Integrins, cancer and snake toxins (mini-review)

REVIEW ARTICLE

Integrins, cancer and snake toxins (mini-review)

Rádis-Baptista G.

Department of Biochemistry and Molecular Biology, Federal University of Ceará, Picí Campus, Fortaleza, Ceará, Brazil

^{Correspondence}

ABSTRACT

Integrins encompass a family of transmembrane heterodimeric proteins of adhesion that maintain cells attached to other cells and to the extracellular matrix (ECM). Integrins work as bi-directional mechanotransducers, conveying mechanical signal from outside to inside the cell through a cascade of phosphorylation signals. On the other hand, the signal from inside to outside controls the strength and affinity of integrin adhesion. As proteins of focal contact, integrins are involved in diverse cell functions, such as cell activation, migration, growth, and survival. In the development of neoplastic disease and metastatic tumor, integrins can influence cancer invasiveness and progression, as well as mediate the formation of new blood vessels (angiogenesis). Diverse snake venom toxins have the ability to interact with multiple integrins, what results in inhibition of cell attachment, inhibition of angiogenesis, and induction of apoptotic death of tumor and vascular endothelial cells. The aim of this review is to present data about snake venom toxins that bind to integrins and evoke antiangiogenesis and antitumoral effects.

Key words: integrin, angiogenesis, cancer, apoptosis, snake toxin, snake venom C-type lectin, metalloprotease, disintegrin.

INTEGRINS

Integrins constitute a large family of focal adhesion proteins, in other words, transmembrane proteins of adhesion that keep cells attached to the extracellular matrix; the latter composed of an intricate network of proteins and polysaccharides on the cell surface (41). Integrins work as receptors for several types of cell matrix proteins, for example collagen, fibronectin, lamin, vitronectin, and fibrinogen (50).

The integrins family comprises, in mammals, at least 24 members of heterodimeric transmembrane proteins, which are formed by the noncovalent association of two glycoproteins: one a subunit out of 19 and one b subunit from 9 types. The diversity of the integrins family is further increased not only by alternative mRNA splicing of some a and b subunits, but also by their post-translational modification (49, 110). As example of integrin assembly, b₁ subunit associates with twelve subtypes of the a subunit. Thus, a1b1, a2b1, a3b1, a4b1, a5b1, a6b1, a7b1, a8b1, a9b1, a10b1, a11b1, and avb1 are found on almost all the vertebrate cells. In contrast, av subunit, where v stands for vitronectin receptor, associates with more than one b subunit, making up avb1, avb3, avb5, avb6, and avb8. Multiple integrins recognize several cell matrix proteins, many of which usually contain the consensus motif of arginine-glycine-aspartic acid (RGD). Extracellular divalent cations, such as Ca²⁺ and Mg²⁺, influence the specificity and affinity of integrins binding to their ligands (8, 102)

In addition to participating in the cell matrix attachment, clustering of integrin at the sites of cell contact can activate intracellular signaling through focal adhesion kinase (FAK or pp125^FAK) and via other associated proteins (18, 97). FAK also associates with a number of other adapter, signaling, and cytoskeletal proteins, such as paxillin, Graf (GTPase regulator associated with FAK), cytohesin-1, Grb2, p130^CAS, a-actinin, filamin, and talin (17, 92, 97). This way, signaling from the outside to inside the cell ("outside-in" integrin signaling) induces FAK to assemble in complexes of proteins that intermediate the signal transduction. For example, once the accessory proteins paxillin and talin induce (auto-) phosphorylation of numerous tyrosine residues, in FAK cytoplasmic tails, FAK acts as a docking site for SH2 domains of signaling proteins, like Src and phosphatidylinositol 3-kinase (PI 3-kinase) (17, 92). In turn, Src protein mediates the phosphorylation of the SH3 domain-containing adapter protein, p130^CAS, which promotes Crk binding and downstream signaling for cell migration. On the other hand, Grb2 directly binding to FAK, in response to integrin activation, initiate Ras/Erk/MAP kinase pathway, and, consequently, cell proliferation in normal and pathological states (17, 82, 83, 87). FAK is also implicated in controlling cell cycle progression via Jun NH2-terminal kinase (JNK) and preventing apoptosis through a pathway involving the proteins kinase C, phospholipase A2, and p53 (5, 76).

