RESUMO
Disintegrins, a family of snake venom protein, which are capable of modulating the activity of integrins that play a fundamental role in the regulation of many physiological and pathological processes. The main purpose of this study is to obtain the recombinant disintegrin (r-DI) and evaluate its biological activity. In this study, we explored a high-level expression prokaryotic system and purification strategy for r-DI. Then, r-DI was treated to assay effects on cell growth, migration, and invasion. The affinity for the interactions of r-DI with integrin was determined using Surface plasmon resonance (SPR) analyses. The r-DI can be expressed in Escherichia coli and purified by one-step chromatography. The r-DI can inhibit B16F10 cells proliferation, migration, and invasion. Also, we found that r-DI could interact with the integrin αIIbß3 (GPIIb/IIIa). The r-DI can be expressed, purified, characterized through functional assays, and can also maintain strong biological activities. Thus, this study showed potential therapeutic effects of r-DI for further functional and structural studies.
Assuntos
Desintegrinas , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Animais , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/isolamento & purificação , Desintegrinas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Camundongos , Viperidae/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Linhagem Celular Tumoral , Expressão Gênica , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos/química , Venenos de Crotalídeos/genética , Crotalinae , Serpentes PeçonhentasRESUMO
Snake venoms are a potential source of various enzymatic and non-enzymatic compounds with a defensive role for the host. Various peptides with significant medicinal properties have been isolated and characterized from these venoms. Few of these are FDA approved. They inhibit tumor cells adhesion, migration, angiogenesis and metastasis by inhibiting integrins on transmembrane cellular surfaces. This plays important role in delaying tumor growth, neovascularization and development. Tumor targeting and smaller size make them ideal candidates as novel therapeutic agents for cancer treatment. This review is based on sources of these disintegrins, their targeting modality, classification and underlying anti-cancer potential.
Assuntos
Antineoplásicos/uso terapêutico , Desintegrinas/uso terapêutico , Integrinas/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Venenos de Serpentes/metabolismo , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/isolamento & purificação , Desintegrinas/efeitos adversos , Desintegrinas/isolamento & purificação , Humanos , Integrinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de SinaisRESUMO
In the soft treatment of cancer tumours, consequent downregulation of the malignant tissue angiogenesis constitutes an efficient way to stifle tumour development and metastasis spreading. As angiogenesis requires integrin-promoting endothelial cell adhesion, migration, and vessel tube formation, integrins represent potential targets of new therapeutic anti-angiogenic agents. Our work is a contribution to the research of such therapeutic disintegrins in animal venoms. We report isolation of one peptide, named Dabmaurin-1, from the hemotoxic venom of snake Daboia mauritanica, and we evaluate its potential anti-tumour activity through in vitro inhibition of the human vascular endothelial cell HMECs functions involved in tumour angiogenesis. Dabmaurin-1 altered, in a dose-dependent manner, without any significant cytotoxicity, HMEC proliferation, adhesion, and their mesenchymal migration onto various extracellular matrix proteins, as well as formation of capillary-tube mimics on MatrigelTM. Via experiments involving HMEC or specific cancers cells integrins, we demonstrated that the above Dabmaurin-1 effects are possibly due to some anti-integrin properties. Dabmaurin-1 was demonstrated to recognize a broad panel of prooncogenic integrins (αvß6, αvß3 or αvß5) and/or particularly involved in control of angiogenesis α5ß1, α6ß4, αvß3 or αvß5). Furthermore, mass spectrometry and partial N-terminal sequencing of this peptide revealed, it is close to Lebein-1, a known anti-ß1 disintegrin from Macrovipera lebetina venom. Therefore, our results show that if Dabmaurin-1 exhibits in vitro apparent anti-angiogenic effects at concentrations lower than 30 nM, it is likely because it acts as an anti-tumour disintegrin.
