151. [Inhibitory effect of human mitochondria-targeted MPG recombinant on proliferation of human non-small cell lung cancer multidrug-resistant cell line A549/DDP]
- Author
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Shi-Cang, Yu, Gui-Sheng, Qian, Yu-Ying, Li, Wei-Zhong, Lu, Jin, Li, and Gui-Jun, Huang
- Subjects
Lung Neoplasms ,Apoptosis ,Transfection ,Drug Resistance, Multiple ,DNA Glycosylases ,Mitochondria ,Drug Resistance, Neoplasm ,Carcinoma, Non-Small-Cell Lung ,Cell Line, Tumor ,Gene Targeting ,Humans ,RNA, Messenger ,Cisplatin ,Gene Fusion ,Cell Proliferation - Abstract
Multidrug resistance is the key obstacle to the improvement of chemotherapy effect of lung cancer. This study was to construct eukaryotic expression vector of human mitochondria-targeted N-methylpurine DNA glycosylase (MPG), and explore its inhibitory effect on proliferation of human non-small cell lung cancer multidrug-resistant cell line A549/DDP.Manganese-superoxide dismutase mitochondria-targeted sequence-MPG fusion gene (mito-MPG) was constructed through splicing by overlap extension (SOE). Recombinant eukaryotic expression vector pCMV-Script/mito-MPG was constructed by molecule-cloning technique, and then transfected into A549/DDP cells. In the stably transfected cells which were screened out by G418, the expression of mito-MPG mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR); its expression in separated and purified mitochondria was detected by Western blot. The proliferation of A549/DDP cells was detected by trypan blue exclusion trial. Cell cycle distribution was analyzed by flow cytometry.The mito-MPG fusion gene was confirmed by DNA sequencing,the recombinant pCMV-Script/mito-MPG was confirmed by restrictive endonuclease digestion and DNA sequencing. mito-MPG mRNA and protein were detected in the cells transfected with pCMV-Script/mito-MPG (MPG group), but not in the cells transfected with pCMV-Script (P group) and untransfected cells (C group). The cell double time were 72.6 h in C group, 73.5 h in P group, and 98.9 h in MPG group. Cell cycle blockage and subdiploid peak were found in MPG group. The proliferation indexes were 51.3% in C group, 54.3% in P group, and 26.1% in MPG group.pCMV-Script/mito-MPG could be constructed and transfected into mitochondria of A549/DDP cells successfully, and inhibit proliferation and induce apoptosis of A549/DDP cells.
- Published
- 2006