Chenodeoxycholic acid (CDCA)
Chenodeoxycholic acid (CDCA, purity greater than 98%) was obtained and purified from the bile of geese in our laboratory as described by Z. W. Yan [41], and it was identified by 1H NMR and 13C NMR.
Cell culture
The human lung cancer cell lines, A549 and H1650, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 1% penicillin/streptomycin and 10% foetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), at 37 °C in a 5% CO2 atmosphere.
Cell viability assay
A549 and H1650 cells were seeded into 96-well plates at a density of 2×103 cells per well in 100 μl of medium with 10% FBS and cultured for 24 hours. Different concentrations of CDCA were then added to the medium for 24 hours followed by CCK8 treatment. The viability of cells was measured at 450 nm by a spectrophotometer (Bio–Rad, CA, USA).
Western blot assay
Total protein of A549 and H1650 cells was collected and extracted using RIPA lysis buffer (Jinkelong Biotechnology Co., Ltd., Beijing, China), containing protease inhibitor cocktail and a protein phosphatase inhibitor (Beyotime Institute of Biotechnology, Haimen, China), followed by centrifugation at 10,000 g for 10 min at 4 °C. Following electrophoresis, the samples were then transferred to PVDF membranes, and the membranes were incubated with rabbit anti-integrin α5, rabbit anti-integrin β1, rabbit anti-FAK and rabbit anti-p-FAK primary antibodies (Cell Signaling Technology, Inc.) overnight at 4 °C. The membranes were then incubated with the indicated secondary antibodies (Santa Cruz Biotechnology, Inc., SC-2005) for 1 h at room temperature. Detection was performed using an electrochemiluminescence kit (Pierce; Thermo Fisher Scientific, Inc.). β-actin (Santa Cruz Biotechnology, Inc.) was used as an internal control.
RNA sequencing
After treatment with CDCA at a concentration of 0.4 mM or in total medium containing 1% DMSO for 24 h, cells were lysed with 1 ml of RNAiso Plus (TaKaRa, Osaka, Japan). RNA sequencing was performed on the Illumina HiSeqXTen sequencing platform (NovelBio Bio-Pharm Technology Co. Ltd., Shanghai). The dosing group and control group were evaluated in a 3:3 ratio.
Quantitative real-time polymerase chain reaction (qRT–PCR)
For the detection of apoptosis-related gene expression in A549 and H1650 cells regulated by CDAC, we first extracted total RNA using TRIzol reagent (Invitrogen, CA, USA), and the RNA concentration was determined by a NANODROP 2000 (NanoDrop, Wilmington, DE, USA). Total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT–PCR) experiments were conducted using a SYBR Green Premix Ex Taq II kit (Takara, Japan) according to the manufacturer’s instructions. The primers were synthesized by the Shanghai Biotechnology Company, and the corresponding sequences of the primers are shown in Table 1. Each group was composed of three duplicate wells. The relative mRNA expression levels were normalized to β-actin, and the △△Ct method was applied to calculate the relative quantity of mRNA.
Cell apoptosis
For the detection of apoptosis induced by CDCA, an apoptosis kit (BD) and flow cytometry or fluorescence microscopy were used. In short, for flow cytometry detection, trypsin digestion was terminated by the addition of the original complete medium, and the harvested cells were centrifuged at 1000 g for 5 min. Then, 100 μl of binding buffer containing 5 μl of Annexin V-FITC and propidium iodide (PI) was added to the resuspended cells at room temperature for 15 min in the dark, and apoptosis was detected using flow cytometry (Thermo Fisher Scientific, Waltham, MA, USA) and analysed using FlowJo V10.0.7 (BD). For fluorescence microscopy detection, cells were washed twice with PBS, and diluted Hoechst 33342 and PI were added followed by incubation for 20 min at 4 °C. Imagers were then acquired by fluorescence microscopy.
Wound-healing assay
A wound-healing assay was performed to analyse cell migration. Briefly, A549 and H1650 cells (1.0×105) were seeded into 6-well plates and cultured to 100% confluence. Subsequently, the complete medium was replaced with serum-free RPMI-1640 medium, and cells were then incubated for 6 h at 37 °C. The confluent cell monolayer was then scratched with a sterile 100 µl pipette tip and treated with CDCA. The wounds were visualized at 0 h and after incubation with CDCA for 12 hours and 24 hours using an inverted microscope, and images were acquired at 40× magnification.
