Prediction of drug targets: The following two databases were used to predict targets of scoparone activity: SwissTargetPrediction (http://www.swisstargetprediction.ch/), a database for predicting drug targets based on the similarity of two- or three-dimensional structures with known compounds; and STITCH (http://stitch.embl.de/, Version:5.0), a platform for searching known and predicted interactions between compounds and proteins, containing 9643763 proteins from 2031 species.
Prediction of pancreatic cancer targets: The following two databases were used to predict pancreatic cancer-related targets: GeneCards (https://www.genecards.org, Version:5.0), a searchable comprehensive database that integrates data from over 150 sources; and Comparative Taxonomic Database (CTD, http://ctdbase.org, updated 10/28/2020), which provides information on interactions or relationships between compounds and genes, compounds and proteins, compounds and diseases, or genes and diseases. Targets with a score greater than the median were screened out, and the duplicates were obtained as pancreatic cancer-related targets. In addition, the overlaps of drug and disease targets were the effective targets of scoparone to treat pancreatic cancer.
Enrichment analysis of potential targets: WebGestalt (http://www.webgestalt.org, updated 01/14/2019) is an online website focusing on functional enrichment analysis. Biological processes, cellular composition, and molecular function were selected for Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) was selected for pathway analysis for the predicted pancreatic cancer-related targets of scoparone.
Protein-protein interaction network: STRING (https://string-db.org/cgi/input.pl/, Version 11.0) is an online database for predicting protein-protein interactions (PPIs). The predicted targets were uploaded into the STRING database to obtain the PPI network, which was then imported into Cytoscape 3.7.2, an open software platform for constructing data analysis, integration, and visualization networks. The software plugins Cytohubba and Network Analyzer[30,31] were used to analyze the network characteristics, in which the degree of the nodes represents the importance of the proteins in the network. The higher the degree value, the darker the node color.
Expression patterns of candidate hub genes: Gepia (http://gepia.cancer-pku.cn/) is a database that can be used for differential expression analyses between tumor and normal tissues as well as survival and correlation analyses. The expression patterns of candidate hub genes identified by pathway and PPI network analyses were evaluated using Gepia.
Cell culture: Pancreatic cancer cell lines Capan-2 and SW1990 were respectively purchased from American Type Culture Collection (ATCC, Manassas, VA, United States) and the cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Capan-2 cells were cultivated in RPMI-1640 medium (Biological Industries, Beit HaEmek, Israel) and SW1990 cells in L15 medium (Biological Industries) mixed with 10% fetal bovine serum (FBS, Biological Industries) in a 5% CO2 incubator at 37 °C.
Cell viability assay: Capan-2 and SW1990 cells were seeded onto 96-well plates at a density of 4000 cells/well. After 12 h, the medium was replaced with different scoparone (Sigma-Aldrich, St. Louis, MO, United States) concentrations (0, 0.5, 1, 5, 10, 50, 80, 100, 200, 300, 400, 600, and 800 μmol/L) for 48 h. Cell viability was measured with the CCK-8 assay kit (ApexBio Technology, Houston, TX, United States) according to the instructions. Next, the optical density (OD) value at 450 nm was measured, and the IC50 value was calculated. Cells were then inoculated again with 100, 200, and 400 μmol/L scoparone (1/2 IC50, IC50, and 2 IC50) for 24, 48, and 72 h, and the OD value was measured as compared to the control group (0 μmol/L scoparone with the same amount of DMSO solution of 400 μmol/L scoparone).
Wound healing assay: Capan-2 and SW1990 cells were pretreated with 0 (Con), 100, 200, and 400 μmol/L scoparone for 24 h. A vertical wound was drawn in the cells with a 200-μL sterile pipette tip. After washing with phosphate buffer saline (PBS) (Biological Industries), serum-free culture medium was added, and images of the wound were taken at 0, 24, 48, and 72 h using an Eclipse microscope (Nikon Corporation, Tokyo, Japan).
Transwell assay: Capan-2 and SW1990 cells were pretreated with 0, 100, 200, and 400 μmol/L scoparone for 24 h, following which the cells were resuspended in serum-free medium. A 200-μL aliquot of cell suspension (2.5 × 105 cells/mL) was added to the upper layer of the cell culture chamber, while 600-μL medium containing 10% FBS was added to the lower level and then incubated in a 5% CO2 incubator at 37 °C for 16-24 h. The membrane of the upper level was fixed with 4% paraformaldehyde for 2 min, permeated with 100% methanol for 20 min, and then stained with 0.2% crystal violet at 25 °C for 15 min. Under an Eclipse microscope (Nikon Corporation), five fields (200 × magnification) were randomly observed and imaged.
The cell invasion assay was conducted by adding 100 μL of Matrigel (Corning Inc., New York, NY, United States) diluted at a 1:8 ratio in a serum-free medium to the upper level, followed by incubation overnight at 37 °C. The other steps were performed as described in the migration assay above.
