Peptide VGB4 (2HN-KQLVIKPHGQILMIRYPSSQLEM-COOH) was synthesized by Shine Gene Biotechnology (Shanghai, China), purified by high performance liquid chromatography (HPLC) to 90% purity, analyzed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF), and approved by electrospray ionization mass spectrometry (ESI–MS) analysis (see also )
Antibodies and reagents
Anti-p53 (sc-126), anti-PTEN (sc-7974), goat anti-mouse IgG-FITC (sc-2010), anti-β-Actin (sc-47778), anti-Bcl2 (sc-509), anti-BAX (sc-20067), anti-Cytochrome c (sc-13156), anti-XIAP (sc-55551) and goat anti-mouse IgG-PE (sc-3738) were from SANTA CRUZ BIOTECHNOLOGY, INC., Santa Cruz, California, USA. Anti-AKT phospho S473 (#9271), anti-Caspase 3 (#9662) and anti-Caspase 9 (#9502) were from Cell Signaling Technology, Danvers, Massachusetts, USA; DAPI (D9542), Propidium iodide (P4170), RIPA buffer, polyvinyl difluoride (PVDF) membranes (IPVH00010) and anti-AKT (SAB4500800) was from Sigma (St Louis, MO, USA). Annexin V-FITC Apoptosis Staining / Detection Kit (ab14085) was purchased from Abcam, Cambridge, UK.
Cell lines and cell culture
The human lung adenocarcinoma cell line A549 (NCBI code, C137) and the human colon adenocarcinoma cell line HT29 (NCBI code, C466) were purchased from the National Cell Bank, Pasteur Institute of Iran. A549 and HT29 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Life Technologies, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma, St. Louis, Missouri, USA), and 100 units/ml penicillin-streptomycin (Gibco/Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO2.
MTT cell proliferation assay
For cell growth analysis, the cells were seeded into 96-well plates at a density of 2×103 cells/well and allowed to grow in DMEM medium supplemented with 10% FBS for 24 h. Subsequently, the medium was replaced with the fresh medium containing 2% FBS, 200 ng/ml VEGFA (Sigma, St. Louis, Missouri, USA), and increasing concentrations (0.09, 0.18, 0.37, 0.55, 0.74 and 0.93 µM) of VGB4 or (0.93 µM) scr. After 24 h incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma, St. Louis, Missouri, USA) was added for 4 h at 37°C. Then, the intracellular formazan crystals were solubilized with dimethyl sulfoxide (DMSO) and the absorbance of the colored solution was read at 570 nm using ELISA plate reader (Bio Rad, USA).
Colony formation assay
To further evaluate proliferation and efficacy of VGB4, colony formation assay was conducted on A549 and HT29 cell lines. 2000–3000 cells/well were seeded on 6-well plates and allowed to adhere overnight. The next day medium was replaced with fresh medium containing 200 ng/ml VEGFA and concentrations (0.74 and 0.93 µM) of VGB4 or (0.93 µM) scr. The cells were grown for 11 days with media changes every 3 days. The colonies were fixed with 4% formaldehyde, and stained with crystal violet (0.1%).
Wound-scratch assay was performed to evaluate the migration rate of HT-29 and A549 cells treated with concentrations (0.74 and 0.93 µM) of VGB4 or (0.93 µM) scr in the presence of 200 ng/ml VEGFA. To accomplish this, 2×103 cells/well were seeded in 96-well plates, and cultured until the cells reached 90 % confluence. A sterile 200-µl pipette tip was used to create the wound in the monolayer of cells in each well. The detached cells were washed off with phosphate-buffered saline (PBS). After 24 h, the rate of wound recovery was monitored using an inverted microscope (Olympus BX-50, Japan) and a digital camera (Canon, C4742-95, Japan).
Flow Cytometry Analysis
To quantify cell apoptosis, Annexin V/propidium iodide (PI) staining was performed on HT29 and A549 cell lines. The cells were treated with VGB4 (0.74 and 0.93 µM) or scr (0.93 µM) for 24h in the presence of 200 ng/ml VEGFA, and then scraped and digested with trypsin/EDTA. Digested cells were centrifuged at 1500 rpm for 5 min at 4 ºC. Subsequently, the cells were washed two times with cold PBS, and resuspended in binding buffer at a cell density of 1 × 106/ml, and stained with Annexin V-fluorescein isothiocyanate (FITC) and PI for 10 min at room temperature in the dark. The cell suspensions were analyzed on a FACSCalibur flow cytometer (BD Biosciences, NJ, USA).
Immuno fluorescence staining
3 × 104 cells were seeded in a 12-well plate, and incubated overnight at 37°C. Then, the cells were treated with VGB4 (0.74 and 0.93 µM) or scr (0.93 µM). After 24 h of incubation, the cells were fixed with 3.7% formaldehyde for 10 min at room temperature, washed two times in PBS containing 0.05% tween 20 (PBS-T), permeabilized with 0.2% Triton X-100 for 5 min at room temperature. After two times washing in PBS-T, blocking buffer containing 1% BSA/ 10% normal goat serum/0.3 M glycine in PBS-T was added for 1 h at room temperature. The cells were reacted with anti-p53 and anti-PTEN primary antibodies overnight at 4°C, washed three times, and reacted with Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG secondary antibody and Phycoerythrin (PE)-labeled goat anti-mouse IgG secondary antibody, respectively, in the dark situation at 37°C for 1 h. Finally, the cells were counterstained with DAPI solution. Fluorescence was detected by a fluorescence microscope.
A549 or HT29 cells from VGB4-treated, scr-treated groups and control group were lysed with RIPA buffer containing protease and phosphatase inhibitor cocktail (Sigma, St. Louis, Missouri, USA). Equal volumes of cell lysates from each group were resolved by 12 % sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). After transferring the proteins to PVDF membrane and blocking with 5% non-fat milk, the membrane was incubated with the primary antibodies including anti-p53, anti-PTEN, anti-phospho-AKT, anti-total AKT, anti-Bcl2, anti-BAX, anti-Cytochrome c, anti-XIAP, anti-Caspase 3, anti-Caspase 9, and anti-β-Actin at 4°C overnight. In the next step, the membrane was incubated with secondary HRP-conjugated anti-mouse antibody at room temperature for 1 h. β-Actin was used as the internal reference. The quantification of protein density was performed using Image J software (NIH Image, National Institutes of Health; online at: https://rsbweb.nih.gov/ij/).
Data were represented as the mean ± standard deviation (SD). Statistical analyses were carried out by GraphPad Prism7 software. Data normality was evaluated using Kolmogorov–Smirnov test. The significant difference between groups was assessed via Unpaired Student’s t-test. P < 0.05 was considered to indicate a statistically significant difference.