Molecular cloning and functional characterization of a cell-permeable superoxide dismutase targeted to lung adenocarcinoma cells. Inhibition cell proliferation through the Akt/p27kip1 pathway.
In clinical oncology, many trials with superoxide dismutase (SOD) have failed to demonstrate antitumor ability and in many cases even caused deleterious effects because of low tumor-targeting ability. In the current research, the Nostoc commune Fe-SOD coding sequence was amplified from genomic DNA. In addition, the single chain variable fragment (ScFv) was constructed from the cDNA of an LC-1 hybridoma cell line secreting anti-lung adenocarcinoma monoclonal antibody. After modification, the SOD and ScFv were fused and co-expressed, and the resulting fusion protein produced SOD and LC-1 antibody activity. Tracing SOD-ScFv by fluorescein isothiocyanate and superoxide anions (O2*-) in SPC-A-1 cells showed that the fusion protein could recognize and enter SPC-A-1 cells to eliminate O2*-. The lower oxidative stress resulting from the decrease in cellular O2*- delayed the cell cycle at G1 and significantly slowed SPC-A-1 cell growth in association with the dephosphorylation of the serine-threonine protein kinase Akt and expression of p27kip1. The tumor-targeting fusion protein resulting from this research overcomes two disadvantages of SODs previously used in the clinical setting, the inability to target tumor cells or permeate the cell membrane. These findings lay the groundwork for development of an efficient antitumor drug targeted by the ScFv.
Lu
Min
M
Institute of Biochemistry, Zhejiang University, Hangzhou, 310027, China.
Gong
Xingguo
X
Institute of Biochemistry, Zhejiang University, Hangzhou, 310027, China. Electronic address: gongxg@zju.edu.cn.
Lu
Yuwen
Y
Institute of Biochemistry, Zhejiang University, Hangzhou, 310027, China.
Guo
Jianjun
J
Institute of Biochemistry, Zhejiang University, Hangzhou, 310027, China.
Wang
Chenhui
C
Institute of Biochemistry, Zhejiang University, Hangzhou, 310027, China.
Pan
Yuanjiang
Y
Institute of Chemical Biology and Pharmaceutical Chemistry, Zhejiang University, Hangzhou, 310027, China. Electronic address: panyuanjiang@zju.edu.cn.
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D049109:Cell Proliferation; D003001:Cloning, Molecular; D050760:Cyclin-Dependent Kinase Inhibitor p27; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D051058:Oncogene Protein v-akt / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
D000970:Antineoplastic Agents; C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013482:Superoxide Dismutase; D051058:Oncogene Protein v-akt