RESUMO
Summary: The homocysteine (Hcy)-induced tissue factor (TF) expression in human vascular smooth muscle cells (VSMCs) and the effect of Hey on the activity of nuclear factor-kappaB (NF-κB) and the expression of inducible nitric oxide synthase (iNOS) were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC (NF-κB inhibitor). Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Westem blot was carried out to detect the expression of NF-κB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hey at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF protein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-κB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hey could significantly induce the expression of TF in VSMCs and enhance the activation of NF-κB, subsequently mediate TF gene expression and protein synthesis. NF-κB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.
RESUMO
The effects of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblas- toma 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 pSU- PER vector. Transfection of TFsiRNA-pSUPER was performed using lipofectamine2000. The cytotox- icity 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 trans- fection of TFsiRNA-pSUPER on SK-N-MC cells in a dose-dependent manner. Inhibition of TF sig- nificantly 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 ex-posed to 1 μg/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 doxorubi- cin-induced apoptosis in human nearoblastoma 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.