Purification And Anti-tumor Metastasis Characterization In Vitro Of A Disintegrin From Gloydius Brevicaudus Venom

Tumor metastasis is a very perplex course. It involves the interaction of the tumor cells, vascular endothelium cells, lymphoid endothelium cells and extracellular matrix (ECM) and also does it include the aggregation of blood platelets. Disintegrin which contains the amino acid sequence Arg-Gly-Asp(RGD) or Lys-Gly-Asp(KGD), originate of snake and leech venom. RGD is the common sequence which exists in ECM and cell membrane such as fibrinogen,fibronectin,thrombospondin,osteopontin and von willebrand factor(vWF) etc. It plays a great role in the course of tumor invasion. Disintegrin just stimulate the mutual amino acid sequence and take precedence to interfere with the interaction of the tumor cells and ECM to attain the aim of inhibitting tumor invasion and metastasis. This study was designed to isolate and purify disintegrin from Gloydius brevicaudus venom and analyse its characterization and anti-tumor adhesion,migration and invasion activities.1.Purification and characterization of the disintegrin from Gloydius brevicaudus venom(GBV)Seven fractions(Ⅰ～Ⅶ) were isolated from GBV by Gel filtration chromatography on Superdex 75 . The fractionⅣinhibited the platelet aggregations induced by ADP with molecular weight lower than 10000 dalton, so it suggested as a contained disintegrin component. A disintegrin named GBVⅣ4 was purified from the fraction by sephadex G-25 gel filtration, 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) . GBVⅣ4 is composed of 70 amino acid sequence with RGD active region and molecular weight 7442 dalton as assayed by Mass Spectrography. Characteration of GBVⅣ4 is consistent with meta-chain disintegrin's(70 amino acid sequence, six pairs of disulfide bond). Retrieved by Genbank , GBVⅣ4 has high homology with other disintegrins .Above all, we conclude that GBVⅣ4 is a new kind of disintegrn.2. The effects of GBVⅣ4 on the human platelet aggregation.According to the Born's method, the effects of GBVⅣ4 on platelet aggregation induced by ADP and thrombin were assayed. The results showed that GBVⅣ4 dose-dependently inhibited ADP- and thrombin-induced platelet aggregation with IC50 0.339 and 0.577μg/ml respectively.3. The effects of GBVⅣ4 on B16 cells'adhesive capability to Fibronection Cell-matrix adhesion assay was used to study the adhesive ability of B16 cells.With phase microscope observation we found the B16 cells looked like epidermoid cells and were polygonal, but the B16 cells treated with GBVⅣ4 undergo remarkable morphological change :the cells turned to ground,formed gaps and multicelluar aggregation. According to MTT method, different concentration of GBVⅣ4 showed inhibitory effects, and the inhibited effects was dose dependent, the inhibition ratio was 59.34% at the concentration of 16μg/ml.4. The effects of GBVⅣ4 on B16 cells'migration on Fibronection.The migration of B16 cells was observed by erasion trace test.The results showed that GBVⅣ4 could inhibit B16 cells from moving on Fibronection effectively.5. The effects of GBVⅣ4 on B16 cells'invasion on MatrigelTranswell chamber assay was applied to measure the effect of GBVⅣ4 on invasion of B16 cells. After 48 h, the invasion of B16 cells was less than those in control groups, and the number of invasive cells was dose dependent.ConclusionsA disintegrin GBVⅣ4 was purified from Gloydius brevicaudus venom with molecular weight 7442 dalton, 70 amino acid sequence and RGD active region. It was a potent platelet aggregation inhibitor. GBVⅣ4 has strong activity of anti-tumor cell adhesion , migration and invasion.