Mecanismos de resistência à morte por Anoikis mediada por óxido nítrico em células de melanoma humano
Data
2022-09-02
Autores
Nascimento, Igor Ribeiro [UNIFESP]
Orientadores
Monteiro, Hugo Pequeno [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
O melanoma caracteriza-se pelo crescimento descontrolado dos melanócitos. E, embora seja menos comum do que outros tipos de câncer de pele (American Cancer Society, 2021) é a forma mais mortal entre eles, exibindo padrões de desenvolvimento extremamente agressivos. Sua taxa de sobrevida em 5 anos é inferior a 5% para os pacientes com melanomas metastáticos para os linfonodos. Um processo de metástase bem-sucedido está associado à capacidade de resistir à morte por perda de adesão ao substrato (anoikis), que melanomas e outros tumores adquirem durante seu desenvolvimento. Dentre uma série de fatores que podem influenciar a morte por anoikis, o papel do óxido nítrico (NO) ainda foi pouco explorado. O NO produzido ou ofertado em baixas concentrações estimula a proliferação de células aderidas a matriz extracelular (MEC).Por outro lado, nestas mesmas condições, o NO produzido ou ofertado em concentrações elevadas pode promover a morte celular. No entanto, em células onde a adesão a MEC foi impedida com indução de morte por anoikis, sabe-se pouco sobre o papel do NO. Nossos primeiros resultados evidenciaram que a utilização de uma superfície de cultivo celular revestida com 20mg/ml de Poli-Hema promovia total incapacidade de adesão das células A375 e SK-MEL-28 de melanoma humano. Estas condições promoviam a morte por anoikis nas células das duas linhagens. As células das duas linhagens quando em suspensão entravam em anoikis em diferentes proporções: Células A375 com mortalidade de 58,6% e células SK-MEL-28 com mortalidade de 34,3%. Utilizamos inicialmente a oferta de NO a partir do doador Detanonoato e a retirada de NO com o uso do seu sequestrador específico, CPTIO. Os resultados obtidos com esta abordagem nos levaram a procurar entender um possível papel para o NO produzido endógenamente pelas duas linhagens. Fizemos uma estimativa de produção endógena de NO e os resultados obtidos mostraram que essa produção nas células A375 se mantinha elevada estando as células aderidas ou mantidas em suspensão. Para as células SK-MEL-28 a produção de NO se encontrava elevada nas células aderidas e diminuída nas células em suspensão. Quando as células eram tratadas com o inibidor geral das enzimas NO sintases (NOS) a viabilidade das células A375 mantidas em suspensão caía de 41,4% para 10,6%, enquanto que para as células SK-MEL-28 em suspensão a viabilidade se mantinha inalterada, de 65,7% para 65,5%. Estes resultados demonstraram que NO desempenha papel muito importante na resistência a anoikis apresentada pelas células A375. Por outro lado parece não influenciar na resistência a anoikis apresentada pelas células SK-MEL-28. Com base nesses resultados investigamos as possíveis fontes de NO nas células A375. Nossos resultados sugeriram que uma possível fonte endógena de NO nestas células, utilizável na resistência a anoikis, poderia ser a isoforma NOS3. Em relação aos impactos do NO na expressão das proteínas diretamente relacionadas a anoikis como Bim e a forma clivada/ativa de Caspase 3, não observamos maiores alterações nas células das duas linhagens. Quando analisamos as expressões de proteínas reguladoras da anoikis, Caveolina-1 e a proteína tirosina quinase do complexo de adesão focal (FAK), observamos uma diminuição nos níveis de expressão de Caveolina-1 nas células das duas linhagens mantidas em suspensão. Entretanto, não se observam para as duas linhagens, diferenças entre as três condições experimentais analisadas: células em suspensão; células em suspensão tratadas com PBS (diluente do inibidor das NOSs); células em suspensão tratadas com L-NAME. Os níveis de expressão e de fosforilação da proteína FAK diminuíram nas células A375 mantidas em suspensão e tratadas com L-NAME. Mais ainda, observamos consistentemente em células A375 que as expressões de b-actina e a-tubulina diminuíam quando estas células eram mantidas em suspensão e tratadas com L-NAME. Concluindo, os resultados obtidos neste estudo sugerem a existência de uma via de sinalização celular associada à resistência a anoikis em melanomas humanos de sítio primário, mediada pelo NO. Esta via seria constituída pela proteína tirosina quinase FAK e pelas proteínas constituintes da MEC, b-actina e a-tubulina.
