-
Biological & Pharmaceutical Bulletin Jul 2007Beta-sitosterol is the main dietary phytosterol found in plants and has been shown to inhibit proliferation and induce apoptosis in human solid tumors such as colon and...
Beta-sitosterol is the main dietary phytosterol found in plants and has been shown to inhibit proliferation and induce apoptosis in human solid tumors such as colon and breast cancers. However, the mechanism by which beta-sitosterol induces apoptosis is not completely understood in leukemic cells. This study investigated the mechanism of apoptosis induced by beta-sitosterol in human leukemic U937 cells. beta-Sitosterol induced cytotoxicity and apoptosis in U937 cells in a concentration dependent manner, as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis, and flow cytometry analysis. The increase in apoptosis induced by beta-sitosterol was associated with down-regulation of Bcl-2, degradation of poly-(ADP-ribose) polymerase (PARP) and phospholipase C (PLC)-gamma1 protein, and activation of caspase-3. beta-Sitosterol induced apoptosis was not associated with changes in the expression of Bcl-xL, Bax, or inhibitor of apoptosis proteins (IAPs). z-DEVD-fmk, a caspase-3 specific inhibitor, blocked caspase-3 activation and PARP degradation, and significantly attenuated beta-sitosterol-induced apoptosis. This suggests that caspase-3 activation is partially essential for beta-sitosterol-induced apoptosis. Bcl-2 overexpression also significantly blocked caspase-3 activation and the decrease in PARP cleavage by beta-sitosterol, and effectively attenuated the apoptotic response to beta-sitosterol. These results show that beta-sitosterol potently induces apoptosis in U937 cells and that beta-sitosterol-induced apoptosis is related to the selective activation of caspase-3 and induction of Bax/Bcl-2 ratio.
Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Cell Proliferation; Enzyme Activation; Humans; Proto-Oncogene Proteins c-bcl-2; Sitosterols; U937 Cells; bcl-2-Associated X Protein
PubMed: 17603173
DOI: 10.1248/bpb.30.1317 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... May 2023To 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...
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. 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. 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 (<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) %] (<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 (<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) (<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) (<0.05) . 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.
Topics: Humans; U937 Cells; RUNX1 Translocation Partner 1 Protein; Leukemia; Core Binding Factor Alpha 2 Subunit; Oncogene Proteins, Fusion; Leukemia, Myeloid, Acute
PubMed: 37550185
DOI: 10.3760/cma.j.issn.0253-2727.2023.05.003 -
Cancer Immunology, Immunotherapy : CII Mar 2012Severe immune suppression is frequent in late-stage tumor patients and promotes tumor immune evasion and subsequent tumor progression. Regulatory T cells (Treg) are... (Clinical Trial)
Clinical Trial
Severe immune suppression is frequent in late-stage tumor patients and promotes tumor immune evasion and subsequent tumor progression. Regulatory T cells (Treg) are major suppressors of anti-tumor immune responses. Therefore, targeting of Treg has become a key goal of anti-tumor therapy. Several preclinical and clinical observations suggest that Treg can be depleted by cyclophosphamide. Over a period of 3 months, we investigated the effect of metronomic low-dose cyclophosphamide on Treg numbers, suppressive capacity and proliferation on endogenous anti-tumor T-cell responses and on their correlation to clinical outcome in 12 patients with treatment-refractory metastasized breast cancer who received single-agent 50 mg cyclophosphamide p.o. daily. Cyclophosphamide treatment initially caused a significant reduction in circulating Treg by more than 40% (P = 0.002). However, Treg numbers completely recovered during the treatment due to increased proliferative activity and maintained their suppressive capacity. Treg depletion coincided with a strong increase in breast tumor-reactive T cells (P = 0.03) that remained at high levels during the whole period. Numbers of tumor-reactive T cells but not of Treg correlated with disease stabilization (P = 0.03) and overall survival (P = 0.027). We conclude that metronomic low-dose cyclophosphamide only transiently reduces Treg but induces stable tumor-specific T-cell responses, which correlate with improved clinical outcome in advanced-stage breast cancer patients.
