Avaliação da ação da melatonina em células-tronco tumorais mamárias RE-positivas tratadas com disruptores estrogênicos: verificação da via do receptor de estrógeno mediada pelo gene OCT 4
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Data
2016-05-06
Autores
Lopes, Juliana Ramos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As neoplasias mamárias são os tumores de maior ocorrência em mulheres no mundo. A alta incidência deste tipo tumoral deve-se a um conjunto de fatores, entre eles o uso de hormônios, como o estrógeno e a exposição a produtos químicos, entre eles o Bisfenol A (BPA). Apesar da introdução de tratamentos mais efetivos para o câncer de mama, muitas vezes ocorre recorrência e/ou crescimento do tumor, invasão e metástase. As “células-tronco tumorais” (CTTs) vem sendo estudadas em tumores humanos nos últimos anos, consideradas as responsáveis pela recorrência do tumor e resistência a terapias. Devido ao determinante papel das CTTs, é importante a identificação de fatores que controlam sua proliferação. Estudos identificaram o fator de transcrição OCT4 (octamer-binding transcription factor 4), como um dos responsáveis pela auto-renovação e manutenção da pluripotência das células-tronco. Dessa forma, o estudo de potenciais fármacos que atuem sobre esta população específica no câncer de mama tem especial interesse. A melatonina, parece apresentar efeitos oncostáticos no entanto, pouco se sabe sob sua ação terapêutica sobre as células-tronco tumorais mamárias. Dessa forma, o objetivo deste estudo foi avaliar o efeito da melatonina sobre a regulação do marcador de células-tronco OCT4 pelo receptor de estrógeno em células-tronco tumorais mamárias, após o tratamento com estrógeno e bisfenol A. Para o estudo, as células de câncer de mama humano MCF-7 e MDA-MB-231 foram submetidas ao cultivo tridimensional de mamosferas, representando as células-tronco. As CTTs foram cultivadas em suspensão celular ou em crescimento independente de ancoragem. Simultaneamente, as mamosferas foram tratadas com 1 mM melatonina, 10 nM estrógeno e/ou com 10 µM bisfenol A. Ao final, foi avaliado o efeito da melatonina, estrógeno e bisfenol A sobre o crescimento celular, através da contagem do número de células, mensuração do tamanho das mamosferas e por meio do ensaio de viabilidade celular MTT. Além disso, a expressão gênica e proteica do OCT4 e do receptor de estrógeno (RE) foi avaliada por PCR em tempo real, western blotting e imunocitoquímica, respectivamente. Foi avaliada também a ligação do receptor de estrógeno ao gene OCT4 através do Ensaio de Imunoprecipitação da Cromatina (ChIP). Foi observado que o aumento no número e no tamanho das mamosferas induzidos pelo estrógeno ou BPA foi reduzido pelo tratamento com melatonina (P<0,05). Além disso, a ligação do receptor de estrógeno ao gene OCT4 foi reduzida, acompanhada por uma redução na expressão do OCT4 e do receptor de estrógeno (P<0,05). Nossos resultados demonstram que a melatonina é eficaz no controle da proliferação das células-tronco tumorais mamárias in vitro, atuando na via do receptor de estrógeno, demonstrando seu potencial uso terapêutico no câncer de mama.
