RESUMO
The effects of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma were investigated. The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was constructed by inserting specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TFsiRNA-pSUPER was performed using lipofectamine2000. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest33342 and counted under fluorescence inverted microscope. It was found that human neuroblastoma cell line SK-N-MC expressed high level of TF. Knockdown of the TF expression was achieved by transfection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-dependent manner. Inhibition of TF significantly decreased the viability of transfected SK-N-MC cells treated with different concentrations of doxorubicin. Cleavage of Caspase-3 and PARP was enhanced in transfected SK-N-MC cells with down-regulation of TF. TFsiRNA treatment significantly increased the number of apoptotic cells in transfected SK-N-MC cells as compared with those control cells (P<0.05) when these cells were exposed to 1 mug/mL doxorubicin for 8 h. These results suggested that knockdown of the TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells. Over-expression of TF might contribute to chemotherapy resistance in human neuroblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.
Assuntos
Apoptose , Regulação para Baixo , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , RNA Interferente Pequeno , Tromboplastina/biossíntese , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de TempoRESUMO
The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.
Assuntos
Apoptose/genética , Arsenicais/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Óxidos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , SurvivinaRESUMO
OBJECTIVE: To explore the role of PTEN gene in the regulation of tissue factor (TF) expression in human neuroblastoma cells. METHODS: Expression of PTEN or TF was determined by Western blotting. Transcription of TF was examined by RT-PCR. PTEN gene expressing vector pCMV-PTEN was transfected with Lipofectamine2000. Phosphorylation of AKT was inhibited by LY294002 and then examined by Western blotting. RESULTS: Human neuroblastoma cell line SK-N-SH was PTEN-positive and expressed low level TF, whereas an other neuroblastoma cell line SK-N-MC was PTEN-negative but expressed high level TF. TF level was downregulated in SK-N-MC cells by enforced expression of PTEN in a dose dependent manner. Inhibition of TF was achieved along with inactivation of AKT. Furthermore treatment with PI3K/AKT inhibitor LY294002 also resulted in decrease of TF expression in a dose-dependent manner. CONCLUSION: Expression of TF is inhibited by PTEN gene via inactivating PI3K/AKT pathway, loss of PTEN might be the explanation of aberrant high-level TF in human neuroblastoma. It may be at least one of the mechanisms by which loss of PTEN expression confers to cancer progression.
Assuntos
Neuroblastoma/metabolismo , PTEN Fosfo-Hidrolase/genética , Tromboplastina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Neuroblastoma/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tromboplastina/genética , TransfecçãoRESUMO
Patients with multiple myeloma (MM) have increased bone marrow angiogenesis, but the angiogenic properties of myeloma cells and the mechanism of MM-induced angiogenesis have not been completely clarified. The brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, have been identified as critical factors in the regulation of vessel formation. In this study, we demonstrate that patients with MM had increased BDNF and vascular endothelial growth factor (VEGF) in their peripheral blood. We also found in particular that a decreased BDNF level was correlated with the remission of MM. BDNF was expressed by the human myeloma cell line RPMI8226 and primary myeloma cells, and TrkB was expressed by human umbilical vein endothelial cells (HUVEC) at the protein levels. In a coculture system, we observed that both RPMI8226 cells and primary myeloma cells induced the migration and formation of a net-like structure in HUVEC. The anti-BDNF monoclonal antibody significantly but partially restrained the angiogenesis effect of MM cells. Moreover, in an experimental model of angiogenesis in vivo, BDNF and VEGF significantly promoted vessel formation in Matrigel plug compared to the control. These effects were also blocked by anti-BDNF monoclonal antibody. Finally, our in vitro results were supported by the in vivo finding in human myeloma xenograft NOD/SCID models. Anti-BDNF mAb treatment resulted in inhibition of tumor growth, decreased vessel density, and tumor necrosis. Our study suggested that the BDNF/TrkB signaling pathway could be involved, at least in part, in MM-induced angiogenesis.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Mieloma Múltiplo/patologia , Neovascularização Patológica , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Pessoa de Meia-Idade , Modelos Biológicos , Transplante de Neoplasias , Receptor trkB/metabolismoRESUMO
To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.