Thus, the "outside-in" signaling mediated by integrins is implicated in several cellular functions such as cell adhesion and migration, cell growth and survival, cell differential and development, tissue repair, hemostasis, and even apoptosis (8, 50, 113).

The signal from the extracellular environment into the cell, i.e. "the outside-in signaling", is not the only transduced by integrins. "Inside-out signaling pathway", in which signals inside the cell are mediated by phosphorylation of the integrin cytoplasmic tail, also operates in the regulation of the integrin affinity and adhesion strength in the site of focal contact (3, 29, 108). The inside-out signaling is also particularly significant in the activation of platelets and white blood cells (9).

Most of the recent advances on the role of integrins (and some of their ligands) functions in vertebrate development processes came with the use of knockout mutation, mainly in mouse model (27, 49). As compiled by De Arcangelis and Georges-Laburesse (27) and others (35, 103), the inactivation of the b1 integrin gene expression, which corresponds to the lack of more than ten integrins, results in embryonic lethality. By working on the a subunit genes silencing, more precise integrin functions were revealed, including their role in the formation of cerebral vasculature, in the maintenance of muscle integrity, and in the development of the nervous system.

INTEGRINS AND CANCER

Tumor or neoplasm is an abnormal mass of tissue that results when cells divide without control. When tumor cells acquire the ability to invade surrounding tissues (metastasis), they become malignant, and the metastatic or malignant tumor is referred as cancer. Cancer cells can invade nearby tissues and spread through the bloodstream and lymphatic system to other parts of the body. There are several main types of cancer. Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is cancer that starts in blood-forming tissues, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream. Lymphoma and multiple myeloma are cancers that begin in the immune system cells (75).

As pointed out, integrins operate as mechanotransducer, integrating essential functions in cell development and in physiological processes, by regulating cell-cell and cell-matrix contacts. It should be noted that sites of contact between integrins and ECM, the focal adhesions, are prominent regions of signal transduction via phosphorylation. Signaling through integrin clusters and FAKs, at focal adhesions, is essential for normal cell growth and migration. Loss of ECM contacts and normal cytoskeletal structure can stimulate cancer cells growth, whereas restoration of ECM adhesion reverses this tendency (93). For example, activation of integrin a3b1 down-regulates E-cadherin-mediated adhesion, causing loss of cell-cell adhesion and junctional communication, as well as enhancing invasiveness of malignant tumor cells (55). Obviously, reduction of adhesion and increasing of motility give cancer cells the capacity of invasion and mestatasis. In fact, in some invasive tumors, cells over-expressing FAKs or other integrin-associated protein (e.g., integrin-linked kinase, p59^ILK) lose their contact and adherence with ECM, and become highly motile (43, 80), although the detachment of cell anchorage from ECM induces apoptosis in vitro and in physiological conditions. Therefore, an increase of FAK expression and downstream signaling through tyrosine phosphorylation are correlated with the behavior of cell proliferation, survival, migration, and progression to an invasive phenotype (90, 91, 113).

The development of cancer is not a simple pathological process in which a single participant in a pathway has a primordial role in the disease progression. In this context, integrins not only mediate some direct effects in cancer fates due to their involvement in cellular response to chemical signaling and attachment, but also have an indirect participation by conveying molecular signaling for the development of new blood vessels, which will nourish the growing tumor mass.

