Apo-BLf with a purity of 97% was from NutriScience Innovations, LCC (Connecticut, USA). Apo-BLf was saturated with iron to obtain Holo-BLf according to a method described previously ; iron in Holo-BLf was 93% and it was quantified by an enzymatic automated method (MicroTech Laboratories, Mexico). LA sodium salt with a purity of 99% and tetramethylrhodamine (TRITC)-conjugated phalloidin were from Sigma-Aldrich (Merck KGaA). Anti-vimentin antibody (Ab), anti-E-cadherin Ab and anti-focal adhesion kinase (FAK) Ab were from Santa Cruz Biotechnology (Sta. Cruz, CA, USA). Anti-paxillin Ab was from Abcam® (Waltham, MA, USA). Phospho-specific Ab to tyrosine (Tyr)-397 of FAK (anti-FAK-p-Tyr397) was from Invitrogen (Camarillo, CA, USA). Anti-actin Ab was from R&D Systems, Inc (Minneapolis, MN, USA)
Cell lines and culture
MDA-MB-231 and MCF-7 breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 3.7 g/l sodium bicarbonate and antibiotics. Cultures were incubated under a humidified atmosphere containing 5% CO2 and 95% air at 37 ºC. MDA-MB-231 cells were FBS-starved in DMEM for 24 h and MCF-7 cells were FBS-starved in DMEM for 18 h before treatment with BLf, FBS and/or LA.
Cultures of MDA-MB-231 and MCF-7 cells were washed twice with phosphate-buffered saline (PBS), equilibrated in DMEM for 30 min and treated for various times and concentrations of Apo-BLf, Holo-BLf, FBS or LA. After stimulation, conditioned media were collected and cells were solubilized in 0.5 ml ice-cold RIPA buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X100, 1% sodium deoxycholate, 1.5 mM MgCl2, 0.1% SDS and 1 mM PMSF). Protein concentration of samples was determined by the micro-Bradford protein assay (Bio-Rad, USA).
Western blot (WB) analysis
Equal amounts of protein were separated using 10% SDS-PAGE separating gels followed by transfer to nitrocellulose membranes. Membranes were blocked for 2 h at room temperature using 5% non-fat dried milk in PBS pH 7.2/0.1% Tween 20 (wash buffer), and incubated overnight at 4 ºC with primary Ab. Membranes were washed three times with wash buffer and incubated with secondary Ab conjugated to horseradish peroxidase for 2 h at room temperature. After washing three times with wash buffer, immunoreactive bands were visualized using WB luminol reagent and exposition to an autoradiography film. For quantification, autoradiograms were scanned and bands were analyzed using the ImageJ software v. 1.52e (NIH, USA).
Cultures were treated for 2 h with 12 µM mitomycin C to inhibit proliferation during the experiment. Cell cultures were scratched, washed twice with PBS and supplemented with FBS-free DMEM with or without BLf, FBS and LA for 48 h at 37 ºC. Cultures were photographed using an inverted microscope coupled to a camera. Images from ≥ 3 fields per experimental condition were acquired and analyzed using the ImageJ software v. 1.52e (NIH, USA).
Inserts of 24-well plates were covered with 30 ml Matrigel (3 mg/ml) and incubated overnight at 37 ºC. Next, MDA-MB-231 cells (1×105) were plated on Matrigel of each insert (Upper chamber) in fresh FBS-free DMEM. Lower chamber contained 600 ml FBS-free DMEM without or with Holo-BLf. Plates were incubated for 48 h at 37 °C under a humidified atmosphere with 5% CO2 and 95% air. After incubation, cells and Matrigel on the upper surface of membranes were removed with cotton swabs, and the cells on the lower surface of membranes were washed with PBS and fixed with methanol for 5 min at room temperature. Quantification of invaded cells was determined by staining of membranes with crystal violet (0.5%) in PBS for 15 min at room temperature and elution of dye with 300 ml acetic acid (10%). Absorbance of collected solution was measured at 600 nm.
Conditioned media were concentrated using 5,000 Da Centricon® filters (EDM Millipore). Equal volumes of non-heated conditioned medium and sampler buffer (2.5% SDS, 2% sucrose, 4 µg/ml phenol red) were mixed and loaded into 8% polyacrylamide gels copolymerized with gelatin (1 mg/ml). After electrophoresis at 72 V for 2 h, gels were rinsed twice for 30 min with 2.5% Triton X-100, and incubated in assay buffer (50 mM Tris–HCl pH 7.4, 5 mM CaCl2) at 37 °C for 48 h. Gels were fixed and stained with a solution of Coomassie Brilliant Blue G-250 (0.25%) dissolved in acetic acid (10%) and methanol (30%). Proteolytic activity was identified as clear bands against the background stain of undigested substrate in the gel. Controls of MMP-2 and MMP-9 secretions were included, which were prepared by treatment of MDA-MB-231 cells with 400 mg/dl ethanol and 100 ng/ml PDB for 24 h at 37 ºC respectively [19, 20].
Immunofluorescence confocal microscopy
MDA-MB-231 cells were grown on coverslips, washed with PBS, equilibrated in FBS-free DMEM and treated with Holo-BLf and/or FBS for 20 min at 37 °C. Cells were fixed with paraformaldehyde (4%) for 20 min, permeabilized with 0.5% Triton X-100 for 20 min and blocked with blocking solution (0.5% gelatin, 1 mM CaCl2, 0.5 mM MgCl2 in PBS) for 20 min at room temperature. Cells were incubated for 12 h at 4 °C with anti-paxillin Ab (1:1000) followed by FITC-labeled anti-mouse secondary Ab for 2 h at room temperature. Staining of fibrillar actin was performed by incubation of cells for 2 h at room temperature with TRITC-conjugated phalloidin. Cells were mounted on glass slides using Vectashield and analyzed by confocal microscopy (Model TCS SP2; Leica Microsystems, Inc). Images were analyzed by using the ImageJ software v. 1.52e (NIH, USA).
Data are expressed as mean ± SD of at least three independent experiments, and were analyzed using one-way ANOVA and Dunnett’s multiple comparison test. Statistical probability of P<0.05 was considered significant.