Efeito da melanina e do oxigenio singlete na morte celular e fluxo de cálcio em células Melan-A e B16-F10

• Main Author: Aliprandini, Eduardo
• Other Authors: Universidade Federal do Paraná. Setor de Ciências Biológicas. Programa de Pós-Graduação em Ciências (Bioquímica)
• Format: Others
• Language: Portuguese
• Published: 2018
• Subjects: Teses; Melanoma; Melanina; Bioquímica
• Online Access: http://hdl.handle.net/1884/22552

Orientadora : Prof. Glaucia Regina Martinez === Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 12/02/2010 === Bibliografia: 65-74 === Área de concentração: Bioquímica === Resumo === Resumo: O melanoma e um tipo de cancer bastante relevante ja que as opcoes de tratamento eficazes sao limitadas. A presenca da melanina protege os individuos de pele escura contra os efeitos da radiacao solar, a principal causa de formacao do melanoma pela geracao de especies reativas de oxigenio (ROS). Porem, a melanina tambem pode ter um papel duplo, que e a de gerar especies reativas durante sua sintese que podem prejudicar a celula. Portanto, o objetivo deste trabalho foi a avaliacao das caracteristicas de morte celular causadas por uma ROS, o oxigenio singlete (1O2), nas celulas de melanoma murino B16-F10 com e sem estimulo para producao de melanina e nas celulas de melanocito murino Melan-a. O estimulo para a sintese de melanina foi obtido incubando-se as celulas por 48 horas com meio RPMI 1640 enriquecido com 400 ƒÊmol/L de L-tirosina e 10 mmol/L de cloreto de amonio. A concentracao de melanina aumentou em mais de nove vezes nas celulas B16-F10 e as celulas Melan-a tiveram aumento de menos de duas vezes. Foi utilizado o endoperoxido DHPNO2 10 mmol/L por 2 horas para a geracao de 1O2. Essa condicao causou queda na viabilidade avaliada pelo metodo do MTT para 78,0% nas celulas B16-F10, 70,2% nas B16-F10 estimuladas (B16-F10 Y) e 79,3% nas Melan-a. O ensaio foi feito apos 24 horas do inicio do tratamento com DHPNO2 e a viabilidade caiu para 49,7% nas B16-F10, 53,3% nas B16- F10 Y e 72,5% nas Melan-a. A avaliacao da morte celular utilizando laranja de acridina e brometo de etidio mostrou que apos o tratamento por 2 horas, somente as celulas B16- F10 tiveram aumento significativo na quantidade de celulas em apoptose, e as B16-F10 Y tiveram leve queda na quantidade de celulas viaveis, com tendencia ao aumento de celulas em apoptose. As celulas Melan-a nao tiveram diferenca entre os tratamentos. A liberacao do citocromo c foi determinada por HPLC e mostrou-se que apos 2 horas, as celulas B16-F10 tratadas com 1O2 tiveram mais citocromo c liberado para o citoplasma comparado com o controle. Nos demais grupos, nao houve alteracao com o tratamento. Porem, as celulas controle com mais melanina tiveram maior liberacao de citocromo comparado com o controle das celulas nao estimuladas, mostrando que as celulas estavam sofrendo algum dano inerente da sintese de melanina. A analise da fragmentacao do DNA apos 2 e 24 horas mostrou que nao houve aparecimento de quebras caracteristicas de apoptose pelo tratamento com 1O2 em nenhum dos grupos testados. As celulas B16-F10 Y controle apresentaram DNA fragmentado inespecificamente, representado como um arraste no gel de agarose, que nao foi alterado pelo tratamento. A razao entre ADP e ATP foi quantificada para avaliar o estado energetico da celula, que pode refletir algumas caracteristicas de morte. Nenhuma das celulas teve diferenca estatistica apos o tratamento de 2 horas com 1O2, mas foi observado que as razoes ADP/ATP das celulas controle B16-F10 com e sem estimulo apresentaram valores acima do valor considerado para celulas viaveis/proliferativas. Os resultados da celula Melan-a foram bem proximos dos valores ditos normais. O fluxo de calcio tambem foi avaliado e o 1O2 foi capaz de liberar calcio das reservas intracelulares para o citoplasma nas celulas B16-F10, sendo que nas celulas estimuladas, o aumento do calcio citoplasmatico foi menor, indicando a possivel recaptacao do calcio pela melanina. As celulas Melan-a nao sofreram grandes alteracoes na quantidade de calcio liberado para o citoplasma. Nao houve diferenca na liberacao de AIF em nenhuma das celulas. Em conjunto, os resultados mostram que a sintese da melanina estimulada pela suplementacao do meio foi deleteria as celulas, pois causou fragmentacao