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PubMed2005

Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity.

Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.

Angiogenesis Inhibitors↗
PubMed2002

Altered expression of ADAMs (A Disintegrin And Metalloproteinase) in fibrillating human atria.

BACKGROUND: ADAMs (A Disintegrin And Metalloproteinase) are ectoproteases that have recently been reported to be expressed in cardiac tissue. Although they are known to regulate cell-cell and cell-matrix interactions, their pathophysiological role in various cardiac diseases is unclear. The purpose of the present study was to determine whether structural remodeling of the atria during atrial fibrillation (AF) is associated with altered ADAM expression. METHODS AND RESULTS: Atrial tissue samples of 30 patients undergoing open-heart surgery were examined. Fifteen patients had persistent AF (> or =6 months), and the remaining 15 patients had no history of AF. ADAM9, ADAM10, and ADAM15 expression was analyzed quantitatively at the mRNA and protein levels. ADAM expression was localized by immunohistochemistry. ADAM expression was correlated with amounts of integrins beta1 and beta3. The amount of ADAM10 protein more than doubled during AF (82+/-15 versus 36+/-8 U; P<0.01). Amounts of ADAM15 protein (102+/-12 versus 40+/-6 U; P<0.01) and mRNA (24.0+/-5.6 versus 10.5+/-2.5 U; P<0.05) increased significantly during AF compared with sinus rhythm. ADAM9 protein was not detected in any sample. ADAM/integrin ratios showed an increase of 4- to 6-fold (P<0.05) in patients with AF who had significantly dilated atria (4.94+/-0.6 versus 4.3+/-0.7 cm; P<0.05). ADAM/integrin ratios correlated with atrial diameter. CONCLUSIONS: AF is associated with an increase in the expression of ADAM10 and ADAM15. Enhanced ADAM-dependent disintegrin and metalloproteinase activity may be a molecular mechanism that contributes to the dilation of fibrillating human atria.

ADAM Proteins↗
PubMed2000

ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

ADAM Proteins↗
PubMed2000

ADAMTS-1: A cellular disintegrin and metalloprotease with thrombospondin motifs is a target for parathyroid hormone in bone.

PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.

ADAM Proteins↗
PubMed2002

A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium.

The cellular slime mold, Dictyostelium discoideum is a non-metazoan organism, yet we now demonstrate that a disintegrin domain-containing protein, the product of the ampA gene, plays a role in cell type specification. Disintegrin domain-containing proteins are involved in Notch signaling in Drosophila and C. elegans via an ectodomain shedding mechanism that depends on a metalloprotease domain. The Dictyostelium protein lacks a metalloprotease domain. Nonetheless, analysis of cell type specific reporter gene expression during development of the ampA null strain identifies patterning defects that define two distinct roles for the AmpA protein in specifying cell fate. In the absence of a functional ampA gene, cells prematurely specify as prespore cells. Prestalk cell differentiation and migration are delayed. Both of these defects can be rescued by the inclusion of 10% wild-type cells in the developing null mutant aggregates, indicating that the defect is non-cell autonomous. The ampA gene is also demonstrated to be necessary in a cell-autonomous manner for the correct localization of anterior-like cells to the upper cup of the fruiting body. When derived from ampA null cells, the anterior-like cells are unable to localize to positions in the interior of the developing mounds. Wild-type cells can rescue defects in morphogenesis by substituting for null cells when they differentiate as anterior-like cells, but they cannot rescue the ability of ampA null cells to fill this role. Thus, in spite of its simpler structure, the Dictyostelium ampA protein carries out the same diversity of functions that have been observed for the ADAM and ADAMTS families in metazoans.

Animals↗
PubMed2005

Structure-function correlations of snake venom disintegrins.

Disintegrins represent a family of polypeptides released in the venoms of Viperidae and Crotalidae snakes (vipers and rattlesnakes) by the proteolytic processing of multidomain metalloproteinases, which selectively block the function of beta(1) and beta(3) integrin receptors. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information of new structural determinants involved in integrin recognition that may be useful in both, basic and clinical research. The role of the composition, conformation and concerted dynamics of the integrin inhibitory loop and the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Amino Acid Sequence↗
PubMed2001

Contortrostatin, a homodimeric disintegrin, actively disrupts focal adhesion and cytoskeletal structure and inhibits cell motility through a novel mechanism.

