Intracellular IL-23 Receptor (IL-23R) Is a Regulator of Mitotic Spindle and Centrosome Formation and Is Essential for AML Viability

IL-23 receptor (IL-23R) is a heterodimeric cytokine cell surface receptor that is traditionally expressed on T cells. The cytokine, IL-23, is secreted by dendritic cells and macrophages in response to inflammatory stimuli. Here, we identify a novel function and localization for IL-23R in which intracellular IL-23R is necessary for AML viability and acts as a critical regulator of the mitotic spindle. We analyzed gene ontologies that were upregulated in AML samples compared to normal hematopoietic cells. Amongst upregulated gene ontologies, the mitotic spindle ontology was enriched in AML samples. Further analysis of all protein coding genes and their correlation to the mitotic spindle ontology surprisingly returned IL-23R as a top hit. Given that IL-23R is not known to be expressed in AML, we confirmed the presence of IL-23R protein by immunoblotting in 14 of 20 primary AML patient samples, while it was undetectable in bulk (n=5) and CD34+ sorted (n=3) normal hematopoietic cells. Consistent with previous investigations, we confirmed cell surface localization of IL-23R in double negative T cells by flow cytometry and confocal microscopy. In contrast, only small amounts of IL-23R were present on the cell surface of AML cells. Instead, IL-23R was detected intracellularly in the cytoplasm and nucleus of AML cell lines, as well as primary AML cells (including the stem cell fraction). We demonstrated intracellular localization using flow cytometry, confocal microscopy, and immunoblotting of subcellular fractions. To ensure we were detecting genuine IL-23R protein, we probed for IL-23R using 4 different antibodies targeting 4 different epitopes of the receptor whilst using 4 methods of detection (immunoblotting, flow cytometry, confocal microscopy, and immunoprecipitation). We also demonstrated intracellular localization of the IL-23R heterodimer subunit, IL12Rβ1, which is known to bind the IL-23R subunit to form the fully functional IL-23 receptor. To elucidate the function of intracellular IL-23R, we performed BioID mass spectrometry to identify proteins that interact with IL-23R. Compared to controls, we identified 61 proteins that preferentially interacted with IL-23R. 36 of those 61 proteins are known cytoplasmic or nuclear localized proteins. Pathway analysis of those interacting proteins identified the mitotic spindle as a top pathway corroborating with our bioinformatics analysis. Proximity Ligation Assay (PLA) and confocal microscopy verified that endogenous IL-23R protein interacted with mitotic spindle associated proteins, NUMA, TMEM201, TACC1, and BAG6 in OCI-AML2 cells and primary AML samples. Consistent with our PLA results, we demonstrated IL-23R co-localized with the mitotic spindle and centrosomes in AML cell lines and primary patient samples. Knockdown and knockout of IL-23R in AML cells led to the dysregulation of the mitotic spindle with multipolarity, lagging chromosomes, and spindle orientation errors. Knockdown of IL-23R reduced cell proliferation and viability in OCI-AML2, TEX, K562, NB4, and U937 cells compared to non-targeting controls. IL-23R knockdown also reduced the engraftment efficiency of TEX leukemia cells in murine bone marrow. Subsequent knockdowns of IL-23R in primary AML cells demonstrated decreased clonogenic growth and reduced engraftment into the marrow of immune deficient mice. In contrast, knockdown of IL-23R in normal human cord blood cells did not impair their engraftment into murine bone marrow. Finally, we analyzed hematopoietic cells and stem cells in IL-23R -/- mice and found that knockout of IL-23R did not decrease blood counts or the abundance and function of normal hematopoietic cells. In summary, we discovered a novel intracellular localization and function for IL-23R in AML. IL-23R regulates mitotic spindle and centrosome formation and is necessary for AML cell viability. We have thus identified a new biological function for IL-23R and a potential therapeutic target for AML.