Assuntos
Inibidores da Angiogênese/isolamento & purificação , Desintegrinas/isolamento & purificação , Neovascularização Patológica/prevenção & controle , Venenos de Víboras/química , Viperidae , Sequência de Aminoácidos , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/farmacologia , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Humanos , Neovascularização Patológica/patologia , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/farmacologiaRESUMO
Disintegrins are low molecular weight cysteine-rich proteins (4-14 kDa) that are isolated mainly from viperid snake venom. Due to their potential as lead compounds for binding and blocking integrin receptors, snake venom disintegrins have become one of the most studied venom protein families. The aim of this study was to obtain disintegrins from C. totonacus venom and evaluate their capability to bind and block integrin receptors. The C. totonacus disintegrin fraction (totonacin) represents two disintegrin isoforms obtained from C. totonacus venom. These disintegrins showed extracellular-matrix (ECM) protein adhesion and migration inhibitory effects on MDA-MB-231 and HMEC-1 cells. Totonacin (3 µM) inhibited MDA-MB-231 cell adhesion to the ECM proteins, fibronectin, vitronectin, and laminin by 31.2, 44.0, and 32.1, respectively. Adhesion inhibition to fibronectin, vitronectin, and laminin observed on HMEC-1 cells was 42.8, 60.8, and 51%, respectively. In addition, totonacin (3 µM) significantly inhibited MDA-MB-231 and HMEC-1 cell migration (41.4 and 48.3%, respectively). Totonacin showed more potent cell adhesion inhibitory activity toward vitronectin in both cell lines. These results suggest a major affinity of totonacin toward αVß3, α8ß1, αVß5, αVß1, and αIIbß3 integrins. In addition, the inhibitory effect observed on MDA-MB-231 and HMEC-1 cell migration reinforces the evidence of an interaction between these disintegrins and αVß3 integrin, which plays a key role in migration and angiogenesis.
Assuntos
Venenos de Crotalídeos/química , Desintegrinas/farmacologia , Proteínas de Répteis/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Crotalus , Desintegrinas/isolamento & purificação , Humanos , Proteínas de Répteis/isolamento & purificação , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND: Disintegrins from snake venoms bind with high specificity cell surface integrins, which are important pharmacological targets associated with cancer development and progression. OBJECTIVE: In this study, we isolated a disintegrin from the Porthidium lansbergii lansbergii venom and evaluated its antitumoral effects on breast cancer cells. METHODS: The isolation of the disintegrin was performed on RP-HPLC and the inhibition of platelet aggregation was evaluated on human platelet-rich plasma. The inhibition of cell adhesion was also evaluated in vitro on cultures of cell lines by the MTT method as well as the inhibition of breast cancer cell migration by the wound healing assay. The binding of the disintegrin to integrin subunits was verified by flow cytometry and confocal microscopy. Finally, inhibition of angiogenesis was assessed in vitro on HUVEC cells and the concentration of VEGF was measured in the cellular supernatants. RESULTS: The disintegrin, named Lansbermin-I, is a low molecular weight protein (< 10 kDa) that includes an RGD on its sequence identified previously. Lansbermin-I showed potent inhibition of ADP and collagen-induced platelet aggregation on human plasma and also displayed inhibitory effects on the adhesion and migration of breast cancer MCF7 and MDA-MB 231cell lines, without affecting nontumorigenic breast MCF-10A and lung BEAS cells. Additionally, Lansbermin-I prevented MCF7 cells to adhere to fibronectin and collagen, and also inhibited in vitro angiogenesis on human endothelial HUVEC cells. CONCLUSION: Our results display the first report on the antitumor and anti-metastatic effects of an RGDdisintegrin isolated from a Porthidium snake venom by possibly interfering with α2 and/or ß1-containing integrins. Thus, Lansbermin-I could be an attractive model to elucidate the role of disintegrins against breast cancer development.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Venenos de Crotalídeos/farmacologia , Desintegrinas/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Desintegrinas/química , Desintegrinas/isolamento & purificação , Relação Dose-Resposta a Droga , Feminino , Humanos , Integrinas/análise , Integrinas/metabolismo , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Relação Estrutura-Atividade , Viperidae , Cicatrização/efeitos dos fármacosRESUMO
