Cell lines and Reagents
Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Cell Bank of Representative Culture Preservation Committee of Chinese Academy of Sciences (Shanghai, China). HUVECs were cultured in HUV-EC-C medium and kept in an incubator at 37°C with 5% CO2.
Lyc was purchased from MedChemExpress (HY-N0288) and prepared as described before. Sunitinib and PDGF-AA were also purched from MedChemExpress (HY-10255A; HY-P70598).
Cell viability was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (3000 cells/well) were seeded in a 96-well plate overnight and then exposed to different concentrations of Lyc (and/or sunitinib) and incubated for 24 and 48 h. A total of 20 µL of MTT solution (5 mg/mL, Sigma, M5655) were added to each well and the cells were incubated for another 4 h at 37°C. The supernatant was then removed and 200 µL of DMSO were added to each well to dissolve the precipitate. Absorbance was measured using a spectrophotometer (BIOBASE, EL10A) at 490 nm.
Cells were seeded in six-well plates at a density of 1000 cells/well. On the next day, cells were treated with Lyc and/or sunitinib. Ten days after the treatment, cells were fixed with methanol for 15 min and then staixned with 0.1% crystal violet. Finally, stained cell colonies were photographed with a digital camera and analyzed using ImageJ (National Institutes of Health (NIH), Bethesda, USA).
Cells cultured in a Petri dish and treated with Lyc and/or sunitinib for a range of time points were collected and incubated with 5 µL of Annexin V and 10 µL of PI for 15 min in the dark. The samples were then evaluated using flow cytometry and the data were analyzed using FlowJo (TreeStar, USA).
For the migration assay, 200 µL of HUV-EC-C medium containing cells (2.0 x 104 cells/chamber) and different concentrations of Lyc (and/or sunitinib) were seeded in an upper Transwell chamber (Corning 3422, USA). HUV-EC-C medium containing 25 ng/mL PDGF-AA was added to the lower chamber. Non-migrated cells from the upper membrane were removed after incubation for 24 h at 37°C. The migrated cells were stained with 0.1% crystal violet and photographed under a light microscope at x 200. Migrated cells were analyzed quantitatively using ImageJ.
Cells (2 x 105 cells/well) were seeded in six-well plates. As the cells became sub-confluent, a straight cell-free wound was scratched with a 10 µL pipette tip. Cells were then washed twice with PBS and incubated in serum-free medium containing different concentration of Lyc and/or sunitinib. Cell scratches were observed and measured at different time points. Cell migration distances were analyzed quantitatively using ImageJ.
HUVECs were plated at a density of 1 x 104 cells/well in 96-well flat-bottomed plates after Matrigel (BD Biosciences, USA) pre-coating at 37°C for 30 min. Thereafter, HUVECs were treated with different concentrations of Lyc and/or sunitinib for 4 h. After exposure, HUVECs tubes and branches were photographed under a light microscope at x 200. The formed branches were analyzed quantitatively using ImageJ.
Chick Chorioallantoic Membrane (CAM) Model
Five fresh 10-day-old fertile eggs were cleaned with alcohol and then incubated at 37°C and 60-80% humidity in an egg incubator. The shell was cut to create a small window (20 x 20 mm2) and the shell membrane was removed with sterile forceps to expose the chorioallantoic membrane. A small rubber ring was placed on the chorioallantoic membrane and different concentrations of Lyc and/or sunitinib were added into the ring and incubated. At baseline and 8, 16 h later, the ring area was photographed by a scanner and the images were analyzed using Image-Pro Plus 6.0 (Epix, USA).
Lyc’s structural formula was drawn using Chemdraw (ChemBioOffice, CambridgeSoft, USA) and uploaded to PharmMapper (http://220.127.116.11/pharmmapper/) to obtain possible Lyc targets. The target names were then corrected to the official symbol using UniProt (https://www.uniprot.org/). Gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis[30, 31] of the Lyc-related targets were performed using WebGestalt (http://www.webgestalt.org/). Biological processes, molecular functions, and cellular components of the target were visualized using the ggplot2 package in R. Angiogenesis-related targets were obtained using GeneCards (www.genecards.org/) and intersected to the predicted targets in order to get possible Lyc targets for angiogenesis-inhibiting. Finally, target names were submitted to the Cytoscape software (http://www.cytoscape.org) to visualize the protein-protein interaction networks.
Western blotting was conducted as we described before. Briefly, proteins treated with different concentrations of drugs were extracted from the cells and quantified. Equal amounts of protein were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.5% Tween-20) for 40 min at room temperature and incubated overnight at 4°C with primary antibodies (p-PDGFRα, Cell Signaling Technology, 3166, etc). The membranes were then washed in TBST and incubated with secondary antibodies (zsbio, ZB-2301) for 40 min. After extensive washing, membranes were visualized using the enhanced chemiluminescence reagent. Final images were analyzed using ImageJ.
Immunofluorescence Staining Asssy
HUVECs administered with different concentrations of Lyc were grown at a 24-well plate after stimulated by PDGF-AA, fixed with 4% paraformaldehyde for 15 min and blocked with 5% bovine serum albumin for 1 h at room tempature. Sequently, primary antibody (p-PDGFRα, Thermofisher, 44-1000G) was applied to incubate the cells overnight and incubated with Alexa Fluor 488 secondary antibody (Thermofisher, A-11008) for 2 h. Finally, cells were incubated with hoechst (Thermofisher, 33258) for 5 min and visualized under an inverted fluorescent microscope.
Molecular docking was performed using Schrödinger software, which included Protein Preparation Wizard, LigPrep, and Glide modules. PDGFRα crystal structure (PDB ID: 6JOK) was prepared from RCSB and optimized with the Protein Preparation Wizard. LigPrep was used to generate multiple conformational states for Lyc ligand molecules. Docking between Lyc and PDGFRα with standard precision (SP) was performed by Glide[36, 37]. The rootmean-square deviation (RMSD) value was calculated from superimposed ligands to examine docking parameters that were capable of reproducing a similar conformation to that of the co-crystal at the active site of PDGFRα.
PDGFRα Enzymatic Assay
PDGFRα enzymatic assay was performed using the Kinase Activity Assay Kit (Abace, Beijing, China) following the manufacturer's protocol. Purified human PDGFRα protein was treated with Lyc at different concentrations in a black 384-well plate. The fluorescence intensity was measured by an automatic microplate reader (Promega, USA).
Biacore Assay for Surface Plasmon Resonance (SPR) Analysis
Surface plasmon resonance (SPR) affinity experiment[38, 39] for drug-target interaction analysis was conducted employing a Biacore 100 T biosensor detector (GE healthcenter, USA). Different concentrations of Lyc were injected to protein (Human PDGFRα Protein, His, Strep II Tag Protein [H5252]) and blank channels. The experiment was conducted at 25°C, the supernatant flow rate was 20 µl/min. 10 mM acetate was employed as immobilization buffer and 1 x PBS as running buffer.
CElluar Thermal Shift Assays (CETSAs)
CETSAs were conducted to detect the direct binding between Lyc and PDGFRα in celluar. Briefly, cells were pre-treated with 6 uM of Lyc for 48 h, chilled on ice, washed with PBS containing protease inhibitor and then transferred into 1.5 ml PCR tubes and heated for 3 min at appropriate temperature. Subsequently, cells were lysed, seperated and detected by western blot assays.
Results were analyzed using SPSS software (IL, USA). All experiments were independently repeated at least three times and data were presented as the mean ± SD. Student’ s two-tailed t-test and one-way ANOVA were performed to determine statistical significance between different groups. Differences were considered significant when p-values < 0.05.