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

Abstract

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 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 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.

Keywords

tissue factor / neuroblastoma / apoptosis / chemotherapy

References

[1] VersteegH. H., RufW.. Emerging insights in tissue factor-dependent signaling events. Semin Thromb Hemost, 2006, 32: 24-32

[2] FangJ., SongS. J.. Nonthrombotic function of tissue factor. Zhonghua Xue Ye Xue Za Zhi (Chinese), 2002, 24(3): 184-186

[3] BrummelkampT. R., BernardsR., AgamiR.. A system for stable expression of short interfering RNAs in mammalian cells. Science, 2002, 296: 550-553

[4] HolenT., AmarzguiouiM., WiigerM. T., et al.. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res, 2002, 30: 1757-1766

[5] PeskinA. V., WinterbournC. C.. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta, 2000, 293: 157-166

[6] ForsterY., MeyeA., AlbrechtS., et al.. Tissue factor and tumor: clinical and laboratory aspects. Clin Chim Acta, 2006, 364: 12-21

[7] FangJ., WeiW. N., XiaL. H., et al.. The effects of tissue factor/activated factor VII complex on the invasion and metastasis of human ovarian cancer. Zhonghua Xue Ye Xue Za Zhi (Chinese), 2004, 25(9): 523-527

[8] RakJ., MilsomC., MayL., et al.. Tissue factor in cancer and angiogenesis: the molecular link between genetic tumor progression, tumor neovascularization, and cancer coagulopathy. Semin Thromb Hemost, 2006, 32: 54-70

[9] VermeulenK., Van BockstaeleD. R., BernemanZ. N.. Apoptosis: mechanisms and relevance in cancer. Ann Hematol, 2005, 84: 627-639

[10] SorensenB. B., RaoL. V., TornehaveD., et al.. Antiapoptotic effect of coagulation factor VIIa. Blood, 2003, 102: 1708-1715

[11] VersteegH. H., SpekC. A., RichelD. J., et al.. Coagulation factors VIIa and Xa inhibit apoptosis and anoikis. Oncogene, 2004, 23: 410-417

[12] MizutaniH., Tada-OikawaS., HirakuY., et al.. Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide. Life Sci, 2005, 76: 1439-1453

[13] KimD., ChengG. Z., LindsleyC. W., et al.. Targeting the phosphatidylinositol-3 kinase/Akt pathway for the treatment of cancer. Curr Opin Investig Drugs, 2005, 6: 1250-1258