Transwell assay
A549 and H1650 cells were added to the upper chamber of the Transwell for the migration assay. For the invasion assay, the upper chamber was precoated with Matrigel. The lower chamber contained 800 μl of medium with 20% FBS. Subsequently, cells were treated with CDAC for 24 hours. Cells in the lower chamber were fixed with methanol for 20 min and then stained with crystal violet. The stained cells were counted under an inverted microscope and imaged at 100× magnification.
Immunofluorescence
A549 and H1650 cells were plated onto coverslips and treated with CDAC for 24 h. Cells were fixed with 5% paraformaldehyde solution and incubated with 1% Triton X-100 PBS solution for 20 min at room temperature. After washing with PBS, nonspecific binding sites were blocked with 5% nonfat milk in PBS for 1 hour at room temperature. Cells were then incubated with the anti-p-FAK primary antibody overnight at 4 °C. After washing, cells were incubated with secondary antibody solution (Alexa Fluor® 633 goat anti-rabbit) for 1 h in the dark at room temperature. Nuclei were stained with DAPI (1 μg/mL) for 15 min at room temperature. Images were then acquired using a Leica SP8 confocal microscope under standardized conditions.
Molecular docking
To simulate the molecular-level interaction between integrin α5β1 and its potential inhibitor, CDCA, a molecular docking simulation was performed using the AutoDock-vina (version 1.1.2) program. The three-dimensional structure of integrin α5β1 was downloaded from the Protein Data Bank (PDB ID:4wk4), and the ligand structure of CDCA was constructed and energy-minimized by the Chemoffice package. Before docking simulation, crystal ligands and water molecules in integrin α5β1 were removed by PyMOL 1.7, and AutoDockTools was used to prepare the pdbqt files of the protein and ligand for docking. The docking box was centred at the interface of the α- and β-subunits with the box dimension set to 32 Å in all directions. All other docking parameters were set as default, and the pose with the lowest affinity value was selected for further analysis.
Molecular dynamics (MD) simulation
MD was performed using the sander module implemented in the Amber 18 suite with the ff14SB force field used for the protein system and the GAFF force field used for the ligand. Hydrogen atoms and sodium ions (to neutralize charge) were added to the protein with the leap utility. The simulation system was immersed in a truncated octahedral box full of TIP3P explicit water extended 10 Å outside the protein on all sides. The initial structure of the complex was treated as follows: (a) water molecules and counter ions were relaxed to minimize energy during 10,000 minimization steps (5,000 steepest descent steps, SD; and 5,000 conjugate-gradient steps, CG) with the protein and ligand restrained with a force constant of 500 kcal/mol∙Å2; and (b) the whole system was then minimized without restraints during 10,000 minimization steps (5,000 SD and 5,000 CG). After energy minimization, the system was gradually heated in the NVT ensemble from 0 to 300 K over 50 ps using the Berendsen coupling algorithm. This procedure was followed by 50 ps of NPT simulations at 300 K and 1 atm pressure using the Langevin dynamics algorithm. After equilibration, a 10000 ps production MD simulation was performed. A time step of 2.0 fs was used, and coordinates of the system were saved every 20 ps. All processing and trajectory analyses were performed using CPPTRAJ or VMD programs.
In vivo tumour xenograft animal model
Female BALB/c nude mice (3–4 weeks old weighing 16–20 g) were obtained from the Experimental Animal Center of Soochow University. A total of 1.5×106 A549 cells were inoculated subcutaneously into the flanks, and the female mice were randomly divided into the following two groups (six mice per group): a DMSO control group and a CDCA group (50 mg·kg-1). When the tumour weight reached nearly 100 mm³, DMSO or CDCA was administered to mice every 4 days via intraperitoneal injection. Tumour growth was evaluated by volume (V), which was calculated using the following formula: V = L (tumour length) *W (tumour width)2/2.
Statistical analysis
Results are presented as the mean ± SD. All statistical analyses were performed with GraphPad Prism 5.02 (GraphPad Software) and SPSS 16.0 software (SPSS, Inc.). The statistical significance of differences among multiple groups was evaluated using one-way ANOVA. Significant differences between two groups (parametric) were analysed using Student's t-test, and significant differences between two groups (non‑parametric) were analysed by the Mann‑Whitney U test. P < 0.05 was considered to indicate a statistically significant difference. All experiments were repeated three times independently.