Mechanism of Proliferation and Apoptosis of Acute Promyelocytic Leukemia Cell Line NB4 Induced by TPA

To investigate the effect of phorbol-12-myristate-13-ace-tate (TPA) on the proliferation and apoptosis of acute promyelocytic leukemia cell line NB4 and its molecular mechanism. The effect of different concentrations of TPA on the proliferation of NB4 cells at different time points was detected by CCK-8 assay. The morphological changes of NB4 cells were observed by Wright-Giemsa staining. The cell cycle and apoptosis of NB4 cells after TPA treatment were detected by flow cytometry. The mRNA expressions of NB4 cells after TPA treatment were analyzed by high-throughput microarray analysis and real-time quantitative PCR. Western blot was used to detect the protein expression of CDKN1A, CDKN1B, CCND1, MYC, Bax, Bcl-2, c-Caspase 3, c-Caspase 9, PIK3R6, AKT and p-AKT. Compared with the control group, TPA could inhibit the proliferation of NB4 cells, induce the cells to become mature granulocyte-monocyte differentiation, and also induce cell G1 phase arrest and apoptosis. Differentially expressed mRNAs were significantly enriched in PI3K/AKT pathway. TPA treatment could increase the mRNA levels of CCND1, CCNA1, and CDKN1A, while decrease the mRNA level of MYC. It could also up-regulate the protein levels of CDKN1A, CDKN1B, CCND1, Bax, c-Caspase 3, c-Caspase 9, and PIK3R6, while down-regulate MYC, Bcl-2, and p-AKT in NB4 cells. TPA induces NB4 cell cycle arrest in G1 phase and promotes its apoptosis by regulating PIK3/AKT signaling pathway.

Pyrrolidine Dithiocarbamate Enhances the Cytotoxic Effect of Arsenic Trioxide on Acute Promyelocytic Leukemia Cells.

More than 95% patients with acute promyelocytic leukemia (APL) carry the PML-RARα fusion oncoprotein. Arsenic trioxide (ATO) is an efficacious therapeutic agent for APL, and the mechanism involves the binding with PML and degradation of PML-RARα protein. Pyrrolidine dithiocarbamate (PDTC) demonstrates the function of facilitating the cytotoxic effect of ATO. To investigate whether PDTC is potential to enhance the cytotoxic effect of ATO to APL cells by acting on PML-RARα oncoproteins. Inhibitory effects of drugs on cell viability were examined by CCK-8 test, and apoptosis was evaluated by flow cytometry. Western blotting and immunofluorescence assays were used to explore the mechanism. PDTC improved the effect of ATO on inhibiting proliferation of NB4 cells in vitro. Further, PDTC-ATO promoted apoptosis and cell cycle arrest in NB4 cells. The expression of caspase- 3 and Bcl-2 was reduced in PDTC-ATO-treated NB4 cells, while cleaved caspase-3 and Bax was up-regulated. Furthermore, less PML-RARα expression were found in PDTC-ATO-treated NB4 cells than that in NB4 cells treated with ATO singly. Laser confocal microscopy showed that protein colocalization of PML and RARα was disrupted more significantly by PDTC-ATO treatment than that with ATO monotherapy. In conclusion, PDTC enhanced the cytotoxic effect of ATO on APL, and the mechanism was, at least in part, related to the promotion of ATO-induced degradation of PML-RARα fusion protein via forming a complex PDTC-ATO.

Critical role of MXRA7 in differentiation blockade in human acute promyelocytic leukemia cells

The biology of the matrix remodeling associated 7 (MXRA7) gene had been ill-defined. Inspired by public datasets and domestic findings concerning MXRA7, we hypothesized that MXRA7 might be involved in the pathogenesis of leukemia. This study aimed to investigate the role and effect of MXRA7 expression in acute promyelocytic leukemia (APL) cells differentiation and behavior. Bioinformatics of public datasets revealed MXRA7 mRNA was expressed highly in acute myeloid leukemia (AML), especially in APL. High expression of MXRA7 was associated with poor overall survival of AML patients. We confirmed MXRA7 expression was upregulated in APL patients and cell line. Knockdown or overexpression of MXRA7 did not affect the proliferation of NB4 cells directly. Knockdown of MXRA7 in NB4 cells promoted drug induced cell apoptosis, while overexpression of MXRA7 had no obvious influence on the drug induced cell apoptosis. Lowering MXRA7 protein levels in NB4 cells promoted ATRA-induced cell differentiation possibly through decreasing PML-RARα level and increasing PML and RARα levels. Correspondingly, overexpression of MXRA7 showed the consistent results. We also demonstrated that MXRA7 altered the expression of genes involved in leukemic cell differentiation and growth. Knockdown of MXRA7 upregulated the expression levels of C/EBPB, C/EBPD and UBE2L6, and downregulated the expression levels of KDM5A, CCND2 and SPARC. Moreover, knockdown of MXRA7 inhibited the malignancy of NB4 cells in NOD-SCID mice model. In conclusion, this study demonstrated that MXRA7 influences the pathogenesis of APL via regulation of the cell differentiation. The novel findings about the role of MXRA7 in leukemia not only shed lights on the biology of this gene, but also proposed this gene as a new target for APL treatment.