Breast neoplasms are the most frequent tumors in women worldwide. The high incidence of this tumor type is due to a number of factors, including the use of hormones such as estrogen and exposure to chemicals, including bisphenol A (BPA). Despite the introduction of more effective treatments for breast cancer often occurs recurrence and/or tumor growth, invasion and metastasis. Recently, it was discovered the existence of "cancer stem cells" (CSCs) in human tumors, being responsible for tumor recurrence and resistance to therapy. Due to the important role of CSCs, the identification of factors that control its proliferation is important. Studies have identified the transcription factor OCT4 (octamer-binding transcription factor 4), as the one responsible for self-renewal and maintenance of pluripotency of stem cells. Thus, it has become interesting the study of potential drugs that target this specific population in breast cancer. Melatonin, seems to have oncostatic effects, however, little is known about its therapeutic action on breast cancer stem cells. Thus, the aim of this study was to evaluate the effect of melatonin on the regulation of the stem cell marker, OCT4, by the estrogen receptor in breast cancer stem cells, after treatment with estrogen and bisphenol A. For the study, human breast cancer cells MCF-7 and MDA-MB-231 cells were submitted to three dimensional growth of mammospheres, representing the stem cells. The CSCs were grown in cell suspension or anchorage independent growth. Simultaneously, mammospheres were treated with 1 mM melatonin, 10 nM estrogen and/or 10 uM bisphenol A. In the end, it was evaluated the effects of melatonin, estrogen and bisphenol A on cell growth by counting the number of cells, measuring the size of mammospheres and by the MTT cell viability assay. Furthermore, the gene and protein expression of OCT4 and estrogen receptor (ER) was evaluated by real time PCR, western blotting and immunocytochemistry, respectively. It was also evaluated the binding of ER to OCT4 by Chromatin Immunoprecipitation Assay (ChIP). It was observed that the increase in the number and size of mammospheres induced by estrogen and/or BPA was reduced by melatonin treatment (P<0.05). Furthermore, binding of the ER to OCT4 gene was reduced, accompanied by a reduction in expression of OCT4 and ER (P<0,05). Our results demonstrate that melatonin is effective in controlling the proliferation of breast cancer stem cells in vitro, acting on estrogen receptor pathway, demonstrating their potential therapeutic use in breast cancer.
Breast neoplasms are the most frequent tumors in women worldwide. The high incidence of this tumor type is due to a number of factors, including the use of hormones such as estrogen and exposure to chemicals, including bisphenol A (BPA). Despite the introduction of more effective treatments for breast cancer often occurs recurrence and/or tumor growth, invasion and metastasis. Recently, it was discovered the existence of "cancer stem cells" (CSCs) in human tumors, being responsible for tumor recurrence and resistance to therapy. Due to the important role of CSCs, the identification of factors that control its proliferation is important. Studies have identified the transcription factor OCT4 (octamer-binding transcription factor 4), as the one responsible for self-renewal and maintenance of pluripotency of stem cells. Thus, it has become interesting the study of potential drugs that target this specific population in breast cancer. Melatonin, seems to have oncostatic effects, however, little is known about its therapeutic action on breast cancer stem cells. Thus, the aim of this study was to evaluate the effect of melatonin on the regulation of the stem cell marker, OCT4, by the estrogen receptor in breast cancer stem cells, after treatment with estrogen and bisphenol A. For the study, human breast cancer cells MCF-7 and MDA-MB-231 cells were submitted to three dimensional growth of mammospheres, representing the stem cells. The CSCs were grown in cell suspension or anchorage independent growth. Simultaneously, mammospheres were treated with 1 mM melatonin, 10 nM estrogen and/or 10 uM bisphenol A. In the end, it was evaluated the effects of melatonin, estrogen and bisphenol A on cell growth by counting the number of cells, measuring the size of mammospheres and by the MTT cell viability assay. Furthermore, the gene and protein expression of OCT4 and estrogen receptor (ER) was evaluated by real time PCR, western blotting and immunocytochemistry, respectively. It was also evaluated the binding of ER to OCT4 by Chromatin Immunoprecipitation Assay (ChIP). It was observed that the increase in the number and size of mammospheres induced by estrogen and/or BPA was reduced by melatonin treatment (P<0.05). Furthermore, binding of the ER to OCT4 gene was reduced, accompanied by a reduction in expression of OCT4 and ER (P<0,05). Our results demonstrate that melatonin is effective in controlling the proliferation of breast cancer stem cells in vitro, acting on estrogen receptor pathway, demonstrating their potential therapeutic use in breast cancer.
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Palavras-chave
Câncer de mama, Células-tronco tumorais, POU5F1, Melatonina, Bisfenol A, Estrógeno, Esferóides, Breast neoplasms, Neoplastic stem cells, Melatonin, Bisphenol A., Estroge, Spheroids