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Previous issue date: 2007-12-14 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this study, a BCR-ABL expressing human chronic myelogenous leukaemia cell line (K562) was used to investigate the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians. CvL inhibited the growth of K562 cells with an IC50 value of 70 g/ml, but was ineffective to normal human peripheral blood lymphocytes in the same range of concentrations tested (180 g/ml). Cell death occurred after 72 h of exposure to the lectin and with sign of apoptosis as analysed by DAPI staining. Investigation of the possible effectors of this process showed that cell death occurred in the presence of Bcl-2 and Bax expression, and involved a caspase-independent pathway. Confocal fluorescence microscopy indicated a major role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished the cytotoxic effect of CvL. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and downmodulation of pRb, suggesting that CvL is capable of cell cycle arrest. Collectively, these findings suggest that cathepsin B acts as death mediator in CvL-induced cytotoxicity possibly in a still uncharacterized connection with the membrane death receptor pathway / Neste trabalho, a linhagem K562 de c?lulas de leucemia miel?ide cr?nica, expressando a prote?na oncog?nica BCR-ABL, foi usada como modelo para investigar a atividade antitumoral da lectina CvL purificada da esponja marinha Cliona varians. CvL inibiu o crescimento de c?lulas K562 com um IC50 de 70 g/mL, mas n?o afetou a viabilidade celular de linf?citos normais de sangue perif?rico humano no mesmo intervalo de concentra??es testadas (1 80 g/mL). A morte celular ocorreu ap?s 72 horas de exposi??o ? lectina e com altera??es nucleares t?picas de apoptose como analisado pela fluoresc?ncia de DAPI. Investiga??o dos poss?veis efetores deste processo mostrou que a morte celular ocorreu sem ativa??o de caspases e na presen?a de express?es aumentadas de Bcl-2 e Bax. O fato de CvL desencadear a libera??o de catepsina B, como evidenciado pela microscopia de fluoresc?ncia, e do inibidor E-64 bloquear completamente a morte celular induzida por CvL, sugerem papel central dessa protease lisossomal na ativa??o de uma via alternativa de morte celular. CvL tamb?m induziu o aumento de express?o do receptor de morte TNFR-1 e a diminui??o dos n?veis de NFκB. Estes efeitos foram acompanhados pelo aumento significativo na express?o de p21 e pela modula??o negativa de pRb, mostrando que CvL foi capaz de bloquear a progress?o do ciclo celular. Juntos, estes dados sugerem que catepsina B age como mediador da citotoxicidade induzida por CvL possivelmente atrav?s de uma conex?o ainda n?o caracterizada com a via dos receptores de morte
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/12523 |
| Date | 14 December 2007 |
| Creators | Moura, Gioconda Emanuella Diniz de Dantas |
| Contributors | CPF:29706106391, http://lattes.cnpq.br/7890362793618911, Rocha, Hugo Alexandre de Oliveira, CPF:76111830449, http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4799567J8&dataRevisao=null, Sales, Maur?cio Pereira de |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Bioqu?mica, UFRN, BR, Bioqu?mica; Biologia Molecular |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| 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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