Snake venom disintegrins update: insights about new findings

ABSTRACT Snake venom disintegrins are low molecular weight, non-enzymatic proteins rich in cysteine, present in the venom of snakes from the families Viperidae, Crotalidae, Atractaspididae, Elapidae, and Colubridae. This family of proteins originated in venom through the proteolytic processing of metalloproteinases (SVMPs), which, in turn, evolved from a gene encoding an A Disintegrin And Metalloprotease (ADAM) molecule. Disintegrins have a recognition motif for integrins in their structure, allowing interaction with these transmembrane adhesion receptors and preventing their binding to proteins in the extracellular matrix and other cells. This interaction gives disintegrins their wide range of biological functions, including inhibition of platelet aggregation and antitumor activity. As a result, many studies have been conducted in an attempt to use these natural compounds as a basis for developing therapies for the treatment of various diseases. Furthermore, the FDA has approved Tirofiban and Eptifibatide as antiplatelet compounds, and they are synthesized from the structure of echistatin and barbourin, respectively. In this review, we discuss some of the main functional and structural characteristics of this class of proteins and their potential for therapeutic use.

Role of crotoxin in coagulation: novel insights into anticoagulant mechanisms and impairment of inflammation-induced coagulation

Snake venom phospholipases A2 (svPLA2) are biologically active toxins, capable of triggering and modulating a wide range of biological functions. Among the svPLA2s, crotoxin (CTX) has been in the spotlight of bioprospecting research due to its role in modulating immune response and hemostasis. In the present study, novel anticoagulant mechanisms of CTX, and the modulation of inflammation-induced coagulation were investigated. Methods: CTX anticoagulant activity was evaluated using platelet poor plasma (PPP) and whole blood (WB), and also using isolated coagulation factors and complexes. The toxin modulation of procoagulant and pro-inflammatory effects was evaluated using the expression of tissue factor (TF) and cytokines in lipopolysaccharide (LPS)-treated peripheral blood mononuclear cells (PBMC) and in WB. Results: The results showed that CTX impaired clot formation in both PPP and WB, and was responsible for the inhibition of both intrinsic (TF/factor VIIa) and extrinsic (factor IXa/factor VIIIa) tenase complexes, but not for factor Xa and thrombin alone. In addition, the PLA2 mitigated the prothrombinase complex by modulating the coagulation phospholipid role in the complex. In regards to the inflammation-coagulation cross talk, the toxin was capable of reducing the production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, and was followed by decreased levels of TF and procoagulant activity from LPS-treated PBMC either isolated or in WB. Conclusion: The results obtained in the present study recognize the toxin as a novel medicinal candidate to be applied in inflammatory diseases with coagulation disorders.(AU)

Bothrops moojeni L-amino acid oxidase induces apoptosis and epigenetic modulation on Bcr-Abl+ cells

Resistance to apoptosis in chronic myeloid leukemia (CML) is associated with constitutive tyrosine kinase activity of the Bcr-Abl oncoprotein. The deregulated expression of apoptosis-related genes and alteration in epigenetic machinery may also contribute to apoptosis resistance in CML. Tyrosine kinase inhibitors target the Bcr-Abl oncoprotein and are used in CML treatment. The resistance of CML patients to tyrosine kinase inhibitors has guided the search for new compounds that may induce apoptosis in Bcr-Abl+ leukemic cells and improve the disease treatment. Methods: In the present study, we investigated whether the L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) (i) was cytotoxic to Bcr-Abl+ cell lines (HL-60.Bcr-Abl, K562-S, and K562-R), HL-60 (acute promyelocytic leukemia) cells, the non-tumor cell line HEK-293, and peripheral blood mononuclear cells (PBMC); and (ii) affected epigenetic mechanisms, including DNA methylation and microRNAs expression in vitro. Results: BmooLAAO-I induced ROS production, apoptosis, and differential DNA methylation pattern of regulatory apoptosis genes. The toxin upregulated expression of the pro-apoptotic genes BID and FADD and downregulated DFFA expression in leukemic cell lines, as well as increased miR-16 expression - whose major predicted target is the anti-apoptotic gene BCL2 - in Bcr-Abl+ cells. Conclusion: BmooLAAO-I exerts selective antitumor action mediated by H2O2 release and induces apoptosis, and alterations in epigenetic mechanisms. These results support future investigations on the effect of BmooLAAO-I on in vivo models to determine its potential in CML therapy.(AU)

BthTX-I from Bothrops jararacussu induces apoptosis in human breast cancer cell lines and decreases cancer stem cell subpopulation