Leucurogin is an ECD disintegrin-like protein, cloned from Bothrops leucurus venom gland. This new protein, encompassing the disintegrin region of a PIII metalloproteinase, is produced by recombinant technology and its biological and functional activity was partially characterized in this study. Biological activity was characterized in vitro using human fibroblasts. Functional activity of leucurogin was analysed in vitro and in vivo with murine B16F10 Nex-2 and human melanoma BLM cells. The results show that leucurogin inhibits cellular processes dependent on collagen type I. In a competition assay with collagen, leucurogin inhibits, in a dose-dependent manner, the adhesion of fibroblast to collagen. At 10⯵M leucurogin reduces adhesion (40%) and migration (70%) of hFb and inhibits migration (32%) and proliferation (65%) of BLM cells. At 2.5⯵M leucurogin inhibits 80% cell proliferation of B16F10 Nex-2 melanoma cells. At 4.8⯵M leucurogin inhibits, in vitro, the vascular structures formation by endothelial cells by 66%. Leucurogin, injected intraperitoneally, i.p. (5 µg/animal, two-month old C57/Bl6 male mice) on alternate days for 15 days, inhibits lung metastasis of B16F10 Nex-2â¯cells by 70-75%. In the treatment of human melanoma, grafted intradermally in the nude mice flank, leucurogin (7.5⯵g/kg in alternate days during 17 days) inhibits tumor growth by more than 40%. Leucurogin can be considered a promising agent for melanoma treatment.
Assuntos
Venenos de Crotalídeos/química , Desintegrinas/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Bothrops/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/isolamento & purificação , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Masculino , Melanoma/patologia , Metaloproteases/química , Metaloproteases/isolamento & purificação , Camundongos , Proteínas Recombinantes/químicaRESUMO
The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.
Assuntos
Antineoplásicos/isolamento & purificação , Venenos de Crotalídeos/química , Desintegrinas/isolamento & purificação , Metaloproteases/isolamento & purificação , Fosfolipases A2/isolamento & purificação , Trimeresurus/fisiologia , 5'-Nucleotidase/química , 5'-Nucleotidase/isolamento & purificação , 5'-Nucleotidase/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fracionamento Químico/métodos , Cromatografia Líquida de Alta Pressão , Venenos de Crotalídeos/isolamento & purificação , Desintegrinas/química , Desintegrinas/farmacologia , Humanos , Concentração Inibidora 50 , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/isolamento & purificação , L-Aminoácido Oxidase/farmacologia , Lectinas Tipo C/química , Lectinas Tipo C/isolamento & purificação , Espectrometria de Massas , Metaloproteases/química , Metaloproteases/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/farmacologia , Fosfolipases A2/química , Fosfolipases A2/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/isolamento & purificação , Diester Fosfórico Hidrolases/farmacologia , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologiaRESUMO
Investigating new antimicrobial and antiparasitic components from Viperidae venoms represents an alternative therapeutic strategy. In this study, we report the characterization of a disintegrin isolated from Cerastes cerastes venom, exhibiting antiparasitic activity on Leishmania infantum promastigotes. Indeed, isolated disintegrin, referred to Disintegrin_Cc, induced 84.75% of parasiticidal activity and deep morphological alterations on the parasites. SDS-PAGE analysis indicated that this disintegrin was homogenous. This dimeric disintegrin of 14,193.97 Da contains an RGD domain and four intramolecular disulfide bridges. It presents a high percentage of identity with other related snake disintegrins. Predicted 3D structure indicated that this peptide shares partial homology with well-known active antimicrobial peptides. Disintegrin_Cc inhibited 80% of arachidonic acid-induced platelet aggregation. The obtained results suggest that the isolated molecule plays a dual role as a disintegrin and as an anti-leishmanial compound. This component could be useful as a drug in the treatment of leishmaniasis.
