CC5 and CC8, two homologous disintegrins from Cerastes cerastes venom, inhibit in vitro and ex vivo angiogenesis.

Angiogenesis constitutes a fundamental step in tumor progression. Thus, targeting tumour angiogenesis has been identified to be promising in cancer treatment. In this work, CC5 and CC8, two highly homologous disintegrins isolated from the venom Cerastes cerastes viper from the south of Tunisia, were assessed for their anti-angiogenic effect by testing their ability to interfere with viability, adhesion, migration and angiogenesis of Human Microvascular Endothelial Cells, HMEC-1 and HBMEC. We found that CC5 and CC8 displayed pro-apoptotic potential in HMEC-1 cells. Anoïkis like induced by these two disintegrins was evidenced by cell detachment, down regulation of FAK/AKT/PI3K axis and caspase activation. In addition, both CC5 and CC8 exhibited in vitro anti-adhesive, anti-migratory and anti-proliferative effects on endothelial cells HBMEC. These effects appeared to require RGD and/or WGD loops disintegrin. CC5 and CC8 also inhibited tube-formation on matrigel and displayed potent anti-angiogenic activities as assessed ex vivo, using both the embryo chick chorioallantoic membrane model (CAM) and rat aortic ring assay. Altogether our results demonstrate that CC5 and CC8, are potent inhibitors of angiogenesis, by disrupting αvß3 and α5ß1 binding. The use of CC5 and/or CC8 could provide a beneficial tool to inhibit abnormal angiogenesis and to induce cancer regression.

Antitumoral potential of Tunisian snake venoms secreted phospholipases A2.

Phospholipases type A2 (PLA2s) are the most abundant proteins found in Viperidae snake venom. They are quite fascinating from both a biological and structural point of view. Despite similarity in their structures and common catalytic properties, they exhibit a wide spectrum of pharmacological activities. Besides being hydrolases, secreted phospholipases A2 (sPLA2) are an important group of toxins, whose action at the molecular level is still a matter of debate. These proteins can display toxic effects by different mechanisms. In addition to neurotoxicity, myotoxicity, hemolytic activity, antibacterial, anticoagulant, and antiplatelet effects, some venom PLA2s show antitumor and antiangiogenic activities by mechanisms independent of their enzymatic activity. This paper aims to discuss original finding against anti-tumor and anti-angiogenic activities of sPLA2 isolated from Tunisian vipers: Cerastes cerastes and Macrovipera lebetina, representing new tools to target specific integrins, mainly, α5ß1 and αv integrins.

PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells.

A novel Kunitz-type serine proteinase inhibitor, termed PIVL, was purified to homogeneity from the venom of the Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric polypeptide chain cross-linked by three disulfide linkages with an isotope-averaged molecular mass of 7691.7 Da. The 67-residue full-length PIVL sequence was deduced from a venom gland cDNA clone. Structurally, PIVL is built by a single Kunitz/BPTI-like domain. Functionally, it is able to specifically inhibit trypsin activity. Interestingly, PIVL exhibits an anti-tumor effect and displays integrin inhibitory activity without being cytotoxic. Here we show that PIVL is able to dose-dependently inhibit the adhesion, migration and invasion of human glioblastoma U87 cells. Our results also show that PIVL impairs the function of αvß3 and to a lesser extent, the activity of αvß6, αvß5, α1ß1 and α5ß1 integrins. Interestingly, we demonstrate that the (41)RGN(43) motif of PIVL is likely responsible for its anti-cancer effect. By using time lapse videomicroscopy, we found that PIVL significantly reduced U87 cells motility and affected cell directionality persistence by 68%. These findings reveal novel pharmacological effects for a Kunitz-type serine proteinase inhibitor.

Leberagin-C, A disintegrin-like/cysteine-rich protein from Macrovipera lebetina transmediterranea venom, inhibits alphavbeta3 integrin-mediated cell adhesion.

Leberagin-C, a new member of the disintegrin-like/cysteine-rich (D/C) family, was purified to homogeneity from the venom of Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric protein with a molecular mass of 25,787 Da. Its complete sequence of 205 amino acid residues was established by cDNA cloning. The leberagin-C shows many conserved sequences with other known D/C proteins, like the SECD binding sites and a pattern of 28 cysteines. It is the first purified protein from M. lebetina transmediterranea with only two disintegrin-like/cysteine-rich domains. Leberagin-C is able to inhibit platelet aggregation induced by thrombin and arachidonic acid with IC(50) of 40 and 50 nM respectively. It was also able to inhibit the adhesion of melanoma tumour cells on fibrinogen and fibronectin, by interfering with the function of alphavbeta3 and, to a lesser extent, with alphavbeta6 and alpha5beta1 integrins. To our knowledge, leberagin-C is the sole described D/C protein that does not specifically interact with the alpha2beta1 integrin. Structure-activity relationship study of leberagin-C suggested that there are some important amino acid differences with jararhagin, the most studied PIII metalloprotease from Bothrops jararaca, notably around the SECD motif in its disintegrin-like domain. Other regions implicated in leberagin-C specificities could not be excluded.

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration.

Two non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes.