Purification and bioactivity of a novel angiogenesis inhibitor extracted from the cartilage of Dasyatis akajei

In the early 1980s,shark cartilage has been revealed to contain a protein,an angiogenesis inhibitor that significantly inhibits the development of blood vessels that nourish solid tumors,thereby restraining tumor growth.Since then,it has generated intense interest in both public and medical circles. The aim of this study was to determine the method of isolation and characterization of the angiogenesis inhibitor,and identify the bioactivity of angiogenesis inhibitor derived from the Dasyatis akajei cartilage.In this view,we study in detail the method of purification and characterization the bioactivity of a novel angiogenesis inhibitory factor derived from the Dasyatis akajei cartilage(DCAIF-Ⅰ).By the 1.0 mol·L~(-1) guanidinium chloride extract of Dasyatis akajei cartilage,the extraction was then purified by Hitrap DEAE FF ion exchange chromatography,Superdex 75 10/300 GL gel filtration,and reverse-phase high performance liquid chromatography.The bioactivity of the products obtained is identified by the model of inhibiting the formation of the blood vessels of the chorioallantoic membrane of chicken embryo.Statistical analysis of blood vessels of DCAIF-Ⅰwas used for quantitative analysis of the inhibitory effect of DCAIF-Ⅰ. The pure inhibitor was homogeneous as a single band on a coomasie brilliant blue-stained 12% SDS-PAGE gel electrophoresis.DCAIF-Ⅰwas a novel angiogenesis inhibitory factor which has a molecular weight about of 62 ku.The results of bioactivity identification of angiogenesis inhibitory factor indicates that the large area of blood vessels in active substance groups have heavy loss of color,vascular structure blurred with broken branches,accompanied by the decreased density of vessels.In control group,the leaflike vascular net is clear and grew radiatively.Quantitative analysis blood vessels of the chorioallantoic membrane of chick embryos(CAM)indicated that DCAIF-Ⅰcan strongly inhibit the angiogenesis in the chorioallantoic membrane of chick embryos(CAM),1μg of DCAIF-Ⅰinhibited angiogenesis in 56% of the eggs.There is also a positive relation between the dosage and the effect.The results from the present study indicated that the DCAIF-Ⅰfrom Dasyatis akajei cartilage has angiogenesis inhibitory effect,and there is also a positive relation between the concentration and inhibitory effect.Therefore it is hoped that this angiogenesis inhibitor may provide a novel treatment for patients with malignancies,and perhaps even for those with nonmalignancies.It will be helpful for our knowledge of the molecular mechanisms of this angiogenesis inhibitor from Dasyatis akajei.