Cells culture and drug treatment
Human PCC lines PANC-1 and Patu8988, which have strong proliferative and invasive abilities [18], were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 100 U ml− 1 penicillin and 100 µg ml− 1 streptomycin (Invitrogen). PANC-1 and Patu8988 cells were plated at a density of 1 × 106 and preincubated for 24 h at 37 °C, in the complete medium containing 5% FBS at approximately 70% confluence in culture plates. After 24 h, the complete medium was replaced with serum-free medium for 24 h before drug treatment. PANC-1 and Patu8988 cells were treated with 50 or 100 µM fraxetin (Fig. 1A, CAS#: 574-84-5, Purity: ≥98% by HPLC, Yuanye Biotechnology, Shanghai, China) with or without colivelin (CAS#: 867021-83-8, MedChemExpress, NJ, USA).
Cell Counting Kit-8 assay
To measure the viability of PANC-1 and Patu8988 cells, the Cell Counting Kit-8 (CCK-8) for cell proliferation and cytotoxicity assay kit (Dojindo, Shanghai, Japan) was performed as per the manufacturer’s instructions. Firstly, cells were plated in DMEM medium for 24 h followed by incubation in 96-well plates. Secondly, incubated cells were added to the plates at a density of 5 × 103 cells/well and then were treated with different concentrations of fraxetin for 24 h. At appropriate time points, 10 µl of this reagent was added per well and incubated for another 2 h. Finally, the absorbance at 450 nm was determined. The cell viability was calculated by comparing the experimental cells with untreated control cells. All experiments were repeated at least in triplicate.
Cell apoptosis analysis
PANC-1 and Patu8988 cells were cultured in DMEM with different concentrations of fraxetin for 24 h, and then were collected by centrifugation. For apoptosis analysis, Annexin V-FITC (Multi Science, Hangzhou, China) was added to resuspended cells at room temperature, and incubated for 15 min in the dark. Next, propidium iodide (PI, Multi Science) was added to resuspended cells, and incubated for another 5 min in the dark. Finally, cell apoptosis was analyzed using flow cytometry (Ex = 488 nm; Em = 530 nm, BD FACSVerse™, BD Biosciences, San Jose, CA, USA).
Real-time cellular analysis
PANC-1 or Patu8988 cells were seeded at a density of 4 × 103 cells/well in cell culture E16-Plate (ACEA Biosciences, San Diego, CA, USA), and then cellular growth index was recorded by Label-free Real-time Cellular Analysis System (RTCA; Roche, Penzberg, Germany) automatically.
Transwell invasion assay
A transwell chamber (Costar, New York City, NY, USA) assay was performed in a 24-well plate. The transwell inserts were coated with 150 µl Matrigel at 37˚C for 2 h. PANC-1 and Patu8988 cells (4 × 105) were collected and resuspended in a serum-free medium supplemented with fraxetin. Cells were incubated in the upper chamber, and the lower chamber was infused with 500 µl DMEM containing 10% FBS. The transwell plate was incubated at 37˚C for 24 h, and then removed the gel and cells in the upper chamber. After formalin fixation, crystal violet (Sigma-Aldrich, USA) was used to stain the membrane for 10 min. Finally, the number of invaded cells in six randomly selected fields was counted using a microscope (Leica Microsystems, Wetzlar, Germany).
Colony formation assay
PANC-1 or Patu8988 cells were plated in 6-well plates at a density of 1 × 103 cells/well for 24 h, and then were treated with fraxetin. After treatment for 24 h, the culture medium was placed with DMEM for 14 days. Colonies were fixed with formaldehyde for 30 min and stained with crystal violet (Sigma-Aldrich). Finally, cell colonies were counted using a microscope (Leica Microsystems).
Wound healing assay
PANC-1 or Patu8988 cells were seeded in 6well plates and maintained at 37˚C for 24 h. The culture area was scratched using a crystal pipette tip to make a linear gap among the cells. Next, the detached cells washed away with PBS and then added different concentrations of fraxetin. Cells were allowed to fill the gap, and after 24 h, images of the culture area were captured using a microscope (Leica Microsystems).
Immunocytochemical staining
Immunofluorescence staining was performed according to our previous report [19]. Firstly, PANC-1 and Patu8988 cells treated with fraxetin were grown on glass coverslips for 24 h, and then fixed with 4% formaldehyde for 30 min. Secondly, cells were permeabilized with 0.1% Triton X-100, and blocked in 4% normal goat serum for 1.5 h. PANC-1 and Patu8988 cells were incubated overnight at 4 °C with primary antibodies against Ki67 (1:150, Abcam, Cambridge, MA, USA), Ref1 (1:200, Abcam), STAT3 (1:200, Cell Signaling Technology, CST, Beverly, MA, USA), E-cadherin (1:200, Abcam), and Vimentin (1:200, Abcam) followed by incubation at room temperature with secondary antibody (1:400) for 1 h. Finally, cells were stained with 4'6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China), and Ki67-positive cells were counted using a microscope (Leica Microsystems).
Glucose metabolism assay
The intact cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XF96 Extracellular Flux Analyser (Seahorse Bioscience, North Billerica, MA, USA). In short, 1.0 × 104 of PANC-1 and Patu8988 cells were seeded into 96-well cell plates and incubated overnight at 37 ℃, 5% CO2. Both cells were pretreated with or without different concentrations of fraxetin for 24 hours. Simultaneously, the calibration plates were incubated overnight at 37 ℃ in a non-CO2 incubator. Then both cell mediums were replaced with assay medium, once the probe calibration was completed, the cell plate replaced the probe plate. The analyzer plotted the value of OCR followed by injection of the compounds sequentially as follows: oligomycin (inhibitor of ATP synthase; 2.5 µM), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupler of OXPHOS; 2 µM), rotenone (inhibitor of complex I; 0.25 µM) and anti-mildew A (inhibitor of complex III; 0.25 µM) (n = 8). ECAR was evaluated after continuous injection of glucose (10 mM), oligomycin (1 µM) and 2-Deoxyglucose 2-Deoxyglucose (2-DG, 50 mM) (n = 8). After completing the test, the BCA Protein Assay Kit was performed to determine protein concentration to normalize OCR and ECAR according to the manufacturer’s instructions.