In truth, it was realized more than 100 years ago that angiogenesis occurs in tumors. In the early 70's, Dr. Judah Folkman proposed that tumor growth and metastasis are dependent on angiogenesis and that detaining new blood vessel formation might be an effective therapeutical strategy to arrest tumor growth (36). It was recognized that tumor expansion outside the limit of 1 to 2 mm in size is extremely dependent on the recruitment of new blood vessels. The angiogenesis process is regulated in a tissue by the balance between angiogenic factors and inhibitors, and involves activation of several receptors on endothelial vascular cells, including integrins.

INTEGRIN AND VASCULAR DEVELOPMENT

In both vasculogenesis and angiogenesis, vessel formation is a dynamic, complex biological process that depends on interactions with regulatory factors and adhesive substrates. Although both processes utilize practically common molecular components in the vessel development, they have different origins. Angiogenesis is the process of growth of new capillaries from pre-existing blood vessels and, in contrast, vasculogenesis only develops during embriogenesis from pluripotent blood cells (86).

In mammals, angiogenesis is involved in both physiological and pathological processes, and multicellular organisms must recruit new blood vessels to grow beyond the limit of oxygen diffusibility (100 – 200 µm). In normal tissue growth, such as in embryonic development, there is a dependence on new vessel formation, while menstrual cycle and wound healing are characterized by transient neovascularization from quiescent vascular endothelial cells (101).

Proper activation of angiogenesis depends on the proliferation, adhesion, migration, and maturation of endothelial cells. Several factors contribute to each individual process, and the balance between angiogenic factors and endogenous angiogenic inhibitors is the key component affecting the development of new vessels. For example, one of the major players in angiogenesis initiation is the powerful stimulator vascular stimulator growth factor (VEGF). This growth factor is under the control of the hypoxia-inducible factor (HIF), and is up-regulated under hypoxic or ischemic condition (39). VEGF is abundantly produced by hypoxic tumor cells and by several cell lines of the immune system, although the host cells also contribute to tumor angiogenesis (11, 72, 104, 109).

As emphasized, integrins are transmembrane proteins that mediate cell-cell and cell-ECM adhesion and bi-directional signal transduction, and thus they participate in many aspects of vasculogenesis and angiogenesis (reviewed in 85). At least seven integrin heterodimers are present in endothelial cells: a1b1, a2b1, a3b1, a5b1, a6b1, avb3, and avb5, and their activation and differential expression are finely regulated in response to several angiogenic factors (46, 94, 95, 96, 111). In the development and remodeling of vascular system, these integrins are directly involved in matrix assembling, in which they interact with ECM proteins and mediate cell attachment and migration. In addition, through the inside-out and outside-in bi-directional signaling and in association with other non-receptor tyrosine kinases, they also mediate agonist affinity and specificity, gene expression, shaping, and survival of vascular cells (34, 60, 97).

Several pathological conditions are associated with abnormal angiogenesis. These include arteriosclerosis, scar keloids, psoriasis, endometriosis, obesity, rheumatoid arthritis, inflammatory and infectious processes, diabetes, retinopathy, tumor growth, and metastasis, among others (16, 37). Thus, the inhibition of angiogenesis was adopted as a therapeutic strategy for the treatment of cancer and other diseases, in which neovascularization is one of the pathological components (14, 38, 87). As recently reviewed (15), more than 60 angiogenesis inhibitors are being evaluated for their anticancer effects in human patients. These compounds encompass integrin antagonists, blockers of VEGF-, FGF-, and PDGF-receptors, natural and synthetic matrix metalloproteases (MMPs), and monoclonal antibodies, among others.

INTEGRINS, CANCER, AND SNAKE TOXINS

As mentioned above, integrins play significant roles in angiogenesis, in addition to the signal transduction that affects cell behaviors, like cancer spreading. Therefore, one strategy to undermine cancer progression is to target integrins either on tumor cells or on vascular endothelial cells that are recruited by cancer. One particular importance in targeting integrins is that they are differentially distributed in adult human tissue. For example, a high level of avb3 integrin is expressed in endothelial cells, which are exposed to various growth factors, or in cells undergoing normal or pathological neovascularization (34). It is noteworthy that avb3 and avb5 integrins are also expressed in some types of invasive tumors, correlating with cancer aggressiveness. In addition, up- or down-regulation of integrin expression sub-types determines the incidence of such diseases and the patient prognosis (87).