Breast cancer is the neoplasm with both the highest incidence and mortality rate among women worldwide. Given the known snake venom cytotoxicity towards several tumor types, we evaluated the effects of BthTX-I from Bothrops jararacussu on MCF7, SKBR3, and MDAMB231 breast cancer cell lines. Methods: BthTX-I cytotoxicity was determined via MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide assay. Cell death was measured by a hypotonic fluorescent solution method, annexin-V-FITC/propidium iodide staining and by apoptotic/autophagic protein expression. Cancer stem cells (CSCs) were quantified by flow cytometry using anti-CD24-FITC and anti-CD44-APC antibodies and propidium iodide. Results: BthTX-I at 102 µg/mL induced cell death in all cell lines. The toxin induced apoptosis in MCF7, SKBR3, and MDAMB231 in a dose-dependent manner, as confirmed by the increasing number of hypodiploid nuclei. Expression of pro-caspase 3, pro-caspase 8 and Beclin-1 proteins were increased, while the level of the antiapoptotic protein Bcl-2 was diminished in MCF7 cells. BthTX-I changed the staining pattern of CSCs in MDAMB231 cells by increasing expression of CD24 receptors, which mediated cell death. Conclusions: BthTX-I induces apoptosis and autophagy in all breast cancer cell lines tested and also reduces CSCs subpopulation, which makes it a promising therapeutic alternative for breast cancer.(AU)

BjussuLAAO-II induces cytotoxicity and alters DNA methylation of cell-cycle genes in monocultured/co-cultured HepG2 cells

The use of animal venoms and their toxins as material sources for biotechnological applications has received much attention from the pharmaceutical industry. L-amino acid oxidases from snake venoms (SV-LAAOs) have demonstrated innumerous biological effects and pharmacological potential against different cancer types. Hepatocellular carcinoma has increased worldwide, and the aberrant DNA methylation of liver cells is a common mechanism to promote hepatic tumorigenesis. Moreover, tumor microenvironment plays a major role in neoplastic transformation. To elucidate the molecular mechanisms responsible for the cytotoxic effects of SV-LAAO in human cancer cells, this study aimed to evaluate the cytotoxicity and the alterations in DNA methylation profiler in the promoter regions of cell-cycle genes induced by BjussuLAAO-II, an LAAO from Bothrops jaracussu venom, in human hepatocellular carcinoma (HepG2) cells in monoculture and co-culture with endothelial (HUVEC) cells. Methods: BjussuLAAO-II concentrations were 0.25, 0.50, 1.00 and 5.00 μg/mL. Cell viability was assessed by MTT assay and DNA methylation of the promoter regions of 22 cell-cycle genes by EpiTect Methyl II PCR array. Results: BjussuLAAO-II decreased the cell viability of HepG2 cells in monoculture at all concentrations tested. In co-culture, 1.00 and 5.00 μg/mL induced cytotoxicity (p < 0.05). BjussuLAAO-II increased the methylation of CCND1 and decreased the methylation of CDKN1A in monoculture and GADD45A in both cell-culture models (p < 0.05). Conclusion: Data showed BjussuLAAO-II induced cytotoxicity and altered DNA methylation of the promoter regions of cell-cycle genes in HepG2 cells in monoculture and co-culture models. We suggested the analysis of DNA methylation profile of GADD45A as a potential biomarker of the cell cycle effects of BjussuLAAO-II in cancer cells. The tumor microenvironment should be considered to comprise part of biotechnological strategies during the development of snake-toxin-based novel drugs.(AU)

Antivenomic approach of different Crotalus durissus collilineatus venoms

Our group has previously performed a proteomic study verifying that individual variations can occur among Crotalus durissus collilineatus venoms. These variations may lead to differences in venom toxicity and may result in lack of neutralization of some components by antivenom. In this way, this study aimed to evaluate the Brazilian anticrotalic serum capacity in recognizing twenty-two Crotalus durissus collilineatus venoms, as well as their fractions. Methods: The indirect enzyme-linked immunosorbent assay (ELISA) was chosen to evaluate the efficacy of heterologous anticrotalic serum produced by Instituto Butantan (Brazil) in recognizing the twenty-two Crotalus durissus collilineatus venoms and the pool of them. Moreover, the venom pool was fractionated using reversed-phase fast protein liquid chromatography (RP-FPLC) and the obtained fractions were analyzed concerning antivenom recognition. Results: Evaluation of venom variability by ELISA showed that all venom samples were recognized by the Brazilian anticrotalic antivenom. However, some particular venom fractions were poorly recognized. Conclusion: This study demonstrated that the Brazilian anticrotalic serum recognizes all the different twenty-two venoms of C. d. collilineatus and their fractions, although in a quantitatively different way, which may impact the effectiveness of the antivenom therapy. These results confirm the need to use a pool of venoms with the greatest possible variability in the preparation of antivenoms, in order to improve their effectiveness.(AU)

Cytotoxic and pro-apoptotic action of MjTX-I, a phospholipase A2 isolated from Bothrops moojeni snake venom, towards leukemic cells

