[Inhibition of tissue factor by siRNA enhances doxorubicin-induced apoptosis in human neuroblastoma].
OBJECTIVE
To investigate the regulation of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma.


METHODS
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.
Fang
Jun
J
Department of Hematology, Affiliated Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Molecular Targeting Laboratory of Hubei Province, Wuhan 430022, China.
Tang
Hao
H
Xia
Ling-Hui
LH
Zhou
Mu-Xiang
MX
Chen
Yan
Y
Wei
Wen-Ning
WN
Hu
Yu
Y
Song
Shan-Jun
SJ
D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013925:Thromboplastin / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D034741:RNA, Small Interfering; D004317:Doxorubicin; D013925:Thromboplastin; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3