OBJECTIVE: To investigate and analyze the effect of CXC chemokine receptor 1/2 (CXCR1/2) targeting inhibitor Reparixin combined with cytarabine (Ara-C) on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family, while exploring the accompanying molecular mechanism, providing scientific basis and reference for new molecular markers and targeted therapy for AML. METHODS: Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin, Ara-C alone or in combination, and the cell morphology was observed under an inverted microscope; Wright-Giemsa staining was used to detect cell morphological changes; CCK-8 method was used to detect cell proliferation; the ability of cell invasion was detected by Transwell chamber method; the ability of colony formation was detected by colony formation assay; cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry; monodansylcadaverine(MDC) staining was used to detect cell autophagy; the expression of apoptosis, autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Reparixin could inhibit the proliferation, invasion, migration and clone formation ability of U937 cells. Compared with the single drug group, when U937 cells were intervened by Reparixin combined with Ara-C, the malignant biological behaviors such as proliferation, invasion and colony formation were significantly decreased, and the levels of apoptosis and autophagy were significantly increased (P<0.01). After Reparixin combined with Ara-C intervenes in U937 cells, it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2, and also hydrolyze and activate Caspase-3, thereby inducing cell apoptosis. Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱ and Beclin-1 proteins in U937 cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P<0.01). MDC result showed that the green granules of vesicles increased significantly, and a large number of broken cells were seen (P<0.01). Reparixin combined with Ara-C can significantly inhibit the phosphorylation level of PI3K, AKT and NF-κB signaling molecule, inhibit the malignant biological behavior of cells by inhibiting the activation of PI3K/AKT/NF-κB pathway, and induce programmed cell death. Ara-C intervention in U937 cells had no effect on the expression of CXCR family (P>0.05). The expression of CXCR1, CXCR2, and CXCR4 mRNA could be down-regulated by Reparixin single-agent intervention in U937 cells (P<0.05), and the expression of CXCR2 was more significantly down-regulated than the control group and other CXCRs (P<0.01). When Reparixin and Ara-C intervened in combination, the down-regulated levels of CXCR1 and CXCR2 were more significant than those in the single-drug group (P<0.01), while the relative expressions of CXCR4 and CXCR7 mRNA had no significant difference compared with the single-drug group (P>0.05). CONCLUSION Reparixin combined with Ara-C can synergistically inhibit the malignant biological behaviors of U937 cells such as proliferation, invasion, migration and clone formation, and induce autophagy and apoptosis. The mechanism may be related to affecting the proteins expression of Bcl-2 family and down-regulating the proteins expression of CXCR family, while inhibiting the PI3K/AKT/NF-κB signaling pathway.
Humans ; U937 Cells ; Cytarabine/therapeutic use* ; Receptors, Interleukin-8A ; NF-kappa B ; Proto-Oncogene Proteins c-akt ; Phosphatidylinositol 3-Kinases ; Leukemia, Myeloid, Acute/genetics* ; Apoptosis ; Cell Proliferation ; Apoptosis Regulatory Proteins ; Proto-Oncogene Proteins c-bcl-2 ; RNA, Messenger ; Cell Line, Tumor

Objective: To investigate the effect of the AML1-ETO (AE) fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression. Methods: The doxycycline (Dox) -dependent expression of the AE fusion gene in the U937 cell line (U937-AE) were established using a lentivirus vector system. The Cell Counting Kit 8 methods, including the PI and sidanilide induction, were used to detect cell proliferation, cell cycle-induced differentiation assays, respectively. The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing. Results: ①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells. ②Cell proliferation slowed down and the cell proliferation rate with AE expression (3.47±0.07) was lower than AE non-expression (3.86 ± 0.05) after inducing the AE fusion gene expression for 24 h (P<0.05). The proportion of cells in the G(0)/G(1) phase in the cell cycle increased, with AE expression [ (63.45±3.10) %) ] was higher than AE non-expression [ (41.36± 9.56) %] (P<0.05). The proportion of cells expressing CD13 and CD14 decreased with the expression of AE. The AE negative group is significantly higher than the AE positive group (P<0.05). ③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence, nuclear factor kappa-light-chain-enhancer of activated B cells, interferon-α/γ, and other inflammatory response and immune regulation signals after AE expression. ④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE. The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing, propionyl L-carnitine, wherein those with AE expression (0.46±0.13) were lower than those with AE non-expression (1.00±0.27) (P<0.05). The metabolite concentration of some long-chain fatty acid acylcarnitine increased in cells with AE expressing tetradecanoyl carnitine, wherein those with AE expression (1.26±0.01) were higher than those with AE non-expression (1.00±0.05) (P<0.05) . Conclusion: This study successfully established a leukemia cell model that can induce AE expression. The AE expression blocked the cell cycle and inhibited cell differentiation. The gene sets related to the inflammatory reactions was significantly enriched in U937-AE cells that express AE, and fatty acid metabolism was disordered.
Humans ; U937 Cells ; RUNX1 Translocation Partner 1 Protein ; Leukemia/genetics* ; Core Binding Factor Alpha 2 Subunit/genetics* ; Oncogene Proteins, Fusion/genetics* ; Leukemia, Myeloid, Acute/genetics*

