RESUMO
Phospholipases A2 (PLA2) represent a superfamily of enzymes widely distributed in living organisms, with a broad spectrum of pharmacological activities and therapeutic potential. Anti-angiogenic strategies have become one of the main tools in fighting cancer. In this sense, the present work reports the inhibition of tumor angiogenesis induced by Asp-49 BthTX-II using in vitro, ex vivo and in vivo approaches. We demonstrate that BthTx-II inhibited cell adhesion, proliferation, and migration of human umbilical vein endothelial cells (HUVEC), as well as caused a reduction in the levels of endothelial growth factor (VEGF) during in vitro angiogenesis assays. BthTx-II was also able to inhibit the sprouting angiogenic process, by the ex vivo germination assay of the aortic ring; in addition, this toxin inhibited the migration and proliferation of HUVEC in co-culture with triple-negative breast cancer cells (e.g., MDA-MB-231 cells). Finally, in vivo tumor suppression and anti-angiogenic activities were analyzed using MDA-MB-231 cells with Matrigel injected into the chorioallantoic membrane of chicken embryo (CAM) for 7 days treatment with BthTx-II, showing a considerable reduction in vessel caliber, on the size and weight of tumors. Together, these results suggest an important antiangiogenic and antitumor role for BthTx-II, as a potential prototype for the development of new tools and antitumor drugs in cancer therapy.
Assuntos
Bothrops , Venenos de Crotalídeos , Neoplasias de Mama Triplo Negativas , Animais , Bothrops/metabolismo , Embrião de Galinha , Venenos de Crotalídeos/farmacologia , Fosfolipases A2 do Grupo II , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fosfolipases A2/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Although crotoxin B (CB) is a well-established catalytically active secretory phospholipase A2 group IIA (sPLA2-IIA) myotoxin, we investigated its potential stimulatory effect on myogenesis with the involvement of prostaglandins (PGs) produced by cyclooxygenase (COX)-1 and -2 pathways. Myoblast C2C12 were cultured in proliferation or commitment protocols and incubated with CB followed by lumiracoxib (selective COX-2 inhibitor) or valeryl salicylate (selective COX-1 inhibitor) and subjected to analysis of PG release, cell proliferation and activation of myogenic regulatory factors (MRFs). Our data showed that CB in non-cytotoxic concentrations induces an increase of COX-2 protein expression and stimulates the activity of both COX isoforms to produce PGE2, PGD2 and 15d-PGJ2. CB induced an increase in the proliferation of C2C12 myoblast cells dependent on PGs from both COX-1 and COX-2 pathways. In addition, CB stimulated the activity of Pax7, MyoD, Myf5 and myogenin in proliferated cells. Otherwise, CB increased myogenin activity but not MyoD in committed cells. Our findings evidence the role of COX-1- and COX-2-derived PGs in modulating CB-induced activation of MRFs. This study contributes to the knowledge that CB promote early myogenic events via regulatory mechanisms on PG-dependent COX pathways, showing new concepts about the effect of sPLA2-IIA in skeletal muscle repair.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Crotoxina/farmacologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo II/farmacologia , Proteínas de Membrana/metabolismo , Desenvolvimento Muscular/efeitos dos fármacos , Mioblastos Esqueléticos/efeitos dos fármacos , Neurotoxinas/farmacologia , Prostaglandinas/metabolismo , Animais , Linhagem Celular , Camundongos , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/enzimologia , Fator Regulador Miogênico 5/metabolismo , Miogenina/metabolismo , Fator de Transcrição PAX7/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: The treatment for snakebites is early administration of antivenom, which can be highly effective in inhibiting the systemic effects of snake venoms, but is less effective in the treatment of extra-circulatory and local effects. To complement standard-of-care treatments such as antibody-based antivenoms, natural and synthetic small molecules have been proposed for the inhibition of key venom components such as phospholipase A2 (PLA2) and PLA2-like toxins. Varespladib (compound LY315920) is a synthetic molecule developed and clinically tested aiming to block inflammatory cascades of several diseases associated with high PLA2s. Recent studies have demonstrated this molecule is able to potently inhibit snake venom catalytic PLA2 and PLA2-like toxins. METHODS: In vivo and in vitro techniques were used to evaluate the inhibitory effect of varespladib against MjTX-I. X-ray crystallography was used to reveal details of the interaction between these molecules. A new methodology that combines crystallography, mass spectroscopy and phylogenetic data was used to review its primary sequence. RESULTS: Varespladib was able to inhibit the myotoxic and cytotoxic effects of MjTX-I. Structural analysis revealed a particular inhibitory mechanism of MjTX-I when compared to other PLA2-like myotoxin, presenting an oligomeric-independent function. CONCLUSION: Results suggest the effectiveness of varespladib for the inhibition of MjTX-I, in similarity with other PLA2 and PLA2-like toxins. GENERAL SIGNIFICANCE: Varespladib appears to be a promissory molecule in the treatment of local effects led by PLA2 and PLA2-like toxins (oligomeric dependent and independent), indicating that this is a multifunctional or broadly specific inhibitor for different toxins within this superfamily.
