Research ArticleZinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin☆
Introduction
Breast cancer is a major health threat to women with high mortality worldwide. Mortality from breast cancer is not caused by the primary tumour, but rather, from tumour growth in secondary locations, breast cancer metastasis [1]. Thus strategies to reduce metastases in breast cancer patients are important in the battle against breast cancer.
To metastasize cancer cells must undergo a phenotypic transition from epithelial cells to mesenchymal cells. Mesenchymal cells have greater motility and are capable of gaining access to the blood circulation or lymphatic system by penetrating through the basement membrane. Necessary for cancer cells to migrate is their ability to attach to the extra-cellular matrix (ECM). Integrins are the primary mediators of cell to ECM adhesion and are therefore important in cell migration during embryogenesis, wound healing and cancer metastasis. Presently, there are a total of 24 known distinct integrins. Each integrin consists of an α- and a β-subunit forming a heterodimer [2]. The expression of integrins is cell-type specific and each integrin recognizes a particular ECM protein.
Many members of the integrin β1 family (e.g. α5/β1, the fibronectin receptor) have been implicated in cancer metastasis. Blocking of integrin α5/β1 reduces the adhesion of a highly metastatic breast cancer cell line to a model endothelium [3]. Interestingly, mammary tumours bearing disrupted integrin β1 gene exhibit significantly reduced capacity to metastasize to the lung in mice [4]. This evidence supports a role of integrin α5/β1 in promoting breast cancer metastasis.
The role of zinc in integrin-mediated adhesion of breast cancer cells and its role in the process of breast cancer metastasis are essentially unknown. Zinc has been shown to activate integrin β2 to mediate the adhesion of monocytic cells to fibrinogen [5]. Furthermore, Kagara et al. [6] reported an association between elevated expression of ZIP10, a zinc importer, in cancer breast tissue and lymph node metastases. Knockdown of ZIP10 in MDA-MB-231 cells reduces cellular zinc uptake and migration, suggesting that zinc is needed for breast cancer cell migration. In contrast, an elevated expression of ZIP6, another zinc importer, is associated with a longer relapse free and an overall increase in survival of breast cancer patients with invasive ductal carcinoma [7]. Attenuation of ZIP6 in breast cancer T47D cells reduces intracellular zinc concentration and E-cadherin expression, suggesting that zinc inhibits the epithelial-to-mesenchymal transition, a necessary step in preparing breast cancer cells for metastasis [8]. Collectively, these reports showed that intracellular zinc status could influence breast cancer metastasis; however, whether intracellular zinc functions as a promoter or inhibitor in breast cancer metastasis is controversial.
Integrin structurally contains three distinct divalent cation-binding sites [9]. Two binding sites are located close to the binding site where integrin interacts with its ligand and are occupied by manganese or magnesium [9], [10]. Presence of manganese or magnesium is essential for integrin-ligand binding and their presence is required for cell adhesion and cancer metastasis [11], [12], [13]. The third binding site appears to be occupied only by calcium and is involved in the stabilization of the bound integrin head to its ligand [10]. Calcium promotes adhesion in a leukemic cell line [13], but inhibits adhesion in other cell lines [12], [14]. These divalent cation-binding sites are, therefore, vital to integrin-mediated cancer metastasis.
We hypothesized that, zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. The objective was to establish the role of zinc in the migration of breast cancer MDA-MB-231 cells. Our observations showed that zinc functioned as an inhibitor of MDA-MB-231 cell migration on fibronectin through inhibiting magnesium-dependent integrin-, likely integrin α5/β1-, mediated adhesion, suggesting that zinc could function as an inhibitor of integrin-mediated breast cancer metastases.
Section snippets
Cell culture
Breast carcinoma MDA-MB-231 cells (ATCC; passage 39–45) were routinely maintained in a regular medium (DMEM [Dulbecco's modified eagle medium;Invitrogen] supplemented with fetal bovine serum [FBS; 10%; Invitrogen], glucose [4,500 mg/L] and l-glutamine [584 mg/L], sodium pyruvate [110 mg/L] and penicillin streptomycin) at 37°C, 5% CO2. Initial seeding density was 5×105 cells/T-75 flask or 100 mm cell culture-treated Petri dishes.
Zinc-depleted medium
To prepare the zinc-depleted medium, FBS was treated with
Zinc status and growth unaffected
Total cellular zinc content in MDA-MB-231 cells was not affected by zinc treatments (Table 1). The abundance of LIPZ in cells in the 10 μM group was significantly higher than that in cells in the control, 2.5 and 5 μM groups but was similar to cells in the 25 μM group (P<.05). Zinc treatment had no effect on total cell number and cell viability (Table 1).
Metastatic potential reduced
The metastatic potential of the MDA-MB-231 cells was assessed by a combination of single cell migration distance and migration rate in a
Zinc inhibited integrin-specific migration potential on fibronectin
In this study, zinc at adequate and supplementation levels significantly reduced both the distance of single cell migration of MDA-MB-231 cells and their migration rate in a confluent monolayer on a fibronectin matrix. Single cell migration distance assesses the individual cell invasiveness. In contrast, migration in a confluent monolayer is more representative of the migration ability of cancer cells in vivo where the ability of cancer cells to migrate is first dependent on their ability to
Acknowledgments
This study was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (Z. Xu).
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Sylvia Lymburner, Sarah McLeod, Markus Purtzki, Calvin Roskelley and Zhaoming Xu have no conflicts of interest or financial ties to disclose.