RESUMO
Snake venom metalloproteinases (SVMPs) are predominant in viperid venoms, which provoke hemorrhage and affect hemostasis and thrombosis. P-I class enzymes consist only of a single metalloproteinase domain. Despite sharing high sequence homology, only some of them induce hemorrhage. They have direct fibrin(ogen)olytic activity. Their main biological substrate is fibrin(ogen), whose Aα-chain is degraded rapidly and independently of activation of plasminogen. It is important to understand their biochemical and physiological mechanisms, as well as their applications, to study the etiology of some human diseases and to identify sites of potential intervention. As compared to all current antiplatelet therapies to treat cardiovascular events, the SVMPs have outstanding biochemical attributes: (a) they are insensitive to plasma serine proteinase inhibitors; (b) they have the potential to avoid bleeding risk; (c) mechanistically, they are inactivated/cleared by α2-macroglobulin that limits their range of action in circulation; and (d) few of them also impair platelet aggregation that represent an important target for therapeutic intervention. This review will briefly highlight the structure-function relationships of these few direct-acting fibrinolytic agents, including, barnettlysin-I, isolated from Bothrops barnetti venom, that could be considered as potential agent to treat major thrombotic disorders. Some of their pharmacological advantages are compared with plasmin.
Assuntos
Fibrinolíticos/farmacologia , Hemostasia/efeitos dos fármacos , Metaloproteases/farmacologia , Venenos de Serpentes/enzimologia , Serpentes , Sequência de Aminoácidos , Animais , Domínio Catalítico , Fibrinolíticos/química , Fibrinolíticos/uso terapêutico , Humanos , Metaloproteases/química , Metaloproteases/uso terapêutico , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Malignant melanoma is a less common but highly dangerous form of skin cancer; it starts in the melanocytes cells found in the outer layer of the skin. Jararhagin toxin, a metalloproteinase isolated from Bothrops jararaca snake venom acts upon several biological processes, as inflammation, pain, platelet aggregation, proliferation and apoptosis, though not yet approved for use, may one day be employed to treat tumors. METHODS: B16F10 murine melanoma cells were treated with jararhagin (jara), a disintegrin-like metalloproteinase isolated from Bothrops jararaca snake venom, and jari (catalytic domain inactivated with 1,10-phenanthroline). Viability and adhesion cells were evaluated by MTT assay. The expression of caspase-3 active, phases of the cell cycle and apoptosis were assessed by flow cytometry. We analyze in vivo the effects of jararhagin on melanoma growth, apoptosis and metastasis. RESULTS: The tumor cells acquired round shapes, lost cytoplasmic expansions, formed clusters in suspension and decreased viability. Jari was almost 20 times more potent toxin than jara based on IC50 values and on morphological changes of the cells, also observed by scanning electron microscopy. Flow cytometry analysis showed 48.3% decrease in the proliferation rate of cells and 47.2% increase in apoptosis (jara) and necrosis (jari), following 1.2 µM jara and 0.1 µM jari treatments. Caspase-3 activity was increased whereas G0/G1 cell cycle phase was on the decline. Proliferative rate was assessed by staining with 5,6-carboxyfluoresceindiacetate succinimidyl ester, showing a significant decrease in proliferation at all concentrations of both toxins. CONCLUSIONS: In vivo treatment of the toxins was observed reduction in the incidence of nodules, and metastasis and antiproliferative inhibition capacity. This data strengthens the potential use jararhagin as an anti-neoplastic drug.
Assuntos
Antineoplásicos/uso terapêutico , Bothrops , Venenos de Crotalídeos/uso terapêutico , Melanoma/tratamento farmacológico , Metaloendopeptidases/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Melanoma/metabolismo , Metaloendopeptidases/isolamento & purificação , Metaloendopeptidases/farmacologia , Metaloproteases/farmacologia , Metaloproteases/uso terapêutico , Camundongos , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Neoplasias Cutâneas/metabolismo , Veneno de Bothrops jararaca , Melanoma Maligno CutâneoRESUMO
As metaloproteinases da matriz (MMPs) são uma família de endopeptidades cálcio e zinco dependentes que participam da degradação de praticamente todos os componentes da matriz extracelular. Com o pressuposto de que estas enzimas podem estar relacionadas à progressão da erosão dental e que os agentes ácidos causadores da erosão podem influenciar na ativação das MMPs, o objetivo desse estudo in vitro foi avaliar a influência do pH sobre a atividade funcional das MMP-2 e -9 presentes na dentina coronária e radicular humana. O pó das dentinas coronária e radicular, provenientes de terceiros molares inclusos recém extraídos foi obtido separadamente e submetido ao protocolo de extração das proteínas, com ácido fosfórico a 1%. Após, o extrato contendo as proteínas e o pó de dentina parcialmente desmineralizado foram incubados em uma das respectivas soluções: solução 2 mM de APMA (acetato de 4-aminofenilmercúrio / grupo controle) ou em uma das soluções tampão (fosfato de potássio 0.1 M) com diferentes pHs (2.5, 4.5, 5.0, 6.0 e 7.0). Após a incubação, as proteínas foram separadas por eletroforese, em um gel de poliacrilamida copolimerizado com gelatina, para a avaliação da atividade gelatinolítica das MMPs, por zimografia. Esta análise foi realizada em triplicada e os zimogramas obtidos ao final foram avaliados por densitometria. A quantificação das bandas observadas nos zimogramas foi realizada pelo programa de imagens ImageJ e os dados avaliados de maneira descritiva e qualitativa. Para a avaliação do conteúdo de colágeno solubilizado, o pó de dentina parcialmente desmineralizado e incubado nos respectivos pHs (n = 8) foi mantido em um tampão de incubação durante 24h, e o conteúdo de hidroxiprolina (HYP) liberado neste meio foi mensurado em espectrofotômetro