Assuntos
Arsenicais/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Óxidos/farmacologia , Trombomodulina/biossíntese , Tromboplastina/biossíntese , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Trióxido de Arsênio , Humanos , Leucemia Promielocítica Aguda/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Trombomodulina/genética , Tromboplastina/genética , Tromboplastina/metabolismo , Células Tumorais CultivadasRESUMO
A new, convenient, and rapid method for kinetic measurement of human fibrinolysis was established. The alteration of absorbance (A) in the process of blood coagulation and lyses was automatically scanned and recorded using a UV2000 spectrophotometer connected to a computer. The parameters of human fibrinolysis kinetics were established. Urokinase at 20 U/mL was the optimal concentration used. There was significant difference in fibrinolysis kinetics and plasma plasminogen concentration between 22 normal subjects and 27 patients with acute myeloblastic leukemia (P<0.05 and <0.01 respectively). The coefficience of variation was (5.24+/-1.51)%. This method could also be used to measure the plasma fibrinogen concentration at the same time. It was concluded that this method was stable and was capable of providing dynamic, direct experimental data and multiparemeters for clinicians. It was also valuable in evaluating the anti-and pro-fibrinolytic capcity of patients' plasmas, allowing for monitoring of therapy, choice of drugs and adjustment of drug concentrations.
Assuntos
Protocolos Clínicos , Fibrinólise/fisiologia , Leucemia Mieloide Aguda/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Adolescente , Adulto , Coagulação Sanguínea/efeitos dos fármacos , Estudos de Casos e Controles , Linhagem Celular Tumoral , Venenos de Crotalídeos/análise , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , Monitoramento de Medicamentos , Feminino , Fibrinogênio/análise , Fibrinogênio/metabolismo , Fibrinólise/efeitos dos fármacos , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta , Trombina/análise , Trombina/metabolismo , Trombina/farmacologia , Fatores de Tempo , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/análise , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
OBJECTIVE: To investigate the regulation of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma. METHOD: The expression of TF was examined by Western blotting. TF siRNA-pSUPER plasmid was constructed by inserting a specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TF siRNA-pSUPER was performed using lipofectamine 2000. The activation of caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope. RESULTS: (1) Human neuroblastoma cell line SK-N-MC expressed high level of TF. (2) Downregulation of TF expression was achieved by transfection of TF siRNA-pSUPER into SK-N-MC cells in a dose-dependent manner. (3) Cleavage of caspase-3 and PARP was increased in transfected SK-N-MC cell with down-regulation of TF. (4) TF siRNA treatment at 1 microg/ml for 8 h significantly increased apoptotic cell number in transfected SK-N-MC cells compared to that in non-transfected cells (P < 0.05) while exposing to 1 microg/ml doxorubicin for 8 h. CONCLUSIONS: Downregulation of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells.
Assuntos
Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Neuroblastoma/patologia , Interferência de RNA , Tromboplastina/genética , Apoptose/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Neuroblastoma/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , Tromboplastina/metabolismo , TransfecçãoRESUMO
The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow microenvironment, which support proliferation and survival of malignant plasma cells. Previous study defined a brain-derived neurotrophic factor-tyrosine kinase receptor B (BDNF/TrkB) axis in myeloma and autocrine growth stimulation by BDNF in various tumor cells. We examined the biological effects of BDNF on MM cells. Using a reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry, we observed that both BDNF and its high-affinity receptor TrkB are expressed by MM cell lines (RPMI 8226, U266, and KM3) and primary MM cells. Functional studies revealed that BDNF was a potent growth factor for MM. BDNF (5-500 ng/mL) had strong proliferative effects on both MM cell lines and primary MM cells, shown by [(3)H]thymidine incorporation assay. BDNF (12.5-200 ng/mL) also induced migration of MM cells, as indicated by the Transwell migration assay. Together, our data indicate that BDNF is a potent myeloma growth and chemotactic factor and suggest that the BDNF/TrkB pathway is a potential therapeutic target in MM.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Divisão Celular/efeitos dos fármacos , Mieloma Múltiplo/patologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Resveratrol has been proposed to act as a chemopreventive agent in numerous epidemiologic studies and has been shown to inhibit proliferation of various tumor cells in vitro. In the present study, we investigated the antitumor effects of resveratrol on multiple myeloma (MM) cells and the mechanisms involved. Our findings indicated that resveratrol inhibited proliferation of tumor cells in a dose- [corrected] dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and [3H]thymidine incorporation assay. Resveratrol also enhanced the inhibitory effect of dexamethasone on the growth of MM cells by MTT assay. Flow cytometric analysis demonstrated that resveratrol arrested the cells at the G1 and S phases of the cell cycle. Because nuclear factor-kappaB (NF-kappaB) plays a key role in cell survival and proliferation of human MM cells, we tested