Objective: To investigate the prognostic significance of interferon regulatory factor 9 (IRF9) expression and identify its role as a potential therapeutic target in acute promyelocytic leukemia (APL) . Methods: The gene expression profile and survival data applied in the bioinformatic analysis were obtained from The Cancer Genome Atlas and Beat acute myeloid leukemia (AML) cohorts. A dox-induced lentiviral system was used to induce the expression of PML-RARα (PR) in U937 cells, and the expression level of IRF9 in U937 cells treated with or without ATRA was examined. We then induced the expression of IRF9 in NB4, a promyelocytic leukemia cell line. In vitro studies focused on leukemic phenotypes triggered by IRF9 expression. Results: ①Bioinformatic analysis of the public database demonstrated the lowest expression of IRF9 in APL among all subtypes of AML, with lower expression associated with worse prognosis. ②We successfully established a PR-expression-inducible U937 cell line and found that IRF9 was downregulated by the PR fusion gene in APL, with undetectable expression in NB4 promyelocytic cells. ③An IRF9-inducible NB4 cell line was successfully established. The inducible expression of IRF9 promoted the differentiation of NB4 cells and had a synergistic effect with lower doses of ATRA. In addition, the inducible expression of IRF9 significantly reduced the colony formation capacity of NB4 cells. Conclusion: In this study, we found that the inducible expression of PR downregulates IRF9 and can be reversed by ATRA, suggesting a specific regulatory relationship between IRF9 and the PR fusion gene. The induction of IRF9 expression in NB4 cells can promote cell differentiation as well as reduce the colony forming ability of leukemia cells, implying an anti-leukemia effect for IRF9, which lays a biological foundation for IRF9 as a potential target for the treatment of APL.
Cell Differentiation ; Humans ; Interferon-Stimulated Gene Factor 3, gamma Subunit/metabolism* ; Leukemia, Myeloid, Acute/drug therapy* ; Leukemia, Promyelocytic, Acute/genetics* ; Oncogene Proteins, Fusion/metabolism* ; Phenotype ; Tretinoin/therapeutic use* ; U937 Cells

OBJECTIVE: To investigate the antileukemia activity of phosphatidylinositol-3 kinase (PI3K) inhibitor ZSTK474 on human leukemia cell line U937. METHODS: MTT, soft agar assay, flow cytometric analysis and western blot were used to detect the effect of ZSTK474 on U937 cell proliferation, tumorigenicity, cell cycle, cell apoptosis and phosphorylation levels of the key factor of PI3K/AKT pathway. Chou-Talalay method was used to evaluate the combination of ZSTK474 with Cytarabine or Homoharringtonine. RESULTS: PI3K inhibitor ZSTK474 could inhibit the proliferation and tumorigenicity of U937 cell, induce G CONCLUSION ZSTK474 can inhibit the pathway of PI3K/AKT, ZSTK474 alone or in combination with Homoharringtonine shows potential antileukemia activity on U937 cells.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Glycogen Synthase Kinase 3 beta ; Humans ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Triazines ; U937 Cells

