Efeitos da melatonina sobre a polarização de macrófagos do colostro e níveis de citocinas em co-cultura com células de tumor de mama (MCF-7)

Abstract

Melatonin is a hormone that has several physiological functions, among them we have antiinflammatory and anti-tumor effects. Studies suggest that macrophages, which represent one of the main components infiltrated in the tumor microenvironment and apparently contribute to its progression and dissemination in breast cancer, can be reprogrammed to act as a potent antitumor immune response due to its plasticity characteristic. Thus, the aim of this work was to evaluate the influence of the hormone melatonin on macrophage polarization (M1 profile/M2 profile) of colostrum from healthy mothers in co-culture with breast tumor cells (MCF-7) at 2 and 24 hours of incubation. For this purpose, colostrum samples were collected from 12 volunteer mothers monitored by the Public Health System of Barra do Garças/MT. Colostrum macrophages were obtained by Ficoll-Paque density gradient and maintained in culture for 2 and 24 hours, treated or not with melatonin [100 ng/mL] and in the presence or absence of MCF-7 cells. For immunophenotyping and polarization identification, cells were labeled with two sets of monoclonal antibodies and analyses were performed by flow cytometry. Macrophages expressing CD197+CD86+ were defined with M1-type macrophages, while CD197-CD86+ or CD14+CD163+ cells were defined as M2-type macrophages. Analyses of cytokine secretion in the supernatants of the cultures and co-cultures were also performed. We observed an increase in expression of colostrum CD14+CD163+ macrophages not treated with melatonin after 24 hours of incubation and that the expression of CD14+CD163+ when cocultured with MCF-7 was similar regardless of incubation time or melatonin treatment. The expression of M1 macrophages was lower after 2 hours in culture, and the treatment of cells with melatonin increased the expression of M1 to percentages similar to M2. After 24 hours in culture, independently of the treatment with melatonin, there was an increase of M1 and a reduction of M2 macrophages. Furthermore, there was a positive correlation between untreated and melatonin-treated M2 macrophages. In the co-culture with melatonin, the expression of M1 and M2 cells was reduced after 24 hours of incubation, but at the 2-hour time point, regardless of melatonin treatment, no difference in M1 and M2 expression was observed. Furthermore, in the co-culture with melatonin, increased secretion of interleukin 8 and a reduction in TNF-α concentration was observed. These data suggest that colostrum macrophages differentiate into M1 and M2, and that melatonin was able to increase the percentages of M1, reinforcing that these cells are probably involved in protecting the organism through microbicidal and phagocytic activity and that possibly melatonin is promoting some balance of the inflammatory and non-inflammatory picture managed by these cells. The reduction observed in the expression of both macrophages phenotypes in the 24 hours incubation under melatonin action suggests that this hormone may inhibit the formation of inflammatory infiltrates caused by M1 macrophages in the tumor microenvironment and that the incubation time was a determining factor in cell expression. Furthermore, melatonin was able to reduce TNF-α secretion in coculture with MCF-7 cells, reinforcing the potential of this hormone as a therapeutic strategy for cancer.