RESUMO
Alternagin-C (ALT-C) is a disintegrin-like peptide purified from Rhinocerophis alternatus snake venom with the property of inducing vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, and angiogenesis. Therefore, this protein could be interesting as a new approach for ischemic heart diseases, an imbalance between myocardial oxygen supply and demand, leading to cardiac dysfunction. We investigated the effects of a single dose of alternagin-C (0.5â¯mgâ¯kg-1, via intra-arterial), after 7â¯days, on hypoxia/reoxygenation challenge in isolated ventricle strips and on morphological changes and density of blood vessels of the heart, using fish as an alternative experimental model. ALT-C treatment provided protection of cardiomyocytes against hypoxia/reoxygenation-induced negative inotropism. ALT-C also stimulated angiogenesis and improved excitation-contraction coupling during hypoxic conditions. Our results provide a new insight into a functional role of ALT-C against hypoxia/reoxygenation-induced cardiomyocyte injury pointing out to a potential therapeutic strategy for ischemia-related diseases.
Assuntos
Bothrops , Cardiotônicos/uso terapêutico , Venenos de Crotalídeos/química , Modelos Animais de Doenças , Desintegrinas/uso terapêutico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Proteínas de Répteis/uso terapêutico , Indutores da Angiogênese/administração & dosagem , Indutores da Angiogênese/uso terapêutico , Animais , Aquicultura , Cardiotônicos/administração & dosagem , Caraciformes , Colágeno/metabolismo , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Desintegrinas/administração & dosagem , Acoplamento Excitação-Contração/efeitos dos fármacos , Proteínas de Peixes/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Coração/fisiopatologia , Injeções Intra-Arteriais , Contração Miocárdica/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas de Répteis/administração & dosagemRESUMO
To examine the toxicological effect of saxatilin, a disintegrin isolated from the venom of a Korean snake (Gloydius saxatilis), recombinant saxatilin was highly expressed as a biologically active form in Pichia pastoris, and was successfully purified to homogeneity from the culture broth supernatant. The molecular and biological properties of the recombinant protein were the same as those of its natural form. Plasma half-life of the protein in rat was determined to 13.8 min. The maximum tolerated dose of the recombinant saxatilin was examined in ICR mice. The determined LD(50) values were 400 and 600 mg/kg of the body weight of a male and female mouse, respectively. To investigate the repeated dose toxicity of saxatilin in mice, the test item was intravenously administered to groups of ICR mice every day for 4 weeks. We observed a decrease in locomotor activity, piloerection, and crouching in clinical findings, a decrease of red blood cells (RBCs) in hematology, and hyperplasia of the spleen in histology related to administration of the test item. These results suggest that the target organ of intravenous administration of the test item is the spleen. The no adverse effect level (NOAEL) in this test for both males and females is considered to be 3mg/kg. Our results also indicate that recombinant saxatilin is non-toxic at an administration dose with an anti-platelet effect, and might be a potential anti-adhesion therapeutic agent for thrombosis, cancer, restenosis, cataract, and osteoporosis.
Assuntos
Desintegrinas/administração & dosagem , Desintegrinas/toxicidade , Viperidae , Animais , Desintegrinas/química , Desintegrinas/metabolismo , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Regulação da Expressão Gênica , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Masculino , Camundongos , Camundongos Endogâmicos ICR , Tamanho do Órgão/efeitos dos fármacos , Organismos Geneticamente Modificados , Pichia/genética , Pichia/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Baço/efeitos dos fármacos , Baço/patologia , Urina , Redução de Peso/efeitos dos fármacosRESUMO
The aim of this feasibility study was to test the ability of fluorescent nanodiamond particles (F-NDP) covalently conjugated with bitistatin (F-NDP-Bit) to detect vascular blood clots in vivo using extracorporeal near-infrared (NIR) imaging. Specifically, we compared NIR fluorescence properties of F-NDP with N-V (F-NDPNV) and N-V-N color centers and sizes (100-10,000 nm). Optimal NIR fluorescence and tissue penetration across biological tissues (rat skin, porcine axillary veins, and skin) was obtained for F-NDPNV with a mean diameter of 700 nm. Intravital imaging (using in vivo imaging system [IVIS]) in vitro revealed that F-NDPNV-loaded glass capillaries could be detected across 6 mm of rat red-muscle barrier and 12 mm of porcine skin, which equals the average vertical distance of a human carotid artery bifurcation from the surface of the adjacent skin (14 mm). In vivo, feasibility was demonstrated in a rat model of occlusive blood clots generated using FeCl3 in the carotid artery bifurcation. Following systemic infusions of F-NDPNV-Bit (3 or 15 mg/kg) via the external carotid artery or femoral vein (N=3), presence of the particles in the thrombi was confirmed both in situ via IVIS, and ex vivo via confocal imaging. The presence of F-NDPNV in the vascular clots was further confirmed by direct counting of fluorescent particles extracted from clots following tissue solubilization. Our data suggest that F-NDPNV-Bit associate with vascular blood clots, presumably by binding of F-NDPNV-Bit to activated platelets within the blood clot. We posit that F-NDPNV-Bit could serve as a noninvasive platform for identification of vascular thrombi using NIR energy monitored by an extracorporeal device.
Assuntos
Bioengenharia/métodos , Diagnóstico por Imagem , Desintegrinas/química , Raios Infravermelhos , Nanodiamantes/química , Peptídeos/química , Trombose/diagnóstico , Animais , Artérias Carótidas/patologia , Modelos Animais de Doenças , Desintegrinas/administração & dosagem , Fluorescência , Humanos , Infusões Intravenosas , Masculino , Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Venenos de Serpentes , SuínosRESUMO
Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4-16kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell-cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin αvß3, a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified αvß3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin αvß3.
Assuntos
Adesão Celular/efeitos dos fármacos , Desintegrinas/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/biossíntese , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismoRESUMO
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
Despite significant research in this area, metastatic breast cancer remains a disease with a poor prognosis. Until an effective therapy is developed, it is imperative that new treatment modalities be investigated. In this report, we describe an effective method for delivery of a novel snake venom disintegrin, contortrostatin (CN), in an orthotopic, xenograft model of human mammary cancer in immunodeficient mice. CN (Mr 13,500) is a homodimeric disintegrin isolated from venom of the Southern Copperhead snake. The homodimer possesses two Arg-Gly-Asp sites, which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. Although our laboratory has previously described the antitumor activity of CN in a mouse model of human mammary cancer, the method of delivery, daily intratumor injection, was not translatable to clinical application. We now describe a clinically relevant method of administering CN, liposomal delivery (LCN). A unique liposomal system has been designed for i.v. administration of a biologically active protein with full retention of biological activity. Pharmacokinetics, biodistribution, platelet reactivity, and immunogenicity of LCN were determined and compared with similar characteristics of native, unencapsulated CN. There are several advantages to liposomal delivery of CN: (1) LCN has a significantly prolonged circulatory half-life compared with native CN; (2) LCN is passively accumulated in the tumor; (3) LCN has no platelet reactivity; and (4) LCN is not recognized by the immune system. Finally, antiangiogenic activity is an important component of CN's mechanism of antitumor action. We have demonstrated that i.v. delivery of LCN leads to potent antiangiogenic activity in the orthotopic, xenograft human mammary tumor model.