Experimental Study Of The Regulation Of Tissue Factor On Doxorubicin-induced Apoptosis In Human Glioblastoma

Part 1 Tissue Factor/FVII Regulates Doxorubicin-induced Apoptosis in Human Glioblastoma via Activating PI3K/Akt SignalingObjective: To investigate the role of tissue factor(TF) in chemotherapeutic reagent–induced apoptosis on human glioblastoma and to explore its mechanism. Methods: The expression of TF was examined by Western blotting. The cytotoxicity of doxorubicin was determined by WST assay. The activation of Caspase-3 and PARP induced by adoxorubicin were tested by Western blotting. Results: Human glioblastoma cell line U373MG expressed high level of TF while LN-229 was with low-TF level. The chemotherapeutic reagent doxorubicin revealed stronger cototoxic effect on high-TF U373MG cells than low-TF LN-229 cells. Enforced strong expression of TF was achieved by transfection of TF-pcDNA3 combinant on LN-229 cells in a dose-dependent manner. Enforced TF expression in transfected LN-229 cells not only impaired the doxorubicin-induced cleavage of Caspase-3 and PARP, but also inhibited the cytotoxic effect of doxorubicin. Furthermore, activation of Akt was strong in high-TF U373MG cells but weak in low-TF LN-229 cells. Incubation of factor VII(FVII) with enforced TF-expressing LN-229 cells increased the phosphorylation of Akt in a time-dependent manner. Conclusions: These results suggest that over-expression of TF on glioblastoma could inhibit doxorubicin-induced apoptosis. Interaction of FVII and TF activates the downstream PI3K/Akt pathway. Tumor-derived over-expression of TF might play a role in chemotherapy resistance in glioblastoma, at lest in part, by activating PI3K/Akt-mediated survival and anti-apoptotic mechanism through the interaction of TF/FVII signaling. Part 2 Construction of TFsiRNA Expression Vector and Study on its Inhibitive Effect in Human GlioblastomaObjective: To construct the specific tissue factor(TF) siRNA expressive vector and study the inhibitive effect in human glioblastoma. Methods: TFsiRNA-pSUPER plasmid was construscted by inserting specific 19-NT silencing sequence targeting TF gene into pSUPER vector. Potential construct were screened by restrictive enzyme digestion reaction and then identified by gene sequencing. Transfection of TFsiRNA-pSUPER was performed using lipofectamine2000. The expression of TF was examined by Western blotting. Results: The constructed TFsiRNA-pSUPER was identified by gene sequencing. Human glioblastoma cell line U373MG expressed high level of TF. Knockdown of TF expression was achieved by transfection of TFsiRNA-pSUPER on U373MG cells in a dose-dependent manner. Conclusions: These results suggest that TFsiRNA-pSUPER has been constructed successfully and can knockdown TF in high TF-expressive human glioblastoma cell line U373MG. TFsiRNA-pSUPER can be used as a potential means of inhibition of TF expression especially while studying its non-coagulative function.Part 3 The Effect of Downregulation of Tissue Factor on Doxorubicin -induced Apoptosis in Human GlioblastomaObjective: To explore the effect of downregulation of tissue factor(TF) on doxorubicin–induced apoptosis in human glioblastoma. Methods: The expression of TF was examined by Western blotting. TFsiRNA-pSUPER plasmid was construscted 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 were tested by Western blotting. The apoptotic cells were stained by Hochest33342 and counted under fluorescence inverted microscope. Results:①Human glioblastoma cell line U373MG expressed high level of TF.②Knockdown of TF expression was achieved by transfection of TFsiRNA-pSUPER on U373MG cells in a dose-dependent manner.③Inhibition of TF significantly decreased the viability of transfected U373MG cells while treating with increasing concentration of doxorubicin.④Cleavage of Caspase-3 and PARP, which are the main effectors of doxorubicin-induced apoptosis, enhanced in transfected U373MG cell with down-regulation of TF.⑤TFsiRNA treatment significantly increased the apoptotic cell number in transfected U373MG cells contrasted to those control cells(P<0.05) while exposing to 1μg/ml doxorubicin for 8h. Conclusions: These results suggest that knockdown of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human glioblastoma cells. Over-expression of TF might contribute to chemotherapy resistance in human glioblastoma and its progression, at lest in part, by regulating doxorubicin-induced apoptosis.