Assuntos
Antiparasitários/farmacologia , Desintegrinas/farmacologia , Leishmania infantum/efeitos dos fármacos , Proteínas de Répteis/farmacologia , Venenos de Víboras/química , Viperidae/fisiologia , Sequência de Aminoácidos , Animais , Antiparasitários/química , Antiparasitários/isolamento & purificação , Sobrevivência Celular , Biologia Computacional , Sequência Conservada , Dimerização , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/isolamento & purificação , Sistemas Inteligentes , Ontologia Genética , Leishmania infantum/crescimento & desenvolvimento , Peso Molecular , Filogenia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Répteis/química , Proteínas de Répteis/genética , Proteínas de Répteis/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Venenos de Víboras/enzimologiaRESUMO
Metastasis involves the migration of cancer cells from a primary tumor to invade and establish secondary tumors in distant organs, and it is the main cause for cancer-related deaths. Currently, the conventional cytostatic drugs target the proliferation of malignant cells, being ineffective in metastatic disease. This highlights the need to find new anti-metastatic drugs. Toxins isolated from snake venoms are a natural source of potentially useful molecular scaffolds to obtain agents with anti-migratory and anti-invasive effects in cancer cells. While there is greater evidence concerning the mechanisms of cell death induction of several snake toxin classes on cancer cells; only a reduced number of toxin classes have been reported on (i.e., disintegrins/disintegrin-like proteins, C-type lectin-like proteins, C-type lectins, serinproteases, cardiotoxins, snake venom cystatins) as inhibitors of adhesion, migration, and invasion of cancer cells. Here, we discuss the anti-metastatic mechanisms of snake toxins, distinguishing three targets, which involve (1) inhibition of extracellular matrix components-dependent adhesion and migration, (2) inhibition of epithelial-mesenchymal transition, and (3) inhibition of migration by alterations in the actin/cytoskeleton network.
Assuntos
Desintegrinas/farmacologia , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Venenos de Serpentes/farmacologia , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Desintegrinas/isolamento & purificação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , HumanosRESUMO
Current therapies available for the treatment of human osteosarcoma, an aggressive bone tumor, are insufficient. To examine an alternative approach of integrin-based anti-osteosacoma strategy, acurhagin-C, a Glu-Cys-Asp (ECD)-disintegrin, was isolated and evaluated for its application in combination with two potent inhibitors of basic fibroblast growth factor (bFGF) and interleukin-8 (IL-8). The investigation of human osteosarcoma MG-63 cells pre-incubated with a FGF receptor-1 (FGFR-1) blocker AZD4547, a CXC-chemokine receptor-1/-2 (CXCR1/2) antagonist reparixin, and acurhagin-C via two given modes of separation and combination was executed. Detected by flow cytometry, integrins-α2/-α5/-αv/-ß1, FGFR-1, CXCR1 and CXCR2 constitutively express on the resting membrane. However, bFGF/IL-8-activated MG-63 cells only statistically enhanced the surface exposure of integrins-α5/-ß1, FGFR-1 and CXCR2. In activated MG-63 cells, acurhagin-C targeting integrin-α5 not only might potentiate the inhibitory effect of AZD4547 and reparixin on the surface expression of integrin-α5, FGFR-1 and CXCR2, but also acurhagin-C used alone remained effectively to diminish the surface exposure of those targeted receptors. Hence, a complicated crosstalk mechanism should be involved in the membrane interactions. Furthermore, co-administration of acurhagin-C with AZD4547 and reparixin also showed to have the synergistic suppression toward cell proliferation and the gene expression of matrix metalloproteinase-2. Also, the administration of three-in-one mode could nearly abrogate the cellular attachment onto collagen-IV- and fibronectin-coated wells, as well as penetration into Matrigel-barrier. These data supported an ECD-disintegrin acurhagin-C targeting integrin-α5 upon combined used with AZD4547 and reparixin may become a promising therapeutic approach for attenuating osteosarcoma development.
Assuntos
Benzamidas/farmacologia , Desintegrinas/farmacologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Piperazinas/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Benzamidas/química , Proliferação de Células/efeitos dos fármacos , Desintegrinas/química , Desintegrinas/isolamento & purificação , Humanos , Piperazinas/química , Pirazóis/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Sulfonamidas/química , Células Tumorais CultivadasRESUMO
Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases.