It is well known that some snake venoms contain toxins with the ability to bind to integrins and interfere with cell-cell and cell-ECM adhesion by integrin binding. For instance, the integrins a2b1 (glicoprotein Ia/IIa, GPIa/IIa) and aIIbb3 (GPIIb/IIIa) expressed on platelet membranes, are the targets for several snake toxins, which inhibit or promote platelet aggregation (1, 2). Among the toxins from snake venom that bind to integrin a2b1 (GPIa/IIa) promoting platelet aggregation are aggretin, from Calloselasma rhodostoma (21); bilinexin, from Agkistrodon bilineatus (32); crovidisin, from Crotalus viridis (61); EMS16, from Echis multisquamatus (48); jararhagin, from Bothrops jararaca (28, 52); and rhodocetin, from Calloselasma rhodostoma (112). After binding to the integrin, the induction of platelet activation by some of these toxins initiates a cascade of phosphorylation conducted by p125^FAK and p72^SYC (serine-tyrosine kinase, SYK), as reported for aggretin (74).

Some snake toxins acting on the integrin aIIbb3, which causes inhibition of platelet aggregation, are reported: barbourin, from Sistrurus m. barbouri (89); CC5 and CC8, from Cerastes cerastes (12); crotavirin, from Crotalus viridis (62); echistatin, from Echis carinatus (40, 44); eristotatin, from Eristocophis macmahoni (71); piscivostatin, from Agkistrodon piscivorous piscivorous (77); Sal-C and salmosin, from Agkistrodon halys brevicaudus (22, 59); saxatilin, from Gloydius saxatilis (47); Schistatin, from Echis carinatus (107).

It should be noted that most snake toxins acting on these integrins belong to the families of C-type lectins, disintegrins, and metalloproteases. Snake venom C-type lectins contain the conserved carbohydrate recognition domain (CRD) of other animal C-type lectin and share significant primary structure similarities with them, but they not necessarily bind to carbohydrate molecules nor require calcium ions for their activity. Unlike the classic C-type lectins, those from snake venoms are generally heterodimeric, with two subunits, alpha and beta, and some of them are multimeric heterodimers (2, 23, 31). Metalloproteases constitute prominent components of viperid and crotalid venom with the ability to hydrolyze extracellular matrix proteins, interfere with cell-ECM and cell-cell adhesion, and consequently cause hemorrhage. High molecular weight (20 to 100 kDa) metalloproteases are referred as metalloproteinase/disintegrin-like/cysteine (MDC) rich proteins, since they have a N-terminal catalytic site, a disintegrin-like interacting domain (usually RGD), and a hydrophobic cell-cell fusion domain in cysteine-rich C-terminus (53). Disintegrins are low molecular weight, non-enzymatic, polypeptides, rich in residues of cysteine, containing, in general, the RGD motif with the ability to interact with integrins on the cell membrane (53, 66).

It is noteworthy that ECM-degrading metalloproteases including members of the metzincins family, such as ADAMs (a disintegrins and metalloproteinase domain) and adamalysins-like enzymes, constitute extrinsic regulators of epithelial tumor progression (7). In fact, proteolysis of ECM components, including growth factor precursors embedded (sequestered) in or anchored to ECM, is a key process during cell adhesion, growth and differentiation; tissue morphogenesis; wound healing; and conversion of benign tumors into invasive malignant metastatic cancer (4, 30, 81).

