Phosphoinositide 3-Kinase/Akt and Nuclear Factor-κB Are Involved in Staphylococcus Aureus-induced Apoptosis in U937 Cells

Objective To explore the mechanisms involved in Staphylococcus aureus （S. aureus） invading human monocytic U937 cells. Methods S. aureus were added to U937 cells at multiplicity of infections （MOI） of 20：1 for 0, 15, 30, 60, and 90 minutes, respectively. Cell apoptosis was analyzed with Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate （FITC）/propidium iodide （PI） flow cytometry analysis. Akt and nuclear factor-κB （NF-κB） activities were detected by Western blotting. Results Infection of U937 cells with S. aureus induced rapid cell death in a time-dependent manner, and the cells displayed characteristic features of apoptosis. S. aureus-induced apoptosis was associated with a prominent downregulation of activated （phosphorylated） Akt and NF-κB. The inhibition of phosphorylated Akt by LY294002 led to the inhibition of NF-κB in a dose-dependent manner. Inhibition of Akt with LY294002 caused further increase in apoptosis of U937 cells. Conclusions S. aureus can stimulate the apoptosis of U937 ceils. S. aureus induces apoptosis of U937 cells by inhibiting Akt-regulated NF-κB.

Resveratrol Inhibits the Secretion of Vascular Endothelial Growth Factor and Subsequent Proliferation in Human Leukemia U937 Cells

This study examined the effect of resveratrol on the secretion of vascular endothelial growth factor （VEGF） and subsequent proliferation of human leukemia U937 cells, and explored the mechanisms involved. Human leukemia U937 cells were treated with resveratrol of different concentrations （12.5-200 μmol/L） for different time lengths （12-48 h）. The proliferation of the U937 leukemic cells was determined by MTT assay. Apoptosis was observed by Annexin- V-FIFC/PI double staining and flow cytometry （FCM）. Cells cycle was analyzed by PI staining and FCM. The content of VEGF was determined by ELISA. Human umbibical vein endothelial cells were examined for vasoformation in vitro after exposures to resveratrol of various concetrations. The results showed that resveratrol inhibited the proliferation of U937 leukemia cells in a dose- and time-dependent manner. Resveratrol induced apoptosis and S-phase cell cycle arrest in human leukemic U937 cells. Resveratrol inhibited the secretion of VEGF in U937 cells. Resveratrol inhibited the vasoformation of human vein endothelial cells in a dose-dependent manner. It was concluded that resveratrol could down-regulate the secretion of VEGE induce apoptosis and suppress the proliferation of U937 cells.

Inhibiting effect of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides on VEGF expression in U937 cells

Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.

Phagocytosis of IgA Immune Complexes by Human U937 Cells

In order to study FcαRⅠ mediated phagocytosis of IgA immune complexes by U937 cells, antigen 8.9NIP/BSA was labeled with FITC and reacted with anti-NIP IgA or anti-NIP IgG antibody to form immune complexes (ICs). They were then incubated with phorbol 12-myristate 13-acetate (PMA) stimulated U937 cells.The phagocytosed ICs were quantified by flow cytometry. The results was that the expression of FcαRⅠ on U937 cells was higher than that of FcγRⅠ, FcγRⅡ and FcγRⅢ. After stimulation by PMA, expression of FcαRⅠ on U937 cells was markedly upregulated and the phagocytosis of IgA ICs was enhanced. FcαRⅠ mediated specific IgA phagocytosis was stronger than FcγRⅠ and FcγRⅡ mediated IgG phagocytosis. Complement receptors, CR1 and CR3, enhanced U937 cell phagocytosis of IgA ICs. It concludes that FcαRⅠ mediated strong phagocytosis of IgA ICs.