Integrins play a major role in the regulation of cell motility. They physically link the extracellular environment to the cytoskeleton and participate in large protein complexes known as focal adhesions. In this report, it is demonstrated that treatment of tumor cells with the homodimeric disintegrin contortrostatin induces integrin-mediated tyrosine phosphorylation events and causes severe disruptions in the actin cytoskeleton and disassembly of focal adhesion structures without affecting cellular adhesion to a reconstituted basement membrane. Included in this disruption is the tyrosine phosphorylation and altered subcellular localization of FAK. Through use of transfected 293 cells with specific integrin expression profiles and anti-alphavbeta3 mAbs, we demonstrate that these events are mediated exclusively by the alphavbeta3 integrin and are likely the result of contortrostatin-mediated crosslinking of this receptor at the cell surface, since monovalent disintegrins, flavoridin or echistatin do not induce such effects. Further, it is shown that contortrostatin potently inhibits motility in cells expressing the alphavbeta33 integrin. The results of this study describe a novel integrin-mediated mechanism by which cell motility can be inhibited and suggest an alternative approach to therapeutic intervention for cancer invasion and metastasis.

Animals↗
PubMed2004

The transmembrane CXC-chemokine ligand 16 is induced by IFN-gamma and TNF-alpha and shed by the activity of the disintegrin-like metalloproteinase ADAM10.

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-gamma and TNF-alpha, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-gamma and TNF-alpha synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.

ADAM Proteins↗
PubMed2000

Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom.

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin alpha(5)beta(1) antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.

Amino Acid Sequence↗
PubMed2000

Molecular cloning and characterization of a human metalloprotease disintegrin--a novel marker for dendritic cell differentiation.

The 1alpha,25-dihydroxyvitamin D(3) (1,25- [OH](2)VD(3)) modulates the differentiation of monocytic cell lines and monocytes (MOs) in vitro. Up to now several target genes of 1,25(OH)(2)VD(3) have been described in monocytic cell lines; however, little is known about target genes in primary MOs. With the Differential Display technique, we found a transcript up-regulated by 1,25(OH)(2)VD(3) in short-term cultured human blood MOs, which we called MADDAM (metalloprotease and disintegrin dendritic antigen marker; EMBL/GenBank/DDBJ accession no. Y13786). Northern blot analysis confirmed this result and revealed a signal of MADDAM messenger RNA (mRNA) at about 7.5 kilobases (kb). Long-term culture (more than 20 hours) of MOs during macrophage (MAC) differentiation led to a rapid and complete down-regulation of MADDAM expression. In contrast, MADDAM expression was maintained in MOs differentiated along the dendritic cell (DC) pathway and induced in CD34(+)-derived DCs. In addition, in situ hybridization revealed signals of MADDAM mRNA in follicles of human lymph nodes and MADDAM mRNA was detected in freshly isolated human blood-DCs by reverse transcription-polymerase chain reaction (RT-PCR). By means of a database search, we found that MADDAM is a member of the ADAM (a metalloprotease and disintegrin) family, the human homologue to murine meltrin-beta (ADAM 19). From these data, we conclude that MADDAM is an important marker for the differentiation and characterization of DCs and the distinction between MACs and DCs. (Blood. 2000;96:732-739)

ADAM Proteins↗
PubMed2000

Contortrostatin, a snake venom disintegrin, induces alphavbeta3-mediated tyrosine phosphorylation of CAS and FAK in tumor cells.

Contortrostatin is a homodimeric disintegrin that inhibits platelet aggregation and cell adhesion to extracellular matrix proteins by blocking integrins. The effect of contortrostatin on integrin-mediated signaling in tumor cells was investigated by studying tyrosine phosphorylation events and activation of specific signaling molecules. We found that at concentrations as low as 1 nM, soluble contortrostatin activates integrin signals leading to increased tyrosine phosphorylation of FAK and CAS, and that these signals are abolished by inhibiting Src family kinases. Using transfected 293 cells expressing specific integrins, it was determined that contortrostatin-generated signals are mediated exclusively by the alphavbeta3 integrin. This observation was extended by showing that cells lacking alphavbeta3, but expressing alphavbeta5 and alpha5beta1, do not respond in this way to contortrostatin treatment. In cells expressing alphavbeta3, blocking contortrostatin binding with antibodies against alphavbeta3 completely abrogates contortrostatin signals. Monovalent disintegrins echistatin and flavoridin were incapable of affecting tyrosine phosphorylation alone, but when added simultaneously with contortrostatin, completely inhibited contortrostatin-initiated signals. We propose that the homodimeric nature of contortrostatin imparts the ability to crosslink alphavbeta3 integrins, causing Src activation and hyperphosphorylation of FAK and CAS. This activity may represent a novel mechanism by which tumor cell motility can be inhibited.

