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Previous issue date: 2016-10-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O c?ncer ? um termo utilizado para representar um conjunto de mais de 200 patologias, incluindo tumores malignos de diferentes localiza??es. V?rios s?o os mecanismos que contribuem para a carcinog?nese: sinal proliferativo sustentado, desregula??o da energia celular, evas?o a apoptose, indu??o a angiog?nese, replica??o ilimitada, entre outros. Dentre os principais tipos de c?ncer existentes, o c?ncer de pele se destaca: surge nos melan?citos e ? o mais frequente no Brasil, correspondendo a 30% de todos os tumores malignos registrados no Pa?s. Melanomas em est?gio inicial podem, na maioria das vezes, ser tratados apenas com cirurgia, por?m os c?nceres mais avan?ados requerem outros tratamentos. Neste trabalho, um inibidor de tripsina do tipo Kunitz foi purificado de sementes da leguminosa Mimosa regnellii Benth (ITJ), parcialmente caracterizado e avaliado quanto sua toxicidade frente a linhagens de c?lulas tumorais, atuando especificamente com um IC50 de 0,65 ?M em linhagem celular B16-F10, n?o apresentando toxicidade frente a linhagens de c?lulas n?o transformadas. Sua capacidade de induzir morte celular pela via de apoptose em c?lulas de melanoma de camundongo B16-F10 tamb?m foi avaliada, atrav?s de citometria de fluxo com os marcadores Anexina V-FITC/PI, induzindo cerca de 45% das c?lulas a apoptose. Al?m disso, o inibidor tamb?m foi avaliado quanto a sua capacidade de: alterar o potencial de membrana mitocondrial, visualizado por experimentos em citometria de fluxo utilizando a sonda Rodamina123 e microscopia confocal com o marcador Mitotracker Red, onde foi capaz de alterar de forma significativa o ??m; Liberar esp?cies reativas de oxig?nio e nitrog?nio, atrav?s de sondas espec?ficas visualizadas por t?cnicas de microscopia, causando libera??o de ROS na concentra??o de IC50, por?m n?o influenciando libera??o de ERNs; Liberar c?lcio citos?lico, evento que influencia na ativa??o de apoptose, com efeito significativo em c?lulas B16-f10; Inibir atividade angiog?nica de c?lulas endoteliais de coelho, atrav?s de experimentos de inibi??o de forma??o de novos vasos em matrigel, an?lise da express?o de VEGF por t?cnicas de western Blotting e redu??o da express?o de IL-6 analisado por microscopia confocal; Inibir o processo de migra??o celular em ensaio de indu??o de ferimento e an?lise em microscopia e, por fim, a alterar a morfologia celular de B16-F10, analisada por incuba??o com anticorpos espec?ficos para componentes da matriz extracelular e filamentos intermedi?rios das c?lulas de melanoma, realizados em microscopia de fluoresc?ncia. Todos esses resultados reunidos favorecem a proposi??o de um poss?vel mecanismo de a??o de ITJ na indu??o de morte celular por apoptose em c?lulas B16-F10, onde o inibidor atuaria inicialmente aumentando os n?veis de c?lcio citos?lico e ROS, alterando posteriormente a express?o de p53 em 36h de incuba??o, que agiriam alterando o metabolismo mitocondrial, ativando vias de apoptose dependentes da participa??o de caspases; ITJ tamb?m atuaria inibindo processos migrat?rios at? 18 horas de exposi??o, al?m de influenciar de forma tardia na inibi??o do processo angiog?nico in vitro. Estes resultados sugerem que ITJ apresenta potencial para ser utilizado como f?rmaco em tratamento adjuvante contra melanomas, devido a sua especificidade e baixa dosagem quando comparado a outras mol?culas bioativas. / Cancer is a term used to represent a set of more than 200 diseases, including malignant tumors of different localizations. There are several mechanisms that contribute to carcinogenesis: sustained proliferative signals, deregulation of cellular energy, evasion of apoptosis, angiogenesis induction and unlimited replication, among others. Among the main types of cancer, skin cancer stands out: arises in melanocytes and is the most common in Brazil, accounting for 30% of all malignant tumors registered in the country melanomas at an early stage can, in most cases,. It is treated with surgery, but the most advanced cancers require other treatments. In this work a Kunitz-type trypsin inhibitor was purified from Mimosa regnellii Benth (ITJ) legume seeds, partially characterized and evaluated for their toxicity front tumor cell lines, specifically acting with an IC50 of 0.65 ?M in B16-F10 cell line, showing no toxicity compared to non-transformed cell lines. Its ability to induce cell death by apoptosis pathway in mouse B16-F10 melanoma cells was evaluated by flow cytometry with Annexin V-FITC / PI markers, inducing about 45% apoptosis of cells. In addition, the inhibitor was also evaluated for their ability to: change the mitochondrial membrane potential, visualized by flow cytometry experiments using Rhodamine123 probe and confocal microscopy with Mitotracker Red marker, which was able to significantly change the ??m; Release of ROS and RNs through specific probes visualized by microscopy techniques, causing release of ROS in the concentration of IC50, but not influencing release RNS; Liberation of cytosolic calcium, an event that influences the apoptosis activation, with significant effect on B16-F10 cells; Inhibition of angiogenic activity on rabbit endothelial cells through experiments of inhibition of new vessel formation in Matrigel, analysis of VEGF expression by western blotting techniques and reduction of IL-6 expression analyzed by confocal microscopy; Inhibition of cell migration process in wound induction assay and microscopy analysis and, finally, to alter the cellular morphology of B16-F10 analyzed by incubation with specific antibodies to extracellular matrix components and intermediate filaments of melanoma cells, conducted in fluorescence microscopy. All these combined results favor the proposal of a possible ITJ action mechanism in the induction of cell death by apoptosis in B16-F10 cells, where the inhibitor initially act by altering the p53 expression in 36h of incubation, increasing calcium cytosolic levels and ROS, which would act changing the mitochondrial metabolism, activating dependent apoptosis pathways of caspase participation; ITJ also act by inhibiting migration processes up to 18 hours of exposure, as well as influence belatedly in inhibiting the angiogenic process in vitro. These results suggest that ITJ has the potential to be used as a drug adjuvant treatment for melanomas, due to their specificity and low-dose when compared to other bioactive molecules.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/22059 |
| Date | 25 October 2016 |
| Creators | Rab?lo, Luciana Maria Ara?jo |
| Contributors | 41305655400, Dore, Celina Maria Pinto Guerra, 03383860418, Lanza, Daniel Carlos Ferreira, 05372896663, Rocha, Hugo Alexandre de Oliveira, 76111830449, Migliolo, Ludovico, 04312172463, Santos, Elizeu Antunes dos |
| Publisher | PROGRAMA DE P?S-GRADUA??O EM BIOQU?MICA, UFRN, Brasil |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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