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Desintegrinas/administração & dosagem , Desintegrinas/uso terapêutico , Lipossomos/administração & dosagem , Venenos de Serpentes/química , Agkistrodon , Animais , Plaquetas/metabolismo , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Progressão da Doença , Desintegrinas/imunologia , Desintegrinas/farmacocinética , Humanos , Injeções Intravenosas , Radioisótopos do Iodo , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Ligação Proteica , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND PURPOSE: Acurhagin, a member of versatile metalloproteinase disintegrins from Agkistrodon acutus venom, has been identified as a platelet aggregation inhibitor, previously. Here, acurhagin-C, the C-terminal Glu-Cys-Asp (ECD)-containing fragment of acurhagin, was evaluated for its biological activities and potential applications in anti-angiogenic therapy. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were treated with acurhagin-C to assay effects on viability, apoptosis, adhesion, migration, invasion, proliferation and angiogenesis. The recognition site and signalling involved for the interactions of acurhagin-C with HUVEC were determined using flow cytometric, electrophoresis and immunoblotting analyses. KEY RESULTS: Acurhagin-C decreased viability and induced apoptosis in HUVEC. It also dose-dependently inhibited HUVEC adhesion to immobilized extracellular matrices fibronectin, collagen I and vitronectin with respective IC(50) values of approximately 0.6, 0.3 and 0.1 microM. Acurhagin-C prevented migration and invasion of HUVEC through vitronectin- and Matrigel-coated barriers respectively. Furthermore, acurhagin-C attenuated fibroblast growth factor-2-primed angiogenesis both in vitro and in vivo, and specifically blocked the binding of anti-alphavbeta3 monoclonal antibody 23C6 to HUVEC in an ECD-dependent manner. However, purified alphavbeta3 also dose-dependently bound to immobilized acurhagin and acurhagin-C with a saturable pattern. Interference with integrin alphavbeta3-mediated functions and promotion of caspase-3 activation by acurhagin-C affected morphology of HUVEC and induced apoptosis. CONCLUSIONS AND IMPLICATIONS: Acurhagin-C elicited endothelial anoikis via disruption of alphavbeta3/focal adhesion kinase/phosphatidylinositol 3-kinase/Akt survival cascade and subsequent initiation of the procaspase-3 apoptotic signalling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/efeitos dos fármacos , Desintegrinas/farmacologia , Integrina alfaVbeta3/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Anoikis/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Desintegrinas/administração & dosagem , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Fragmentos de Peptídeos/administração & dosagem , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
Integrins alpha(1)beta(1) and alpha(2)beta(1) are highly expressed on the microvascular endothelial cells, and blocking of their adhesive properties significantly reduced the VEGF-driven neovascularization ratio and tumor growth in animal models. Hence, inhibitors of the alpha(1)beta(1) and alpha(2) beta(1) integrins, alone or in combination with antagonists of other integrins involved in angiogenesis (eg. alpha (v)beta(3), alpha(v)beta(5), and alpha(6)beta(4)), may prove beneficial in the control of tumor neovascularization. Viperidae snakes have developed in their venoms an efficient arsenal of integrin receptor antagonists. KTS-(obtustatin, viperistatin, lebestatin) and RTS- (jerdostatin) disintegrins represent viper venom peptides that specifically block the interaction of the alpha(1)beta (1) integrin with collagens IV and I in vitro and angiogenesis in vivo. The possible therapeutic approach towards tumor neovascularization by targeting the alpha (5)beta (1), alpha(v)beta(5) and alpha (v)beta(3) integrins with RGD-bearing disintegrins has been explored in a number of laboratories. Here we discuss structure-function correlations of the novel group of specific (K/R)TS-disintegrins targeting the alpha(1)beta(1) integrin.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Desintegrinas/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Integrina alfa1beta1/antagonistas & inibidores , Peptídeos/administração & dosagem , Venenos de Víboras/administração & dosagem , Inibidores da Angiogênese/química , Inibidores da Angiogênese/metabolismo , Animais , Desintegrinas/química , Desintegrinas/metabolismo , Humanos , Integrina alfa1beta1/metabolismo , Relação Estrutura-Atividade , Venenos de Víboras/químicaRESUMO