Topics: Adult; Aged; Antineoplastic Agents, Alkylating; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cyclophosphamide; Dose-Response Relationship, Drug; Female; Humans; Interferon-gamma; Lymphocyte Count; Middle Aged; Neoplasm Metastasis; Survival Analysis; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Time Factors; Treatment Outcome; U937 Cells
PubMed: 21915801
DOI: 10.1007/s00262-011-1106-3 -
Molecular Medicine Reports May 2016Neutrophil elastase (NE) is a neutrophil‑derived serine proteinase with specificity for a broad range of substrates. NE has been reported to be associated with the...
Neutrophil elastase (NE) is a neutrophil‑derived serine proteinase with specificity for a broad range of substrates. NE has been reported to be associated with the pathogenesis of several conditions, particularly that of pulmonary diseases. Previous studies have shown that NE can cleave the pro‑myelocyte ‑ retinoic acid receptor‑alpha chimeric protein and is important for the development of acute pro‑myelocytic leukemia. To further elucidate the role of NE in acute pro‑myelocytic leukemia, the present study successfully constructed a lentiviral vector containing the NE gene (LV5‑NE), which was transfected into NB4 acute pro‑myelocytic leukemia cells. The effects of NE overexpression in NB4 cells were detected using a Cell-Counting Kit‑8 assay, flow cytometry and western blot analysis. The results showed that NE significantly promoted the proliferation of NB4 cells, inhibited cell apoptosis and apoptotic signaling, and led the activation of Akt. In an additional experiment, a vector expressing small hairpin RNA targeting NE was constructed to assess the effects of NE knockdown in U937 cells. Western blot analysis revealed that apoptotic signaling was increased, while Akt activation was decreased following silencing of NE. The results of the present study may indicate that NE activates the phosphoinositide-3 kinase/Akt signaling pathway in leukemia cells to inhibit apoptosis and enhance cell proliferation, and may therefore represent a molecular target for the treatment of pro‑myelocytic leukemia.
Topics: Apoptosis; Cell Proliferation; Enzyme Activation; Humans; Leukemia; Leukocyte Elastase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; U937 Cells
PubMed: 27035679
DOI: 10.3892/mmr.2016.5051 -
Blood Feb 1990Macrophage colony-stimulating factor (recombinant human M-CSF) given as a single intravenous injection to Lewis rats induces a dose-dependent peripheral monocytosis,...
Macrophage colony-stimulating factor (recombinant human M-CSF) given as a single intravenous injection to Lewis rats induces a dose-dependent peripheral monocytosis, neutrophilia, and lymphopenia. The monocytosis peaks at 28 to 32 hours with a seven- to eightfold increase in the number of circulating monocytes and promonocytes. The peripheral monocytosis is accompanied by a slight increase in marrow blasts, promonocytes, and monocytes. A monocytopenia reaching a nadir at 15 minutes precedes the monocytosis, suggesting that M-CSF activates circulating monocytes and causes intravascular margination. The M-CSF-induced neutrophilia and lymphopenia are relatively mild in magnitude, are observed between 2 and 16 hours after injection, and are no longer evident at later time-points. The monocytosis was at least partially inhibited by dexamethasone. M-CSF-induced monocytosis most likely reflects a direct effect of M-CSF on marrow monocyte precursor proliferation, maturation, and release, whereas the neutrophilia and lymphopenia may reflect indirect effects mediated by the known ability of M-CSF to cause the release of other cytokines.
Topics: Animals; Cell Count; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Injections, Intravenous; Leukocytes; Lymphocytes; Macrophage Colony-Stimulating Factor; Male; Monocytes; Neutrophils; Rats; Rats, Inbred Lew; Recombinant Proteins
PubMed: 2405921
DOI: No ID Found -
The Journal of Biological Chemistry Aug 2005The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display...
