| Most patients with cancer die not because of the tumor in the primary site, but rather because it has spread to other sites. Tumor metastasis process consists of several sequential steps, and all of which must be successfully completed. These steps include detachment of cancer cells from their original localization, survival of the cells in the circulation, arrest in a new organ, invasion of cancer cells into the surrounding tissue and colonization and growth at distant sites in the organism.The plasminogen activation(PA) system play a key role in tumor cell invasion and metastasis. The PA system mainly includes urokinase(uPA), urokinase plasminogen activator inhibitor type1, 2 ( PAI-1 and PAI-2) , plasminogen and urokinase receptor (uPAR). uPA is synthesized and secreted by tumor cells and normal cells, and interacts with a specific cell surface receptor (uPAR) thereby focalizing enzymatic activity to the cell surface. The activity of uPA is controlled by plasminogen activator inhibitors type-1 and type-2. Upon the action of urokinase, plasminogen can be cleaved and turned into plasmin, which is involved in breakdown of basement membranes and extracellular matrix during the process of tumor cell invasion and metastasis. Substantial data demonstrates that there is a strong clinical value of the PA system in predicting disease recurrence and survival in cancer patients, and there is a great hope that substances designed to target the PA system will be administered in cancer patients therapy.So we express and purify a bifunctional fusion protein(ATF-PAI2CD) that can not only competitively binds uPAR on tumor cell surfaces, but also inhibit the serine proteinase activity of uPA. The recombinant protein consists of amino-terminal fragment of uPA and mutant of PAI-2. The recombinant fusion protein, was purified by protocols including precipitation by ammonium sulfate, Sephadex G-75 gel filtration and Q-Sepharose ion-exchange chromatography. Finally, 1.1 mg of purified fusion protein were obtained from 1 L culture of Pichia pastoris. The purity and specific activity of fusion protein were 95% and 1.0×104 IU/mg, respectively. While in prokaryotic expression system, 50 mg of purified fusion protein were obtained from 1 L fermentation culture of E.coli. The purity and specific activity of fusion protein were 90% and 1.2×104 IU/mg, respectively. The purified fusion protein can efficiently inhibit serine proteinase activity of uPA, and was found to bind human tumor cells via uPAR.In the present study, we evaluated the effects of fusion protein on 95D lung cancer cells invitro and in vivo. Our results support the hypothesis that fusion protein blocks tumor invasion and motility by inhibiting localized pericellular proteolysis. Treatment of 95D lung cancer cells with fusion protein resulted in a dose-dependent decrease in tumor-cell invasion through Matrigel and the fusion protein was much more effective than PAI2CD; In addition, treatment of 95D lung cancer cells with fusion protein resulted in a significant decrease of expression level of extracellular regular protein kinase (ERK1/2), and increase of adhesion ability to fibronectin which was comfirmed by cell adhesion assay. For in vivo studies, BALB/c (nu/nu) mice female were inoculated with human 95D lung cancer cells (5 × 106) into the subcutaneous scapular. From this time, intraperitoneal administration of NaCl, ATF-PAI2CD fusion protein (0.5, 5, 50μg) was repeated daily until autopsy. Autopsy was performed at 70 days. Lungs and lymph nodes were removed, lung and lymphatic metastasis was identified by HE stain.The high-concentration of fusion protein caused a significant reduction in tumor volume and weight; however, these antitumor effects were significantly greater in animals receiving fusion protein which demonstrated a 67.9% ± 4.2% reduction in tumor growth as compared with control animals. The number of lymphatic metastasis was significantly reduced in high- and middle- concentration of fusion protein-treated animals, whereas low- concentrat...
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