OBJECTIVE: To detect the expression of different transcripts of lactamase β（LACTB) gene in leukemic cell lines. METHODS: NCBI website and DNAstar software were used to detect the Bioinformatics analysis of LACTB. The expression of different transcripts of LACTB gene in leukemic cell lines (THP-1, HL60, K562, U937, Jurkat and Raji) was detected by reverse transcription PCR (RT-PCR), DNA and clone sequencing; the expression of different transcripts of LACTB gene in leukemic cell lines was detected by Quantitative Real-time PCR. RESULTS: There were a variety of splicing isomers in LACTB, and it could produce a variety of protein isomers with conserved N-terminal and different C-terminal, moreover, there were many splice isoforms of LACTB in leukemia cell lines, and there were different expression patterns in different cell lines, including XR1, V1, V2 and V3. The expression of total LACTB showed high in HL60 cells, while low in Raji cells, and the difference was statistically significant (P<0.05). The V1 was high expression in U937 cells but low in Raji cells, and the difference was statistically significant (P<0.05). V2 was high expression in HL60 cells but lowly in Raji cells, and the difference was statistically significant (P<0.05). The expression of V3 was low in THP-1 cells, which was significantly different as compared with that in normal bone marrow (P<0.05). CONCLUSION The reaserch found that there are many splice isomers of LACTB in leukemic cell lines, and there are different expression patterns in different cell lines.
Alternative Splicing ; HL-60 Cells ; Humans ; Leukemia/genetics* ; Membrane Proteins/genetics* ; Mitochondrial Proteins/genetics* ; RNA Splicing ; U937 Cells ; beta-Lactamases/genetics*

OBJECTIVE: To investigate the effects of high dose vitamin C on proliferation and apoptosis of acute myeloid leukemia (AML) cell lines including HL-60, U937 and primary CD34 leukemia cells in AML. METHODS: CD34 cells were sorted by using immunomagnetic cell sorting system, then the primary CD34 leukemia cells, including HL-60 and U937 cell lines were cultured in vitro. Cells in each group were treated with different concentrations of vitamin C, the survival rate of cells was determined by MTT assay, the apoptosis rate of cells was evaluated by Annexin V/PI double staining, the expression of apoptotic proteins-including cleaved caspase 3, cleaved caspase-9 and cleaved PARP were detected by Western blot. RESULTS: The proliferation of HL-60 and U937 cells could be inhibited by high dose vitamin C, which showed a concentration-dependent manner (r=-0.9664; r=-0.9796). HL-60 and U937 cells were treated with different concentrations of vitamin C (8 and 20 mmol/L) for 24 hours, respectively, it was found that with the increasing of vitamin C concentration, cell apoptosis rate was significantly increased (r=0.9905; r=0.9971), and the expression of apoptosis related proteins including cleaved caspase 3, cleaved caspase-9 and cleaved PARP was aslo significantly increased with the increasing of concentration. In addition, it was found that with or without the mutation of TET2, high dose vitamin C could inhibit the proliferation (r=-0.9719; r=-0.9699) and promote the apoptosis (r=0.9998; r=0.9901) of primary CD34 leukemia cells in AML, which showed a dose-dependent manner, but it showed no effect on the proliferation (r=-0.2032) and apoptosis (r=0.1912) of normal CD34 cells. CONCLUSION High dose vitamin C can inhibit the proliferation and promote the apoptosis of acute myeloid leukemia cells, and selectively kill primary CD34 leukemia cells in AML.
Apoptosis ; Ascorbic Acid ; Cell Proliferation ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; U937 Cells

BACKGROUND: The roots of Scutellaria baicalensis Georgi (Labiatae) have been widely used in traditional medicine for treatment of various diseases. In this study, we investigated the effects of ethanol extracts of S. baicalensis roots (EESB) on the growth ofn human leukemia U937 cells. METHODS: The effect of EESB on cell viability was measured by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apoptosis was determined using 4,6-diamidino-2-phenyllindile staining and flow cytometry. The effects of EESB on the expression of regulatory proteins of apoptosis and phosphatidyl inositol 3-kinase (PI3K)/Akt signaling were determined by Western blotting. Caspase activity and mitochondrial membrane potential (MMP) were measured using flow cytometric analysis.
Apoptosis ; Blotting, Western ; Caspase 8 ; Caspase 9 ; Cell Survival ; Down-Regulation ; Ethanol ; Flow Cytometry ; Humans ; Leukemia ; Ligands ; Medicine, Traditional ; Membrane Potential, Mitochondrial ; Phosphatidylinositols ; Receptors, Death Domain ; Scutellaria baicalensis ; Scutellaria ; U937 Cells ; Up-Regulation

