The Anti-angiogenic And Molecular Mechanism Research Of Recombinant Kallistatin

Angiogenesis is a complex process involving endothelial cell activation,proliferation, migration and the damage of vascular basement membrane, theformation of new blood vessels and vascular network, and connected with an existingvascular network. The angiogenesis becomes necessary for nutrient supply in thegrowth of solid tumors. With the proposed concept of tumor angiogenesis, thedevelopment of anti-angiogenesis drugs has become an important research directionof oncology drug. As a target for the treatment of tumor angiogenesis, thedevelopment and application of efficient tumor angiogenesis inhibitors in anti-tumortherapy become one of the main directions of the development of anti-cancer drugs.Kallistatin (Kal) is a tissue kallikrein binding protein which has a unique serineprotease inhibitor activity. Study found their participation in the development andprogression of hypertension, inflammation, angiogenesis, cancer and other diseases.Kallistatin will be a protein drugs used in clinical application in future. Currently theability of anti-tumor angiogenesis and anti-tumor growth of Kal has beendemonstrated, but the exact molecular mechanism of action is unclear. In this study,the recombinant Kallistatin protein (rhKal) was acquired by the fermentation of yeast.The activity and molecular mechanism of anti-tumor angiogenesis was verified byin vivo and in vitro experiments.Firstly, we activated the genetic engineering Pichia of pPIC9-Kal/GS115, thenexpressed rhKal by Methanol induction Medium. We collected the supernatant aftercentrifugal. The high purity of rhKal protein was obtained by further isolation,purification by using Phenyl sepharose, Heparin Sepharose and G-25sepharosechromatography. Secondly, the biological activities of human recombinant Kallistatinon HUVEC proliferation, migration, invasion, a small tube generation stimulated byVEGF were observed by experimental study in vivo and in vitro. After that, the affectof rhKal on the VEGFR-2receptor activity and downstream signaling pathways alsobe studied in order to understand the anti-angiogenesis molecular mechanisms ofrhKal. Thirdly, in order to clear the anti-tumor angiogenesis of rhKal, we observed the effects of MCF-7induced HUVEC angiogenesis after treated with rhKal in vitroand in vivo experiments. We further studied the relationship between the NF-κBsignaling pathway and the anti-tumor angiogenesis activity of rhKal. We firstlydetected the expression of P65, I-κBα, phosphorylated I-κBα, phosphorylated IKK ofHUVEC cell and tumor cells after treated with different concentrations rhKal. Wefurther detected the P65, P50nuclear translocation change by western-blotting assay.The effects on P65transcription activation was observed by report gene technology.Finally, in order to validate the relationship between NF-κB pathway andanti-angiogenic effect by Kallistatin, we constructed the p65gene eukaryoticexpression plasmids and transfected into MCF-7breast cancer and HUVEC cells.We also observed the impact of exogenous p65gene on apoptosis, invasion,anti-angiogenic activity of Kallistatin.The above results show that rhKal significantly inhibited VEGF-inducedangiogenesis and the mechanism may be partly though block the VEGFR-2signalpathway. The rhKal can also inhibit angiogenesis stimulated by breast cancer cells invitro and in vivo. The molecular mechanism may be blocking the NF-κB signalingpathway, thereby inhibiting the expression of its downstream target genes. Inconclusion, the study shows rhKal has good anti-angiogenic effect. The rhKal is avery promising application on development anti-angiogenesis protein drug.