The parasitic protozoan Leishmania specifically manipulates the expression of host macrophage genes during initial interactions, as revealed by mRNA differential display reverse transcription-PCR and cDNA microarray analyses. The genes that are down-regulated in mouse (J774G8) or human (U937) macrophages upon exposure to Leishmania include small RNA transcripts from the short interspersed element sequences. Among the short interspersed element RNAs that are down-regulated is 7SL RNA, which is the RNA component of the signal recognition particle. Because the microbicidal functions of macrophages profoundly count on vesicular protein transport processes, down-regulation of 7SL RNA may be significant in the establishment of infection by Leishmania in macrophage phagolysosomes. To evaluate whether down-regulation of 7SL RNA results in inhibition of signal recognition particle-mediated vesicular protein transport processes, we have tested and found that the targeting of proteins to the endoplasmic reticulum and plasma membrane and the secretion of proteins by macrophages are compromised in Leishmania-infected J774G8 and U937 cells. Knocking down 7SL RNA using small interfering RNA mimicked the effect of exposure of macrophages to Leishmania. The overexpression of 7SL RNA in J774G8 or U937 cells made these cells resistant to Leishmania infection, suggesting the possible biological significance of down-regulation of 7SL RNA synthesis in the establishment of infection by Leishmania. We conclude that Leishmania down-regulates 7SL RNA in macrophages to manipulate the targeting of many proteins that use the vesicular transport pathway and thus favors its successful establishment of infection in macrophages.
Topics: Animals; Cell Differentiation; Cell Membrane; DNA, Complementary; Down-Regulation; Endoplasmic Reticulum; Gene Expression Profiling; Gene Expression Regulation; Humans; Leishmania; Leishmaniasis; Macrophages; Mice; Protein Transport; RNA; RNA, Messenger; RNA, Small Cytoplasmic; RNA, Small Interfering; Reverse Transcriptase Polymerase Chain Reaction; Signal Recognition Particle; Signal Transduction; Time Factors; Trypanosoma brucei brucei; U937 Cells; Up-Regulation
PubMed: 15955815
DOI: 10.1074/jbc.M504162200 -
Blood Feb 2006TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6, a frequent target of chromosome translocation in human leukemia. Although both proteins are...
TEL2/ETV7 is highly homologous to the ETS transcription factor TEL/ETV6, a frequent target of chromosome translocation in human leukemia. Although both proteins are transcriptional inhibitors binding similar DNA recognition sequences, they have opposite biologic effects: TEL inhibits proliferation while TEL2 promotes it. In addition, forced expression of TEL2 but not TEL blocks vitamin D3-induced differentiation of U937 and HL60 myeloid cells. TEL2 is expressed in the hematopoietic system, and its expression is up-regulated in bone marrow samples of some patients with leukemia, suggesting a role in oncogenesis. Recently we also showed that TEL2 cooperates with Myc in B lymphomagenesis in mice. Here we show that forced expression of TEL2 alone in mouse bone marrow causes a myeloproliferative disease with a long latency period but with high penetrance. This suggested that secondary mutations are necessary for disease development. Treating mice receiving transplants with TEL2-expressing bone marrow with the chemical carcinogen N-ethyl-N-nitrosourea (ENU) resulted in significantly accelerated disease onset. Although the mice developed a GFP-positive myeloid disease with 30% of the mice showing elevated white blood counts, they all died of T-cell lymphoma, which was GFP negative. Together our data identify TEL2 as a bona fide oncogene, but leukemic transformation is dependent on secondary mutations.
Topics: Alkylating Agents; Animals; Bone Marrow; Bone Marrow Transplantation; Carcinogens; Cell Transformation, Neoplastic; Ethylnitrosourea; Female; Gene Expression; HL-60 Cells; Hematopoiesis; Humans; Lymphoma, T-Cell; Male; Mice; Proto-Oncogene Proteins c-ets; Repressor Proteins; Transcription Factors; Translocation, Genetic; U937 Cells; ETS Translocation Variant 6 Protein
PubMed: 16234363
DOI: 10.1182/blood-2005-03-1196 -
IUBMB Life Aug 1999Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating...