Leukemia Cells Resistant to Glutamine Deprivation Express Glutamine Synthetase Protein

Objective:Low glutamine levels have been shown in tumor environments for several cancer subtypes. Therefore, it has been suggested that cancer cells rewire their metabolism to adopt low nutrient levels for survival and proliferation. Although glutamine is a non-essential amino acid and can be synthesized de novo, many cancer cells including malignant hematopoietic cells have been indicated to be addicted to glutamine. This study aimed to investigate the proliferation of leukemia cell lines in glutamine-deprived conditions.Materials and Methods:Cell proliferation of K562, NB-4, and HL-60 cells was determined by calculating cell numbers in normal vs. low glutamine media. Changes in mRNA expressions were investigated using qRT-PCR. The glutamine synthetase (GS)-encoding GLUL gene was knocked out (KO) in HL-60 cells using the CRISPR/Cas9 method and protein expression was evaluated with immunoblotting.Results:The proliferation of all cell lines was decreased in glutamine-deprived medium. GS protein expression was increased in glutamine-limited medium although the mRNA level did not change. Increased protein expression was confirmed with inhibition of new protein synthesis by treating cells with cycloheximide. To further investigate the role of GS protein, the GS-encoding GLUL gene was KO in HL-60 cells using the CRISPR/Cas9 method. GS KO cells proliferated less compared to control cells in glutamine-limited medium.Conclusion:Our results indicate that upregulated GS protein expression is responsible for glutamine addiction of leukemia cell lines. Exploiting the genetic and metabolic mechanisms responsible for GS protein expression could lead to the identification of new anti-cancer drug targets.

The Combination of Gefitinib With ATRA and ATO Induces Myeloid Differentiation in Acute Promyelocytic Leukemia Resistant Cells.

In approximately 15% of patients with acute myeloid leukemia (AML), total and phosphorylated EGFR proteins have been reported to be increased compared to healthy CD34+ samples. However, it is unclear if this subset of patients would benefit from EGFR signaling pharmacological inhibition. Pre-clinical studies on AML cells provided evidence on the pro-differentiation benefits of EGFR inhibitors when combined with ATRA or ATO in vitro. Despite the success of ATRA and ATO in the treatment of patients with acute promyelocytic leukemia (APL), therapy-associated resistance is observed in 5-10% of the cases, pointing to a clear need for new therapeutic strategies for those patients. In this context, the functional role of EGFR tyrosine-kinase inhibitors has never been evaluated in APL. Here, we investigated the EGFR pathway in primary samples along with functional in vitro and in vivo studies using several APL models. We observed that total and phosphorylated EGFR (Tyr992) was expressed in 28% and 19% of blast cells from APL patients, respectively, but not in healthy CD34+ samples. Interestingly, the expression of the EGF was lower in APL plasma samples than in healthy controls. The EGFR ligand AREG was detected in 29% of APL patients at diagnosis, but not in control samples. In vitro, treatment with the EGFR inhibitor gefitinib (ZD1839) reduced cell proliferation and survival of NB4 (ATRA-sensitive) and NB4-R2 (ATRA-resistant) cells. Moreover, the combination of gefitinib with ATRA and ATO promoted myeloid cell differentiation in ATRA- and ATO-resistant APL cells. In vivo, the combination of gefitinib and ATRA prolonged survival compared to gefitinib- or vehicle-treated leukemic mice in a syngeneic transplantation model, while the gain in survival did not reach statistical difference compared to treatment with ATRA alone. Our results suggest that gefitinib is a potential adjuvant agent that can mitigate ATRA and ATO resistance in APL cells. Therefore, our data indicate that repurposing FDA-approved tyrosine-kinase inhibitors could provide new perspectives into combination therapy to overcome drug resistance in APL patients.

MiR-139-5p Regulates the Proliferation of Acute Promyelocytic Leukemia Cells by Targeting MNT.