Paradoxically, it has been reported the ability of some snake venom components, belonging to the metalloprotease/disintegrin family, to inhibit in vitro cell migration, adhesion/detachment, proliferation and in vivo cancer progression. For instance, vascular apoptosis-inducing proteins 1 and 2 (VAP1 and VAP2), from the venom of western diamondback rattlesnake (Crotalus atrox), as their names suggest, induce in vitro apoptotic death of vascular endothelial cells, and the integrins a3b1 and a6b1 are involved (69, 70). Crovidisin, a PIII snake venom metalloproteinase purified from western rattlesnake (Crotalus viridis) venom, causes detachment of ROS 17/2.8 osteosarcoma cells, but not of primary cultured osteoblasts from extracellular matrix proteins. The proteolysis of extracellular matrix proteins by crovidisin was implicated in the selective detachment (106).

At this point, it would be necessary to clarify the mechanisms of the apparently contradicting roles of metalloproteases in the facilitation of cancer progression or in the restrain of tumor evolution. It was verified that some proteolytic fragments of ECM proteins act as inhibitors of neoplastic growth (7, 42). The explanation is validate by the remarkable works of O'relly and collaborators (78, 79) that isolated plasminogen and collagen XVIII fragments able to block angiogenesis and the spreading of metastasis. Thus metalloproteases can, not only promote cancer proliferation and invasion, but also suppress tumor growth by generating inhibitory proteolytic fragments that can interact to transmembrane receptors, such as integrins and other adhesion proteins, (over-) expressed in cancer and in vascular endothelial cells.

Snake toxins belonging to the families of C-type lectins and disintegrins, acting on cell migration, adhesion and proliferation, have also been isolated and characterized. Consequently, proteolysis of ECM components is not the sole essential mechanism by which snake toxins commit the cell fate. For instance, contorstrostatin, a homodimeric disintegrin from Agkistrodon contortix contortix venom, induces protein phosphorylation in T24 human bladder cancer cells via integrin-mediated signaling, which includes the integrin-FAK-adapter protein p130^CAS. In contrast, the monomeric disintegrins echistatin and flavoridin from saw-scaled viper (E. carinatus) and habu snake (Trimeresurus flavoviridis) venoms, respectively, do not induce protein phosphorylation (85). Salmosin is a disintegrin purified from the venom of the Korean snake (Agkistrodon halys brevicaudus). In addition to antagonizing the platelet integrin GPIIb-IIIa, it interacts with the integrin avb3 and inhibits the proliferation of bovine capillary endothelial (BCE) cells induced by the basic fibroblast growth factor (bFGF). In BCE cells treated with salmosin, focal adhesion kinase (FAK) was dephosphorylated and the expression of paxillin and p130^CAS was decreased, but PI3 kinase, and other FAK adapter proteins are deregulated, suggesting that salmosin induces apoptosis of BCE cells by inactivating FAK-dependent integrin signaling pathways (45).

Several other disintegrins are able to interfere with experimental tumor metastasis. Eristostatin, from the venom of the leaf-nosed viper (Eristocophis macmahonii), bound to integrins on each B16F1 melanoma cell injected into C57BL/6 mice and reduced the number of growing cells (73). This disintegrin requires the RGDW motif, as well as an intact C-terminus to interact with both platelets and melanoma cell lines, which include 1205-LU, WM164, C8161, and MV3 (71).

Likewise, albolabrin, from the venom of the green pit viper (Trimeresurus albolabris); barbourin, from the dusky pigmy rattlesnake (Sistrurus miliarius barbouri) venom; and echistatin, inhibited the formation of experimental lung metastases induced by IV injection of B16F10 murine melanoma cells into C57BL/6 mice (10). Trigramin, isolated from the venom of the Indian green tree viper (Trimeresurus gramineus) snake, has the RGD motif and inhibits the adhesion of human melanoma cells to fibronectin and fibrinogen (58). In addition, trigramin and rhodostomin (a disintegrin from the venom of the Malayan pit viper - Calloselasma rhodostoma) prevent platelet aggregation induced by SW-480 tumor cell, derived from a primary human colon adenocarcinoma, and by MCF-7 cells, a metastatic human breast carcinoma line (19, 20).