Disintegrins are soluble peptides found in snake venom. They bind to Arg-Gly-Asp (RGD)-responsive integrins with high affinity (nM range) and block integrin function. Contortrostatin (CN), the disintegrin from southern copperhead venom, is a homodimer with a molecular weight of 13,500. Each chain contains 65 amino acids with an Arg-Gly-Asp motif. CN has anti-invasive and anti-adhesive activity on tumor cells and endothelial cells in vitro, and binds to integrins alphavbeta3, alphavbeta5, and/or alpha5beta1. In vivo studies using the human metastatic breast cancer cell line MDA-MB-435, in an orthotopic xenograft model in nude mice, revealed that CN has potent anti-tumor and anti-angiogenic activity. Recent studies have employed an intravenous liposomal delivery procedure. Liposomal delivery of CN has also been shown to provide effective in vivo anti-tumor and anti-angiogenic activity in a human ovarian cancer animal model.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Desintegrinas/farmacologia , Venenos de Serpentes/química , Inibidores da Angiogênese/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Desintegrinas/administração & dosagem , Desintegrinas/uso terapêutico , Feminino , Humanos , Lipossomos/administração & dosagem , Lipossomos/uso terapêutico , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Transplante Heterólogo , Resultado do TratamentoRESUMO
Contortrostatin (CN) (Mr 13,500 Da) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix (Southern Copperhead) snake and displays two RGD motifs (one on each chain), which modulate its interaction with integrins on tumor cells and angiogenic vascular endothelial cells. In previous studies, we have shown that native CN administered in a liposomal formulation exhibits potent anti-angiogenic and tumor growth inhibitory activities. Current isolation of the protein from crude venom is difficult and prohibitively expensive for translation into the clinic. In this report, we describe a method amenable to large-scale production of a soluble monomeric form of recombinant CN with biologic activity; the protein is expressed directly in the cytoplasm of an engineered bacterial system with an expression yield of approximately 20 mg/l of culture. We present here the in vitro assays as well as the anti-tumor and anti-angiogenic evaluation of liposomal recombinant CN in an orthotopic, xenograft model of human breast cancer.
Assuntos
Inibidores da Angiogênese/biossíntese , Antineoplásicos/síntese química , Clonagem Molecular/métodos , Desintegrinas/genética , Desintegrinas/uso terapêutico , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Desintegrinas/administração & dosagem , Sistemas de Liberação de Medicamentos , Escherichia coli/genética , Humanos , Lipossomos/uso terapêutico , Proteínas Recombinantes , Transplante Heterólogo , Resultado do TratamentoRESUMO
The influence of laminin-1 (LN) and tenascin-C (TN), extracellular matrix molecules expressed spatially and temporally along the neural growth route from spiral ganglion (SG) neurons to the cochlear sensory cells, was evaluated in cultured SG explants from postnatal day 4 rats. Increasing concentrations of LN resulted in a strong, dose-dependent increase in the length of neurites and in a higher number of neural processes, while varying TN concentrations had relatively minor effects on both parameters. The results suggest differential receptor activation by LN and TN. When explants grown on LN were treated with Kistrin, an inhibitor of the alphavbeta3 integrin, the LN-induced increase in neurite length was reduced in a dose-dependent manner. However, the number of extending neurites was not affected, indicating that different receptors mediate this response, perhaps by increasing neuronal survival.
Assuntos
Desintegrinas/farmacologia , Laminina/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Peptídeos/farmacologia , Gânglio Espiral da Cóclea/metabolismo , Animais , Técnicas de Cultura , Desintegrinas/administração & dosagem , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Peptídeos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Tenascina/metabolismoRESUMO
OVCAR-5 is a human epithelial carcinoma cell line of the ovary, established from the ascitic fluid of a patient with progressive ovarian adenocarcinoma without prior cytotoxic treatment. The unique growth pattern of ovarian carcinoma makes it an ideal model for examining the anticancer activity of contortrostatin (CN), a homodimeric disintegrin from southern copperhead venom. FACS analysis revealed that OVCAR-5 is integrin alphavbeta3 negative, but alphavbeta5 positive. CN effectively blocks the adhesion of OVCAR-5 cells to several extracellular matrix proteins and inhibits tumor cell invasion through an artificial basement membrane. In a xenograft nude mouse model with intraperitoneal introduction of OVCAR-5 cells, intraperitoneal injection of CN was used for therapy. Tumor dissemination in CN-treated versus control groups was studied by gross examination, and antiangiogenic potential was examined by factor VIII immunohistochemistry and image analysis. CN not only significantly inhibited ovarian cancer dissemination in the nude mouse model, but it also dramatically prevented the recruitment of blood vessels to tumors at secondary sites.