SVMP affecting tumor cells
Tumorigenesis and metastasis are two processes with inter-related mechanisms. These include tumor growth and angiogenesis, detachment of tumor cells from the primary tumor, followed by migration through the local connective tissue and penetration into the circulation (intravasation). Once in the blood stream, tumor cells interact with circulating blood cells, arrest in the microvasculature of target organs, then extravasate and secondary proliferate. During each of these steps, integrin-mediated adhesion, migration, proliferation and survival of tumor cells and angiogenic endothelial cells play crucial roles [1,2].
Integrins are a family of heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions. These cell adhesion molecules are composed by non covalent association of α and β subunits. Although 18 α and 8 β subunits have been described, only 24 different combinations have been identified to date . Specific integrin heterodimers preferentially bind distinct ECM proteins. The repertory of integrins present on a given cell dictates the extent to which that cell will adhere to and migrate on different matrices. Several integrins, among others αv and α5β1, recognize the RGD sequence on their respective ligands. Other adhesive sequences in ECM proteins have also been observed, including the EILDV and REDV sequences that are recognized by integrin α4β1 in an alternatively spliced form of fibronectin . On ligation to the ECM, integrins cluster in the plane of the membrane and recruit various signalling and adaptor proteins to form structures known as focal adhesions .
Integrin expression can also vary considerably between normal and tumor tissue. Most notably, integrins αvβ3, α5β1 and αvβ6 are usually expressed at low or undetectable levels in most adult epithelia but can be highly up-regulated in some tumors. Expression levels of some integrins, such as α2β1, decrease in tumor cells; potentially increasing tumor cell dissemination . The integrin αvβ3 is particularly important for tumor growth and invasiveness . The receptor plays a major role in neo-vessels formation, its expression being strongly up-regulated in endothelial cells and specifically required during angiogenesis stimulated by basic fibroblast growth factor (bFGF) and tumor necrosis factor-α [7,8]. αvβ3 is functionally involved in the malignant spread of various tumor cell types such as breast carcinoma, prostate carcinoma and melanoma, and supports tumor cell adhesion and migration through endothelium  and matrix proteins [10,1]. Blocking αvβ3 is therefore expected to have a broad impact in cancer therapy and diagnosis. In the last decade, several clinical trials evaluating the efficacy of αvβ3 blockers have led to encouraging results. Thus, MEDI-522 (Vitaxin), a humanized antibody derived from the mouse LM609 monoclonal antibody, was recently reported to give positive results in a phase II trial enrolling patients with stage IV metastatic melanoma . Cilengitide is an inhibitor of both αvβ3 and αvβ5 integrins; it is currently being tested in phase II trials in patients with lung and prostate cancers  and in phase II and Phase III trials studying their role against glioblastoma are currently underway.
In addition to their role in tumor cells, integrins are also important for the host cellular response against cancer. Endothelial cells, fibroblasts, pericytes, bone marrow-derived cells, inflammatory cells and platelets all use integrins for various functions, including angiogenesis, desmoplasia and immune response.
Nature has been a source of medicinal products for thousands of years among which snake venoms form a rich source of bioactive molecules such as peptides, proteins and enzymes with important pharmacological activities. International research and development in this area, based on multidisciplinary approaches including molecular screening, proteomics, genomics and pharmacological
2. Snake venom protein families
2.1. The Snake Venom Metalloproteinases (SVMP)
Metalloproteinases are among the most abundant toxins in many
Most of the functional activities of SVMPs are associated with hemorrhage or the disruption of the hemostatic system, which are primarily mediated by the proteolytic activity of the M domain. SVMPs cause hemorrhage by disturbing the interactions between endothelial cells and the basement membrane through the degradation of endothelial cell membrane proteins (e.g., integrin, cadherin) and basement membrane components (e.g., fibronectin, laminin, nidogen, type IV collagen) . Blood coagulation proteins (e.g., fibrinogen, factor X, prothrombin) are also targets of their proteolytic activities.
In the other side, it was reported that several SVMPs inhibited integrin-mediated adhesion of cancer cells on ECM proteins (table 1). BaG, a dimeric PIII class of SVMP from
|VAP1, VAP2||α3,α6,β1||Induce apoptosis of HUVEC||[31,36]|
|HV1||-||Inhibits adhesion of HUVEC and induces apoptosis|||
|Halysase||α1β1;α5β1||Inhibits proliferation and Induces apoptosis of HUVEC|||
|VLAIPs||-||Inhibits proliferation and Induces apoptosis of HUVEC|||
|Graminelysin||α1β1;α5β1||Inhibits proliferation and Induces apoptosis of HUVEC|||
|BaG||α5β1||Inhibits adhesion of K562 cells|||
|TSV-DM||-||Inhibits cell proliferation and induces transient cell morphologic changes of endothelial cells.|||
Several apoptosis-inducing proteins have been purified from hemorrhagic snake venom, such as VAP1 and VAP2 (
2.2. The disintegrins
Disintegrins are a family of non-enzymatic and low molecular weight proteins derived from viper venom [37-39]. They are able to inhibit platelet aggregation and interact with adhesion molecules in particular integrins in a dose-dependent manner. They have a K / RTS sequence which is known as the RGD adhesive loop [37-39]. Their primary structure shows a strong conservation in the arrangement of cysteines . Most disintegrins represent the C-terminal domain of metalloproteinases PIIa, d and e classes and are released into the venom by proteolytic cleavage [40,37,38]. A minority of these proteins exist as D / C domains from the class of SVMPs PIIIb.