One of the C-type lectins with the ability to inhibit the integrin-mediated attachment to ECM components of various tumor cell lines is lebectin. Lebectin was isolated from the venom of Levantine viper (M. lebetina). It was also able to block cell migration towards fibronectin, and prevented invasion of fibrin gels by tumor cells. Moreover, lebectin showed an inhibitory effect on tumor cell proliferation (88).

Most snake venom toxins exert their inhibitory effects on cell adhesion, migration and proliferation through the interaction with integrins expressed on the cell surface. In fact, contorstrostatin binds to the integrins aIIbb3, a5b1, and avb3, and inhibits adhesion and invasion of cancer cells (118). The disintegrins echistatin, flavoridin (from Trimerusurus flavoviridis venom), and kistrin (from Calloselasma rhodostoma), bind with high affinity to immobilized avb3 in solid phase assay (51). Eristostatin seems to interfere with a4b1-VCAM binding, thus it strongly inhibits lung colonization of MV3 cells in nude mice injected with tumor cells (25). EMS16, another snake venom C-type lectin, isolated from Echis multisquamatus, selectively binds to the a2I domain of a2b1 integrin and inhibits collagen-induced platelet aggregation. EMS16 also binds to the integrin a2 transfected K562 cells and inhibits the human umbilical vein endothelial cells (HUVEC) migration in collagen I gel (64). Rhodostomin blocks the integrin avb3 on human vascular endothelial cells thus inhibiting distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF) and suppressing murine melanoma tumor growth in vivo. To verify the binding of rhodostomin to the integrin avb3, a monoclonal avb3 antibody (mAb) selectively inhibited the binding of rhodostomin to both naive and bFGF-primed HUVECs (115).

A striking number of papers have been published on snake venom toxins, which cover the range of specificities to every mammalian integrin subtype, including the integrins a1b1, a3b1, a4b1, a5b1, a5b3, a6b1, a6b4, a7b1, a9b1, avb3, ab5, and aM(b2). Most importantly, snake venom toxins interact with integrins according to their specificity and interfere with cell adherence, invasiveness, and survival, but to different extent.

For instance, halysase, a monomeric DECD-metalloprotease isolated from the venom of halys viper (Gloydius halys), strongly inhibits the proliferation of human umbilical vein of endothelial cells in a dose-dependent manner and inhibits the adhesion of these cells to extracellular matrix proteins through the interaction with integrins a1b1 and a5b1. It is also able to induce apoptosis of endothelial cells by activating caspase-3 and decreasing the level of BcL-X(L)/Bax (116). Lebein-1 (known as lebein) and lebein-2, two RGD-containing desintegrins purified from the venom of Vipera lebetina, strongly interacts with the integrins a3b1, a6b1, and a7b1, but not with the collagen-binding a1b1 and a2b1 integrins. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, the interaction seemed to be RGD-independent and, in some way, the toxins mimic a yet unknown integrin-binding structure of laminins (33). Three non-RGD heterodimeric disintegrins, VLO5, EO5, and EC3, from the venom of Vipera lebetina obtusa, Echis ocellatus, and E. carinatus, respectively, are potent inhibitors of a4b1, a4b7, and a9b1 integrins expressed in leukocytes (6). Furthermore, it was showed that VLO5, and other five novel disintegrins, VLO4, VB7, VA6, EO4, and EMS11, from the venom of V. l. obtusa, V. berus, V. ammodytes, Echis ocellatus, and E. multisquamatus, blocked the adhesion of the a4b1 integrin to vascular cell adhesion molecule 1 (VCAM-1) with high specificity. While the desintegrin EMS11 inhibited both a5b1 and a4b1 integrins with almost the same degree of specificity (13). The heterodimeric MLDG-disintegrins (non-RGD) EC3 and EC6, from E. carinatus venom, exhibited specificity towards the integrins a4b1 and a9b1. Both EC3 and EC6 were potent inhibitors of a9b1 mediated adhesion to VCAM-1 and of neutrophils migration across the layer of endothelial cells activated by tumor necrosis factor (65). Snake venom disintegrins capable of interfering with integrin aM were also characterized, but they evoke responses other than cell detachment and apoptosis. For example, jarastatin, a monomeric RGD-disintegrin from the venom of jararaca (Bothrops jararaca), inhibts neutrophil chemotaxis induced by fMet-Leu-Phe. Comparison of JT and EC3 indicated that both chemotactic disintegrins induce the activation of FAK and PI 3-kinase. However, they are different in that, while JT delays apoptosis of neutrophil via activation of Erk-2, EC3 inhibits Erk-2 signaling and holds a proapoptotic activity (24). HF3 is another component of B. jararaca venom, which mediated cell function by means of interaction with the integrin aM. HF3 belongs to the class P-III and contains the typical disintegrin-like/cysteine-rich domains. It was shown that both native and recombinant HF3 increase aMb2-mediated phagocytosis of opsonized-zymosan particles by macrophages (100).

