Experimental Study Of Gene Therapy For Colon Cancer By Replication-defective Recombinant Adenovirus Harboring Human Endostatin

BackgroundNeovascularization plays a critical role in solid tumor rapid growth and metastasis. Various researches indicate that angiogenesis inhibitors are highly potent in suppression angiogenesis which enable the tumor mass remaining in a dormant state,and such a therapy usually does not form an acquired drug resistance.Thus,the treatment would be expected effective if angiogenesis inhibitors were administered combined with radiotherapy , chemotherapy or operation. Endostatin is a new potent vascular endothelial growth inhibitor, it possesses powerful antitumor activity in animal study, but it needs a large dosage and repeat injection in treat,and a great deal preparation of Endostatin is quite difficult,so it will makes a lot of inconvenience in its future clinical application.ObjectiveThis study is to construct a adenovirus vector which contains human Endostatin at first and then make adenovirus with high liters through packaging.lt explores the possibility of genetherapy to use adenovirus which can transfect Endostatin through anti-colon cancer experimental study both in vitro and in vivo.MethodsThe single chain human Endostatin cDNA was inserted into MCS of recombinant adenovirus shuttle vectors pAdCMV-Linkl. The recombinant adenovirus vector, pAdCMV-IL3-Endo, was cotransfected into 293 cells together with pBHG10 by lipofectamine, and recombinant adenovirus, which named Ad-ILs-Endo, were generated by homologous recombination.The recombinant adenovirus was amplified and purified, the titers was then detected. When SW620 Cells were infected with advenovirus, gene transfer efficiency was analyzed by X-gal staining. The ability of the vectors to express Endostatin proteins was determined by Western blot. Thesupernatant's affection on the proliferation of three different kinds of vascular endothelial cells were observed. Wild-type SW620 cells and cells infected with Ad-IL3-Endo/AdCMVLacZ were inoculated at the flank of nude mice,the growth of tumors were observed and the volumes were calculated. Ad-IL3-Endo were injected into the tumors or the tail veins respectively after local tumors and lung metastases models established.The growth of tumors ,the survival and the number of lung metastases were observed.The Envision system was used to detect the expression of MVD and VEGF in tumor tissues.Results1.The replication-defective recombinant adenovirus contains human Endostatin were generated by homologous recombination.After primary purification the titers of Ad-IL3-Endo was about 1.0 X 108 PFU/ml.2. The infection rate of recombinant adenovirus of SW620 cells in vitro is about 90 %, Endostatin proteins were detected in the supernatant of cells transfected with Ad-IL3-Endo by Western blot.3. SW620 cells infected with Ad-IL3-Endo had the same growth rate as control cells,but its supernatant could inhibite the proliferation of three different kinds of vascular endothelial cells.4.1n animal experiments,^ vivo Endostatin gene delivered into SW620 cells by adenovirus resulted in an efficient inhibition of tumorigenicity in situ, the growth of tumors also reduced.5. After Ad-IL3-Endo were injected into the subcutaneous tumors or the tail veins,the growth of tumors reduced,the survival enhanced and the number of pre-established lung metastases also reduced significantly.6. Compared with control groups, the expression of MVD and VEGF in tumortissues all reduced significantly. ConclusionA good result could get in experimental study of anti-colon cancer with recombinant adenovirus contains human Endostatin.lt had made a good foundation for the further experimental study and future clinical application.