Phosphorylation of the retinoblastoma protein (Rb) during the G1-phase of the mammalian cell division cycle is currently believed to be a controlling element regulating the passage of cells into S-phase. We find, however, that the suspension-grown cell lines U937, L1210, and MOLT-4 contain exclusively hyperphosphorylated Rb. Furthermore, when adherent NIH3T3 cells are grown at very low densities to avoid overgrowth and contact inhibition, they also contain only hyperphosphorylated Rb. NIH3T3 cells exhibit hypophosphorylation when the cells are grown at moderate to high cell densities. We propose that cultures of adherent cells such as NIH3T3, when grown to moderate cell densities, are made up of two populations of cells: (a) cells that are relatively isolated and therefore growing exponentially without contact inhibition, and (b) cells that are growth-inhibited by local cell density or contact inhibition. The common observation in adherent cell lines, that Rb is both hyper- and hypophosphorylated in the G1-phase and only hyperphosphorylated in the S- and G2-phases, is explained by the effects of cell density and contact inhibition. Thus, phosphorylation-dephosphorylation of Rb protein during the G1 phase is not a necessary process during the NIH3T3, L1210, MOLT-4, and U937 division cycles. We propose that phosphorylation-dephosphorylation of Rb is independent of the division cycle and is primarily determined by growth conditions throughout the division cycle.
Topics: 3T3 Cells; Animals; Cell Count; Cell Cycle; Cell Line; G1 Phase; Humans; Leukemia L1210; Mice; Phosphorylation; Retinoblastoma Protein; U937 Cells
PubMed: 10794602
DOI: 10.1080/713803488 -
American Journal of Hematology Mar 2008We previously described a subgroup of immune thrombocytopenic purpura (ITP) patients presenting with recurring transient ischemic attack-like symptoms and progressive...
We previously described a subgroup of immune thrombocytopenic purpura (ITP) patients presenting with recurring transient ischemic attack-like symptoms and progressive cognitive impairment due to small vessel disease (SVD) seen in the brain. They presented minimal bleeding despite thrombocytopenia, and platelet activation was elevated compared to classic ITP. On the hypothesis that the blood-brain barrier (BBB) is compromised in this subgroup, we investigated the effect of plasma from SVD-ITP patients on the transendothelial migration of leukocytes (TEML). Brain microvascular endothelial cells (BMVEC) were grown to confluence on 6.5-microm pore filters and plasma from 10 healthy controls, 20 classic ITP, and 5 SVD-ITP were added and incubated 24 hr. Then 1 x 10(5) monocytes (U937) were added and the number migrated through the EC monolayer after 6 hr was measured by flow cytometry. The effect on TEML of danazol was also assessed. We found that plasma from SVD-ITP but not classic ITP induced 10-fold rise in EC activation marker CD62E and a sevenfold increase in TEML, to 38.5% +/- 12.5% of cells migrated, compared to normal controls (5.6% +/- 1.2%) or classic ITP (6.1% +/- 0.2%), P < 0.001. Preincubation of U937 with endothelial microparticles (EMP) increased TEML by 20.0% +/- 6.4% with SVD-ITP plasma, significantly more than with classic ITP or control plasmas, P = 0.003. Pretreatment of cultures with danazol (100 microg/mL) inhibited TEML by 25% in all wells tested, whether or not EMP were added. In summary, SVD-ITP plasma activates EC and augments TEML, suggesting plasma-mediated BBB dysfunction in this syndrome. Danazol modestly but significantly inhibited TEML.
Topics: Adult; Aged; Brain Ischemia; Cell Migration Assays, Leukocyte; Cells, Cultured; Cerebrovascular Circulation; Endothelium, Vascular; Female; Humans; Male; Microcirculation; Middle Aged; Monocytes; Platelet Count; Purpura, Thrombocytopenic, Idiopathic; Recurrence; Reference Values; Splenectomy; U937 Cells
PubMed: 17876771
DOI: 10.1002/ajh.21061 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Oct 2017To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line...
To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot. ①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (<0.05) , respectively under ATRA exposure. ②The percentages of G(0)/G(1) stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure. ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G(0)/G(1) stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.
Topics: Cell Differentiation; Cell Proliferation; Humans; Mutation; Nuclear Proteins; Nucleophosmin; Tretinoin; U937 Cells
PubMed: 29166739
DOI: 10.3760/cma.j.issn.0253-2727.2017.10.008