Cell culture, reagents and treatments
MDA-MB-231LUC+ cells that stably express firefly luciferase gene (#AKR-231, Cell Biolabs, CA, US) were grown at 37 °C and 5% CO2 in DMEM media (#11995-065, Gibco by Life technologies, NY, US), containing MEM Non-Essential Amino Acids (#11140-050, Thermo Fisher Scientific, MA, US), 10% fetal bovine serum (FBS) (#F2442, Sigma, MO, US), penicillin/streptomycin (#15140-122, Gibco by Life technologies, NY, US and amphotericin B (#400-104, Gemini Bio-products, Ca, US). Cells were cultured in cell culture media (control) or with SP (#1156), CGRP (#1161), NKA (#1152) (all from Tocris Bioscience, Bristol, UK) or their combination for 24, 48, or 72 h. Solutions were prepared in serum free media and replaced daily. No peptidase inhibitor was used.
Migration assay
35 × 103 cells were added to culture-inserts (#81176, Ibidi, Munich, Germany) and cultured for 24 h at 37 °C in 5% CO2. Afterwards, the insert was removed, and cells were allowed to migrate in FBS free media containing vehicle (media), 1, 10, 100 and 1000 nM of SP, CGRP or NKA (when the cells were separately exposed to each NP [dose–response experiments]) or 100 nM of SP, 100 nM of CGRP, 50 nM of NKA or their combination (when the cells were simultaneously exposed to the three NPs). Images obtained of the wound (three images per treatment group) were taken immediately after removing the insert (0 h) and after 18 h using an Olympus-CK2 inverted microscope (10× phase contrast objective) and a USB camera (Dino-Lite, AM4023X). Gap closure was then measured in five different areas of the gap and their values averaged, using DinoCapture 2.0.
Invasion assay
250 × 103 cells were added to collagen-based cell invasion chambers (#ECM551, Millipore, MA, USA) and incubated for 24, 48 or, 72 h (three separate experiments, each in duplicate) at 37 °C in 5% CO2. FBS (10%) was used as a chemo attractant. Solutions containing the NPs (100 nM of SP, 100 nM CGRP, 50 nM of NKA or their combination) or vehicle (media) were replaced every 24 h. After the treatment was completed, noninvasive cells from the interior of the insert were removed. Inserts containing invasive cells were stained, washed, imaged (using a 10× objective and a Nikon E600 upright microscope equipped with a CCD digital camera), and lysed with the extraction buffer provided in the collagen-based invasion assay (50% of Reagent Alcohol [90% ethanol, 5% methanol and 5% isopropanol] in 50 mM acetic acid, pH 4.5). Dye mixture was transferred to a 96-well plate and absorbance at 560 nm was measured using a spectrophotometer (Epoch Microplate Spectrophotometer, BioTek Instruments Inc, Vermont, USA).
Western blot
Cultured cells (control and treated with the NPs) were collected, lysed in homogenization buffer (mammalian cell lysis kit (#MCL-1), Sigma, MO, US) containing a protease inhibitor cocktail (1:1000) and centrifuged. Afterwards, supernatants were collected for immunoblotting (three separate experiments). Protein concentration was determined by a protein assay (#5000006, Bio-Rad Laboratories, CA, US). Twenty microgram of total protein were combined with gel loading buffer, heated to 95 °C for 5 min and separated on 10% Tris–HCl gels (#5671034, Bio-Rad Laboratories, CA, USA). Treated groups were loaded next to its corresponding controls at each time point in each gel. The proteins were transferred to PVDF membranes (#162-0177, Bio-Rad Laboratories, CA, USA), blocked in 5% dry milk in PBS and incubated for 2 h at room temperature or overnight at 4 °C with the primary antibodies Anti-CALCRL (1:500, #PA5-50644, Thermo Fisher Scientific, MA, US); Anti-RAMP1 (1:5000, #156575, Abcam, CA, US), Anti-Neurokinin A Receptor (1:1000, #ab124998, Abcam, CA, US); Anti GAPDH (1: 1000, #MAB374, EMD Millipore, MA, US) or Anti-SP Receptor (1: 500, #ABN1369, EMD Millipore, MA, US), respectively. After washing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:2000, #Sc-2004 or #Sc-2314, respectively, Santa Cruz Biotechnology, TX, US). Signal was visualized using SuperSignal (West Pico or Femto Chemiluminescence Substrate, (#34080 and #34095, respectively, Thermo Scientific, IL, USA) and quantified using an imager (Amersham Imager 600, USA). The ratio of NK1 R, NK2 R, RAMP1, or CALCRL to GAPDH was calculated for each lane and the values of these ratios were normalized to the control group.
Kininogen (HMWK) release
100 × 103 cells were grown in 24-well plates and pretreated after 48 h of culture for up to 1 h (15, 30 and/or 60 min) in serum-free media with the same NPs and concentrations as described above (three separate experiments). Cell culture supernatants were then collected and for HMWK assay using a commercially available ELISA kit (ELISA kit, ab 189574, Abcam, CA, US). Briefly, after preparing duplicates of HMWK standards and samples, they were mixed with the antibody cocktail and incubated at room temperature for 1 h, following the manufacturer’s instructions. After washing all the samples were reacted with the substrate for 10 min afterwards, the reaction was terminated, and the absorbance was read at 450 nm using a spectrophotometer (Epoch Microplate Spectrophotometer, BioTek Instruments Inc, Vermont, USA). A standard curve was generated and the HMWK concentration calculated. The limit of sensitivity of the assay was 8.7 pg/ml, and the coefficient of variation was 7.4%. The final values per group (samples collected after 15, 30, 60 min of treatment with the NPs) were then compared to the vehicle control group.
Statistical analysis
All data were analyzed for normal distribution. Data are presented as mean and standard deviation. Statistical significance was tested using one-way ANOVA, repeated measures ANOVA, or the paired t-test. Post-hoc testing was performed with correction for multiple comparisons as appropriate. Analyses were performed with Origin Lab 9.0. By convention, a two-tailed test was used and P < 0.05 was considered significant for all analyses. Dose–response curves were fitted to a nonlinear regression variable slope equation using GraphPad Prism 6.0 (GraphPad Software, Inc, La Jolla, CA, USA). The mean of each curve was calculated from two independent experiments.