Assuntos
Acetatos/farmacologia , Bothrops/metabolismo , Fosfolipases A2 do Grupo II/antagonistas & inibidores , Indóis/farmacologia , Cetoácidos/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/química , Proteínas de Répteis/antagonistas & inibidores , Animais , Cristalografia por Raios X , Fosfolipases A2 do Grupo II/toxicidade , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas de Répteis/toxicidadeRESUMO
Phospholipases A2 (PLA2s) and PLA2-like proteins are significant components of snake venoms. Some of these proteins act as potent toxins causing muscle necrosis, which may lead to amputation in severe envenomings. Fundamental aspects of the mechanism of action of these toxins are still not completely known. Myotoxin-I is a catalytically active Asp49 PLA2 from the venom of Bothrops asper, a medically relevant pit viper from Central America. Myotoxin-II is a catalytically inactive Lys49 PLA2-homolog also present in the venom of this snake. For the first time, the in vivo cellular localization of these myotoxins was studied in mouse skeletal muscle using immunofluorescence. Results showed that after 5 min of injection in the gastrocnemius muscle, both toxins initially interacted with the sarcolemma, and some colocalization with nuclei was already evident, especially for Mt-II. After 3 h of injection, a significant colocalization with the nuclei was observed for both toxins. These in vivo results confirm the importance of the initial interaction of these toxins with the sarcolemma and furthermore highlight the internalization and interaction of the toxins with nuclei during their pathophysiological activities, as observed in recent studies using cell culture.
Assuntos
Bothrops , Venenos de Crotalídeos , Animais , América Central , Venenos de Crotalídeos/toxicidade , Modelos Animais de Doenças , Fosfolipases A2 do Grupo II , Camundongos , Proteínas de Répteis/toxicidadeRESUMO
Antiangiogenic strategies are promising tools for cancer treatment and several other disorders. In this sense, phospholipases A2 (PLA2s) from snake venom have been described to possess antiangiogenic properties. In this study, we evaluated both in vitro and ex vivo antiangiogenic effects induced by BnSP-7, a Lys49 PLA2 isolated from Bothrops pauloensis snake venom. BnSP-7 was able to inhibit endothelial cell (HUVEC) proliferation, which was indeed confirmed by a modulation of cell cycle progression. Interestingly, BnSP-7 also inhibited the adhesion and migration of HUVECs and blocked in vitro angiogenesis in a VEGF-dependent manner, an important proangiogenic factor. Finally, BnSP-7 was capable of inhibiting sprouting angiogenic process through an ex vivo aortic ring assay. Taken together, these results indicate that BnSP-7 has potent in vitro and ex vivo antiangiogenic effect.
Assuntos
Inibidores da Angiogênese/farmacologia , Fosfolipases A2 do Grupo II/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Proteínas de Répteis/farmacologia , Animais , Aorta/efeitos dos fármacos , Bothrops , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Venenos de Crotalídeos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Bites by viperid snakes belonging to Bothrops genus produce fast and intense local edema, inflammation, bleeding and myonecrosis. In this study, we investigated the role of Myogenic Regulatory Factors (MRFs: MyoD; Myog), negatively regulated by GDF-8 (Myostatin), and ubiquitin-proteasome system pathway (UPS: MuRF-1; Fbx-32) in gastrocnemius muscle regeneration after Bothrops jararacussu snake venom (Bjussu) or its isolated phospholipase A2 myotoxins, BthTx-I (Lys-49 PLA2) and BthTx-II (Asp-49 PLA2) injection. Male Swiss mice received a single intra-gastrocnemius injection of crude Bjussu, at a dose/volume of 0.83â¯mg/kg/20⯵l, and BthTx-I or BthTx-II, at a dose/volume of 2.5â¯mg/kg/20⯵l. Control mice (Sham) received an injection of sterile saline solution (NaCl 0.9%; 20⯵l). At 24, 48, 72 and 96â¯h post injection, right gastrocnemius was collected for protein expression analyses. Based on the temporal expressional dynamics of MyoD, Myog and GDF-8/Myostatin, it was possible to propose that the myogenesis pathway was impacted most badly by BthTx-II followed by BthTx-I and lastly by B. jararacussu venom, thus suggesting that catalytic activity has likely inhibitory role on the satellite cells-mediated reparative myogenesis pathway. Inversely, the catalytic activity seems to be not a determinant for the activation of proteins ubiquitination by MuRF-1 and Fbx-32/Atrogin-1 E3 proteasome ligases, given proteolysis pathway through UPS was activated neither after Bjussu, nor after BthTx-II, but just after the catalytically-inactive BthTx-I Lys-49 PLA2-homologue exposure. The findings of this study disclose interesting perspective for further mechanistic studies about pathways that take part in the atrophy and repair after permanent damage induced by bothropic snakebites.