Apoptosis by flow cytometry: Following the instructions, apoptosis was measured using the PE Annexin V Apoptosis Detection Kit (BD Biosciences, San Jose, CA, United States) by flow cytometry. Capan-2 and SW1990 cells were pretreated with 0, 100, 200, and 400 μmol/L scoparone for 24 h. Cells were rinsed twice with cold PBS after digestion with trypsin without EDTA, followed by resuspension and dilution to 1 × 106 cells/mL with 1 × Annexin V Binding Buffer; 5 μL of PE Annexin V and 5 μL of 7-AAD reagent were added and then incubated in dark place for 15 min. The FACSCalibur flow cytometer (BD Biosciences) was used to measure the apoptosis rate.
Cell cycle by flow cytometry: Following the instructions, cell cycle was measured using PI/RNase Staining Buffer (BD Biosciences) by flow cytometry. Capan-2 and SW1990 cells were pretreated with 0, 100, 200, and 400 μmol/L scoparone for 24 h. Cells were resuspended with cold PBS after digestion and fixed with 70% ethanol overnight at -20 °C and then 500 μL of PI/RNase staining buffer was added to each flow tube and stained in dark place for 15 min. The FACSCalibur flow cytometer (BD Biosciences) was used to measure cell cycle.
Quantitative reverse transcription polymerase chain reaction: TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was used to extract the total RNA, and then the PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc., Beijing, China) was used to reverse transcribe the RNA into cDNA. Using ACTB as an internal reference, real-time polymerase chain reaction (PCR) for AKT1 and MAPK8 was performed using TB Green Premix ExTaq II (Takara Bio) with a LightCycler480 II PCR system (Roche, Basel, Switzerland). The relative expression of mRNA was analyzed using 2-ΔΔCt method. The forward primer sequence of AKT1 was 5'-TGACCATGAACGAGTTTGAGTA-3', and the reverse primer was 5'-GAGGATCTTCATGGCGTAGT AG-3', while the forward primer of MAPK8 was 5'-ACACCACAGAAATCCCT AGAAG-3', and the reverse primer was 5'-GAATTCGATGATCAACTCACGG-3'.
Western blot analysis: Capan-2 and SW1990 cells were pretreated with 0, 100, 200, and 400 μmol/L scoparone for 24 h. RIPA lysis buffer (Beyotime Biotech, Jiangsu, China) was used to lyse the cells and then proteins were separated and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Chicago, IL, United States). After blocking with 5% skimmed milk or 5% bovine serum albumin (Beyotime) for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C and then incubated with secondary antibodies at room temperature for 2 h. Finally, an ECL Western blotting substrate (Tanon Science & Technology Co., Ltd., Shanghai, China) was used to visualize the bands with the Amersham imager 680 chemiluminescence imaging system (Amersham imager 680; GE Healthcare, United States). GAPDH (1:10000; Proteintech Group, Wuhan, China) served as a control for normalization, and primary antibodies against the following antigens were employed: PI3K (1:1000; Cell Signaling Technology, Inc., Danvers, MA, United States), Akt (1:2000; Proteintech Group), p-Akt (1:2000; Cell Signaling Technology), MMP9 (1:1000; Proteintech Group), Bcl-2 (1:2000; Proteintech Group), Bax (1:10000; Proteintech Group), and cleaved caspase-3 (1:1000; Cell Signaling Technology).
Xenograft tumor model in nude mice: The animal experiment was approved by the Animal Ethics Committee of the Institute (Ethical code number: 2020PS766K). Twelve four-week-old BALB/C nude mice were purchased from Beijing Huafukang Bioscience (Beijing, China) and were acclimatized to laboratory conditions (23 °C, 12 h/12 h light/dark, 50% humidity, ad libitum access to food and water) for 2 wk prior to experimentation following the regulations of the guidelines of the Animal Care and Ethics Committee of Shengjing Hospital of China Medical University (Shenyang, Liaoning, China). Nude mice received subcutaneous injections of 100-μL Capan-2 cells (2 × 106). Tumor length (L) and width (W) and mouse weight were measured every second day. Tumor volume was calculated using the following formula: Tumor volume (V) = (L × W2)/2. When V reached approximately 100 mm3 after 7-10 d, 12 nude mice were randomly and equally divided into either a control group or an experimental group. The control group was intraperitoneally injected with 50 μL of normal saline solution, while the experimental group was injected with 50 μL of 200 μmol/L scoparone every 2 d for 3 wk. The nude mice were euthanized by sodium pentobarbital overdose (150 mg/kg) by intraperitoneal injection, following which the tumors were excised, photographed, measured, and weighed.
Immunohistochemistry: Tumor samples were fixed with 4% paraformaldehyde for 48 h and then dehydrated and paraffin-embedded. Tissue sections (3 μm) were deparaffinized, rehydrated, and antigen-repaired. After blocking, sections were incubated with primary antibodies anti-Ki67 (1:200; Abcam, Cambridge, United Kingdom) and anti-PCNA (1:500; Proteintech Group) for 12-16 h at 4 °C, then incubated with the secondary antibody goat anti-rabbit immunoglobulin G at room temperature for 30 min. Sections were stained with 3,3'-diaminobenzidine (DAB), counter-stained with hematoxylin, dehydrated with gradient ethanol, cleared with xylene, sealed with neutral gum, observed, and photographed under an Eclipse microscope (Nikon Corporation).