br Introduction br Iron is a metal that is essential
Iron is a metal that is essential to life, yet it is toxic in excess. In healthy individuals, the metabolic requirement for iron is achieved by a complex homeostatic network strictly regulated at both systemic and cellular levels, in which the organism absorb from diet only the amount of iron necessary to overcome the nonspecific body iron losses [1,2]. Most iron present in human body is incorporated in the hemoglobin of red blood SC 236 for oxygen transport and only about 0.1% of the total body iron is bound to transferrin (Tf), which is the main iron transport protein found in the blood [3,4]. Transferrin plays also a role in maintaining cellular iron homeostasis through regulation of cellular iron intake mediated by the transferrin receptor 1 (TfR1) that bind to Tf on the cell membrane and internalizes it via endocytosis [5,6]. Upon acidification of the endocytotic vesicle, iron is released from Tf, and then forms a cytosolic “labile iron pool” that can be used in the synthesis of different iron-dependent proteins, which have important
roles particularly in mitochondria . The excess of intracellular iron is exported or stored in the protein ferritin .
Perturbations, at least indirect, of iron metabolism in cancer cells have been suspected for a long time [9,10]. For instance, recent reports have shown that the labile iron pool is increased in human breast carcinoma cells compared with normal mammary epithelial cells . This finding supports observations dating from the 80s by which TfR1 is upregulated in many cancer cell types, including breast cancer cells . Consequently, TfR1 has been used as a target for cancer therapy
 and remains a popular strategy for the delivery of anticancer agents . On the other hand, the iron-storage protein ferritin, which sequesters and detoxifies excess of cellular iron, has been also studied as a potential marker of diagnosis and prognosis in breast cancer [12,15,16]. In fact, recent experiments have also revealed important differences on the Fe content of cytosolic ferritin depending on the malignancy of the breast cancer cell line, which indicates a dual role of ferritin in iron homeostasis of breast cancer cells .
E-mail addresses: [email protected] (E. Blanco-González), [email protected] (M. Montes-Bayón).
Therefore, since increasing evidences show that in breast cancer cells, pathways of iron acquisition, efflux, regulation and storage are all perturbed [12,18], it is important to have analytical strategies that permit the exhaustive evaluation of proteins that participate in such pathways. Transferrin in particular, being the main iron incorporation route in cells should be quantitatively addressed in breast cancer cells of different malignancy . In this vein, the determination of Tf in body fluids (e.g. blood serum) is relatively straightforward since the concentration is relatively high (about 3 mg mL−1) and relatively large sample volumes are available. Different analytical methods have been successfully developed for this aim, some of them taking advantage of the specific binding capabilities of Tf for Fe, which permit to stablish a fix Fe: protein stoichiometry (2:1) and quantification of the protein based on Fe detection by inductively coupled plasma-mass spectro-metry (ICP-MS) [20,21]. However, this strategy might not be adequate in cell cultures where the expected Tf concentration is orders of mag-nitude lower than in serum. Thus, the development of selective and sensitive strategies to conduct specific transferrin analysis in this type of samples could be of high interest. In this vein, the use of elemental labelling (p.e. by means of metal-containing nanoparticles or metal chelates) in combination with ICP-MS detection has been a common strategy to allow sensitive determination of specific biomolecules [22,23]. The combination of such elemental labelling methodologies with immunochemical reactions can yield in specific and sensitive al-ternatives for the quantification of proteins in biological samples [24–26].
In this regard, the present work evaluates the use of anti-transferrin antibodies, which have been previously iodinated (as elemental label), for the development of an ICP-MS linked sandwich immunoassay for transferrin determination in cell cultures. The developed strategy will applied to the determination of this protein in breast cancer cells of different malignant phenotype: minimally (MCF-7) and highly (MDA-MB-231) invasive cells.
2. Materials and methods
2.1. Chemicals and reagents
All chemicals were of analytical reagent grade or better. High purity deionized water (18 MΩ cm−1) obtained from a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout this work. Human immunoglobulin G (≥95%) human transferrin (≥98%) and anti-human transferrin polyclonal antibody (T6265) standards were pur-chased from Sigma-Aldrich (St. Louis, MO, USA). The biotinylated anti-transferrin polyclonal antibody was obtained from bioNova Cientifica SL (Madrid, Spain). Thermo Scientific™ Pierce™ iodination beads were obtained from Fisher Scientific SL (Madrid, Spain) and AffiAmino UltraRapid Agarose™ beads (10% bead suspension in 15 mM phosphate pH 7.4, 150 mM NaCl, 20% ethanol) were kindly provided by Lab on a Bead (Uppsala, Sweden). The streptavidin-coated magnetic micro-particles suspension (streptavidin concentration of 0.72 mg mL−1 in hepes–bovine serum albumin buffer, pH 7.4) were obtained from Roche (Roche Diagnostics GmbH, Mannheim, Germany). Tris(hydroxymethyl) aminomethane (Tris) (≥99.8%), hydrochloric acid (36.5–38.0%), nitric acid (65%, Suprapur) and NaI (≥99.5%) used for the antibody iodi-nation reaction were all obtained from Sigma-Aldrich.