Geração de espécies reativas de oxigênio pela NOX4 em células de câncer colorretal humano: papel do óxido nítrico na regulação da NOX4 por s-nitrosilação
Data
2021-08-10
Autores
Teodoro, Ana Caroline de Souza [UNIFESP]
Orientadores
Monteiro, Hugo Pequeno [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
No câncer colorretal (CCR), um pior prognóstico está relacionado à produção das espécies reativas de oxigênio (ROS) peróxido de hidrogênio (H2O2) e ânion superóxido (O2-) e do óxido nítrico (NO). Notadamente, as principais fontes geradoras de NO e ROS nestes tipos de tumor são, respectivamente, as enzimas isoforma induzível da NO sintase (NOS2) e NADPH Oxidase 4 (NOX4). Assim, lançamos a hipótese de que diferentes estágios do CCR transitam, sob o controle de uma modulação temporal da produção de ROS e NO. Parte desta regulação fina pode acontecer a partir de uma ação sinalizadora do NO que introduziria uma modificação pós-tradução na enzima NOX4, a S-nitrosilação de resíduos específicos de cisteína, resultando em inibição da produção de ROS. Utilizando como modelo três diferentes linhagens de tumores de CCR humanos (SW480, SW620 e HCT116), que mimetizam os estágios da progressão tumoral. Inicialmente determinamos as concentrações de ROS e NO nas linhagens celulares mantidas na presença e ausência de soro bovino fetal (SBF) no meio de cultura. A condição de ausência de SBF foi considerada aquela que melhor mimetiza as condições encontradas in vivo pelas células de CCR. A produção de NO foi determinada a partir do acúmulo de seus subprodutos NO2- e NO3- em meio suplementado ou livre de SBF no período de 48 horas. Os resultados mostram que o acúmulo de NO em meio é dependente do tempo, uma vez que há um acúmulo maior de seu produto de oxidação NO2- após 48h. As células SW620, independentemente do tempo de incubação, presença ou ausência de SBF, apresentaram maiores concentrações de NO2- quando comparadas às demais. Ficou demonstrado que as células SW620 produzem mais O2- em comparação às células SW480. Em células SW620, as concentrações de H2O2 foram menores quando comparadas às concentrações desta espécie reativa em células SW480. A produção de H2O2 foi maior nas células SW480. Realizando técnicas de imunodetecção (westernblot, imunoprecipitação), analisamos a expressão proteica das enzimas NOX4 e NOS2 nas linhagens mencionadas onde determinamos que a expressão das duas enzimas, depende da presença ou ausência de SBF no meio de cultura. Para identificar NOX4 s-nitrosilada (SNO-NOX4), realizamos ensaios de ligação por proximidade, e ensaios de imunoprecipitação seguido de western blot revelado com anticorpo anti-biotina, objetivando aumentar as chances de detecção de proteína nitrosilada. Nestes ensaios encontramos SNO-NOX4, nas linhagens estudadas. O conjunto destes resultados sugere que as células SW480 apresentavam SNO-NOX4 em condições basais e que as células SW620 apresentavam SNO-NOX4 associada à elevação dos níveis endógenos de NO, promovida pela retirada de SBF do meio de cultura celular. SNO-NOX4 foi detectada em células HCT116 nas duas condições de cultivo. Mais estudos são necessários para validar nossa hipótese e/ou confirmar que células de CCR metastáticas como as células SW620 teriam SNO-NOX4 como mecanismo de controle negativo para geração de ROS e assim manter a homeostase redox intracelular nestas células.