Effect of Danhong injection on expressions of macrophage scavenger receptor 1 and sub-family A member 1 in human U937 cells

OBJECTIVE： To study the effects of Danhong injec- tion （DHI） on expression of the macrophage scaven- ger receptor 1 （MSR1） and ATP-binding cassette, sub-family A member 1 （ABCA1） genes, which en- code scavenger receptor-A I （SR-AI） and ATP-bind- ing cassette transporter 1 （ABCA1）, respectively, as a potential anti-atherosclerotic mechanism. METHODS： Human U937 cells were stimulated by in- cubation with 100 nM phorbo112-myristate 13-ace- tate （PMA） for 48 h.These stimulated, monocyte-like cells were then incubated for 24 h with 50 mg/L oxi- dized low-density lipoprotein （ox-LDL, to induce foam cell formation）, together with a liver X recep- tor （LXR） agonist or with different DHI concentra- tions. MSR1 and ABCA1 mRNA levels were mea- sured by fluorescence-based quantitative PCR. RESULTS： Compared with control cells （which re- ceived only ox-LDL）, cells treated with both ox-LDL and 10 IJmol/L LXR agonist showed lower MSR1 ex-pression （but this effect was not statistically signifi- cant, P〉0.05） and higher ABCA1 expression （P〈 0.01）. Cells that received ox-LDL and 3 mL/L DHI possessed higher MSR1 mRNA levels than the con- trols, whereas cells treated with ox-LDL and higher DHI concentrations （10, 30 or 60 mL/L） showed low- er MSR1 expression levels （but the differences ob- served between DHI concentration groups were not statistically significant, P〉0.05）. ABCA1 expression in cells treated with ox-LDL and 3, 10 or 30 mL/L DHI was higher than in the control cells, and increased with increasing DHI concentration （P〈0.05）. ABCA1 expression in cells treated with ox-LDL and the highest DHI concentration tested （60 mL/L） was not significantly different from that in the controls. ABCA1 mRNA levels in cells treated with ox-LDL and DHI were similar to, or lower than, those in cells treated with ox-LDL and the LXR agonist. CONCLUSION： DHI does not affect MSR1 mRNA lev- els in ox-LDL-treated U937 cells. However, at certain concentrations （10 and 30 mL/L）, DHI significantly increases ABCA1 mRNA levels. Therefore, the an- ti-atherosclerotic action of DHI might be mediated by an increased expression of ABCA1.

Study on the bone marrow mesenchymal stem cells induced drug resistance in the U937 cells and its mechanism

Background The hematopoietic microenvironment （HM） plays a critical role in malignant cell growth, patient survival, and response to chemotherapy in hematologic malignancies. However, mechanisms associated with this environmental influence remain unclear. In this study, we investigated the role of bone marrow derived mesenchymal stem cells （MSCs） in U937 cell line, the MSCs. to find out the relations between leukemia drug resistance and Methods U937 cells were cultured in suspension or grew adherently with MSCs. The cell growth curve was drawn and the cell cycle was measured by flow cytometry. Apoptosis and sensitivity of U937 to daunoblastina （DNR） were quantified by DNA ladder detection and trypan blue exclusion assays, respectively. The gene expression profile chip technology was used to determine and analyze the changes in apoptosis-related gene expression after adherent culture and the expression of MDR 1 mRNA was assessed by reverse transcriptional polymerase chain reaction （RT-PCR） at the same time. Results In the adherent culture, the proliferation of the U937 cells was inhibited, the G0/G1 phase cells increased （F=64.9726, P〈0.0001）, G2/M phase cells were decreased （F=98.1361, P〈0.0001） and the natural apoptosis rate was decreased （F=24.0866, P〈0.0001） compared with those in the suspended culture. U937 cell viability was enhanced and cell apoptosis was blocked during DNR treatment in adherent culture with MSCs. Thirty-nine differently expressed genes were screened from the 487 apoptosis related genes in the adherent culture U937 cells. Among the 37 upregulated genes, Bcl-XL was upregulated most significantly. Two genes were downregulated. Adherent culture did not induce MDR1 mRNA expression in U937 cells. Conclusions MSCs play a role in modulating the proliferation of U937 cells and response of U937 cells to DNR, and Bcl-XL apoptosis-inhibiting gene may be most important in determining the sensitivity of leukemic cells to treatment, which is not related to MDR1.

