RESUMO
Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.
Assuntos
Venenos de Crotalídeos , Neoplasias da Próstata , Humanos , Masculino , Venenos de Crotalídeos/farmacologia , Venenos de Crotalídeos/metabolismo , L-Aminoácido Oxidase/farmacologia , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Morte Celular , Estresse OxidativoRESUMO
This study reports the isolation of CollinLAAO-I, a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom, its biochemical characterization and leishmanicidal potential in Leishmania spp. CollinLAAO-I (63.1 kDa) was successfully isolated with high purity using two chromatographic steps and represents 2.5% of total venom proteins. CollinLAAO-I displayed high enzymatic activity (4262.83 U/mg/min), significantly reducing after 28 days. The enzymatic activity of CollinLAAO-I revealed higher affinity for hydrophobic amino acids such as L-leucine, high enzymatic activity in a wide pH range (6.0-10.0), at temperatures from 0 to 25 °C, and showed complete inhibition in the presence of Na+ and K+. Cytotoxicity assays revealed IC50 of 18.49 and 11.66 µg/mL for Leishmania (L.) amazonensis and Leishmania (L.) infantum, respectively, and the cytotoxicity was completely suppressed by catalase. CollinLAAO-I significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of both Leishmania species. Furthermore, CollinLAAO-I decreased the parasite capacity to infect macrophages by around 70%, indicating that even subtoxic concentrations of CollinLAAO-I can interfere with Leishmania vital processes. Thus, the results obtained for CollinLAAO-I provide important support for developing therapeutic strategies against leishmaniasis.
Assuntos
Venenos de Crotalídeos , L-Aminoácido Oxidase , Animais , L-Aminoácido Oxidase/química , Venenos de Crotalídeos/química , Crotalus , Venenos de SerpentesRESUMO
Alzheimer's disease (AD) is the most common form of dementia in the elderly, affecting cognitive, intellectual, and motor functions. Different hypotheses explain AD's mechanism, such as the amyloidogenic hypothesis. Moreover, this disease is multifactorial, and several studies have shown that gut dysbiosis and oxidative stress influence its pathogenesis. Knowing that kefir is a probiotic used in therapies to restore dysbiosis and that the bioactive peptides present in it have antioxidant properties, we explored its biotechnological potential as a source of molecules capable of modulating the amyloidogenic pathway and reducing oxidative stress, contributing to the treatment of AD. For that, we used Drosophila melanogaster model for AD (AD-like flies). Identification of bioactive peptides in the kefir sample was made by proteomic and peptidomic analyses, followed by in vitro evaluation of antioxidant and acetylcholinesterase inhibition potential. Flies were treated and their motor performance, brain morphology, and oxidative stress evaluated. Finally, we performed molecular docking between the peptides found and the main pathology-related proteins in the flies. The results showed that the fraction with the higher peptide concentration was positive for the parameters evaluated. In conclusion, these results revealed these kefir peptide-rich fractions have therapeutic potential for AD.
Assuntos
Doença de Alzheimer , Kefir , Acetilcolinesterase/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Antioxidantes/análise , Brasil , Drosophila melanogaster/metabolismo , Disbiose , Simulação de Acoplamento Molecular , Peptídeos/química , ProteômicaRESUMO
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
Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1-116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.
Assuntos
Venenos de Formiga/química , Venenos de Formiga/toxicidade , Formigas/química , Venenos de Crotalídeos/química , Proteínas de Insetos/química , Venenos de Escorpião/química , Animais , BrasilRESUMO
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
The main systemic alterations present in bothropic envenomation are hemostasis disorders, for which the conventional treatment is based on animal-produced antiophidic sera. We have developed a neutralizing antibody against Bothrops pauloensis (B. pauloensis) venom, which is member of the genus most predominant in snakebite accidents in Brazil. Subsequently, we expressed this antibody in plants to evaluate its enzymatic and biological activities. The ability of single-chain variable fragment (scFv) molecules to inhibit fibrinogenolytic, azocaseinolytic, coagulant and hemorrhagic actions of snake venom metalloproteinases (SVMPs) contained in B. pauloensis venom was verified through proteolytic assays. The antibody neutralized the toxic effects of envenomation, particularly those related to systemic processes, by interacting with one of the predominant classes of metalloproteinases. This novel molecule is a potential tool with great antivenom potential and provides a biotechnological antidote to snake venom due to its broad neutralizing activity.
