Establishment Of HIF Mediated Canstatin Gene High Level Expression System And Research On Its Biological Effects On Lung Cancer Angiogenesis

Angiogenesis is essential for tumor's growth and metastasis. Neovascularity brings oxygen and nutrients to and takes metabolite away from tumor cells. Therefore, antiangiogenic therapy may be a good way for tumor dormancy by starving tumor. Many angiogenesis inhibitors have been discovered in succession since the tumor dormancy theory was put forth by Folkman L.. And the characteristics of powerful effect and no observed toxicity of endogenous inhibitors make them feasible for antiangiogenic therapy in cancer. Canstatin is a newly found potent endogenous angiogensis inhibitor. Though the mechanism of action of canstatin remains unknown, its powerful biological activities on endothelial cells have drawn researchers' attention. Meanwhile we also noticed the fact that tumor cells produce plenty of hypoxia inducible factors(HIF), which are transcript factors for hypoxia inducible gene. On the basis of those foundings we designed an HIF mediated canstatin gene high level expression system in order to increase canstatin gene expression in the hypoxia enviroment of tumor in vivo. The system is useful for suppressing tumor progress and also helpful for exploring cancer gene therapy.Methods:l.With RT-PCR methods, Canstatin cDNA was acquired from human liver tissues and cloned into vector pCMV-Script. The recombinant vector was named pCMV-Script-Cans (Vector 1).2.The DNA fragment of hypoxia responsive element (HRE) was inserted into the upper stream of canstatin cDNA in the recombinant vector pCMV-Script-Cans, and the new-formed vector was named hypoxia inducible expression vector pCMV-Script-3HRE-Cans (Vector 2).3.The signal peptide cDNA of carcinoembryonic antigen (CEAS) was cloned by RT-PCR method from cultured lung cancer A549 cells and ligated with canstatin cDNA bySOEing methods. Then, the recombinant gene abbreviated as CEAS-Cans was inserted into pCMV-Script vector, and the new vector was named canstatin secretory vector pCMV-Script -CEAS-Cans (Vector 3).4.With HRE being inserted into the upper stream modulation region of the secretory vector pCMV-Script-CEAS-Cans, a new vector was constructed and named hypoxia induci-ble secretory vector pCMV-Script-3HRE-CEAS-Cans (Vector4).5.The recombinant vectors were transformed into A549 and HUC-EC-C cells by lipo-some separately. The expression of canstatin mRNA in the recombinant vectors transferred cells was detected by Quantitation Real-time RT-PCR. The proliferation, apotosis and cell cycle of the two kinds of cells were measured with the methods of trypan blue exclusion cell count, colony formation test, 3H-thymidine incorporation, electron microscope, TUNEL assay and flow cytometry respectively.6.The secretion of canstatin was examined with the mixed cultivation of vector-transformed lung cancer cells and untransformed human umbilical vein endothelial cells.7.The secretion and activity of canstatin protein were tested by chicken chorio-allantoic membrane (CAM) assay.8.The recombinant vectors were transfered into tumor cells of tumor-bearing nude mouse by particle gun. Microvessel count method (MVC) was employed to evaluate the anti-angiogensis effects of each vector transformed mouse groups.Results:1 .Canstatin cDNA had been cloned from Chinese fetus hypatocytes and normal tissue para-hepatocarcinnoma separately, and ligated to the predigested vector pCMV-Script successfully. The sequence of Chinese canstatin cDNA was analysed and found no differences from that in GenBank. The sequencing result proved that the open reading frames of canstatin in all the recombinant vectors were correct.2.Different copy numbers of canstatin mRNA or CEAS-Canstatin mRNA were detected in the recombinant vectors transformed A549 and HUV-EC-C cells, and the mRNA expression increased in the Vector2\4 transformed cells under anoxic condition. Compared with the naked plasmid transformed cells, the growth and proliferation of recombinant vectors transformed HUV-EC-C cells were inhibited markedly, and G0-G1 phase blocking and apoptosis were observed in those cells, espec...