Melanoma is characterized by the uncontrolled growth of melanocytes. Although it is less common than other types of skin cancer (American Cancer Society, 2021) it is the deadliest form among them, exhibiting extremely aggressive patterns of development. Its 5-year survival rate is less than 5% for patients with metastatic melanomas to the lymph nodes. A successful metastasis process is associated with the ability to resist death from loss of substrate adhesion (anoikis), which melanomas and other tumors acquire during their development. Among several factors that can influence death by anoikis, the role of nitric oxide (NO) has not yet been explored. NO produced or offered at low concentrations stimulates the proliferation of cells attached to the extracellular matrix (ECM). On the other hand, under these same conditions, NO produced or offered at high concentrations can promote cell death. However, in cells where ECM adhesion was impeded with occurrence of anoikis, little is known about the role of NO. Our first results showed that the use of a cell culture surface coated with 20mg/ml of Poly-Hema promoted total incapacity of adhesion of human melanoma A375 and SK-MEL-28 cells. These conditions promoted death by anoikis in cells of both strains. Cells of both strains when in suspension underwent anoikis in different proportions: A375 cells with a mortality of 58.6% and SK-MEL-28 cells with a mortality of 34.3%. We initially used the supplementation of NO from the donor Detanonoate and the withdrawal of NO with the use of its specific scavenger, CPTIO. The results obtained with this approach led us to seek to understand a possible role for the endogenously produced NO by the two strains. After estimating the endogenous production of NO for both cell lines adhered or kept in suspension, we observed that this production in A375 cells remained high in both conditions. For SK-MEL-28 cells, NO production was reduced in cells kept in suspension. When cells were treated with the general inhibitor of NO synthases (NOS) L-NAME, the viability of A375 cells maintained in suspension dropped from 41.4% to 10.6%, while for SKMEL-28 cells in suspension the viability remained unchanged. These results demonstrate that NO plays a very important role in the anoikis resistance presented by A375 cells. On the other hand, it does not seem to influence the anoikis resistance presented by SK-MEL-28 cells. Based on these results, we investigated possible sources of NO in A375 cells. Our results suggested that a possible endogenous source of NO in these cells, usable in anoikis resistance, could be the NOS3 isoform. Regarding the impacts of NO on the expression of proteins directly related to anoikis such as Bim and the cleaved/active form of Caspase 3, we did not observe major changes in the cells of the two strains. When we analyzed the expressions of anoikis regulatory proteins, Caveolin-1 and the focal adhesion complex protein tyrosine kinase (FAK), we observed a decrease in the levels of Caveolin-1 expression in cells of the two cell lines maintained in suspension. However, for both cell lines, no differences were observed between the three experimental conditions analyzed: cells in suspension; cells in suspension treated with PBS (NOS inhibitor diluent); L-NAME treated suspension cells. FAK protein expression and phosphorylation levels decreased in A375 cells maintained in suspension and treated with L-NAME. Furthermore, we consistently observed in A375 cells that beta-actin and alpha-tubulin expressions were decreased when these cells were kept in suspension and treated with L-NAME. In conclusion, the results obtained in this study suggest the existence of a cell signaling pathway mediated by NO and associated with resistance to anoikis in human primary site melanomas. This pathway would be constituted by the FAK protein tyrosine kinase and by the constituent proteins of the ECM, beta-actin and alpha- tubulin.
Melanoma is characterized by the uncontrolled growth of melanocytes. Although it is less common than other types of skin cancer (American Cancer Society, 2021) it is the deadliest form among them, exhibiting extremely aggressive patterns of development. Its 5-year survival rate is less than 5% for patients with metastatic melanomas to the lymph nodes. A successful metastasis process is associated with the ability to resist death from loss of substrate adhesion (anoikis), which melanomas and other tumors acquire during their development. Among several factors that can influence death by anoikis, the role of nitric oxide (NO) has not yet been explored. NO produced or offered at low concentrations stimulates the proliferation of cells attached to the extracellular matrix (ECM). On the other hand, under these same conditions, NO produced or offered at high concentrations can promote cell death. However, in cells where ECM adhesion was impeded with occurrence of anoikis, little is known about the role of NO. Our first results showed that the use of a cell culture surface coated with 20mg/ml of Poly-Hema promoted total incapacity of adhesion of human melanoma A375 and SK-MEL-28 cells. These conditions promoted death by anoikis in cells of both strains. Cells of both strains when in suspension underwent anoikis in different proportions: A375 cells with a mortality of 58.6% and SK-MEL-28 cells with a mortality of 34.3%. We initially used the supplementation of NO from the donor Detanonoate and the withdrawal of NO with the use of its specific scavenger, CPTIO. The results obtained with this approach led us to seek to understand a possible role for the endogenously produced NO by the two strains. After estimating the endogenous production of NO for both cell lines adhered or kept in suspension, we observed that this production in A375 cells remained high in both conditions. For SK-MEL-28 cells, NO production was reduced in cells kept in suspension. When cells were treated with the general inhibitor of NO synthases (NOS) L-NAME, the viability of A375 cells maintained in suspension dropped from 41.4% to 10.6%, while for SKMEL-28 cells in suspension the viability remained unchanged. These results demonstrate that NO plays a very important role in the anoikis resistance presented by A375 cells. On the other hand, it does not seem to influence the anoikis resistance presented by SK-MEL-28 cells. Based on these results, we investigated possible sources of NO in A375 cells. Our results suggested that a possible endogenous source of NO in these cells, usable in anoikis resistance, could be the NOS3 isoform. Regarding the impacts of NO on the expression of proteins directly related to anoikis such as Bim and the cleaved/active form of Caspase 3, we did not observe major changes in the cells of the two strains. When we analyzed the expressions of anoikis regulatory proteins, Caveolin-1 and the focal adhesion complex protein tyrosine kinase (FAK), we observed a decrease in the levels of Caveolin-1 expression in cells of the two cell lines maintained in suspension. However, for both cell lines, no differences were observed between the three experimental conditions analyzed: cells in suspension; cells in suspension treated with PBS (NOS inhibitor diluent); L-NAME treated suspension cells. FAK protein expression and phosphorylation levels decreased in A375 cells maintained in suspension and treated with L-NAME. Furthermore, we consistently observed in A375 cells that beta-actin and alpha-tubulin expressions were decreased when these cells were kept in suspension and treated with L-NAME. In conclusion, the results obtained in this study suggest the existence of a cell signaling pathway mediated by NO and associated with resistance to anoikis in human primary site melanomas. This pathway would be constituted by the FAK protein tyrosine kinase and by the constituent proteins of the ECM, beta-actin and alpha- tubulin.