Assuntos
Bothrops/metabolismo , Testes de Neutralização , Nicotiana/metabolismo , Proteínas Recombinantes/farmacologia , Anticorpos de Cadeia Única/farmacologia , Venenos de Serpentes/toxicidade , Animais , Brasil/epidemiologia , Caseínas/metabolismo , Galinhas , Células Clonais , Reações Cruzadas/imunologia , Fibrinogênio/metabolismo , Geografia , Hemorragia/patologia , Camundongos , Mapas de Interação de Proteínas , Proteólise , Anticorpos de Cadeia Única/isolamento & purificação , Mordeduras de Serpentes/epidemiologiaRESUMO
BACKGROUND: Phospholipases A2 (PLA2) from snake venoms have a broad potential as pharmacological tools on medicine. In this context, strongyloidiasis is a neglected parasitic disease caused by helminths of the genus Strongyloides. Currently, ivermectin is the drug of choice for treatment, however, besides its notable toxicity, therapeutic failures and cases of drug resistance have been reported. BnSP-6, from Bothorps pauloensis snake venom, is a PLA2 with depth biochemical characterization, reporting effects against tumor cells and bacteria. OBJECTIVE: The aim of this study is to demonstrate for the first time the action of the PLA2 on Strongyloides venezuelensis. METHODS: After 72 hours of treatment with BnSP-6 mortality of the infective larvae was assessed by motility assay. Cell and parasite viability was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Furthermore, autophagic vacuoles were labeled with Monodansylcadaverine (MDC) and nuclei of apoptotic cells were labeled with Propidium Iodide (PI). Tissue degeneration of the parasite was highlighted by Transmission Electron Microscopy (TEM). RESULTS: The mortality index demonstrated that BnSP-6 abolishes the motility of the parasite. In addition, the MTT assay attested the cytotoxicity of BnSP-6 at lower concentrations when compared with ivermectin, while autophagic and apoptosis processes were confirmed. Moreover, the anthelmintic effect was demonstrated by tissue degeneration observed by TEM. Furthermore, we report that BnSP-6 showed low cytotoxicity on human intestinal cells (Caco-2). CONCLUSION: Altogether, our results shed light on the potential of BNSP-6 as an anthelmintic agent, which can lead to further investigations as a tool for pharmaceutical discoveries.
Assuntos
Anti-Helmínticos/farmacologia , Venenos de Crotalídeos/farmacologia , Fosfolipases A2/farmacologia , Venenos de Serpentes/farmacologia , Strongyloides/efeitos dos fármacos , Animais , Anti-Helmínticos/química , Anti-Helmínticos/isolamento & purificação , Bothrops , Células CACO-2 , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Relação Dose-Resposta a Droga , Feminino , Humanos , Fosfolipases A2/química , Fosfolipases A2/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Serpentes/química , Venenos de Serpentes/isolamento & purificação , Strongyloides/parasitologia , Relação Estrutura-AtividadeRESUMO
Triple-negative breast cancers (TNBCs) are more aggressive than other breast cancer (BC) subtypes and lack effective therapeutic options. Unraveling marker events of TNBCs may provide new directions for development of strategies for targeted TNBC therapy. Herein, we reported that Annexin A1 (AnxA1) and Cathepsin D (CatD) are highly expressed in MDA-MB-231 (TNBC lineage), compared to MCF-10A and MCF-7. Since the proposed concept was that CatD has protumorigenic activity associated with its ability to cleave AnxA1 (generating a 35.5 KDa fragment), we investigated this mechanism more deeply using the inhibitor of CatD, Pepstatin A (PepA). Fourier Transform Infrared (FTIR) spectroscopy demonstrated that PepA inhibits CatD activity by occupying its active site; the OH bond from PepA interacts with a CO bond from carboxylic acids of CatD catalytic aspartate dyad, favoring the deprotonation of Asp33 and consequently inhibiting CatD. Treatment of MDA-MB-231 cells with PepA induced apoptosis and autophagy processes while reducing the proliferation, invasion, and migration. Finally, in silico molecular docking demonstrated that the catalytic inhibition comprises Asp231 protonated and Asp33 deprotonated, proving all functional results obtained. Our findings elucidated critical CatD activity in TNBC cell trough AnxA1 cleavage, indicating the inhibition of CatD as a possible strategy for TNBC treatment.
Assuntos
Anexina A1/genética , Catepsina D/genética , Simulação de Acoplamento Molecular , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Domínio Catalítico/efeitos dos fármacos , Catepsina D/antagonistas & inibidores , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Pepstatinas/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
This work reports the biological evaluation of a copper complex of the type [Cu(O-O)(N-N)ClO4], in which O-O = 4,4,4-trifluoro-1-phenyl-1,3-butanedione (Hbta) and N-N = 1,10-phenanthroline (phen), whose generic name is CBP-01. The cytotoxic effect of CBP-01 was evaluated by resazurin assay and cell proliferation was determined by MTT assay. DNA fragmentation was analyzed by gel electrophoresis. Cell cycle progression was detected through propidium iodide (PI) staining. Apoptosis and autophagy were determined by, respectively, Annexin V and 7-AAD staining and monodansylcadaverine (MDC) staining. The changes in intracellular reactive oxygen species levels were detected by DCFDA analysis. The copper complex CBP-01 showed in vitro antitumor activity with IC50s values of 7.4 µM against Sarcoma 180 and 26.4 against murine myoblast cells, displaying selectivity toward the tumor cell tested in vitro (SI > 3). An increase in reactive oxygen species (ROS) generation was observed, which may be related to the action mechanism of the complex. The complex CBP-01 may induce DNA damage leading cells to accumulate at G0/G1 checkpoint where, apparently, cells that are not able to recover from the damage are driven to cell death. Evidence has shown that cell death is initiated by autophagy dysfunction, culminating in apoptosis induction. The search for new metal-based drugs is focused on overcoming the drawbacks of already used agents such as acquired resistance and non-specificity; thus, the results obtained with CBP-01 show promising effects on cancer cells.