Bcl-2 siRNA induced apoptosis and increased sensitivity to 5-fluorouracil and HCPT in HepG2 cells.
To investigate the changes in drug sensitivity of Bcl-2 siRNA transfected HepG2 cells. Bcl-2 siRNA and negative siRNA expression vector were constructed and stably transfected into HepG2 cells. RT-PCR and Immunofluorescence were used to detect the target gene expression. Western Blotting was used to detect Bcl-2, Bax and caspase-3 protein expressiom. Drug sensitivity of the cells to 5-fluorouracil (5-FU) and 10-hydroxycamptothecin (HCPT) were analyzed with MTT and flow cytometry. Results were following: (1) the mRNA and protein expression level of Bcl-2 in Bcl-2 siRNA stable transfectants were reduced compared with negative siRNA transfected or untreated cells. Accordingly, Bax protein expression had no change and caspase-3 protein expression showed significantly be up regulated; (2) MTT results showed that Bcl-2 siRNA transfectants had higher cell inhibitory rates after treated with 5-FU or HCPT; (3) flow cytometry results demonstrated that sub G1 population increased in Bcl-2 siRNA transfected cells compared with negative siRNA or untreated cells. After addition 5-FU (1300 mg/l) and HCPT (0.72 mg/l), Bcl-2 siRNA cells showed higher sub G1 population than negative siRNA or untreated cells. siRNA targeting Bcl-2 gene can specifically down-regulate Bcl-2 expression, increased Bax/Bcl-2 ratio expression and caspase-3 activity in HepG2 cells, which lead to increase cells spontaneous apoptosis and sensitize cells to 5-FU or HCPT. Bcl-2 siRNA may be a potential therapy agent against human hepatoblastoma.
Feng
Lan-Fang
LF
Institute of Pharmarcy and Pharmacology, Nanhua University, Hengyang, 421001, China.
Zhong
Miao
M
Lei
Xiao-Yong
XY
Zhu
Bing-Yang
BY
Tang
Sheng-Song
SS
Liao
Duan-Fang
DF
D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002166:Camptothecin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004357:Drug Synergism; D005434:Flow Cytometry; D005472:Fluorouracil / Q000008:administration & dosage / Q000494:pharmacology*; D019254:Genes, bcl-2 / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy / Q000473:pathology; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts; D013844:Thiazoles; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000235:genetics
D000964:Antimetabolites, Antineoplastic; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D051028:bcl-2-Associated X Protein; C028098:10-hydroxycamptothecin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue; D005472:Fluorouracil; D002166:Camptothecin