[Inhibitory effect of human mitochondria-targeted MPG recombinant on proliferation of human non-small cell lung cancer multidrug-resistant cell line A549/DDP].
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yu
Shi-Cang
SC
Institute of Respiratory Disease of PLA, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037, P. R. China.
Qian
Gui-Sheng
GS
Li
Yu-Ying
YY
Lu
Wei-Zhong
WZ
Li
Jin
J
Huang
Gui-Jun
GJ
D017209:Apoptosis; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002945:Cisplatin / Q000494:pharmacology; D045647:DNA Glycosylases / Q000096:biosynthesis* / Q000235:genetics; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D050939:Gene Fusion; D018390:Gene Targeting; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008928:Mitochondria / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D045647:DNA Glycosylases; D002945:Cisplatin