Investigação dos efeitos do extrato metanólico de Smilax officinalis na viabilidade e proliferação de carcinoma mamário e macrófagos murinos e em respostas efetoras de macrófagos in vitro

Abstract

Cancer is characterized by the accumulation of genetic alterations and loss of regulation of normal cellular processes, such as cell proliferation and death. In addition, metabolic reprogramming in tumor cells contributes to the growth, survival, evasion of the immune system and recruitment of cells into the tumor microenvironment. Among the immune cells recruited by the tumor, macrophages are the most abundant and influence tumor progression and antitumor response. Proinflammatory macrophages (M1 phenotype) may assist in tumor destruction, while anti-inflammatory macrophages (M2 profile) contribute to immunosuppression and tumor progression. In macrophages the alterations in the cellular metabolism are related to the proliferation, survival and the different functional phenotypes. In this context, Smilax spp. are used as medicinal plants and have already been associated with immunomodulatory and antitumor activities. The aim of the present study was to investigate the effects of the methanolic extract of Smilax officinalis (SMEx) on the viability and proliferation of mammary carcinoma and murine macrophages, as well as mitochondrial function and effector response of macrophages. Initially, we assessed the influence of SMEx on the viability and proliferation of 4T1 cells (murine mammary carcinoma) and J774A.1 macrophages through the MTT assay, trypan blue exclusion and viable cell count. Our results demonstrate that SMEx had cytotoxic effects on 4T1 cells at lower concentrations compared to macrophages. The effects of SMEx on cell viability were also evaluated by extracellular LDH determination and labeling with propidium iodide (flow cytometry).Treatment with SMEx induced death pathways with plasma membrane ruptures in 4T1 cells. While in macrophages the induction of death by SMEx was observed only at the 50 μg/mL, a higher dose than those toxic to 4T1 cells. Since in macrophages, SMEx at lower doses reduced the metabolism of MTT without interfering with viability and proliferation, we evaluated the mitochondrial activity of these cells. According to the data, SMEx was able to alter mitochondrial function and it may be suggested that it induces metabolic reprogramming. In this way, we investigated the effects of SMEx, in non-toxic concentrations, on macrophages stimulated with IL-4 (M2 phenotype dependent on oxidative metabolism), as well as on LPS-stimulated macrophages (M1 profile with predominantly glycolytic metabolism). Our results suggest that SMEx reduces the levels of anti-inflammatory cytokines and partially recovers the secretion of IL-12 in M2 macrophages. In M1 macrophages SMEx did not alter the production of pro-inflammatory mediators and reduced the secretion of anti-inflammatory cytokines. Taken together, the toxic effects of SMEx on tumor cells associated with their ability to modulate macrophage response suggest that SMEx may contribute to the control of tumor cell growth and stimulate macrophages to an antitumor profile. According to our results, SMEx represents a promising target for new studies aimed at immunotherapeutic strategies for breast cancer.