the effect of resveratrol on NF-kappaB expression by Western blot analysis and immunofluorescence. NF-kappaB was constitutively active in all human MM cell lines examined, and resveratrol down-regulated NF-kappaB expression in all cell lines. Resveratrol also down-regulated the expression of NF-kappaB-regulated gene products by Western blot analysis, gelatin zymography, and enzyme-linked immunosorbent assay, including interleukin-6, Bcl-2, Bcl-xL, XIAP, c-IAP, vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9), which modulates an array of signals controlling cellular survival and proliferation and tumor promotion. Indeed, annexin V-fluoroisothyocyanate and Transwell invasion analyses revealed that incubation of MM cells with resveratrol resulted in apoptotic cell death and inhibition of invasion. In conclusion, these data suggest that resveratrol is an effective in vitro inhibitor of NF-kappaB in human MM cells. Resveratrol plays a role in suppressing the proliferation of MM cells and induces apoptosis, thus providing the molecular basis for the treatment of MM patients with this compound.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Mieloma Múltiplo/genética , NF-kappa B/genética , Estilbenos/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Humanos , Mieloma Múltiplo/patologia , Invasividade Neoplásica/prevenção & controle , ResveratrolRESUMO
To investigate the role of anti-inflammatory cytokine in acute coronary syndrome (ACS), the effect of IL-10 on expression of tissue factor (TF) induced by IL-6 in peripheral blood mononuclear cells (PBMNC) were studied. PBMNC were allowed to culture with rhIL-10 before being stimulated by rhIL-6. One-step recalcification clotting time was used to evaluate procoagulant activity (PCA) of PBMNC. The expression and activity of TF protein were determined by ELISA and cell chromogenic substrate assay. The results showed that the expression of PCA, TF protein and its activity in PBMNC increased significantly after being stimulated by rhIL-6 (P < 0.01). In PBMNC, rhIL-6-induced PCA was regulated by rhIL-10 in different doses. This effect was associated with reduction of TF protein expression and activity by rhIL-10 (P < 0.01). In conclusion, IL-10 down-regulated expression PCA and TF in PBMNC, inhibitory effect of IL-10 on expression and activity of PBMNC TF may be important protective mechanism for ACS, regulation imbalance between inflammatory and anti-inflammatory cytokines may be important factor participating in coronary thrombosis.
Assuntos
Interleucina-10/farmacologia , Interleucina-6/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Tromboplastina/biossíntese , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/genética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
In order to investigate the expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in multiple myeloma patients and the in vitro and in vivo proangiogenic effects of BDNF, the plasma concentrations of BDNF and VEGF in MM patients and control group were determined by ELISA, the effect of BDNF on the in vitro proliferation of human umbilical vein endothelial cells (HUVEC) was examined by MTT assay; the effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effect of BDNF on angiogenesis in vivo. The results demonstrated that the concentration of BDNF was (4.22 +/- 0.64) ng/ml and (2.03 +/- 0.38) ng/ml in MM group and control group, respectively, (P = 0.01). There was also a significant difference between VEGF levels of two groups [(79.35 +/- 13.25) pg/ml vs (34.41 +/- 1.78) pg/ml, P = 0.006]. The levels of BDNF and VEGF correlated significantly (r = 0.430, P = 0.025). BDNF stimulated the migration and tube formation in vitro significantly, although it had no effect on the proliferation of HUVEC. BDNF also stimulated angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. It is concluded that the concentrations of BDNF and VEGF in MM patients' peripheral blood are at high level; BDNF can stimulate the angiogenesis markedly in vitro and in vivo. Therefore, BDNF may act as an important regulator in angiogenesis of MM.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/sangue , Mieloma Múltiplo/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Indutores da Angiogênese/sangue , Indutores da Angiogênese/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/embriologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The plasma levels of inflammatory cytokine interleukin-6 (IL-6) and anti-inflammatory cytokine interleukin-10 (IL-10) in the patients with unstable angina or stable angina were determined and compared. In 30 patients with unstable angina and 22 patients with stable angina, plasma levels of IL-10 and IL-6 were detected by ELISA and plasma lipid parameters by lipid research clinical methods respectively. The results showed plasma levels of IL-10 were significantly lower in unstable angina group than in stable angina group (P = 0.005), while those of IL-6 were significantly increased in unstable angina group as compared with those in stable angina group (P = 0.039). There was a significantly negative correlation between IL-10 and IL-6 in patients with unstable angina (r = -0.41, P = 0.003). In the unstable angina group, IL-6 levels were obviously positively correlated with TC (r = 0.314, P = 0.023), but not with TG and HDL. There were no significant correlations between IL-10 and plasma lipid parameters. It was suggested that the decreased IL-10 and increased IL-6 might be associated with the atheromatous plaque stability and progression of coronary heart diseases. IL-10 may play an important role in preventing coronary vascular lesions.