Cell Adhesion↗
PubMed2000

Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.

ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloprotease-disintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation.

ADAM Proteins↗
PubMed1992

Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity.

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.

Adenosine Diphosphate↗
PubMed2003

Expression of ADAMs (a disintegrin and metalloproteases) and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic adenocarcinomas.

A disintegrin and metalloproteases (ADAMs) are proteins that contain both a disintegrin and metalloprotease domain and have potential implications for the metastasis of human cancer cells via cell adhesion and protease activities. In this study, we analyzed the expression levels of ADAM-9, ADAM-10 and ADAM-17 (TNF-alpha converting enzyme, TACE), and TIMP-3 (tissue inhibitor of metalloproteinase-3) in human prostatic tumor cell lines as well as in clinical patient materials (BPH and tumor tissue samples). Human prostatic tumor cell lines (MDA PCa 2b, LNCaP-C33, -C51, -C81, -Pro5, -Ln3, -C4-2, PC3, and DU145) showed varied levels of expression for ADAM-9, -10 and -17 mRNA. A strong expression of ADAM-17/TACE was further revealed by Western blot analysis in prostatic tumor cell lines. In the case of clinical material, all the tumor samples (8/8) revealed the expression of ADAM-9, -10, and -17 compared to the specimens of benign prostatic hyperplasia (BPH) where 80% of (8/10) samples showed the expression of ADAM-9, 86% (6/7) of ADAM-10, and 30% (3/10) of ADAM-17/TACE. Interestingly, expression of a potent inhibitor of ADAM-17 (TIMP-3) was not detected in any prostatic tumor cell lines (0/9), while TIMP-3 expression was detected in 82% (9/11) of BPH samples. Androgen-sensitive LNCaP-C33 cells exhibited differences in ADAMs regulation by 5alpha-dihydrotestosterone (DHT), while such differences were not detectable in androgen-independent LNCaP-C81 cells. These results suggest that an inverse expression pattern of ADAM-17/TACE and TIMP-3, and the regulation of ADAMs with DHT might play an important role in the pathogenesis of prostate cancer.

ADAM Proteins↗
PubMed2004

Intravenous liposomal delivery of the snake venom disintegrin contortrostatin limits breast cancer progression.

Despite significant research in this area, metastatic breast cancer remains a disease with a poor prognosis. Until an effective therapy is developed, it is imperative that new treatment modalities be investigated. In this report, we describe an effective method for delivery of a novel snake venom disintegrin, contortrostatin (CN), in an orthotopic, xenograft model of human mammary cancer in immunodeficient mice. CN (Mr 13,500) is a homodimeric disintegrin isolated from venom of the Southern Copperhead snake. The homodimer possesses two Arg-Gly-Asp sites, which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. Although our laboratory has previously described the antitumor activity of CN in a mouse model of human mammary cancer, the method of delivery, daily intratumor injection, was not translatable to clinical application. We now describe a clinically relevant method of administering CN, liposomal delivery (LCN). A unique liposomal system has been designed for i.v. administration of a biologically active protein with full retention of biological activity. Pharmacokinetics, biodistribution, platelet reactivity, and immunogenicity of LCN were determined and compared with similar characteristics of native, unencapsulated CN. There are several advantages to liposomal delivery of CN: (1) LCN has a significantly prolonged circulatory half-life compared with native CN; (2) LCN is passively accumulated in the tumor; (3) LCN has no platelet reactivity; and (4) LCN is not recognized by the immune system. Finally, antiangiogenic activity is an important component of CN's mechanism of antitumor action. We have demonstrated that i.v. delivery of LCN leads to potent antiangiogenic activity in the orthotopic, xenograft human mammary tumor model.

Agkistrodon↗
PubMed1994

Contortrostatin, a snake venom disintegrin, inhibits beta 1 integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis.

