Proteômica de células de osteosarcoma humano após tratamento com lectinas de sementes de quiabo
Data
2023-04-18
Autores
Costa, Erika Sayuri Nishiduka 
Orientadores
Tashima, Alexandre Keiji
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Lectinas são proteínas capazes de reconhecer e se ligar a carboidratos presentes na membrana celular com alta especificidade. Células tumorais frequentemente possuem alterações no padrão de glicosilação da superfície celular, que podem ser seletivamente reconhecidos por lectinas de plantas. Aelki representa um conjunto de isolectinas da semente do quiabo (Abelmoschus esculentus L. Moench) que apresentou em estudos anteriores ação antitumoral seletiva contra células de câncer de mama humanos (MCF7) e baixa toxicidade contra uma linhagem não tumorigênica de células de fibroblastos (CCK-1059 sk). A investigação da atividade dar AelKI poderia ser ampliada a tumores de outras origens histológicas, incluindo tipos raros como o osteosarcoma. Este trabalho investigou a atividade antitumoral in vitro da AelKI, contra células de osteosarcoma humano (U-2 OS). O proteoma das células tratadas com AelKI foi comparado com o das células não tratadas, visando estabelecer alvos moleculares e vias bioquímicas afetados pelo tratamento. O extrato de AelKI foi preparado a partir das sementes do quiabo e purificado por cromatografia de troca iônica. A citotoxicidade de AelKI sobre as células U-2 OS foi avaliada para a determinação das concentrações para incubação, e dois níveis equivalentes a IC25 e IC40 foram selecionadas para o tratamento. Foi realizada a análise proteômica quantitativa de células U-2 OS, pelo método sem marcação. Ensaios de immunoblotting foram utilizados para verificação de marcadores das vias apoptótica e autofágica. AelKI reduziu a viabilidade da população celular em cerca de 90% nas concentrações a partir de 2 micromolar, apresentando IC50=0,26 micromolar. A análise proteômica revelou em torno de 2.200 proteínas quantificadas em todos os grupos, das quais 41 foram diferencialmente abundantes (PDA). Em termos de vias bioquímicas alteradas, as PDAs foram principalmente relacionadas à apoptose, autofagia, metabolismo energético, vesiculação intracelular e replicação de DNA. O ensaio de immunoblotting revelou que marcadores de apoptose (Caspase-3, Caspase-9, Bax e Bcl-2) tiveram seus níveis alterados pelo tratamento, indicando ativação da via intrínseca. Em relação à via autofágica, não houve alteração significativa no nível de expressão dos marcadores testados. Conjuntamente, os resultados apresentados indicam que AelKI induz a morte celular de células de osteosarcoma humano U-2 OS pela ativação da via apoptótica intrínseca, sugerindo o seu potencial como modelo para o desenvolvimento de novos agentes terapêuticos antitumorais.
Lectins are proteins with ability to recognize and attach to carbohydrates on cell membrane with high specificity. Tumoral cells often present altered glycosylation pattern on cell surface, that can be selectively recognized by plant lectins. AelKI represents a group of isolectins extracted from okra seeds (Abelmoschus esculentus L. Moench) that presented antitumoral activity against human breast cancer cells (MCF7) and low toxicity in non-tumorigenic fibroblastic cells (CCK-1059 sk). The investigation of this activity could be expanded to cells from different histological origins, including rare cancer types such as osteosarcoma. In this study the in vitro antitumoral activity of AelKI was investigated against a human osteosarcoma cell line (U-2 OS). The proteome of AelKI- treated cells was compared with that of untreated cells, aiming to unveil the molecular targets and biochemical pathways affected by the treatment. AelKI extract was prepared from okra seeds and purified by ion-exchange chromatography. AelKI cytotoxicity against U-2 OS cells was evaluated to determine the incubation concentrations, and two levels equivalent to IC25 and IC40 were selected for treatment. After treatment, quantitative proteomics analysis of U-2 OS cells was performed by label-free method. Immunoblotting assays were used to check apoptotic and autophagy markers. The results showed a 90% reduction in cell growth at 2 micromolar of AelKI, and the IC50 was 0.26 micromolar. Proteomics analysis revealed around 2,200 proteins quantified in all groups, from which 41 were differentially abundant proteins (DAPs). In terms of affected biochemical pathways, the main DAPs were related to apoptosis, autophagy, energy metabolism, intracellular vesicles, and DNA replication pathways. Immunoblotting assays revealed modulation of apoptotic markers (cleaved caspase-3, caspase-9, Bax, and Bcl-2) evidencing the intrinsic pathway activation. In contrast, no significative stimulation of autophagy was evidenced. Overall, the data indicates that AelKI induces cell death in human osteosarcoma U-2 OS by activating the intrinsic apoptosis pathway. This suggests that AelKI has the potential to serve as a scaffold for the development of novel antitumoral therapies.
Lectins are proteins with ability to recognize and attach to carbohydrates on cell membrane with high specificity. Tumoral cells often present altered glycosylation pattern on cell surface, that can be selectively recognized by plant lectins. AelKI represents a group of isolectins extracted from okra seeds (Abelmoschus esculentus L. Moench) that presented antitumoral activity against human breast cancer cells (MCF7) and low toxicity in non-tumorigenic fibroblastic cells (CCK-1059 sk). The investigation of this activity could be expanded to cells from different histological origins, including rare cancer types such as osteosarcoma. In this study the in vitro antitumoral activity of AelKI was investigated against a human osteosarcoma cell line (U-2 OS). The proteome of AelKI- treated cells was compared with that of untreated cells, aiming to unveil the molecular targets and biochemical pathways affected by the treatment. AelKI extract was prepared from okra seeds and purified by ion-exchange chromatography. AelKI cytotoxicity against U-2 OS cells was evaluated to determine the incubation concentrations, and two levels equivalent to IC25 and IC40 were selected for treatment. After treatment, quantitative proteomics analysis of U-2 OS cells was performed by label-free method. Immunoblotting assays were used to check apoptotic and autophagy markers. The results showed a 90% reduction in cell growth at 2 micromolar of AelKI, and the IC50 was 0.26 micromolar. Proteomics analysis revealed around 2,200 proteins quantified in all groups, from which 41 were differentially abundant proteins (DAPs). In terms of affected biochemical pathways, the main DAPs were related to apoptosis, autophagy, energy metabolism, intracellular vesicles, and DNA replication pathways. Immunoblotting assays revealed modulation of apoptotic markers (cleaved caspase-3, caspase-9, Bax, and Bcl-2) evidencing the intrinsic pathway activation. In contrast, no significative stimulation of autophagy was evidenced. Overall, the data indicates that AelKI induces cell death in human osteosarcoma U-2 OS by activating the intrinsic apoptosis pathway. This suggests that AelKI has the potential to serve as a scaffold for the development of novel antitumoral therapies.