Assuntos
Venenos de Crotalídeos/enzimologia , Desintegrinas/metabolismo , Metaloproteases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Desintegrinas/isolamento & purificação , Humanos , Metaloproteases/genética , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência de AminoácidosRESUMO
The vast amounts of toxins within the venom of snakes, while known to cause medical emergencies, display various biological functions. Trans-pecos copperhead (Agkistrodon contortrix pictigaster) crude venom separated by cation-exchange chromatography showed several fractions with fibrinolytic, hemorrhagic, gelatinase and platelet activities. Venom fractions 1, 2, 4, 5, and 12-17 contained fibrinolytic activity. Venom fractions 1, 2, 5 and 12-14 had hemorrhagic activity. Fractions 1, 2, 12, 13 and 17 contained gelatinase activity. Reverse-Phase C18 High Performance Liquid Chromatography was also used to purify and isolated disintegrins from this venom. Anti-platelet aggregation activity of the C18 fractions collected and performed on whole human blood showed that they inhibited platelet aggregation in presence of several agonists. Results from both SDS-PAGE and N-terminal sequencing determined that pictistatin 1 obtained from the Trans-Pecos copperhead venom was a dimeric disintegrin, and pictistatin 2 was a heterodimeric disintegrin. The molecules with anti-platelet activity could be considered in the development of more effective drugs, for numerous blood-related diseases such as stroke, heart attacks, thrombosis, and other medical conditions. In this study, we are presenting the first report of the purification, isolation, and partial characterization of two new dimeric disintegrins isolated from the venom of trans-pecos copperhead.
Assuntos
Agkistrodon/metabolismo , Venenos de Crotalídeos/metabolismo , Desintegrinas/isolamento & purificação , Desintegrinas/metabolismo , Sequência de Aminoácidos , Animais , Cátions , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Venenos de Crotalídeos/química , Venenos de Crotalídeos/farmacologia , Desintegrinas/química , Eletroforese em Gel de Poliacrilamida , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacologia , Gelatinases/metabolismo , Humanos , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Multimerização Proteica , Coelhos , Pele/irrigação sanguínea , Pele/efeitos dos fármacosRESUMO
In addition to antiplatelet activity, disintegrin, a small-mass RGD-containing polypeptide, has been shown to exert anti-inflammatory effects but the mechanism involved remains unclear. In this study, we report that trimucrin, a disintegrin from the venom of Trimeresurus mucrosquamatus, inhibits lipopolysaccharide (LPS)-induced stimulation of THP-1 and RAW 264.7 cells. We also investigate the underlying mechanism. Trimucrin decreased the release of proinflammatory cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), nitric oxide, and reactive oxygen species (ROS), and inhibited the adhesion and migration of LPS-activated phagocytes. Trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes such as inducible nitric oxide synthase (iNOS) and COX-2. In addition, its anti-inflammatory effect was associated with the decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, trimucrin concentration dependently inhibited LPS-induced phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. Trimucrin also reversed the DNA-binding activity of NF-κB by suppressing the LPS-induced nuclear translocation of p65 and the cytosolic IκB release. Flow cytometric analyses showed that trimucrin bound to cells in a concentration-dependent manner. The anti-αVß3 mAb also specifically decreased the binding of fluorescein isothiocyanate (FITC)-conjugated trimucrin. Binding assays demonstrated that integrin αVß3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin decreases the inflammatory reaction through the attenuation of iNOS expression and nitric oxide (NO) production by blocking MAP kinase and the NF-κB activation in LPS-stimulated THP-1 and RAW 264.7 cells.