no DNA, liberacao de citocromo c e aumentou a razao ADP/ATP para valores considerados de celula em apoptose. Por outro lado, a presenca da melanina parece ter protegido as celulas da acao do 1O2, pois alguns resultados indicam uma tendencia de melhora dos parametros avaliados. === Abstract: Melanoma is a relevant type of cancer since the options for efficient treatment are limited. The presence of melanin protects the dark-skinned people against the effects of solar radiation, the main cause for melanoma development by the generation of reactive species of oxygen (ROS). However, melanin may also have a role on the generation of reactive species during its synthesis, which may harm the cell. So, the objective of this work was the evaluation of the characteristics of cell death caused by a ROS, the singlet oxygen (1O2) on murine melanoma cells B16-F10 with or without stimulation for synthesis of melanin and on murine melanocytes cells Melan-a. The stimulus for the synthesis of melanin was obtained treating the cells for 48 hours with medium RPMI 1640 supplemented with 400 ìmol/L of L-Tyrosine and 10 mmol/L of ammonium chloride. The concentration of melanin increased more than nine times in the B16-F10 cells and the Melan-a cells increase was almost twice the amount of the control. It was used the endoperoxide DHPNO2 10 mmol/L for 2 hours for the generation of 1O2. This condition caused the decrease in the viability determined by the method of MTT to 78% in B16-F10, 70,2% in B16-F10 that were stimulated (B16-F10 Y) and 79,3% in Melana cells. The test was performed after 24 hours from the beginning of the treatment with DHPNO2 and the viability decreased to 49,7% in B16-F10, 53,3% in B16-F10 Y and 72,5 % in Melan-a. The evaluation of cell death using acridine orange and ethidium bromide showed that after the two-hour treatment, only the B16-F10 cells had a significant increase of the number of apoptotic cells, and the B16-F10 Y cells had a slight decrease of the amount of viable cells, with the tendency of the increase of apoptotic cells. Melan-a cells did not show difference among the treatments. The release of cytochrome c was determined by HPLC and it showed that after two hours, B16-F10 cells had more cytochrome c released to the cytoplasm compared to the control. There was not any alteration for other groups of cells with the treatment. However, the control cells that had more melanin showed increased cytochrome c release compared to the control of the not-stimulated cells, demonstrating that the previous cells were suffering some kind of damage from the melanin synthesis. The analysis of DNA fragmentation revealed the absence of typical apoptosis fragmentation in any of the groups. B16-F10 Y control cells displayed unspecific DNA damage observed as a smear in the agarose gel, which was not altered by 1O2 treatment. The same result was observed after the treatment for 2 and 24 hours. The ratio between ADP and ATP was quantified to evaluate the energetic state of the cell, which may reflect some characteristics of cell death. None of the cells showed results statistically significant after the two-hour treatment 1O2, but it was shown that the values of ADP/ATP ratio of the B16-F10 control cells with and without stimulation were above of the threshold accepted for viable/proliferative cells. The results of Melan-a cells were very close to the values considered normal. The calcium flux was also evaluated and it was evidenced that 1O2 was capable of releasing calcium from the intracellular stores to the cytoplasm in B16- F10 cells, and the release of calcium was lower in B16-F10 Y, indicating the possibility of the binding of the metal to melanin. The Melan-a cells did not showed much increase in the quantities of calcium released to the cytoplasm. There was no difference in the release of AIF in any group. Over all, the results support that the synthesis of melanin that was stimulated by the supplementation of the medium was deleterious to the cells, since it caused DNA fragmentation, release of cytochrome c and increase of the ratio ADP/ATP to values of cells in apoptosis. On the other hand, the presence of melanin seemed to protect the cells against the action of 1O2, because some results indicate a tendency of improvement in the parameters evaluated.