As mentioned, avb3 and avb5 are the main integrins expressed in vascular endothelial cells and in several types of tumor. Toxins targeting avb3 and avb5 integrins have been isolated from viper and pit viper venoms. Accutin, which was purified from the venom of Agkistrodon acutus (or Deinagkistrodon acutus) and is also a member of the disintegrin family, inhibits not only human platelet aggregation by blocking the binding of fibrinogen to the platelet GPIIb/IIIa (integrin aIIbb3), but also the adhesion of HUVEC to immobilized ECM proteins, such as fibrinogen, fibronectin and vitronectin. It also inhibits capillary-like tube formation on Matrigel in a dose- and RGD-dependent manner, and angiogenesis in vivo, when assayed in chick embryo chorioallantoic membranes (CAM) model, while it induces apoptotic DNA fragmentation in HUVEC. All these effects were triggered by toxin binding to the integrin avb3 (114). Contortrostatin, described above, blocks the adhesion of the human epithelial carcinoma cell line of the ovary (OVCAR-5) to extracellular matrix proteins and inhibits tumor cell invasion. In addition, contortrostatin not only inhibited ovarian cancer dissemination significantly, but it dramatically suppressed the recruitment of blood vessels to tumorsin, a xenograft nude mouse model implanted with OVCAR-5 cells (67). Salmosin also binds to the avb3 integrin expressed on the vascular endothelial cell and thus block tumor-induced angiogenesis (54). Likewise, by interacting with avb3 integrin, salmolisin inhibits the proliferation of SK-Mel-2 human melanoma cells and the metastasis of B16 melanoma cells (22, 57). The anti-tumor effect of salmosin was suggested to be due to its antiangiogenic activity, since the recombinant proteins inhibited proliferation of bovine capillary endothelial (BCE) cells and effectively inhibited the migration of highly metastatic B16BL6 mouse melanoma cells. Besides, recombinant salmosin inhibited neovascularization in CAM and in Matrigel implanted subcutaneously into mice (56). Another snake disintegrin able to bind to avb3 integrin and to inhibit adhesion of tumor cells to ECM proteins is triflavin, isolated from the venom of Trimeresurus flavoviridis (99). By the interaction with integrin avb3, triflavin inhibited the in vitro adhesion of HUVECs to ECMs (vitronectin, fibronectin, laminin, and collagen type IV), and suppressed TNFa-induced angiogenesis in CAM assay. Moreover, triflavin inhibits the adhesion of hepatoma cell to extracellular matrices by binding to the integrins avb3, a3b1, and a5b1, expressed on the surface of hepatoma cells (98).