Assuntos
Anti-Inflamatórios/farmacologia , Venenos de Crotalídeos/química , Desintegrinas/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Peptídeos/farmacologia , Fagócitos/efeitos dos fármacos , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/metabolismo , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Desintegrinas/isolamento & purificação , Desintegrinas/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Mediadores da Inflamação/metabolismo , Integrina alfaVbeta3/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Fagócitos/enzimologia , Fosforilação , Ligação Proteica , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Desintegrinas/química , Desintegrinas/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Venenos de Víboras/química , Inibidores da Angiogênese/isolamento & purificação , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Desintegrinas/isolamento & purificação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Ratos , ViperidaeRESUMO
Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg(-1), via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca(2+)-cycling proteins such as SERCA (sarcoplasmic reticulum Ca(2+)-ATPase), PLB (phospholamban), and NCX (Na(+)/Ca(2+) exchanger); 4) abolishment of the typical negative force-frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca(2+) handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction.
Assuntos
Bothrops , Cardiotônicos/farmacologia , Desintegrinas/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Contração Miocárdica/efeitos dos fármacos , Proteínas de Répteis/farmacologia , Animais , Aquicultura , Proteínas de Ligação ao Cálcio/agonistas , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cardiotônicos/administração & dosagem , Cardiotônicos/isolamento & purificação , Caraciformes , Venenos de Crotalídeos/química , Desintegrinas/administração & dosagem , Desintegrinas/isolamento & purificação , Proteínas de Peixes/agonistas , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Ventrículos do Coração/enzimologia , Ventrículos do Coração/metabolismo , Injeções Intra-Arteriais , Distribuição Aleatória , Proteínas de Répteis/administração & dosagem , Proteínas de Répteis/isolamento & purificação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/agonistas , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Fator A de Crescimento do Endotélio Vascular/agonistas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Função Ventricular/efeitos dos fármacosRESUMO
This contribution reports on the first characterization of the venom proteome and the bioactivity screening of Vipera anatolica, the Anatolian Meadow Viper. The crude venom as well as an isolated dimeric disintegrin showed remarkable cytotoxic activity against glioblastoma cells. Due to the rare occurrence and the small size of this species only little amount of venom was available, which was profiled by means of a combination of bottom-up and top-down mass spectrometry. From this analysis we identified snake venom metalloproteases, cysteine-rich secretory protein isoforms, a metalloprotease inhibitor, several type A2 phospholipases, disintegrins, a snake venom serine protease, a C-type lectin and a Kunitz-type protease inhibitor. Furthermore, we detected several isoforms of above mentioned proteins as well as previously unknown proteins, indicating an extensive complexity of the venom which would have remained undetected with conventional venomic approaches.
Assuntos
Espectrometria de Massas/métodos , Proteínas de Répteis/química , Venenos de Víboras/química , Viperidae , Animais , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Desintegrinas/química , Desintegrinas/isolamento & purificação , Desintegrinas/farmacologia , Células HEK293 , Células HeLa , Humanos , Lectinas Tipo C/química , Lectinas Tipo C/isolamento & purificação , Células MCF-7 , Metaloproteases/antagonistas & inibidores , Metaloproteases/química , Metaloproteases/isolamento & purificação , Metaloproteases/farmacologia , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/farmacologia , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Proteínas de Répteis/antagonistas & inibidores , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Serina Proteases/química , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologiaRESUMO
Four disintegrins were isolated from the venoms of the Central American viperid snakes Atropoides mexicanus (atropoimin), Bothrops asper (bothrasperin), Cerrophidion sasai (sasaimin), and Crotalus simus (simusmin). Purifications were performed by reverse-phase HPLC. The four disintegrins have biochemical characteristics, i.e. molecular mass and location of Cys, which allow their classification within the group of medium-size disintegrins. All of them present the canonical RGD sequence, which determines their interaction with integrins in cell membranes. The disintegrins inhibited ADP and collagen-induced human platelet aggregation, with similar IC50s in the nM range. In addition, disintegrins inhibited the adhesion of an endothelial cell line and a melanoma cell line to the extracellular matrix proteins type I collagen, laminin, fibronectin, and vitronectin, albeit showing variable ability to exert this activity. This study expands the inventory of this family of viperid venom proteins, and reports, for the first time, disintegrins from the venoms of species of the genera Atropoides and Cerrophidion.