Effect of Caffeic acid on the Tumor Cells U937 Evaluated by an Electrochemical Voltammetric Method

Electrochemical voltammetric method can;be used to monitor cell health state during its growth. Here we studied the effect of caffeic acid on leukemia cells U937 by the voltammetric behavior of the cells. The result showed that this drug had a negative influence on cell health. which suggests that caffeic acid may be used in inhibition of tumor cells.

C-jun N-terminal Kinase-mediated Signaling Is Essential for Staphylococcus Aureus-induced U937 Apoptosis

Objective To investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism. Methods The human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting. Results S. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells. Conclusions S. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Proteomic profile of human monocytic cells infected with dengue virus

Objective: To identify the changes in the proteome of U937 cells infected with dengue virus(DENV).Methods: In this study, differentiated U937 cultures were infected with two DENV-2strains, one of which was associated with dengue(DENV-2/NG) and the other one with severe dengue(DENV-2/16681), with the aim of determining the cellular proteomic profiles under different infection conditions. Cellular proteins were extracted and separated by two-dimensional electrophoresis, and those proteins with differential expression profiles were identified by mass spectrometry. The obtained results were correlated with cellular viability, the number of infectious viral particles, and the viral DNA/protein quantity.Results: In comparison with non-infected cultures, in the cells infected with the DENV-2/NG strain, nine proteins were expressed differentially(five were upregulated and four were downregulated); in those cultures infected with the DENV-2/16681 strain, six proteins were differentially expressed(two were downregulated and four were upregulated). The downregulated proteins included fatty acid-binding protein, heterogeneous nuclear ribonucleoprotein 1, protein disulfide isomerase, enolase 1, heat shock 70 k Da protein 9, phosphotyrosyl phosphatase, and annexin IV. The upregulated proteins included heat shock 90 k Da protein AA1, tubulin beta, enolase 1, pyruvate kinase,transaldolase and phospholipase C-alpha.Conclusions: Because the monocyte/macrophage lineage is critical for disease pathogenicity, additional studies on these proteins could provide a better understanding of the cellular response to DENV infection and could help identify new therapeutic targets against infection.

The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation

BACKGROUND: CD14 was first described as a differentia- tion antigen on the surface of myeloid lineage cells. It acts as a glycosylphosphatidylinositol ( GPI)-anchored receptor for the complex of lipopolysaccharide (LPS) and plays a key role in the activation of LPS-induced monocytes. The purpose of this study was to observe the expression of CD14 protein and its gene in the human U937 promonocytic cell line when these cells were exposed to 1,25-dihydroxyvita- min D3 ( VitD3 ) and investigate their sensitivity to endo- toxin stimulation. METHODS: U937 cells were exposed to (0.1 μmol) VitD3 for 24 hours and were induced to express the CD14 mRNA gene and CD14 protein, then their responses were observed when they were stimulated with different concentrations of LPS for different time. RESULTS: The U937 cells induced by VitD3 were found to stably express CD14 mRNA and CD14 protein. And CD14 protein enhanced the sensitivity of U937/CD14 cells to li- popolysaccharide ( LPS ) stimulation. NF-ΚB in U937/ CD14 cells can be activated with low concentration of LPS (1 ng/ml-10 ng/ml), the TNF-α mRNA gene was in- duced , and then TNF-α was produced and released into the supernatant of culture. CONCLUSION: VitD3 can induce U937 cell to express the CD14 gene and CD14 protein and enhance the response of this type of cells to LPS stimulation.

