Participação da proteína tirosina quinase src e do óxido nítrico nas vias de sinalização celular estimuladas por estrógeno na linhagem MCF-7 obtida de tumor humano de mama
Arquivos
Data
2015-11-25
Autores
Rosa, Heloisa 
Orientadores
Krieger, Jose Eduardo
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
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Resumo
O receptor de estrógeno tem um papel central no câncer de mama responsivo a estrógeno. Evidências experimentais e clínicas sugerem ainda uma participação do óxido nítrico (NO) e das enzimas óxido nítrico sintases (NOS) na biologia do câncer de mama. A S-nitrosilação de proteínas sinalizadoras como Src quinase, mediada pelo NO, é importante para a progressão do tumor. O objetivo do presente estudo foi descrever uma via de sinalização não-nuclear estimulada por estrógeno que envolve a ativação de óxido nítrico sintase endotelial (eNOS) e de Src quinase em células de carcinoma mamário MCF-7. Para tanto, nós desenvolvemos uma ?isca? molecular a partir da clonagem e expressão da proteína Src quinase recombinante humana contendo uma cauda de Histidina (6xHis-Src). Células MCF-7/6xHis-Src foram tratadas com diferentes concentrações de 17?-estradiol (E2) durante 0, 2, 8, 16 ou 24 horas. Ensaio de Imunofluorescência revelou ativação de Src após tratamento com 100 nM E2. Análise de Western Blotting confirmou que a atividade de Src foi estimulada com 100 nM E2 alcançando sua atividade máxima após 8 e 24 horas de incubação. Níveis intracelulares de NO aumentaram após 24 horas de tratamento das células com 100 nM E2. A S-nitrosilação de Src foi estimulada com 10 nM E2 e inibida por pré-incubação das células com o inibidor de Src, PP1. E2 estimula a proliferação celular, que é inibida por pré-incubação com Fulvestranto (antagonista do receptor de estrógeno), PP1 ou L-N5-(1-Iminoethyl) ornithine (L-NIO) (inibidor da eNOS). Análise baseada em Espectrometria de Massas revelou a presença de proteínas associadas com a proliferação celular após tratamento das células com E2; a pré-incubação das células com inibidores revelou a presença de proteínas associadas com repressão transcricional. Nós concluímos que NO, eNOS e Src quinase desempenham um papel importante na via de sinalização não-nuclear estimulada por E2 associada à proliferação das células MCF-7
The human estrogen receptor plays a major role in breast cancers responsive to this hormone. Increasing experimental and clinical evidence suggest a major role for nitric oxide (NO) and NO synthases (NOS) in breast cancer cell biology. NO-mediated S-nitrosylation of signaling proteins such as Src kinases are relevant to breast tumor progression. The aim of the present study was to describe a non-nuclear signaling pathway stimulated by estrogen which involves the activation of endothelial NOS and Src kinase in MCF-7 cells. To achieve this goal, we developed a molecular "bait" by cloning and expressing human recombinant Src with Histidine tag (6xHis-Src). 6xHis-Src/MCF-7 cells were treated with different concentrations of 17?-estradiol (E2) during 0, 2, 8, 16 or 24 hours. Immunofluorescence assay revealed activation of Src after treatment with 100 nM E2. Western Blot analysis confirmed that Src activity was stimulated with 100 nM E2 with maximum activities after 8 and 24 h incubation. Intracellular levels of NO increased after 24 h incubation of cells with 100 nM E2. S-nitrosylation of Src was stimulated with 10 nM E2 and inhibited by pre-incubation of cells with the Src inhibitor PP1. E2 stimulates cell proliferation which is prevented by pre-incubation with Fulvestrant (estrogen receptor inhibitor), PP1 or L-N5-(1-Iminoethyl) ornithine (L-NIO) (endothelial NOS inhibitor). Mass Spectrometry based analysis revealed the presence of proteins associated with cell proliferation after treatment of the cells with E2; pre-incubation with inhibitors revealed the presence of proteins associated with transcriptional repression. In conclusion, NO/eNOS and Src kinase play a major role in an E2 stimulated non-nuclear signaling pathway associated with the proliferation of MCF-7 breast cancer cells.
The human estrogen receptor plays a major role in breast cancers responsive to this hormone. Increasing experimental and clinical evidence suggest a major role for nitric oxide (NO) and NO synthases (NOS) in breast cancer cell biology. NO-mediated S-nitrosylation of signaling proteins such as Src kinases are relevant to breast tumor progression. The aim of the present study was to describe a non-nuclear signaling pathway stimulated by estrogen which involves the activation of endothelial NOS and Src kinase in MCF-7 cells. To achieve this goal, we developed a molecular "bait" by cloning and expressing human recombinant Src with Histidine tag (6xHis-Src). 6xHis-Src/MCF-7 cells were treated with different concentrations of 17?-estradiol (E2) during 0, 2, 8, 16 or 24 hours. Immunofluorescence assay revealed activation of Src after treatment with 100 nM E2. Western Blot analysis confirmed that Src activity was stimulated with 100 nM E2 with maximum activities after 8 and 24 h incubation. Intracellular levels of NO increased after 24 h incubation of cells with 100 nM E2. S-nitrosylation of Src was stimulated with 10 nM E2 and inhibited by pre-incubation of cells with the Src inhibitor PP1. E2 stimulates cell proliferation which is prevented by pre-incubation with Fulvestrant (estrogen receptor inhibitor), PP1 or L-N5-(1-Iminoethyl) ornithine (L-NIO) (endothelial NOS inhibitor). Mass Spectrometry based analysis revealed the presence of proteins associated with cell proliferation after treatment of the cells with E2; pre-incubation with inhibitors revealed the presence of proteins associated with transcriptional repression. In conclusion, NO/eNOS and Src kinase play a major role in an E2 stimulated non-nuclear signaling pathway associated with the proliferation of MCF-7 breast cancer cells.
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Citação
ROSA, Heloisa. Participação da proteína tirosina quinase src e do óxido nítrico nas vias de sinalização celular estimuladas por estrógeno na linhagem MCF-7 obtida de tumor humano de mama. 2015. 179 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.