Disintegrins are Arg-Gly-Asp-containing proteins that inhibit integrin-mediated cell-cell and cell-matrix interactions. We have purified a disintegrin, contortrostatin, from Agkistrodon contortrix contortrix snake venom that is a potent inhibitor of human metastatic melanoma (M24 met) cell adhesion to extracellular matrix proteins. Contortrostatin inhibits M24 met cell adhesion to type I collagen, vitronectin, and fibronectin with 50% inhibitory concentration values of 20, 75, and 220 nM, respectively. Contortrostatin does not significantly inhibit adhesion of M24 met cells to laminin. 125I-labeled contortrostatin binds to M24 met cells in a saturable and displaceable manner. Scatchard analysis indicates that there are two binding sites for 125I-labeled contortrostatin on the surface of these cells. High affinity binding has a Kd of 3 nM with 165,000 sites/cells low affinity binding has a Kd of 60 nM with 500,000 sites/cell. Immobilized contortrostatin can support adhesion of M24 met cells; this binding is blocked by a monoclonal antibody to the beta 1 integrin subunit and by an antibody to the fibronectin receptor alpha 5 beta 1. The anti-vitronectin receptor (alpha v beta 5) monoclonal antibody which blocks adhesion of M24 met cells to immobilized vitronectin does not block binding of M24 met cells to immobilized contortrostatin. In an in vivo experimental metastasis model system, contortrostatin at 20 micrograms and 100 micrograms inhibits lung colonization of M24 met cells (5 x 10(5)), injected in the tail vein of scid mice, by 51 and 73%, respectively. We conclude that contortrostatin is a potent inhibitor of beta 1 integrin-mediated M24 met cell adhesion in vitro and that it also inhibits lung colonization in vivo.

Animals↗
PubMed1995

Effect of four disintegrins on the adhesive and metastatic properties of B16F10 melanoma cells in a murine model.

Four disintegrins, eristostatin, albolabrin, barbourin and echistatin, injected IV into C57BL/6 mice in combination with B16F10 murine melanoma cells, inhibited formation of experimental lung metastases with ID50s of 0.05, 1.0, 0.9, and 3.7 mumoles per mouse, respectively. When injected 1 h after tumor cells, albolabrin, echistatin and barbourin had the same antimetastatic activity, while eristostatin was not active. Eristostatin (IC50 7-8 nM) was more potent than echistatin (IC50 74-75 nM), barbourin (IC50 46-60 nM), and albolabrin (IC50 130-165 nM) as an inhibitor of murine platelet aggregation induced by ADP or tumor cells. Fibronectin was the best substrate for melanoma cell adhesion (95%), followed by laminin (47%) and vitronectin (24%). Albolabrin was the strongest and eristostatin the weakest inhibitor of cell adhesion to all substrata. Adhesion of melanoma cells to albolabrin, echistatin, and barbourin was partially inhibited by monoclonal antibody against mouse alpha v subunit. This antibody bound to B16F10 melanoma cells in suspension and inhibited binding of fluorescein isothiocyanate (FITC)-labeled disintegrins to these cells, being the most effective with FITC-labeled albolabrin. Our study suggests that a major contribution of eristostatin to inhibition of lung colonization is via preferential binding to platelet alpha IIb beta 3 integrin and blocking tumor cells interaction with platelets. A major contribution of albolabrin, barbourin and echistatin appears to be by interference with other integrin receptors on the tumor cell surface. Albolabrin appeared to inhibit RGD-dependent integrins containing alpha v subunit, such as alpha v beta 3 and alpha v beta 1.

Amino Acid Sequence↗
PubMed1998

Accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as integrin alphavbeta3 antagonist and inducing apoptosis.

Endothelial integrins play an essential role in angiogenesis and cell survival. Accutin, a new member of disintegrin family derived from venom of Agkistrodon acutus, potently inhibited human platelet aggregation caused by various agonists (eg, thrombin, collagen, and, adenosine diphosphate [ADP]) through the blockade of fibrinogen binding to platelet glycoprotein IIb/IIIa (ie, integrin alphaIIbbeta3). In this report, we describe that accutin specifically inhibited the binding of monoclonal antibody (MoAb) 7E3, which recognizes integrin alphavbeta3, to human umbilical vein endothelial cells (HUVECs), but not those of other anti-integrin MoAbs such as alpha2beta1, alpha3beta1, and alpha5beta1. Moreover, accutin, but not the control peptide GRGES, dose-dependently inhibited the 7E3 interaction with HUVECs. Both 7E3 and GRGDS, but not GRGES or Integrelin, significantly blocked fluorescein isothiocyanate-conjugated accutin binding to HUVEC. In functional studies, accutin exhibited inhibitory effects on HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin, and the capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner. In addition, it exhibited an effective antiangiogenic effect in vivo when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced HUVEC apoptotic DNA fragmentation as examined by electrophoretic and flow cytometric assays. In conclusion, accutin inhibits angiogenesis in vivo and in vitro by blocking integrin alphavbeta3 of endothelial cells and by inducing apoptosis. The antiangiogenic activity of disintegrins might be explored as the target of developing the potential antimetastatic agents.

Abciximab↗