Acute promyelocytic leukemia (APL) patients with progressive leukocytosis are more likely to have various complications and poor outcomes. However, the regulatory roles of microRNAs in the leukocytosis of APL have not been clarified. Our study aims to evaluate the effects of miRNAs on leukocytosis during induction therapy of APL patients and explore its potential mechanisms. During induction treatment, patients with white blood cell count higher than 10 × 109/L were divided into leukocytosis group and others were nonleukocytosis group. Using microarray assays, we found that miR-139-5p was significantly downregulated in the leukocytosis group. Elevated expression of miR-139-5p inhibited the proliferation of NB4 cells by arresting the cell cycle and inducing apoptosis. We further identified that MNT was a target of miR-139-5p. miR-139-5p significantly inhibited the proliferation, invasion, and migration function of NB4 cells through targeting MNT. Strategies for regulating miR-139-5p or MNT expression might provide new therapeutic approaches for progressive leukocytosis in APL.

Bioactive peptide isolated from sesame seeds inhibits cell proliferation and induces apoptosis and autophagy in leukemic cells.

Leukemia is the most common type of hematological malignancies. Several natural products including bioactive peptides have been explored and studied for their anti-leukemic activities. In the present study, anti-leukemic peptide, IGTLILM (IM-7), was isolated and identified from the protein hydrolysate of sesame seeds by reverse phase-solid phase extraction, off-gel fractionation and nano LC-MS/MS. The cytotoxic effects of IM-7 were studied in MOLT-4 and NB4 acute leukemic cell lines using an MTT assay. The induction of apoptosis and autophagy was investigated by flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies, respectively. The mRNA alterations of apoptotic and autophagic-related genes were determined by reverse transcription-quantitative PCR. The present study found that IM-7 inhibited the proliferation of MOLT-4 and NB4 cells in dose-dependent manner, but it showed a minimal effect on healthy mononuclear cells. IM-7 activated apoptosis and autophagy through the upregulation of CASP3, ULK1 and BECN1 and the downregulation of BCL2. In addition, IM-7 enhanced the cytotoxic effect of the anti-leukemic drug, daunorubicin. The findings suggested that IM-7 was potent to suppress the proliferation of MOLT-4 and NB4 leukemic cells and induce apoptosis and autophagy through the regulation of caspase 3-Bcl-2 and ULK1-Beclin1, respectively.

NCTD Retards AML HL60 Cell Proliferation via Targeting Hedgehog/SOX2 axis

To investigate the effect of norcantharidin (NCTD) to proliferation of leukemia cells through disrupting key regulators of sonic Hedgehog (SHH) pathway and its downstream transcription factor SOX2. CCK8 was used to detected the HL60 and NB4 cells after inhibited by NCTD, SMO and GLI1 inhibitor for 24 hours. Expression level of SMO, GLI1 and SOX2 in HL60 cells with NCTD treatment was detected by immunoblot. HL60 cells were transfected with pcDNA3.1 plasmid expressing GLI1 or SOX2. Empty vector and pcDNA3. 1-EGFP were divided into negative and positive control group, respectively. The expression of exogenous GLI1 or SOX2 in HL60 cells was confirmed by immunoblot, and growth curve of HL60 cell was checked by CCK8. Proliferation of genetic modified HL60 cells treated by various dose of NCTD was detected. NCTD, SMO/GLI1 inhibitors could inhibit the proliferation of NB4 and HL60 cells in a dose-dependent manner. Compared with solvent (DMSO)-treated control group, NCTD remarkably decreased protein level of SMO, GLI1 and SOX2. GLI1 and SOX2 were overexpressed in HL60 cells as compared with pcDNA3.1 empty vector-transfected group. Growth curve demonstrated significant proliferative advantage of GLI1/SOX2-transfected cells. CCK8 assay indicated that GLI1/SOX2-overexpressed HL60 cells were more resistant to NCTD treatment. NCTD attenuates HL60 proliferation via targeting the Hedgehog/SOX2 axis.

ZnO Q-Dots-Induced Apoptosis Was Coupled with the Induction of PPARγ in Acute Promyelocytic Leukemia Cells; Proposing a Novel Application of Nanoparticles in Combination with Pioglitazone

By the creation of nanoscale-materials, nanotechnology has emerged as one of the focal points of modern medical investigations and nanotechnology-based cancer treatment approaches found their ways into therapeutic strategies. Herein, we evaluated anticancer property and molecular mechanisms of zinc oxide quantum dot nanoparticles (ZnO QD NPs) in the form of nanofluid (NF) in acute promyelocytic leukemia (APL)-derived NB4 cells. ZnO nanofluid (ZnO NF) robustly decreased proliferation of NB4 cells through p21-mediated G1 cell cycle arrest and induced apoptosis probably via ROS-dependent upregulation of FoxO3a and SIRT1. Moreover, our study indicated for the first time that the anti-leukemic effect of ZnO NF was coupled with the up-regulation of peroxisome proliferator-activated receptors gamma (PPARγ) in NB4 cells. Activation of PPARγ using pioglitazone, not only up-regulated the effect the ZnO NF on the expression of anti-apoptotic target genes but also could significantly elevate the expression of pro-apoptotic molecules in NB4 cells, as compared to either agent alone. In conclusion, this study suggested promising anti-leukemic activity of ZnO NF against NB4 cells and proposed a novel mechanism of action through which ZnO NF may induce significant cytotoxicity in APL, either as a single or in combination with PPARγ agonist.

Autophagy and apoptosis induction by sesamin in MOLT-4 and NB4 leukemia cells.

Sesamin, the major furofuran lignan found in the seeds of Sesamum indicum L., has been investigated for its various medicinal properties. In the present study, the anti-leukemic effects of sesamin and its underlying mechanisms were investigated in MOLT-4 and NB4 acute leukemic cells. Leukemic cells were treated with various concentrations of sesamin. Cell viability was determined using an MTT assay. Flow cytometry using Annexin V-FITC/PI staining and anti-LC3/FITC antibodies was applied to detect the level of apoptosis and autophagy, respectively. Reverse transcription-quantitative PCR was performed to examine the alterations in the mRNA expression of apoptotic and autophagic genes. In addition, bioinformatics tools were used to predict the possible interactions between sesamin and its targets. The results revealed that sesamin inhibited MOLT-4 and NB4 cell proliferation in a dose-dependent manner. In addition, sesamin induced both apoptosis and autophagy. In sesamin-treated cells, the gene expression levels of caspase 3 and unc-51 like autophagy activating kinase 1 (ULK1) were upregulated, while those of mTOR were downregulated compared with in the control. Notably, the protein-chemical interaction network indicated that caspase 3, mTOR and ULK1 were the essential factors involved in the effects of sesamin treatment, as with anticancer agents, such as rapamycin, AZD8055, Torin1 and 2. Overall, the findings of the present study suggested that sesamin inhibited MOLT-4 and NB4 cell proliferation, and induced apoptosis and autophagy through the regulation of caspase 3 and mTOR/ULK1 signaling, respectively.

Chidamide acts on the histone deacetylase-mediated miR-34a/Bcl-2 axis to regulate NB4 cell line proliferation and apoptosis.

Acute promyelocytic leukemia (APL), a biologically and clinically distinct variant of acute myelogenous leukemia, is characterized by the fusion of the N-terminus of promyelocytic leukemia protein to the C terminus of retinoic acid receptor alpha, mostly due to chromosomal translocation t(15;17). Chidamide, a synthetic analogue of MS-275 identified from a group of benzamide-type compounds, has been found to have efficient anticancer activity in basic and clinical research studies. However, the concrete role and underlying mechanism of Chidamide in the treatment of APL has not been well characterized. Our data demonstrate that Chidamide inhibited the expression of histone deacetylase (HDAC) to induce apoptosis and suppress proliferation in NB4 cells. Mechanistically, Chidamide increases the expression of miR-34a by suppressing HDAC. Furthermore, B-cell lymphoma-2 (Bcl-2) is a direct target of miR-34a, the expression of which is regulated by miR-34a. Functionally, Chidamide inhibits cell proliferation and promotes apoptosis through miR-34a/Bcl-2. Chidamide exerts its anticancer effect via the HDAC-mediated miR-34a/Bcl-2 axis, providing potential targets for APL therapy.

Immunosuppressive role of CD11b+ CD33+ HLA-DR- myeloid-derived suppressor cells-like blast subpopulation in acute myeloid leukemia.