OBJECTIVE: To investigate the effect of Pim1 expression up-regulation on cell proliferation, apoptosis, chemotaxis and angiogenesis in acute myeloid leukemia (AML) cell line U937, and to explore the possible molecular mechanism involved, finally to estimate the Pim1 expression in primary AML cells. METHODS: GFP-tagged plasmid for Pim1 overexpression and an empty vector plasmid were constructed, and then a stable Pim1 expressed U937 cell line and a control virus-infected U937 cell line were established by a lentiviral vector system. After confirming Pim1 overexpression in U937 cells, proliferation and apoptosis are determined by CCK-8 Kit and flow cytometry respectively. Transwell chemotaxis assay was used to measure the effect of Pim1 overexpression on AML cells. Flow cytometry and confocal microscopy were applied to detect the influence of Pim1 overexpression on phosphorylated CXCR4 (pCXCR4) and its location. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression of angiogenesis and adhesion related genes in AML primary cells. RESULTS: The lentivirus-infected AML cell line with Pim1 overex-pression and the control virusinfected AML cell line were established successfully. The Pim1 overexpression could enhance the proliferation and inhibit the cell apoptosis, moreover accompnied with the increasing expression of cyclin D1, phosphorylated BAD (pBAD) and pCXCR4. After SDF-1 α stimuli, Pim1 overexpression induced AML cell chemotaxis accompanied with p-CXCR4 expression and calcium influx increment. Pim1 overexpression has no effect on angiogenesis. Pim1 mRNA expression was significantly higher in AML patients than the healthy people. CONCLUSION Pim1 plays an important role in the pathogenesis of AML, which not only promotes AML cell proliferation and inhibition of apoptosis, but also enhances the chemotactic ability of leukemia cells, which closely relates with Pim1 phosphorylation of CXCR4 and the increase of intracellular calcium ion influx signals.
Apoptosis ; Cell Proliferation ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Proto-Oncogene Proteins c-pim-1 ; genetics ; Signal Transduction ; U937 Cells

OBJECTIVETo investigate the effect of all transretinoicacid(ATRA) combined with decitabine (5-Aza-2'-deoxycytidine;DAC) on DNA methylation and gene expression of p16INK4a (p16) and retinoic acid receptor β (RARβ), and to explore their combined anti neoplastic effect on U937 cells and newly diagnose delder acute myeloid leukemia(AML) patients.

METHODSThe expression levels of p16 and RARβ were determined by qRT-PCR and Western blot. Methylation-specific PCR was used to analyze their methylation status. WST-1 and flow cytometry were performed to detect growth inhibition, differentiation, apoptosis and cell cycle of U937 cells respectively.

RESULTSThe expression p16 and RARβ was down-regulated by promoter hypermethylation in newly diagnose delder AML patients and U937 cells. Combination treatment of ATRA and DAC induced DNA hypomethylation as well as gene expression of p16 and RARβ, which contributed to the growth inhibition, differentiation, apoptosis and cell cycle arrest of U937 cells. In addition for elder AML patients intolerable to standard chemotherapy, the combination regimen of ATRA and DAC showed antineoplastic activity accompamied by up-regulation of p16 and RARβ expression and decrease of bone marrow blast, moreover the parients showed good tolerence to the reginen.

CONCLUSIONThe regimen of ATRA combined with DAC as the combination therapeutic strategy for inducing differentiation and demethylation possesses the anti-AML potency, and contributes to optimizing the therapeutic strategy for elder AML patients and promoting the clinical prognosis.

Leucocyte extravasation has been known to play an important role in inflammatory reactions including contact dermatitis. Previous studies suggested that CD99 regulates β1 integrin activity and may be a novel therapeutic target molecule for inflammatory diseases. In this study, the effects of CD99-derived peptide, CD99CRIII3, on inflammatory reactions in contact dermatitis mouse model were investigated. CD99CRIII3 decreased β1-integrin activity in human monocytic U937 cells. CD99CRIII3 inhibited the adhesion of U937 monocytes to human umbilical vein endothelial cells and their extravasation through human umbilical vein endothelial cells. CD99CRIII3 reduced inflammation in the phorbol myristate acetate-induced contact dermatitis mice in a dose-dependent manner. These results indicate that CD99CRIII3 suppresses the extravasation of monocytes and inflammatory reactions in the animal model of the contact dermatitis, suggesting that CD99CRIII3 could be a new drug candidate against inflammatory skin diseases.
Animals ; Dermatitis, Contact* ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Mice* ; Models, Animal ; Monocytes* ; Myristic Acid ; Skin Diseases ; U937 Cells