Os dados obtidos foram avaliados estatisticamente por ANOVA 1 fator, seguido do teste de Tukey, com 5% de significância. A análise por zimografia mostrou bandas evidentes correspondente a MMP-2 na sua forma ativa (66kDa) e bandas menos expressivas relacionadas a sua forma latente (72kDa), tanto para a dentina coronária quanto para a radicular, em todos os grupos experimentais. Não foram identificadas bandas correspondentes a MMP-9, para nenhum dos substratos avaliados. As soluções com os menores pHs (2.5, 4.5 e 5.0) resultaram na maior atividade funcional da MMP-2, em comparação as soluções com os maiores pHs (6.0 e 7.0), em ambos os substratos. Para a análise de HYP, os grupos pH 2.5 e pH 4.5 mostraram valores de absorbância abaixo do limite de detecção do aparelho. Para os demais grupos experimentais, tanto para a dentina coronária quanto para a radicular, foram encontradas diferenças estatisticamente significantes entre eles (p < 0,05). O conteúdo de HYP liberada foi maior para o pH 7.0, comparado aos demais grupos (p < 0,05), exceto para o pH 6.0. Não foi encontrada diferença estatisticamente significante entre os grupos pH 6.0, pH 5.0 e controle (p > 0,05). Conclui-se que a atividade funcional da MMP-2 é dependente do pH. Os menores pHs promoveram um aumento na ativação da MMP-2 da dentina coronária e radicular humana
Matrix metalloproteinases (MMPs) are a family of endopeptidades calcium and zinc dependent that participate in the degradation of pratically all components of the extracellular matrix. With the assumption that these enzymes may be related to the progression of dental erosion and acidic agents that cause erosion can influence the activation of MMPs, the aim of this in vitro study was to evaluate the pH-influence on the functional activity of MMP-2 and -9 present in human coronal and root dentin. The powder of root and coronal dentine, from freshly extracted third molars was separately obtained and subjected to protein extraction protocol, with 1% phosphoric acid. After, the extract containing proteins and the partially demineralized powder were incubated in one of the following solutions: 2 mM APMA (4-aminophenylmercuric acetate / control) or one of the buffer solutions (0.1 M potassium phosphate) with different pHs (2.5, 4.5, 5.0, 6.0 and 7.0). After incubation, the proteins were separated by electrophoresis in a polyacrylamide gel copolymerized with gelatin, to evaluate the gelatinolytic activity of MMPs by means of zymography. This analysis was performed in triplicate and the zymograms obtained were evaluated by densitometry. Quantification of the bands observed in zymograms was performed by ImageJ software and the data were evaluated descriptively and qualitatively. To assess the solubilized dentin collagen, the partially demineralized dentin powder treated with different pHs (n=8) was incubated in an artificial saliva for 24 h, and the amount of hydroxyproline (HYP) released in media was measured by spectrophotometer
Data were statistically analyzed by ANOVA one factor, followed by the Tukey's test, with 5% significance. Zymography showed evident bands corresponding to active MMP-2 (66kDa) and less expressed bands related to its latent form (72kDa), for both coronal and root dentin, in all experimental groups. No bands corresponding to MMP-9 were identified, for both substrates. The lowest pH solutions (2.5, 4.5 and 5.0) yielded the higher functional activity than did the highest pH solutions (6.0 and 7.0), for both substrates. For HYP analysis, the groups pH 2.5 and pH 4.5 showed absorbance values below the detection limit of the equipment. For the other groups, for both coronal and root dentin, statistically significant diferences were found between them (p<0.05). The amount of HYP was higher for pH 7.0 than all other groups (p<0.05), except for pH 6.0. No statistical difference was found between pH 6.0, pH 5.0 and control (p>0.05). It can be concluded that the functional activity of MMP-2 is pH-dependent. Low pH solutions are able to increase the activation of human coronal and root MMP-2
Assuntos
Dentina/fisiologia , Erosão Dentária/diagnóstico , Metaloproteases/uso terapêuticoRESUMO
Gram-positive and -negative bacteria are dangerous pathogens that may cause human infection diseases, especially due to the increasingly high prevalence of antibiotic resistance, which is becoming one of the most alarming clinical problems. In the search for novel antimicrobial compounds, snake venoms represent a rich source for such compounds, which are produced by specialized glands in the snake's jawbone. Several venom compounds have been used for antimicrobial effects. Among them are phospholipases A2, which hydrolyze phospholipids and could act on bacterial cell surfaces. Moreover, metalloproteinases and L-amino acid oxidases, which represent important enzyme classes with antimicrobial properties, are investigated in this study. Finally, antimicrobial peptides from multiple classes are also found in snake venoms and will be mentioned. All these molecules have demonstrated an interesting alternative for controlling microorganisms that are resistant to conventional antibiotics, contributing in medicine due to their differential mechanisms of action and versatility. In this review, snake venom antimicrobial compounds will be focused on, including their enormous biotechnological applications for drug development.