Effect of N-tosyl-L-phenylalanylchloromethyl Ketone on Tumor Necrosis Factor-alpha-induced NF-κB Activation and Apoptosis in U937 Cell Line

Summary: To investigate the effect of N-tosyl-L-phenylalanylchloromethyl ketone (TPCK) on tumor necrosis factor-alpha-induced NF-κB activation and apoptosis in U937 cell line, changes and subcellular localization of NF-κB/p65 and IκB-α were observed by fluorescencemicroscopy and expression and degradation of IκB-α by flow cytometry. The apoptosis of U937 cells was measured by flow cytometry and electrophoresis of DNA. Immunolfluorescence assay showed that NF-κB/p65, IκB-α only localized in cytoplasm. After TNF-α stimulation, p65 was localized only in nuclei, and IκB-α was only localized in cytoplasm and decreased. The changes of TNF-α stimulation were specifically inhibited by TPCK. Flow cytometry also revealed the downregulation of IκB-α protein during TNF-α-induced apoptosis and the down-regulation was specifically inhibited by TPCK. Flow cytometry also showed the apoptosis of U937 cells after TNF-α induction. DNA ladder can be detected in cells treated by TNF-α. It is concluded that degradation of IκB-α protein and NF-κB/p65 translocation occur during TNF-α-induced apoptosis of U937 cells, suggesting the activation of NF-κB. TPCK-sensitive protease plays an important role in the degradation of IκB-α protein induced by TNF-α in U937 cells. TPCK sensitive protease also plays an important role in the apoptosis of U937 cells induced by TNF-α.

The Study of Anti-tumor Activity of Trichosanthin by Cyclic Voltammogram

The anti-tumor activity of Trichosanthin (TCS) has been frequently reported in recent years. In our experiments, electrochemical methods were applied to detect the effects of TCS on human leukemia cells U937. 50 mu g/ml TCS treatment for 40 hours can cause irreversible negative effects on the viability of U937 cells. This effect largely depends on the concentration of TCS and the time period of treatment.

The Establishment and Validation of the Human U937 Cell Line as a Cellular Model to Screen Immunomodulatory Agents Regulating Cytokine Release Induced by Influenza Virus Infection

Severe influenza infections are often associated with the excessive induction of pro-inflammatory cytokines,which is also referred to as"cytokine storms".Several studies have shown that cytokine storms are directly associated with influenzainduced fatal acute lung injury and acute respiratory distress syndrome.Due to the narrow administration window,current antiviral therapies are often inadequate.The efforts to use immunomodulatory agents alone or in combination with antiviral agents in the treatment of influenza in animal models have resulted in the achievement of protective effects accompanied with reduced cytokine production.Currently,there are no immunomodulatory drugs for influenza available for clinical use.Animal models,despite being ideal to study the anti-inflammatory responses to influenza virus infection,are very costly and time-consuming.Therefore,there is an urgent need to establish fast and economical screening methods using cellbased models to screen and develop novel immunomodulatory agents.In this study,we screened seven human cell lines and found that the human monocytic cell U937 supports the replication of different subtypes of influenza viruses as well as the production of the important pro-inflammatory cytokines and was selected to develop the cell-based model.The U937 cell model was validated by testing a panel of known antiviral and immunomodulatory agents and screening a drug library consisting of 1280 compounds comprised mostly of FDA-approved drugs.We demonstrated that the U937 cell model is robust and suitable for the high-throughput screening of immunomodulators and antivirals against influenza infection.

N-Terminal Truncation of TACO Inhibits PMA-Induced U937 Cell Adhesion

The effect of TA001-299, the N-terminal truncation of TACO, on phorbol 12-myristate 13-acetate （PMA）-induced U937 cell adhesion was investigated. Full-length TACO and several truncations were overexpressed in U937 cells. The effects of the expressed proteins on U937 cell adhesion mediated by PMA-induced differentiation were observed by fluorescence microscopy. The results show that the overexpression of TACO1-299 inhibits cell adhesion while overexpressions of the other proteins do not have this effect. The actin-binding capability of TACO1-299 was investigated and the results show that TACO1-299 lacks the ability of TACO to bind F-actin. The inhibitive effect of TACO1-299, the functional domain of TACO, suggests that TACO may play a role in cell differentiation mediating adhesion of monoblastic leukemia cells.