Anti-angiogenesis Effect Of Disintegrin From Gloydius Brevicaudus Venom

Disintegrin which contains the amino acid sequence Arg-Gly-Asp(RGD) or Lys-Gly-Asp(KGD),has been implicated as a recognition site in interactions between integrin and its ligands. It inhibits infiltration and migration of the malignant cell by anti-aggregation of platelet aggregation which formed tumor embolus and induced angiogenesis of tumor. This study was designed to isolate and purify disintegrin from Gloydius brevicaudus venom and study its characterization and anti-angiogenesis, anti-tumor metastasis activities.1.Isolation of disintegrin GBV-Ⅳ4 from the venom of Gloydius brevicaudusAccording to the protocol of isolation of disintegrin from Gloydius brevicaudus venom(GBV-) of Lab Snake Venom Research of Fujian Medical University ,we isolated disintegrin(GBV-Ⅳ4) from GBV- by Superdex 75 Gel filtration chromatography, DEAE Sepharose Fast Flow ion-exchange, and Lichrospher C18 reverse chromatography. It was homogeneous as a single band showed on SDS-polyacrylmide gel electrophoresis (SDS-PAGE, Tris-Tricine system) . Effects of GBV-Ⅳ4 on inhibition platelet aggregation induced by ADP were assayed by the Born's method, .2. The effect of GBV-Ⅳ4 on human umbilical vein cells (HUVEC) tube-like structuresThe tube formation assay of HUVEC was used to study anti-angiogenesis of GBV-Ⅳ4. GBV-Ⅳ4 could significantly inhibit the tube formation of HUVEC in a dose-dependent manner. When HUVEC were treated with GBV-Ⅳ4 0.15mg/ml, 0.3mg/ml, 0.6mg/ml or PBS, the number of tube was 15.556±4.925, 9.556±3.046, 2.222±1.563 and 35.667±6.062 respectively. The area (μm2) of tube formation was 1002.198±431, 421.621±52, 118.926±34.003 and 2616.198±312.022 respectively. The perimeter (μm) of tube formation was 435.059±168.221, 180.944±68.178, 71.25±61.26 and 965.431±143.991 respectively.3. The effect of GBV-Ⅳ4 on anti-angiogenesis in the Matrigel plug in miceMatrigel plug assay was used to investigate anti-angiogenesis of GBV-Ⅳ4 in mice. GBV-Ⅳ4 could significantly inhibit microcapsule formation in the matrigel which was induced by b-FGF. When mice were treated with b-FGF, b-FGF + GBV-Ⅳ4 1.25mg/kg , b-FGF + GBV-Ⅳ4 2.5mg/kg or PBS, the number of microcapsule in the matrigel was 17.13±3.20,9.80±1.95 and 2 .33±0.75 and 0.4±0.44 respectively.. 4. The effect of GBV-Ⅳ4 on artificial lung metastasis of B16F1 in miceThe artificial lung metastasis assay was used to measure anti-metastasis of GBV-Ⅳ4. Mice were treated with GBV-Ⅳ4 of 1.25mg/kg, 2.5mg/kg or PBS after injection of B16F1. The metastatic nodes of B16F1 on lung surface were significantly reduced by the GBV-Ⅳ4. The number of metastatic nodes in GBV-Ⅳ4 1.25mg/kg, 2.5mg/kg or PBS treated mice was 118.75±17.44, 73.87±34.21 and 181.14±61.00 respectively. GBV-Ⅳ4 also significantly inhibits the microcapsule genesis in the metastatic node. The number of microcapsule in GBV-Ⅳ4 1.25mg/kg, 2.5mg/kg or PBS treated mice was 24.4±3.36, 11.4±5.77 and 28.40±2.70 77 respectively.ConclusionsDisintegrin (GBV-Ⅳ4 ) could be purified from Gloydius brevicaudus venom by Superdex 75 Gel filtration chromatography, DEAE Sepharose Fast Flow ion-exchange and Lichrospher C18 reverse chromatography. It was a potent platelet aggregation inhibitor and significantly inhibits angiogenesis and metastasis of tumor.