Docosahexaenoic acid reverses the promoting effects of breast tumor cell-derived exosomes on endothelial cell migration and angiogenesis
Introduction
Breast cancer (BC) is by far the most common cause of cancer-associated mortality among women throughout the world. Over recent years, many steps have been taken forward for better BC diagnosis and treatment, although metastatic BC remains unsolvable problems, covering a large number of deaths in patients [1,2]. BC tumor is surrounded by an environment embracing extracellular matrix, tumor-associated vasculature, and a variety of stromal cell types e.g. fibroblasts, adipocytes, endothelial cells, and inflammatory immune cells [3]. As one of the essential members of the tumor microenvironment, stromal cells contribute importantly to the process of tumor development [4] and also angiogenesis [5]. This process of new blood vessel formation contributes critically to the invasive properties and metastatic features of BC cells [6,7].
Extracellular vesicles, e.g. secreted exosomes by tumor cells, are actively involved in the coordination of stromal reprogramming [8]. Exosomes function in intercellular communications by delivering a variety of biomolecules such as proteins, DNA, coding and non-coding RNAs [9,10]. Tumor cell-derived exosomes (TDEs) can carry oncogenic information and exert broad effects on promoting angiogenesis, metastasis, and other cellular events in malignant transformation [11]. As an important content in exosomes, microRNAs (miRNAs, miRs) are small non-coding RNAs (~22 nucleotides in length) playing important roles in gene regulation. They target specific mRNAs through degrading and/or repressing the translation [12,13].
Docosahexaenoic acid (DHA) is known as an important omega-3 fatty acid. This abundant compound in fish oil performs different biological activities including boosting immune function [14], alleviating inflammation [15], and optimizing cellular metabolism [16]. DHA also has anti-cancer functions in BC [17]. The anti-cancer mechanism of DHA is attributed to its anti-angiogenic properties whereby DHA can suppress angiogenic mediators, e.g. vascular endothelial growth factor (VEGF) [18]. The up-regulation of VEGF has been reported in the association of BC invasiveness and metastasis [19]. VEGF is in the first line of promoters of angiogenesis, thus can be considered as an important mitogen with high specificity for endothelial cells (reviewed in [20]). Despite growing knowledge about the therapeutic properties of DHA, unveiling the mechanisms involved in BC requires further investigations.
Considering the biological functions of exosomes, we speculated whether TDEs carry biomolecules that can per se regulate VEGF-related angiogenesis in vascular endothelial cells or not. We also sought to determine whether DHA is able to alter the levels of angiogenesis-associated miRNAs in BC cell-derived exosomes and suppress the migration and angiogenesis of endothelial cells. This information can broaden the horizons toward BC progression and represent potential use for developing new therapeutic strategies targeting intercellular communications in BC.
Section snippets
Cell culture
Human breast adenocarcinoma cell lines (MDA-MB-231 and BT-20), human non-tumorigenic breast epithelial cells (MCF10A), and human umbilical vein endothelial cells (HUVECs) were purchased from the National Cell Bank at Pasteur Institute, Tehran, Iran. All cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) that was supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% penicillin-streptomycin (all from Gibco BRL, Rockville, MD, USA). The cells were incubated at
DHA decreases the viability of BC cells in a specific and time-dependent manner
In order to show the DHA-induced cytotoxic effects on triple-negative BC cells, MDA-MB-231 cells were treated with different concentrations of DHA (12.5, 25, 50, 100, and 200 μM). Afterward, we used the MTT assay to determine cell viability. At 100 μM, DHA decreased 68.72, 46.02, and 34.87% of the viability of MDA-MB-231 cells in 24, 48, and 72 h, respectively. The equivalent volume of the diluent was also tested but did not show any significant effect on cell viability. According to these
Discussion
Over recent years, global efforts have been done to unveil the molecular mechanisms of angiogenesis. The final goal is to discover how to inhibit neovascularization and block the metastatic properties of tumor cells. DHA, as an omega-3 fatty acid, exerts the anti-cancer effects on malignancies, particularly BC. DHA can alter BC exosome secretion and miRNA contents, which in turn bring about the suppression of endothelial tube formation. DHA can also suppress the angiogenesis of endothelial
Conclusion
Taken together, our findings suggest that DHA elicits anti-cancer effects on BC cells through suppressing angiogenesis, particularly through miRNAs. This anti-angiogenic activity might be attributed to modulating pertinent groups of exosomal miRNAs including pro- and anti-angiogenic miRNAs. This information can offer valuable insights into the development of therapeutic strategies against BC tumors based on targeting exosome secretion and the transfer of exosome contents. Some open questions
Declaration of competing interest
The authors declare no competing interests.
Acknowledgments
The authors highly appreciate the help of all colleagues involved in conducting this project.
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