ObjectiveMyeloid‐derived suppressor cells (MDSCs) facilitate tumor growth and development by suppressing T cell function; however, their role in acute myeloid leukemia (AML) remains unclear. Here, we investigated the immunosuppressive role and prognostic value of blasts with an MDSC‐like phenotype.MethodsCD11b+CD33+HLA‐DR− MDSC‐like blasts from bone marrow mononuclear cells of patients with AML were analyzed. To investigate their T cell‐suppressing function, MDSC‐like blasts were isolated using flow cytometry and co‐cultured with CD8+ cytotoxic T cells and NB4 leukemic cells. Treatment outcomes were then compared between the MDSC‐like blasts low (≤9.76%) and high (>9.76%) groups to identify clinical significance.ResultsMDSC‐like blasts showed higher expression of arginase‐1 and inducible nitric oxide synthase. Isolated MDSC‐like blasts significantly suppressed CD8+ T cell proliferation induced by phytohemagglutinin A. NB4 cell proliferation was significantly suppressed upon co‐culture with CD8+ cytotoxic T cells and partially restored upon co‐culture with MDSC‐like blasts. Patients with high MDSC‐like blasts at diagnosis showed substantially shorter overall survival and leukemia‐free survival relative to low MDSC‐like blasts patients, with subgroup analysis showing statistically significant differences in patients not receiving allogeneic hematopoietic stem cell transplantation.ConclusionWe demonstrated that MDSC‐like blasts drive AML‐specific immune‐escape mechanisms by suppressing T cell proliferation and restoring T cell‐suppressed NB4 cell proliferation, with clinically higher fractions of MDSC‐like blasts at diagnosis resulting in poor prognosis.

Inducible expression of a disease-associated ELANE mutation impairs granulocytic differentiation, without eliciting an unfolded protein response

Severe congenital neutropenia (SCN) is characterized by a near absence of neutrophils, rendering individuals with this disorder vulnerable to recurrent life-threatening infections. The majority of SCN cases arise because of germline mutations in the gene elastase, neutrophil-expressed (ELANE) encoding the neutrophil granule serine protease neutrophil elastase. Treatment with a high dose of granulocyte colony-stimulating factor increases neutrophil production and reduces infection risk. How ELANE mutations produce SCN remains unknown. The currently proposed mechanism is that ELANE mutations promote protein misfolding, resulting in endoplasmic reticulum stress and activation of the unfolded protein response (UPR), triggering death of neutrophil precursors and resulting in neutropenia. Here we studied the ELANE mutation p.G185R, often associated with greater clinical severity (e.g. decreased responsiveness to granulocyte colony-stimulating factor and increased leukemogenesis). Using an inducible expression system, we observed that this ELANE mutation diminishes enzymatic activity and granulocytic differentiation without significantly affecting cell proliferation, cell death, or UPR induction in murine myeloblast 32D and human promyelocytic NB4 cells. Impaired differentiation was associated with decreased expression of genes encoding critical hematopoietic transcription factors (Gfi1, Cebpd, Cebpe, and Spi1), cell surface proteins (Csf3r and Gr1), and neutrophil granule proteins (Mpo and Elane). Together, these findings challenge the currently prevailing model that SCN results from mutant ELANE, which triggers endoplasmic reticulum stress, UPR, and apoptosis.

LukS-PV Induces Apoptosis and Inhibits Proliferation Through Methyltransferase SET8 in Human Acute Myeloid Leukaemia Cells

Background: Histone modifiers contribute to leukaemia pathogenesis. LukS-PV is a component of the Panton-Valentine leukocidin (PVL) cytotoxin secreted by Staphylococcus aureus. We previously reported that LukS-PV induces apoptosis in human acute myeloid leukaemia (AML) cells (THP-1 and HL-60). However, it is unclear whether LukS-PV exerts biological effects through epigenetic regulation. We aimed to investigate the epigenetic regulatory mechanisms underlying the effects of LukS-PV. Methods: SET8 expression was quantified through reverse-transcription PCR and western blotting in patient-derived AML cells. To determine whether LukS-PV influences apoptosis and proliferation via SET8, AML cell lines HL-60 and NB-4 were treated with LukS-PV, and SET8 expression was quantified. Chromatin-immunoprecipitation (ChIP) was performed to determine whether LukS-PV regulates H4K20me1 via SET8, and transcriptional targets were determined through ChIP-seq and bioinformatic methods. Findings: Methyltransferase SET8 was upregulated in primary AML blasts and downregulated in LukS-PV-treated AML cells HL-60 and NB4. LukS-PV induced apoptosis and inhibited proliferation by downregulating SET8. Furthermore, LukS-PV regulated H4K20me1 through SET8. Genome-wide analysis identified PIK3CB as the direct target of LukS-PV-mediated H4K20me1. Furthermore, PIK3CB inhibitor GSK2636771 induced apoptosis and inhibited proliferation in HL-60 and NB4 cells. Moreover, LukS-PV induced apoptosis and inhibited proliferation in primary AML blasts. Conclusions: The present results indicate that LukS-PV induces apoptosis and inhibits proliferation in AML cells by downregulating SET8 and regulating downstream molecular targets, suggesting that SET8 is a potential target for AML therapy using LukS-PV for anti-leukaemia treatment. Funding Statement: This work was supported by the Anhui Natural Science Foundation(Grant No.1808085QH259), the Fundamental Research Funds for Central Universities (WK9110000107) and National Natural Science Foundation of China (Grant No.81572065). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee and Institutional Review Board of University of Science and Technology of China, Anhui, China (Approval number: 2019-N(H)-101).