Assuntos
Venenos de Crotalídeos/farmacologia , Fosfolipases A2 do Grupo II/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Venenos de Crotalídeos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfolipases A2 do Grupo II/química , Masculino , Camundongos , Proteínas Musculares/genética , ProteóliseRESUMO
The light-emitting diode (LED) is considered a therapeutic tool due to its anti-inflammatory, analgesic, and wound-healing effects, which occur through angiogenesis, decrease in IL-1ß and IL-6 secretion, and acceleration of the cicatricial process. Snakebites are an important public health problem in tropical regions of the world. LED treatment is a therapeutic tool associated with serum therapy used to minimize the local effects of snakebites, including decrease in creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, myonecrosis, and inflammatory and haemorrhagic responses. In this study, we analysed the photobiomodulation effect of LED on the activation of murine macrophages induced by BthTX-I or BthTX-II isolated from Bothrops jararacussu venom. Photobiomodulation caused an increase in mitochondrial metabolism and a considerable decrease in cytotoxicity in murine macrophages. Moreover, it induced a decrease in reactive oxygen species and nitrogen liberation. However, photobiomodulation caused an increase in macrophage phagocytic capacity and lipid droplet formation. The results of this study corroborated with those of others in an unprecedented way and provide a better understanding of the mechanism of action of photobiomodulation, besides offering a coadjuvant action treatment for the local effects of snakebites, not achieved with serum therapy alone.
Assuntos
Venenos de Crotalídeos/toxicidade , Fosfolipases A2 do Grupo II/toxicidade , Terapia com Luz de Baixa Intensidade , Macrófagos/efeitos dos fármacos , Macrófagos/efeitos da radiação , Animais , Bothrops , Masculino , Camundongos , Mitocôndrias/metabolismo , Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -ß/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Células Espumosas/citologia , Fosfolipases A2 do Grupo II/farmacologia , Músculo Liso Vascular/citologia , Animais , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fenótipo , Ratos , Receptores Depuradores Classe A/metabolismo , Receptores Depuradores Classe E/metabolismoRESUMO
Ophidian accidents are considered an important neglected tropical disease by the World Health Organization. Particularly in Latin America, Bothrops snakes are responsible for the majority of the snakebite envenomings that are not efficiently treated by conventional serum therapy. Thus, the search for simple and efficient inhibitors to complement this therapy is a promising research area, and a combination of functional and structural assays have been used to test candidate ligands against specific ophidian venom compounds. Herein, we tested a commercial drug (acetylsalicylic acid, ASA) and a plant compound with antiophidian properties (rosmarinic acid, RA) using myographic, crystallographic and bioinformatics experiments with a phospholipase A2-like toxin, MjTX-II. MjTX-II/RA and MjTX-II/ASA crystal structures were solved at high resolution and revealed the presence of ligands bound to different regions of the toxin. However, in vitro myographic assays showed that only RA is able to prevent the myotoxic effects of MjTX-II. In agreement with functional results, molecular dynamics simulations showed that the RA molecule remains tightly bound to the toxin throughout the calculations, whereas ASA molecules tend to dissociate. This approach aids the design of effective inhibitors of PLA2-like toxins and, eventually, may complement serum therapy.
Assuntos
Aspirina , Cinamatos , Venenos de Crotalídeos , Depsídeos , Fosfolipases A2 do Grupo II , Simulação de Dinâmica Molecular , Animais , Aspirina/química , Aspirina/farmacologia , Cinamatos/química , Cinamatos/farmacologia , Venenos de Crotalídeos/química , Venenos de Crotalídeos/toxicidade , Cristalografia por Raios X , Depsídeos/química , Depsídeos/farmacologia , Fosfolipases A2 do Grupo II/química , Fosfolipases A2 do Grupo II/toxicidade , Masculino , Camundongos , Estrutura Quaternária de Proteína , Ácido RosmarínicoRESUMO
A need exists to develop specific and clinically useful inhibitors of toxic enzymes present in snake venoms, responsible for severe tissue damage and life-threatening effects occurring in thousands of people suffering envenomations globally. LY315920 (Varespladib, S-5920, A-001), a low molecular weight drug developed to inhibit several human secreted phospholipases A2 (PLA2s), was recently shown to also inhibit PLA2s in whole snake venoms with high potency, yet no studies have examined its direct effect on purified snake venom PLA2s. This work evaluated the ability of LY315920 to neutralize the enzymatic and toxic activities of three isolated PLA2 toxins of structural groups I (pseudexin) and II (crotoxin B and myotoxin I), and their corresponding whole venoms. In vitro, LY315920 inhibited the catalytic activity of these three enzymes upon a synthetic substrate. The drug also blocked their cytotoxic effect on cultured murine myotubes. In mice, preincubation of the toxins or venoms with LY315920, followed by their intramuscular injection, resulted in significant inhibition of muscle damage. Finally, immediate, independent injection of LY315920 at the site of toxin or venom inoculation also resulted in a large reduction of myonecrosis in the case of pseudexin and myotoxin-I, and of Pseudechis colletti and Bothrops asper whole venoms, suggesting a possible method of drug delivery in emergency situations. Present findings add evidence to suggest the possibility of using LY315920 as a field antidote in snakebites, aiming to limit the myonecrosis induced by many venom PLA2s in the clinical setting.