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...

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Bibliographic Details
Published in:Journal of drug targeting 2006, Vol.14 (1), p.21-26
Main Authors: Feng, Lan-Fang, Zhong, Miao, Lei, Xiao-Yong, Zhu, Bing-Yang, Tang, Sheng-Song, Liao, Duan-Fang
Format: Article
Language:English
Subjects:
5-FU
Antimetabolites, Antineoplastic - administration & dosage
Antimetabolites, Antineoplastic - pharmacology
Apoptosis - drug effects
Bcl-2
bcl-2-Associated X Protein - biosynthesis
bcl-2-Associated X Protein - genetics
Biological and medical sciences
Blotting, Western
Camptothecin - administration & dosage
Camptothecin - analogs & derivatives
Camptothecin - pharmacology
Caspase 3
Caspases - biosynthesis
Cell Line, Tumor
Drug Delivery Systems
Drug Synergism
Flow Cytometry
Fluorouracil - administration & dosage
Fluorouracil - pharmacology
General pharmacology
Genes, bcl-2 - genetics
HCPT
HepG2 cells
Humans
Liver Neoplasms - drug therapy
Liver Neoplasms - pathology
Medical sciences
Pharmaceutical technology. Pharmaceutical industry
Pharmacology. Drug treatments
Reverse Transcriptase Polymerase Chain Reaction
RNA interference
RNA, Small Interfering - pharmacology
small interference RNA
Tetrazolium Salts
Thiazoles
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Summary: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.
ISSN:1061-186X
1029-2330
DOI:10.1080/10611860500527947