Assuntos
Angina Instável/sangue , Interleucina-10/sangue , Interleucina-6/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To explore the role of tissue factor/activated factor VII (TF/FVIIa) complex in human ovarian cancer invasion and metastasis. METHODS: (1) Constructed an expression vector of TF, pcDNA3-TF and established a human ovarian cell line A2780/TF expressing high level TF by using molecular cloning and gene transfection techniques. (2) By Boyden chamber assay to count the numbers of A2780 and A2780/TF cells that penetrated the matrigel to the back of PVPF membrane after FVIIa stimulation. (3) BALB/c nude mice were used to establish experimental model of metastasis with A2780 or A2780/TF and the lung tissue sections were examined by microscopy for cancer metastasis. RESULTS: (1) Compared with their parental A2780 cells, A2780/TF cells expressed high level of TF mRNA (3.99 +/- 0.15 vs 0.97 +/- 0.23, P < 0.01) and TF antigen on cell surface \[(48.56 +/- 9.53)% vs (2.73 +/- 1.15)%, P < 0.01\]. (2) After stimulation, the A2780/TF cell number on the back of PVPF membrane increased from basal level 157.3 +/- 19.2 to 447.7 +/- 39.4 (P < 0.01), which could decreased to basal level when coincubated with anti-TF antibody. (3) Cancer metastasis was found in 22.2% of nude mice transplanted with A2780 cells, while in 88.9% of those transplanted with A2780/TF cells. CONCLUSION: TF could promote the invasion and metastasis of human ovarian cancer cells through TF/FVIIa pathway.
Assuntos
Fator VIIa/genética , Neoplasias Ovarianas/patologia , Tromboplastina/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Fator VIIa/fisiologia , Feminino , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Tromboplastina/fisiologia , Transfecção , Transplante HeterólogoRESUMO
Abnormal tissue factor (TF) expression on vascular endothelial cells may account for thrombotic events associated with cardiovascular disease. The transcription factor nuclear factor-kappa B (NF-kappa B) activation plays a key role in endothelial cell injury and TF expression. Disruption of NF-kappa B activation in endothelial cells may inhibit TF expression and be protective in thrombosis. The purpose of the study was to determine whether NF-kappa B transcription factor decoy (TFD) could block TF expression. NF-kappa B TFD was transferred into cultured human umbilical cord vein endothelial cells (HUVEC) by liposomes, and the transfection efficiency was detected by flow cytometry. The effect of NF-kappa B TFD on TF mRNA levels was determined by reverse transcription-polymerase chain reaction. The expression of surface TF antigen was analyzed by flow cytometry. TF activity was studied by measuring enzymatic activation of factor X by the TF-activated factor VII complex. The results suggested that NF-kappa B decoy could be successfully transferred into HUVEC by liposome. The NF-kappa B TFD competed with the endogenous kappa B site sequence in the TF promoter for binding to transcription factor NF-kappa B in tumor necrosis factor-alpha-stimulated HUVEC, which could block the tumor necrosis factor-alpha-induced increase in TF mRNA levels, the upregulation of surface TF antigen and TF activity. This study demonstrated that NF-kappa B decoy could block HUVEC TF gene expression. Targeted genetic disruption of endothelial TF expression by NF-kappa B decoy may provide a possible therapeutic method for cardiovascular and thrombosis disease.
Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Oligodesoxirribonucleotídeos/farmacologia , Tromboplastina/biossíntese , Sítios de Ligação , Ligação Competitiva , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator Xa/biossíntese , Humanos , Lipossomos , Oligodesoxirribonucleotídeos/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Sequências Reguladoras de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/genética , Transcrição Gênica/efeitos dos fármacos , Transfecção , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Veias UmbilicaisRESUMO
OBJECTIVE: To construct the expression vector of human tissue factor (TF), and investigate the influence of TF/coagulant factor VIIa (FVIIa) complex on the transcriptional expression of urokinase plasminogen activator (u-PA) and u-PA receptor (u-PAR) in human ovarian cancer. METHODS: The human TF cDNA was obtained from placenta by RT-PCR and then inserted into eukaryotic expression vector pcDNA3 to obtain the TF-pcDNA3 combinant. This combinant was transfected into human ovarian cancer cell line A2780 by lipofectamine. Stably-transfected cells A2780/TF were screened. A2780 and A2780/TF cell lines were stimulated by FVIIa respectively, and the transcriptional levels of u-PA and u-PAR were examined by RT-PCR. RESULTS: (1) The constructed product was identified as TF-pcDNA3 combinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780/TF cell (transfected cell 3.91 +/- 0.28, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. (3) The u-PA and u-PAR mRNA levels in A2780 cell line did not change significantly after stimulated by FVIIa; (4) While stimulated by FVIIa, the u-PAR mRNA levels in A2780/TF cells increased significantly in both dose-dependent and time-dependent manner, while the u-PA mRNA levels did not change significantly; (5) In the A2780/TF cell line the enhanced expression of u-PAR mRNA by FVIIa was significantly inhibited by coincubated with anti-TF antibody. CONCLUSION: TF/FVIIa complex could up-regulate the transcription of u-PAR in human ovarian cancer cells so as to enhance tumor invasion and metastasis.
Assuntos
Fator VIIa/metabolismo , Neoplasias Ovarianas/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Tromboplastina/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Fator VIIa/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Tromboplastina/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
In order to investigate the association of fibrin monomer polymerization function (FMPF) with traditional cerebrovascular risk factors and ischemic cerebrovascular disease in old people, 1:1 paired case-control comparative study was performed for FMPF and traditional cerebrovascular risk factors on 110 cases of old ischemic cerebrovascular disease and 110 controls matched on age, sex and living condition. The results showed that cerebrovascular risk factors were more prevalent in case group than in control group. In the case group, FMPF was significantly higher than in control group. There was a significant positive correlation between hypertension and fibrin monomer polymerization velocity (FMPV), hypertension and fibrinogen (Fbg), alcohol consumption and Fbg, but no significant correlation between diabetic mellitus, smoking and FMPF was found. Among the parameters of blood lipids, there were significant positive correlations between total cholesterol (TC) and parameters of FMPF to varying degrees, triglycerides (TG) and FMPV, TG and Fbg. Our results also showed there were significant linear trends between TC and FMPV (P < 0.001), TC and Fbg (P = 0.0087), TG and FMPV/Amax (maximum absorbance) (P = 0.0143) respectively. Multiple logistic regression analysis revealed that FMPF in case group remained significantly higher than control group after adjustment of all risk factors that were significant in univariate analysis. It was concluded that there is a possible pathophysiological link between FMPF and cerebrovascular risk factors. An elevated FMPF is associated with ischemic cerebrovascular disease and an independent risk factor of this disease. In old people, detection of FMPF might be a useful screening to identify individuals at increased cerebrothrombotic risk.