Disintegrins can be conveniently divided into five different groups according to their length and the number of disulfide bridges . The first group includes short disintegrins, single polypeptide composed of 49 - 51 amino acids with four disulfide bridges. The second group comprises medium disintegrins containing about 70 amino acids and six disulfide bridges. The third group includes long disintegrins of 83 residues linked by seven disulfide bridges. The disintegrin domains of PIII snake-venom metalloproteinases, containing approx. hundred amino acids with 16 Cysteine residues involved in the formation of eight disulfide bonds, constitute the fourth subgroup of the disintegrin family. Unlike short-, medium- and long-sized disintegrins, which are single-chain molecules, the fifth subgroup is composed of homo and heterodimers. The dimeric disintegrins subunits contain about 67 residues with four disulfide intra-chain bridges and two interchain bridges [42,43].
Although disintegrins are highly homologous, significant differences exist in their affinity and selectivity for integrins, which explains the multitude of effects of these molecules (Table 2).
Disintegrins were first identified as inhibitors of platelet aggregation and were subsequently shown to antagonize fibrinogen binding to platelet integrin αIIbβ3 [44,45]. After that, studies on disintegrins have revealed new uses in the diagnosis of cardiovascular diseases and the design of therapeutic agents in arterial thrombosis, osteoporosis, and angiogenesis-related tumor growth and metastasis (table 2). Triflavin from
|Inhibits adhesion of tumor cells to matrix proteins, cell migration and angiogenesis |||
|Rhodostomin||αvβ3,αvβ5||Inhibits cell migration, invasion of endothelial cells; inhibits angiogenesis |||
|Blocks adhesion, migration invasion of different type of tumor cells|||
|Lebestatin||α1β1||Inhibits migration and angiogenesis|||
|Accurhagin-C||αvβ3||Prevents migration and invasion of endothelial cells; anti-angiogenic activity |||
|Eristostatin||α4β1,other integrin not yet determined||Inhibits cell motility; no effect on cell proliferation or angiogenesis||[59,60]|
|DisBa-01||αvβ3||Anti-angiogenic and anti-metastatic effect on melanoma cells|||
|Leberagin-C||αvβ3||Inhibits cell adhesion of melanoma tumor cells|||
|Accutin||αvβ3||Inhibits angiogenesis |||
Contortrostatin, a disintegrin isolated from the venom of the southern copperhead snake, exhibits anti-cancer activity in a variety of tumor cells [48-50]. It does not display cytotoxic activity
Lebestatin is an example of a non toxic KTS-disintegrin isolated from
There are few reports regarding the effects of ECD-disintegrins on endothelial cell migration. Acurhagin-C, dose-dependently blocked HUVEC migration toward a vitronectin-coated membrane. Furthermore, acurhagin-C elicited endothelial anoïkis
Eristostatin, an RGD-disintegrin from
Since integrin receptors are also quite indiscriminate as they support cell adhesion to several substrates, it seems highly reasonable that the general RGD-disintegrin scaffold of the integrin-binding motif could be employed as a prototype for drug design for new anti-metastatic therapies
2.3. The snake venom phospholipases
PLA2 (EC 18.104.22.168)—are enzymes that catalyze the hydrolysis of sn-2-acyl bond of sn-3-phospholipids, generating free fatty acids and lysophospholipids as products . They are currently classified in 15 groups and many subgroups that include five distinct types of enzymes, namely secreted PLA2 (sPLA2), cytosolic PLA2 (cPLA2), Ca2+ independent PLA2s (iPLA2), platelet-activating factor acetyl-hydrolases (PAF-AH), lysosomal PLA2, and a recently identified adipose-specific PLA2 [65,67]. PLA2 are low molecular weight proteins with molecular masses ranging from 13-19 kDa that generally require Ca2+ for their activities [69,70]. Snake venom sPLA2 are secreted enzymes belonging to only two groups that are based on their primary structure and disulfide bridge pattern [68,71,72]. Those of group I are similar to pancreatic sPLA2 present in mammals, were found in venom of
Despite a high identity of their amino acid sequences, sPLA2 exhibit a wide variety of pharmacological properties such as anticoagulant, haemolytic, neurotoxic, myotoxic, oedema-inducing, hemorrhagic, cytolytic, cardiotoxic, muscarinic inhibitor and antiplatelet activities [63,85-92].
