RESUMEN
Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.
RESUMEN
Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Péptidos de Penetración Celular/aislamiento & purificación , Venenos de Crotálidos/química , Sistemas de Liberación de Medicamentos/métodos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Bothrops/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Cromatografía en Gel , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Hallazgos Incidentales , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Liposomas/química , Liposomas/metabolismo , Peso Molecular , Análisis de Secuencia de Proteína , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Eugenol (EUG) is a major phenolic compound present in clove whose anti-cancer properties have been demonstrated previously. These anti-cancer properties may involves the modulation of different mechanisms, including α-estrogen receptor (αER) in luminal breast cancer cells, COX-2 inhibition in melanoma cells or p53 and caspase-3 activation in colon cancer cells. HYPOTHESIS: EUG promotes a burst in ROS production causing cell-cycle perturbations, mitochondria toxicity and clastogenesis triggering apoptosis in melanoma breast- and cervix-cancer cells in vitro. METHODS: Morphological changes were evaluated through the light- and electronic- microscopy. Cell-cycle, ROS, PCNA and Apoptosis was detected by flow cytometry and clastogenicity was evaluated by Comet-assay. RESULTS: The results obtained herein pointed out that EUG promotes, increasing ROS production leading to abrogation of G2/M of phase of cell-cycle, and consecutively, clastogenesis in vitro. In addition, EUG induces Proliferation Cell Nuclear Antigen (PCNA) downregulation and decreasing in mitochondria potential (ΔΨm). Of note, a Bax up-regulation was also observed on cells treated with EUG. All of these findings cooperate in order to induce apoptosis in cancer cells. CONCLUSION: These promising results presented herein shed new light on the mechanisms of action of EUG suggesting a possible applicability of this phenylpropanoid as adjuvant in anti-cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Eugenol/farmacología , Fase G2/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mutágenos/toxicidad , Neoplasias/patología , Línea Celular Tumoral , Ensayo Cometa , Femenino , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Renal cell carcinoma (RCC) is one of the most lethal urologic cancers and is highly resistant to both radiotherapy and chemotherapy. The recombinant protein Amblyomin-X, characterized as a Kunitz-type protease inhibitor, was obtained from a cDNA library from the salivary glands of the Amblyomma cajennense tick. This paper reports the biological effect of Amblyomin-X on inducing cell death by apoptotic process in vitro. For this purpose, the changes in morphological aspects of cells, the phosphatidylserine exposition and DNA degradation were evaluated after treatment with Amblyomin-X. We found that Amblyomin-X was able to induce apoptosis in Renca cells in a dose-dependent manner. So, the results presented here open perspectives for new researches and developing for Amblyomin-X in the treatment of RCC.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Proteínas y Péptidos Salivales/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Proteínas de Artrópodos , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Neoplasias Renales/patología , Ratones , Proteínas y Péptidos Salivales/administración & dosificaciónRESUMEN
Ticks are blood-feeding arthropods that secrete anticoagulant molecules to maintain the fluidity of the blood during its feeding. Tick saliva has many compounds with biological activities that interact directly with host systems, such as blood clotting, platelet aggregation, cell death, among others. Some reports show that there are proteins with anticancer properties in tick saliva. This paper reports some of the biological roles of the Amblyomma cajennense tick saliva, including Factor Xa and thrombin inhibition, action on platelet aggregation, and also preliminary cytotoxic effects on tumor cell lines. The crude saliva was tested in the coagulation, fibrinolysis and platelet aggregation systems. The protein profile of the crude saliva was examined through anion exchange chromatography performed in a FPLC system. The chromatography separated seven protein fractions (Pools I to VII), which biological activities were evaluated. Moreover, the cytotoxic effects of the crude saliva were evaluated on SK-MEL-28 (melanoma cells) and MIA PaCa-2 (pancreas adenocarcinoma cells) using the MTT assay, flow cytometry and fluorescence microscopy. The crude saliva was able to induce cell death on both cancer cells lines, and, interestingly, the cytotoxic effects were not observed on human fibroblasts, which were used as control. The present work opens perspectives for the characterization and development of new molecules involved in the hemostatic system and in cancer control.