Data on the inhibition of cell proliferation and invasion by the D2A-Ala peptide derived from the urokinase receptor

The data presented in this article are connected to our research article entitled “D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo” (Furlan et al., 2018). These data further extend our understanding of the inhibitory effects of D2A-Ala peptide. Dose-response curve using a wide range of concentrations of D2A-Ala shows that this peptide has no effects per se on proliferation of rat smooth muscle cells (RSMC). However, D2A-Ala dose-dependently inhibits epidermal growth factor (EGF)-induced RSMC proliferation. Kinetics lasting up to seven days revealed that D2A-Ala peptide completely blocked EGF-promoted RSMC proliferation. Moreover, D2A-Ala peptide inhibited invasion of HT 1080 cells towards RSMC.


Value of the data
This data article investigates the effects of an inhibitory peptide, D2A-Ala derived from the human urokinase receptor on proliferation of normal cells, rat smooth muscle cells (RSMC), and invasion of tumoural cells towards normal cells.
Data could be useful guidelines for further design of inhibitory peptides against proliferation and invasion which are some of the hallmarks of malignant neoplastic cells.

Data
In our companion paper, we showed that D2A-Ala peptide inhibits proliferation of human tumoural cells in vitro and tumour growth in vivo [1]. Herein, we extend these results reporting that D2A-Ala inhibits EGF-induced proliferation of normal cells of a different species (rat), i.e. RSMC, and invasion of human tumoural cells towards RSMC. Firstly, RSMC were treated with increasing doses of EGF ranging from 1 ng/ml up to 100 ng/ml and cell proliferation measured after four days of culture ( Fig. 1). Then, RSMC were treated with increasing doses of D2A-Ala ranging from 0.1 pM to 1 μM in the presence or in the absence of the optimal dose of EGF (20 ng/ml), and cell proliferation determined after four days of culture (Fig. 2). Next, RSMC were cultured for up to seven days with the optimal dose of D2A-Ala peptide (100 pM) in the presence or in the absence of EGF (20 ng/ml) (Fig. 3). 0% FCS served as negative control. Cell numbers were determined at the indicated time (Fig. 3). Lastly, Fig. 4 shows the effect of D2A-Ala on invasion of human fibrosarcoma HT 1080 cells towards RSMC.

Cell proliferation assay
The assay was performed as previously described [10]. 20,000 RSMC in DMEM plus 10% FCS were seeded in a 2-cm 2 well of a 24-well plate, cultured overnight, washed twice with PBS pH 7.4, then EGF, D2A-Ala or both were added daily in serum-free medium in each well. Trypsin/EDTA was used to detach the cells, and cell numbers determined under the microscope using a Bürker chamber. RSMC  kept in serum-free medium served as negative control. Data are expressed as mean 7 SD from three experiments performed in triplicate.

Invasion assay
Cell invasion assay was carried out as previously described [10]. RSMC were seeded at 90-100% confluency into the 2-cm 2 -wells of a 24-well plate, cultured for 24 h in DMEM plus 10% FCS, washed with PBS pH 7.4, and further cultured for 24 h in serum-free medium. Then, thick gel layer (100 μl per square centimeter of growth surface) of matrigel (BD Biosciences) were polymerized on the upper side of 8 μm pore-Transwell inserts (Corning), which were positioned into each well of the 24-well plate. 200,000 HT 1080 cells in serum-free medium were plated onto the matrigel, and allowed to migrate for 24 h towards the RSMC in the presence or in the absence of D2A-Ala peptide added in the serum-free medium of the cultured RSMC. Finally, HT 1080 cells remaining on the layer, and the matrigel were removed, and invading cells located on the lower side of filters were fixed in 20% (v/v) methanol, and stained using Diff-Quick solution (Medion Diagnostics). Five high power fields per filter were counted under the microscope (lens 40) in. Results are the mean 7 SD (n ¼ 3). Invasion in the absence of D2A-Ala represents the control, which was given the arbitrary value of 100%.

Statistical analysis
The Prism software served for the calculation of statistical significance using either the Student's t test for pair-wise comparison of treatments, or an ANOVA model for the evaluation of treatments for increasing times or with increasing doses of a reagent.