Nude mouse tumorigenicity
Male nude mice (BALB/c) weighing 20–22 g and 6–8 weeks old were obtained from the Experimental Animal Centre of Wenzhou Medical University (Wenzhou, China). All mice were housed in a humidity-, temperature-, and light-controlled environment. Mice were randomly divided into two groups and each group contained five mice. Then the right thigh root of mice was injected subcutaneously with 3 × 106 PANC-1 cells. The experimental group received intragastric administration of fraxetin (25 mg/kg·d) every three days for a month. In contrast, the control group received intragastric administration of DMSO. Tumor formation in mice was monitored for 30 days, and the length and width were measured every three days. Tumor volume was calculated based on the formula V = (length × width2)/2, where length was always the longest dimension of the tumor. At the end of the experiment, all the mice were euthanized by CO2 asphyxiation. This animal study was approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University, China. The animal experiments were conducted according to all regulatory institutional guidelines for animal welfare (National Institutes of Health Publications, NIH Publications No. 80 − 23) [20].
Histopathological analysis
Tumor specimens from animals embedded in paraffin were cut into 4-µm sections and stained with hematoxylin and eosin (HE, Yuanye Biotechnology, Shanghai, China). Immunohistochemical (IHC) analysis was performed according to a previous report [18]. In brief, 4-µm thick sections were dewaxed with xylene and rehydrated in sequential ethanol. Sections were incubated in 0.1% sodium citrate buffer (pH 6.0) for antigen retrieval, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Beyotime). IHC staining was performed using the following primary antibodies: anti-Ki67 (1:200, Abcam) and anti-E-cadherin (1:200, Abcam). The integrated optical density (IOD) was measured using Image-Pro Plus software (version 6.0, Media Cybernetics, Silver Spring, MD, USA). All samples were semi-quantitatively or quantitatively assessed by two independent investigators in a blinded manner.
Western blot analysis
Whole proteins from the normal pancreatic ductal cell (hTERT-HPEN) and PCCs (PANC-1, Patu8988 and BxPc-3) or PDA tissues treated with fraxetin for 24 h were collected and protein concentrations were determined by double star choline acid protein analysis kit (Beyotime). Total protein (20 µg) of each sample was separated by SDS-PAGE and then was transferred to a polyvinylidene difluoride membrane (PVDF, Solarbio, Beijing, China). After blocking the membranes for 1 h at room temperature with 5% skim nonfat milk, membranes were incubated with the primary antibodies (1:1000), including anti-caspase-3 (Proteintech, Wuhan, China), anti-caspase-8 (Proteintech), anti-Bcl-2 (Proteintech), anti-Bax (Proteintech), anti-N-cadherin (Abcam), anti-Slug (Abcam), anti-JAK2 (Abcam), anti-p-JAK2 (Abcam), anti-STAT3 (CST), anti-p-STAT3 (CST), anti-E-cadherin (Abcam), anti-type Ⅰ collagen (Abcam), anti-GLUT1 (Affinity Biosciences, Cincinnati, OH, USA), anti-VEGFA (Affinity), anti-HIF-1α (Affinity), anti-Ref1 (Abcam), and anti-vimentin (Abcam), overnight at 4 ℃. After washing in TBST (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20), membranes were incubated with secondary antibodies (1:400) for 1 h at room temperature. Finally, the protein bands were visualized using chemiluminescence detection on autoradiographic film and the GADPH antibody (1:8000, Proteintech) was used as the internal reference.
ROS level assay
ROS level assay was performed according to the manufacturer’s instructions (Beyotime). In brief, PANC-1 or Patu8988 cells were seeded in a 6-well plate with a density of about 5 × 104/well. After the cells adhere to the wall, cells were treated with fraxetin for 24 h. On the second day, 2 ml of the dilution (DCFH-DA, 1:1000, Beyotime) was added to each well, incubated at 37 ℃ for 30 min, and then the cells were collected. Images were captured using a fluorescence microscope (Leica Microsystems). In addition, the cells were also resuspended in PBS, and ROS level was detected by flow cytometry (BD Biosciences).
Database analysis
The expression of STAT3 in PDA tissues and adjacent normal tissues in the GEPIA 2 database website (http://gepia2.cancer-pku.cn/#analysis) was analyzed. Besides, the correlation between STAT3 expression and KRAS activity was also evaluated in the GEPIA 2 database. Moreover, the potential target candidates were analyzed using the PharmMapper Server (http://www.lilab-ecust.cn/pharmmapper/index.html) according to a previous report [21].
Molecular docking
Fraxetin structure was downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov) as SDF files and converted into mol2 format using Chimera. The STAT3 crystal structure was downloaded from PDB no. 6NJS as a model (http://www.rcsb.org). Compound and protein were converted into PDBQT format using AudoDockTools and docked using AutoDock Vina. Visualize conformations and interactions were prepared by PyMol, Chimera, and LigPlot+.
Statistical analysis
Data were presented as the means ± standard deviations. All statistical analyses were performed using GraphPad Prism software (version 8.0, GraphPad Software, Inc., La Jolla, CA, USA). A two-sided Student’s t-test was used to analyze differences between the two groups. One-way analysis of variance (ANOVA) with Bonferroni's post-hoc test was used when more than two groups were present. P < 0.05 was considered statistically significant.