There are snake venoms or toxins that are able to inhibit cell adhesion to ECM proteins, induce apoptotic cell death and restrain cell proliferation, of which binding specificities or the venom component responsible for binding are not fully characterized yet. For example, components from Macrovipera lebetina and Cerastes cerastes venoms bind to IGR39 melanoma cells but not to HT29-D4 cells that derived from colonic adenocarcinoma. However, these venoms inhibit the adherence of IGR39 and HT29-D4 to various extracellular matrix proteins (68). Lebectin, a C-type lectin, was then isolated from the venom of M. lebetina (88). Lebectin inhibited the integrin-mediated attachment of various tumor cell lines, and the integrin a5b1 might be the target for lebectin, but it still needs further characterization. Zhao and collaborators (117) demonstrated that the crude venom of western diamond rattlesnake (Crotalus atrox) contains at least one important target-component to integrin b4, and this component is responsible for the early phase of apoptosis in vascular endothelial cells, mediated by integrin signaling and over expression of p53. Lipps (63) had purified from the venoms of Crotalus atrox and Naja naja kaouthia, two cancer inhibitors named atroporin and kaotree, respectively. As noted, atroporin and kaotree inhibited various types of human and animal cancer cells, in low concentrations, and did not cause any damage to normal cells. Both atroporin and kaotree were able to not only prevent the formation, but also induce the regression of ascitic tumors in Balb/C mice inoculated with myeloma cells. The crude venom of Bothrops jararaca and Crotalus durissus terrificus exhibit antitumoral activity against Ehrlich ascite tumor cells. Nevertheless, the antitumoral effect seemed to be mediate by inflammatory response, in which toxin-activated macrophages release tumor necrosis factor and interleukins (26).

Apart of the fundamental research on snake venom proteins, such as metalloproteases, disintegrin and C-type lectins, in the scope of molecular cell biology of tumor, the prospective of the development of a cancer treatment adjuvant from snake toxins is promising. Thus far, clinically significant for the purposes of anticancer therapy, the complexation of integrin-target toxins with liposomes improves their effective therapeutic levels, their circulatory half-life and facilitates their administration. For instance, salmosin gene subcutaneously administered with cationic liposomes in vivo resulted in a systemic expression of the gene product, inhibition of the B16BL6 melanoma cells growth, and suppression of pulmonary metastases (57). Liposome release of contortrostatin, by means of intravenous administration, maintains the potent antiangiogenic activity of naive toxin and restrains breast cancer progression, as demonstrated with a xenograft human mammary tumor model (105).

CONCLUDING REMARKS

It should be noted that the compilation of data concerning to toxins acting specifically on integrins, which are differentially expressed on the membrane of several mammalian cell types, such as platelets, vascular endothelial cells, and mainly immortalized and cancer cells, was aimed. Evidently, much more snake venom toxins acting on the diverse number of integrins can be found. Here some of them were presented to illustrate the specific interaction between target-integrin and snake toxins that can modulate cell function and fate. Collectively, the information offers a vision of the medical and biotechnological potentialities contained in the snake venom per se, and in the particular classes of toxins, like those treated here, useful for cancer research and control.

ACKNOWLEDGEMENTS

I am very thankful to Dr. Kenichi Furihata, at SRL Inc., Tokyo, Japan, for critically read this manuscript and for significant suggestions. I am very grateful to Dr. Tetsuo Yamane, at the Center of Molecular Biology, Nuclear Energy Research Institute, IPEN, São Paulo, Brazil, and Center of Biotechnology of Amazonas, Manaus, Amazonas, Brazil, for the constant and invaluable discussion on Molecular Toxinology and Chemical Ecology. I manifest my gratitude to CAPES, CNPq, and FUNCAP for the financial support.

75 NIC, National Cancer Institute, U.S.National Institute of Health (htpp:// www.cancer.gov)

84 PUBMED, National Center for Biotechnology Information, National Library of Medicine, (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed)

Received: February 2, 2005

Accepted: February 2, 2005

Published online: July 1, 2005

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Publication Dates

• Publication in this collection
  16 Aug 2005
• Date of issue
  Sept 2005

History

• Accepted
  02 Feb 2005
• Received
  02 Feb 2005