A leucemia mieloide crônica (LMC) é uma neoplasia mieloproliferativa BCR-ABL1 + marcada por aumento da mieloproliferação e presença de células leucêmicas resistentes à apoptose. A terapia de primeira linha atual para a LMC é a administração de inibidores da tirosina quinase, mesilato de imatinibe, dasatinibe ou nilotinibe. Embora eficaz no tratamento da LMC, alguns pacientes se tornaram resistentes a essa terapia, levando à progressão da doença e à morte. Assim, a descoberta de novos compostos para melhorar a terapia da LMC ainda é um desafio. Aqui, os destinatários se MjTX-I, uma fosfolipase A 2 isolado a partir de Bothrops moojeni de veneno de cobra, afecta a viabilidade de Bcr-Abl de mesilato de imatinib-resistente + linhas celulares. Métodos: Examinamos o efeito citotóxico e pró-apoptótico de MjTX-I em células K562-S e K562-R Bcr-Abl + e na linha de células HEK-293 não tumorais e células mononucleares de sangue periférico, usando o 3- (4, Brometo de 5-dimetiltiazol-2-il) -2,5-difeniltetrazólio e os métodos de solução fluorescente hipotônica, associados à detecção de ativação de caspases 3, 8 e 9 e clivagem de poli (ADP-ribose) polimerase (PARP). Também analisamos o potencial MjTX-I para modular a expressão de genes relacionados à apoptose em células K562-S e K562-R. Resultados: O MjTX-I diminuiu a viabilidade das células K562-S e K562-R em 60 a 65%, sem afetar a viabilidade das células não tumorais, ou seja, exerceu citotoxicidade seletiva para as linhagens celulares Bcr-Abl + . Em linhas de células leucêmicas, a toxina induziu apoptose, caspases 3, 8 e 9 ativadas, PARP clivada, expressão negativa do gene anti-apoptótico BCL-2 e expressão aumentada do gene pró-apoptótico BAD. Conclusão: O efeito antitumoral de MjTX-I está associado ao seu potencial para induzir apoptose e citotoxicidade em linhagens celulares positivas para Bcr-Abl sensíveis e resistentes ao mesilato de imatinibe, indicando que MjTX-I é um candidato promissor a fármaco para atualizar a terapia de LMC.(AU)

Immunotherapeutic potential of Crotoxin: anti-inflammatory and immunosuppressive properties

For the past 80 years, Crotoxin has become one of the most investigated isolated toxins from snake venoms, partially due to its major role as the main toxic component in the venom of the South American rattlesnake Crotalus durissus terrificus. However, in the past decades, progressive studies have led researchers to shift their focus on Crotoxin, opening novel perspectives and applications as a therapeutic approach. Although this toxin acts on a wide variety of biological events, the modulation of immune responses is considered as one of its most relevant behaviors. Therefore, the present review describes the scientific investigations on the capacity of Crotoxin to modulate anti-inflammatory and immunosuppressive responses, and its application as a medicinal immunopharmacological approach. In addition, this review will also discuss its mechanisms, involving cellular and molecular pathways, capable of improving pathological alterations related to immune-associated disorders.(AU)

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.(AU)

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 g/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 g/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 g/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Potencialização do efeito metemoglobinizante da dapsona em ratos pela N-acetilcisteína / Potentiation of dapsone induced methemoglobinemia by N-acetylcysteine in rats

Dapsona (DDS) (4,4'diaminodifenilsulfona), fármaco de escolha para o tratamento da hanseníase, freqüentemente induz anemia hemolítica e metemoglobinemia. A N-hidroxilação, uma de suas principais vias de biotransformação, é constantemente relacionada com a metemoglobinemia observada com o uso do fármaco. Com o objetivo de prevenir a hemotoxicidade induzida pela DDS, N-acetilcisteína, fármaco precursor de glutationa, foi administrada em associação com DDS em ratos machos Wistar pesando 220-240 g. Os animais foram anestesiados e o sangue coletado da aorta para determinação da concentração plasmática de DDS por CLAE, determinação dos níveis de metemoglobina e de glutationa eritrocitária por espectrofotometria, e avaliação de parâmetros bioquímicos e hematológicos. Os resultados obtidos mostraram que a N-acetilcisteína potenciou o efeito metemoglobinizante da dapsona devido ao aumento de sua concentração plasmática e conseqüente aumento da formação da N-hidroxilamina. Concluímos que as interações medicamentosas com a dapsona exigem estudos individualizados a fim de evitar os efeitos adversos do fármaco.

Dapsone (DDS) (4,4'diaminodiphenylsulfone), the drug of choice for the treatment of leprosy, frequently induces hemolytic anemia and methemoglobinemia. N-hydroxylation, one of the major pathways of biotransformation, has been constantly related to the methemoglobinemia after the use of the drug. In order to prevent the dapsone-induced hemotoxicity, N-acetylcysteine, a drug precursor of glutathione, was administered in combination with DDS to male Wistar rats, weighting 220-240 g. The animals were then anaesthetized and blood was collected from the aorta for determination of plasma DDS concentration by HPLC, determination of methemoglobinemia and glutathione by spectrophotometry, and for biochemical and hematological parameters. Our results showed that N-acetylcysteine enhanced dapsone-induced methemoglobinemia due to increased dapsone plasmatic concentration and consequent increased N-hydroxylamine formation. We concluded that drug interactions with dapsone require individually studies in order to avoid undesirable effects of dapsone.