Effect of combination of all-trans retinoic acid and arsenic trioxide on apoptosis of acute promyelocytic leukemia cells

To study the effect of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) combination treatment on apoptosis of acute promyelocytic leukemia cells (NB4), inflammation and prognosis. The effect of ATRA - ATO combination on the proliferation of NB4 was determined using MTT assay. Apoptosis of NB4 cells was assessed with TUNEL assay. The effect of ATRA-As2O3 combination on the expressions of IL-6 and TNF-α in NB4 cells was determined using ELISA kits, while its effect on the quality of life of 25 acute promyelocytic leukemia patients admitted to our hospital was scored, as an index of prognosis. The combination treatment with ATRA and ATO significantly inhibited the proliferation of NB4 cells and promoted their apoptosis, relative to the model group. In addition, the combination treatment reduced serum IL-6 and TNF-α levels in patients with acute promyelocytic leukemia, and improve their quality of life and survival. Combination treatment with ATRA and ATO significantly inhibits the proliferation of NB4 cells and promotes their apoptosis, and reduces inflammatory responses in patients with acute promyelocytic leukemia, while improving their quality of life and prognosis.

MiR-16 induces G1 cell cycle arrest and apoptosis in acute myeloid leukemia cells

ObjectiveAcute promyelocytic leukemia is a subset of acute myeloid leukemia bearing t (15; 17) chromosomal aberration. The expression of this gene results in PML/RARα recombinant protein. MicroRNAs are a group of small noncoding RNA molecules regulating the expression of genes by targeting their mRNA. MiR-16-1 regulates several cellular functions and acts as a tumor suppressor. The aim of this study was transduction of NB4 cells with miR-16-1 lentiviral as well as evaluation of changing expressions of Bcl-2 gene and apoptosis in cells due to the function of miR-16-1. Materials and methodsIn this study, NB4 cells were transduced with a miR-16-1 lentiviral vector, and cytotoxic effect of this miRNA on cells was investigated using MTT assay. QReal-Time PCR measured the expression level of Bax and Bcl-2 genes before and after treatment. Annexin-PE/7ADD and caspase 3 kits were used to assess the apoptosis induced by increased expression of miR-16-1. ResultsMTT assay showed that miR-16-1 was able to reduce the proliferation and activity of NB4 cells significantly. Study of changing the expression of Bax and Bcl-2 genes in the cells treated with miR-16-1 showed that miR-16-1 had not caused a significant difference. However, miR-16-1 led to the arrest of cells in the G1 phase, and a significant increase was observed in caspase-3 activity in the cells transduced with miR-16-1, which can activate another pathway to induce apoptosis. ConclusionThe data suggest that miR-16-1 induced apoptosis and arrested cell cycle of NB4 cells in G1 phase, which can have therapeutic effects in leukemia.

MiR-15b regulates cell differentiation and survival by targeting CCNE1 in APL cell lines

MicroRNAs have been shown to be involved in various cell processes, including proliferation, apoptosis and differentiation. However, little is known about their function in granulopoiesis. In the present study, overexpression and knockdown experiments revealed that miR-15b was required to block the proliferation of NB4 and HL60 cells and induce them differentiated to granulocyte lineage. Moreover, we identified CCNE1 as a direct target of miR-15b, and demonstrated that CCNE1 was involved in cell differentiation and proliferation in acute promyelocytic leukemia cells. In addition, we demonstrated a novel pathway in which miR-15b regulated cells arrested in the G0/G1 phase and promoted terminal differentiation of cells by targeting CCNE1, which could modulate the cell cycle effort pRb in APL cells. These events blocked cell proliferation and promoted granulocyte differentiation. In conclusion, our data highlighted, for the first time, the important role of miR-15b in myeloid differentiation and suggested the potential role of miR-15b in cancer therapy.