Assuntos
Desintegrinas/química , Desintegrinas/isolamento & purificação , Desintegrinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Venenos de Víboras/química , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Linhagem Celular/efeitos dos fármacos , Costa Rica , Venenos de Crotalídeos/química , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Humanos , Concentração Inibidora 50 , Laminina/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos/química , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/químicaRESUMO
Saxatilin, a novel disintegrin purified and cloned from the venom of the Korean snake Gloydius saxatilis, strongly inhibits activation and aggregation of platelets. Glycoprotein (GP) IIb/IIIa receptor antagonists can resolve thrombus, so saxatilin might also have thrombolytic effects. We investigated the thrombolytic effects of saxatilin in mice using a ferric chloride-induced carotid arterial thrombosis model. Thrombotic occlusion and thrombus resolution were evaluated quantitatively by measuring blood flow in the carotid artery with an ultrasonic flow meter and calculating the degree of flow restoration on a minute-by-minute basis; results were confirmed by histological examination. Saxatilin dissolved thrombi in a dose-dependent manner. Saxatilin at 5 mg/kg restored blood flow to baseline levels. As saxatilin dose increased, time to recanalization decreased. A bolus injection of 10% of a complete dose with continuous infusion of the remaining dose for 60 minutes resulted in effective recanalization without reocclusion. The thrombolytic effect of saxatilin was also demonstrated in vitro using platelet aggregometry by administering saxatilin in preformed thrombi. Bleeding complications were observed in 2 of 71 mice that received saxatilin. Fibrin/fibrinogen zymography and platelet aggregometry studies indicated that saxatilin does not have fibrinolytic activity, but exerted its action on platelets. Integrin-binding assays showed that saxatilin inhibited multiple integrins, specifically α2bß3 (GP IIb/IIIa), α5ß1, αvß3, αvß1, and αvß5, which act on platelet adhesion/aggregation. Saxatilin inhibited multiple integrins by acting on platelets, and was safe and effective in resolving thrombi in mice.
Assuntos
Plaquetas/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Desintegrinas/farmacologia , Fibrinolíticos/farmacologia , Terapia Trombolítica , Trombose/tratamento farmacológico , Animais , Plaquetas/metabolismo , Plaquetas/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Células Cultivadas , Cloretos , Desintegrinas/isolamento & purificação , Relação Dose-Resposta a Droga , Esquema de Medicação , Compostos Férricos , Fibrinolíticos/isolamento & purificação , Hemorreologia , Hemorragia/induzido quimicamente , Integrinas/antagonistas & inibidores , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Agregação Plaquetária/efeitos dos fármacos , Venenos de Serpentes/química , Trombose/induzido quimicamente , Trombose/metabolismo , Trombose/patologiaRESUMO
Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity. Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.
Assuntos
Clonagem Molecular , Desintegrinas/química , Venenos de Víboras/química , Viperidae/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Biologia Computacional , DNA Complementar/biossíntese , Desintegrinas/isolamento & purificação , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Proteoma/química , Proteoma/genética , Viperidae/classificaçãoRESUMO
Disintegrins, a family of polypeptides released in the venoms of viperid snakes (vipers and rattlesnakes) by the proteolytic processing of multidomain metalloproteinases, selectively block the function of ß(1) and ß(3) integrin receptors. Few of the proteins isolated and characterized from snake venoms have proven to be more structural and functional versatile than the disintegrins. Not surprisingly, 25 years after their discovery, our knowledge on the evolutionary history and the molecular determinants modulating the integrin inhibitory activity of disintegrins still remain fragmentary. This paper highlights some seminal contributions, including personal accounts of pioneer authors, related to basic and applied research on disintegrins. Investigators have evaluated disintegrin applications in therapies for a number of pathologies in which integrin receptors play relevant roles, particularly myocardial infarction and inappropriate tumor angiogenesis. Completing the continuing story of the disintegrin family by applying novel research approaches may hold the key to learn how to use deadly toxins as therapeutic agents.