Assuntos
Infarto Cerebral/sangue , Transtornos Cerebrovasculares/sangue , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Hipertensão/complicações , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Polímeros , Fatores de RiscoRESUMO
The objective of this study was to explore tissue factor (TF) expression induced by TNF-alpha in cultured human umbilical vien endothelial cells (HUVEC) and its molecular mechanism. TF expression on the surface of HUVEC, TF mRNA and nuclear factor kappaB (NF-kappaB) in HUVEC were detected by flow cytometry, RT-PCR and Western blot respectively. The results showed that TNF-alpha could enhance TF expression on the surface of HUVEC, the TF expression increase was highly consistent with the increased synthesis of TF mRNA, and the increase of TF expression was lately appeared for several hours. It was also found activation of NF kappaB at the time TF mRNA increase. In conclusion, NF-kappaB could be activated promptly after HUVEC incubated with TNF-alpha, then it was bound to TF promotor to start the TF transcription, TF mRNA expression was upregulated, that leaded to the increase of TF expression on the HUVEC surface and activated the coagulation cascade.
Assuntos
Células Endoteliais/metabolismo , Tromboplastina/genética , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/análise , NF-kappa B/fisiologia , RNA Mensageiro/análise , Veias Umbilicais/metabolismoRESUMO
The objective of this study is to investigate the effect of vaccination with dendritic cells pulsed with survivin antigen on activation of antileukemic T cells, and inhibiting proliferation of leukemic cells. The expression of survivin on acute leukemic cells were detected by cofocal microscopy and immunoprecipitation-Western blot. DCs collected from peripheral blood mononuclear cells were pulsed with survivin purified proteins. Stimulation index (SI) and antileukemia CTL induction were analyzed with (3)H-TdR incorporation and (51)Cr releasing assay, respectively. The phenotype of T cells and DCs were identified by flow cytometry. By immunofluorescence of bone marrow and peripheral blood mononuclear cells, survivin expression was detected in 16 out of 19 AML cases (84.2%). The results showed that survivin fluorescence distribution was in cytoplasm. DCs from peripheral blood mononuclear cells were successfully induced, with typical DC morphologic characteristic. The vaccination with dendritic cells pulsed with survivin antigen dramatically stimulated the proliferation of T cells. The DCs loading survivin activated T cells with higher CD4(+) T(H) ratio as compared with DCs group, T cells activated with DCs expressed CD8 and CD56. Survivin DCs significantly inhibited the growth of leukemic cells in vitro. In conclusion, survivin antigen expressed in the cytoplasm of leukemic cells, leukemic vaccination with DCs pulsed with survivin antigen in vitro inhibited the proliferation of leukemic cells, that may be a pathway for therapy of leukemia.
Assuntos
Células Dendríticas/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Linfócitos T/imunologia , Adulto , Antígenos CD/análise , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Divisão Celular/imunologia , Linhagem Celular , Citotoxicidade Imunológica/imunologia , Células Dendríticas/transplante , Feminino , Citometria de Fluxo , Células HL-60 , Antígenos HLA-DR/análise , Humanos , Imunoterapia Adotiva , Proteínas Inibidoras de Apoptose , Células K562 , Leucemia Monocítica Aguda/tratamento farmacológico , Leucemia Monocítica Aguda/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/imunologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/imunologia , Masculino , Proteínas de Neoplasias , Survivina , Células Tumorais Cultivadas , Vacinação/métodosRESUMO
OBJECTIVE: To investigate the inhibitory effect of NF-kappaB decoy on tissue factor (TF) expression and FVII activation in cultured human umbilical vein endothelial cells (HUVEC), and to explore new methods for prevention and treatment of coronary heart disease. METHODS: NF-kappaB decoy transfection efficiency was detected by flow cytometry, NF-kappaB decoy's mechanism was analyzed by electrophoretic mobility shift assay (EMSA), TF mRNA was detected by RT-PCR, TF antigen expression on the surface of HUVEC by flow cytometry, FVIIa level in plasma incubated with HUVEC stimulated by TNF-alpha by rsTF one stage clotting method. RESULTS: NF-kappaB decoy could be successfully transfected into HUVEC. It could compete with the endogenous kappaB cis sequence element in the regulatory regions of TF promoter to bind transcriptional factor NF-kappaB. It could also significantly inhibit the TF mRNA, TF antigen expression on the cell surface and TF function leading to activation of FVII. CONCLUSION: NF-kappaB decoy could inhibit TF gene expression and FVII activation in cultured HUVEC and might be a potential new strategy for prevention and treatment of coronary heart disease.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator VII/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Tromboplastina/biossíntese , Células Cultivadas , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Oligodesoxirribonucleotídeos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologiaRESUMO
The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.