Recently, PLA2s have been shown to possess anti-tumor and anti-angiogenic properties (Table 3). CC-PLA2-1 and CC-PLA2-2 from
|CCPLA2-1; CCPLA2-2||α5β1,αv||Inhibits migration and adhesion of fibrosarcoma and melanoma cells||[93,94]|
|Bth-A-I-PLA2||-||Anti-tumor activity on adenocarcinoma and leukaemia cells|||
|MVL-PLA2||α5β1,αv||Inhibits adhesion and migration of human microvascular cells and inhibits angiogenesis |||
|BP II||Prothobotrops flavoviridis||-||Induces cell death in human leukaemia cells|||
MVL-PLA2 is a snake venom phospholipase purified from Macrovipera lebetina venom that inhibited adhesion and migration of human microvascular endothelial cells (HMEC-1) without being cytotoxic. Using MatrigelTM and chick chorioallantoic membrane assays, MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both
A cell death activity was discovered in Lysine 49-PLA2 called BPII. It induces caspase-independent cell death in human leukaemia cells regardless of its depressed enzymatic activity .
2.4. The C-type lectins
The C-type lectins are abundant components of snake venom with various function. Typically, these proteins bind calcium and sugar residues. However, the C-type lectin like proteins from snake venom (termed actually snaclec) does not contain the classic calcium/sugar binding loop and have evolved to bind a wide range of physiologically important proteins and receptors .
Snaclecs have a basic heterodimeric structure with two subunits, nearly always linked covalently,
Recently, novel activities of snaclecs were highlighted. They were described for their potential anti-tumor effect by blocking adhesion, migration, proliferation and invasion of different cancer cell lines (Table 4). Among these proteins, EMS16, a heterodimer isolated from the venom of
Lebecetin and lebectin, purified from
Extensive researches have been shown that cell adhesion activities in cancer disease are deregulated. According to this idea, it was also reported that lebectin inhibits these alterations by promoting N-cadherin/catenin complex reorganisation at cell-cell contacts, inducing a strengthening of intercellular adhesion .
Another snaclec, BJcuL isolated from
|Lebecetin, lebectin||α5β1,αv||Inhibits adhesion, migration and invasion of human tumor cells; inhibits angiogenesis|||
|BJcuL||−||Inhibits tumor cell and endothelial cell growth; induces apoptosis of human gastric carcinoma cells; inhibits cell adhesion and actin cytoskeleton disassembly||[111,112]|
|EM16||α2β1||Inhibits adhesion and migration of HUVEC cells|||
3. Potential application of snake venom compounds
Venoms are a rich source of molecules endowed with diverse pharmacological effects. Most part of these molecules act
Until now, no medicine was produced from a native molecule purified from venom. However, several peptidomimetics were designed by basing on the structure of these molecules. The benefits of these peptidomimetics compared to antibodies that can be used for the treatment of certain diseases are: a shorter half-life, reversible inhibition, easier to control a problem and very low immunogenicity. For example, the antihypertensive drug captopril, modelled from the venom of the Brazilian arrowhead viper (
|Capoten ® (Captropil)||Angiotensin converted enzyme (ACE) inhibitor/ high blood pressure||Granted FDA approval|
|Integrilin ® (Eptifibatide)||Platelet aggregation inhibitor/acute coronary syndrome||Granted FDA approval|
|Aggrastat ® (tirofiban)||GPIIb-IIIa inhibitor/ myocardial infarct, refractory ischemia||Approved for use with heparin and aspirin for the treatment of acute coronary syndrome|
|Exanta||Thrombin inhibitor/ arterial fibrillation and blood||Seeking FDA approval|
|Alfimeprase||(Agkistrodon contortrix contortrix)||Thrombolytic/ Acute ischemic stroke, acute peripheral arterial occlusion||Phase III|
|Ancrod ® (viprinex)||Fibrinogen inhibitor/ stroke||Phase III|
|hemocoagulase||Thrombin-like effect and thromboplastin activity/ prevention and treatment of haemorrhage||Phase III|
|Protac/ Protein C activator||Protein C activator/clinical diagnosis of haemostatic disorder||Granted FDA approval|
|Reptilase||Diagnosis of blood coagulation disorder||Granted FDA approval|
|Ecarin||Prothrombin activator/ diagnostic||Granted FDA approval|
Actually, most of the current anticancer therapies (radiotherapy, chemotherapy) are not specific and are targeting at both tumor cells and healthy cells. However, in recent years, new treatments tend to focus on the tumor microenvironment and particularly on the inhibition of tumor angiogenesis. These treatments are based on several active and non toxic proteins from snake venom, as for example contortrostatin from
From the initial discovery of captopril, the first oral ACE inhibitor, to the recent application of disintegrins for the potential treatment of cancer, the various components of snake venoms have never failed to reveal amazing new properties. While the original native snake venom compounds are usually unsuitable as therapeutics, interventions by medicinal chemists as well as scientists and clinicians in pharmaceutical R&D have made it possible to use the snake venom proteins as potential drugs for multiple disorders or scaffolds for drug design.