Down-regulation of Tissue Factor by siRNA Increased Doxorubi-cin-induced Apoptosis in Human Neuroblastoma

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 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.

Down-regulation of tissue factor by siRNA increased doxorubicin-induced apoptosis in human neuroblastoma

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.