In colorectal cancer (CCR), a worse prognosis is related to the production of reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide anion (O2-), and nitric oxide (NO). The main sources of NO and ROS in these types of tumors are, respectively, the inducible isoform of NO synthase (NOS2) and NADPH Oxidase 4 (NOX4). NOX4 produces 85% H2O2 and 15% of O2-. We hypothesized that, different stages of CCR development are under control of a temporal modulation of ROS and NO production. At the initial stage, CCR tumor cells move to next stage experiencing an elevation on their intracellular NO levels. Higher NO levels would promote S-nitrosylation of NOX4 at specific cysteine residues, resulting in partial inhibition of ROS production. Three different human CCR cell lines (SW480, SW620 and HCT116), which mimic the stages of tumor progression, were used in our study. Initially, we determined the concentrations of ROS and NO in cell lines maintained in the presence and absence of fetal bovine serum (FBS) in the culture medium. The condition of absence of FBS was considered the one that best mimics the conditions found in vivo by CCR cells. NO production was determined from the accumulation of its by-products NO2- and NO3- in supplemented or SBF-free medium within 48 hours. Accumulation of NO2- (NO oxidized product) in the medium is time-dependent and this accumulation is greater after 48h. SW620 cells, regardless of incubation time, presence or absence of FBS, showed higher concentrations of NO2- when compared to the other cell lines. SW620 cells also produced higher levels of O2- in both experimental conditions when compared to SW480 cells. Levels of H2O2 produced by SW620 cells were lower when compared to the production of this reactive species in SW480 cells. Performing immune detection techniques (westernblot, immunoprecipitation), we analyzed the protein expression of NOX4 and NOS2 enzymes in the mentioned strains, where we determined that the expression of the two enzymes depends on the presence or absence of FBS in the culture medium. To identify s-nitrosylated (SNO) NOX4, we performed proximity binding assays, and immunoprecipitation assays followed by western blot with anti-biotin antibody (Biotin Switch Assay) to increase our capacity to detect SNO-NOX4. Our results indicate that: (1) SNO-NOX4 is present in HCT116 in the presence or absence of FBS in the culture medium. (2) SW480 cells showed SNO-NOX4 in complete medium (+FBS) suggesting the existence of SNO-NOX4 under basal culture conditions. (3) SW620 cells showed SNO-NOX4 associated with the elevation of endogenous NO levels, promoted by the removal of FBS from the cell culture medium. Further studies are needed to validate our hypothesis and/or confirm that metastatic CCR cells such as SW620 cells would have SNO-NOX4 as a negative control mechanism for ROS generation to maintain intracellular redox homeostasis.
In colorectal cancer (CCR), a worse prognosis is related to the production of reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide anion (O2-), and nitric oxide (NO). The main sources of NO and ROS in these types of tumors are, respectively, the inducible isoform of NO synthase (NOS2) and NADPH Oxidase 4 (NOX4). NOX4 produces 85% H2O2 and 15% of O2-. We hypothesized that, different stages of CCR development are under control of a temporal modulation of ROS and NO production. At the initial stage, CCR tumor cells move to next stage experiencing an elevation on their intracellular NO levels. Higher NO levels would promote S-nitrosylation of NOX4 at specific cysteine residues, resulting in partial inhibition of ROS production. Three different human CCR cell lines (SW480, SW620 and HCT116), which mimic the stages of tumor progression, were used in our study. Initially, we determined the concentrations of ROS and NO in cell lines maintained in the presence and absence of fetal bovine serum (FBS) in the culture medium. The condition of absence of FBS was considered the one that best mimics the conditions found in vivo by CCR cells. NO production was determined from the accumulation of its by-products NO2- and NO3- in supplemented or SBF-free medium within 48 hours. Accumulation of NO2- (NO oxidized product) in the medium is time-dependent and this accumulation is greater after 48h. SW620 cells, regardless of incubation time, presence or absence of FBS, showed higher concentrations of NO2- when compared to the other cell lines. SW620 cells also produced higher levels of O2- in both experimental conditions when compared to SW480 cells. Levels of H2O2 produced by SW620 cells were lower when compared to the production of this reactive species in SW480 cells. Performing immune detection techniques (westernblot, immunoprecipitation), we analyzed the protein expression of NOX4 and NOS2 enzymes in the mentioned strains, where we determined that the expression of the two enzymes depends on the presence or absence of FBS in the culture medium. To identify s-nitrosylated (SNO) NOX4, we performed proximity binding assays, and immunoprecipitation assays followed by western blot with anti-biotin antibody (Biotin Switch Assay) to increase our capacity to detect SNO-NOX4. Our results indicate that: (1) SNO-NOX4 is present in HCT116 in the presence or absence of FBS in the culture medium. (2) SW480 cells showed SNO-NOX4 in complete medium (+FBS) suggesting the existence of SNO-NOX4 under basal culture conditions. (3) SW620 cells showed SNO-NOX4 associated with the elevation of endogenous NO levels, promoted by the removal of FBS from the cell culture medium. Further studies are needed to validate our hypothesis and/or confirm that metastatic CCR cells such as SW620 cells would have SNO-NOX4 as a negative control mechanism for ROS generation to maintain intracellular redox homeostasis.
Descrição
Citação
TEODORO, A C S. Geração de espécies reativas de oxigênio pela NOX4 em células de câncer colorretal humano: papel do óxido nítrico na regulação da NOX4 por s-nitrosilação. São Paulo, 2021. 145 f. Dissertação (Mestrado em Biologia Molecular) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2021.