RESUMO
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.
Assuntos
Peptídeos Penetradores de Células/isolamento & purificação , Venenos de Crotalídeos/química , Sistemas de Liberação de Medicamentos/métodos , Sequência de Aminoácidos , Animais , Transporte Biológico , Bothrops/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Cromatografia em Gel , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Achados Incidentais , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Peso Molecular , Análise de Sequência de Proteína , Espectrometria de Massas em TandemRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Eugenol/farmacologia , Fase G2/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mutagênicos/toxicidade , Neoplasias/patologia , Linhagem Celular Tumoral , Ensaio Cometa , Feminino , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Breast cancer is the world's leading cause of death among women. This situation imposes an urgent development of more selective and less toxic agents. The use of natural molecular fingerprints as sources for new bioactive chemical entities has proven to be a quite promising and efficient method. Here, we have demonstrated for the first time that dillapiole has broad cytotoxic effects against a variety tumor cells. For instance, we found that it can act as a pro-oxidant compound through the induction of reactive oxygen species (ROS) release in MDA-MB-231 cells. We also demonstrated that dillapiole exhibits anti-proliferative properties, arresting cells at the G0/G1 phase and its antimigration effects can be associated with the disruption of actin filaments, which in turn can prevent tumor cell proliferation. Molecular modeling studies corroborated the biological findings and suggested that dillapiole may present a good pharmacokinetic profile, mainly because its hydrophobic character, which can facilitate its diffusion through tumor cell membranes. All these findings support the fact that dillapiole is a promising anticancer agent.
Assuntos
Compostos Alílicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Dioxóis/farmacologia , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Compostos Alílicos/química , Compostos Alílicos/isolamento & purificação , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Sinalização do Cálcio , Caspase 3/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Dioxóis/química , Dioxóis/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Simulação de Dinâmica Molecular , Piper/química , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
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.
RESUMO
Objectives: The reprogramming of cancer cells into induced pluripotent stem cells or less aggressive cancer cells can provide a modern platform to study cancer-related genes and their interactions with cell environment before and after reprogramming. Herein, we aimed to investigate the reprogramming capacity of murine melanoma B16F10 cells. Materials and methods: The B16F10 was transfected using non-viral circular DNA plasmid containing the genes Sox-2, Oct4, Nanog, Lin28 and green fluorescent protein (GFP). These cells were characterized by immunofluorescence, analysis RT-PCR and cell cycle. Results: Our results demonstrated for the first time that reprogramming of B16F10 may be induced using non-viral minicircle DNA containing the four reprogramming factors Oct4, Sox2, Lin 28, Nanog (OSLN) and the GFP reporter gene. The resulting clones are composed by epithelioid cells. These cells display characteristics of cancer stem cells, thus expressing pluripotent stem cell markers and dividing asymmetrically and symmetrically. Reprogrammed B16F10 cells did not form teratomas; however, they showed the suppression of tumourigenic abilities characterized by a reduced tumour size, when compared with parental B16F10 cell line. In contrast to parental cell line that showed accumulation of the cells in S phase of cell cycle, the cells of reprogrammed clones are accumulated in G1 phase. Long-term cultivation of reprogrammed B16F10 cells induces regression of their reprogramming. Conclusions: Our data imply that in result of reprogramming of B16F10 cells less aggressive Murine Melanoma Reprogrammed Cancer Cells may be obtained. These cells represent an interesting model to study mechanism of cells malignancy as well as provide a novel tool for anti-cancer drugs screening.
RESUMO
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 alpha-estrogen receptor (alpha ER) 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 (Delta Psi m). 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. (C) 2016 Elsevier GmbH. All rights reserved.