2.1 Ethical approval.
The protocols of all animal experiments were reviewed and approved by the Research Ethics Committee of Qingdao Municipal Hospital, and all animal experimental studies were conducted in accordance with the ARRIVE guidelines. All experiments in the text were carried out in compliance with the relevant rules and regulations and under the supervision and guidance of the Ethics Committee of Qingdao Municipal Hospital.
2.2 Chemicals and reagents
Tan-IIA (Sigma-Aldrich, purity > 97%). The PI3K agonist 740y-p was purchased from MCE (Shanghai, China), Annexin V-FITC/PI staining kit (absin, abs50001, China), Cell Counting Kit-8 (CCK-8) reagent (APExBIO, K1018, USA), Matrigel glue (BD Biosciences, NJ, USA), and BCA protein analysis kit (Beyotime, Shanghai, China), and ECL reagent (Millipore, Massachusetts, USA). Primary antibodies included anti-PI3K (20584-1-AP, Proteintech, China) and anti-Akt antibody (10176-2-AP, Proteintech, China), anti-mTOR (2983, CST, USA), anti-p-Akt (4060, CST, USA), anti-p-mTOR (5536, CST, USA), anti-Bax (2774, CST, USA), anti- Bcl2 (3498, CST, USA), and anti-Caspase-3 (9662, CST, USA) and anti-Cleaved-Caspase-3 (9661, CST, USA).
2.3 Cell lines and culture conditions
Human Cholangiocarcinoma cell lines, HuCCT-1 and RBE, were purchased from the Chinese Academy of Sciences (Shanghai) Cell Bank. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, Excellbio, USA) and 1% penicillin–streptomycin (HyClone, UT, USA). The cells were cultured at 37°C in an incubator containing 5% CO2.
2.4 Cell viability
The cells were incubated overnight in 96 well plates at a density of 5 × 103 cells per well. Then the cells were treated with different concentrations (0, 5, 10, 20, and 30 µg/mL) of Tan-IIA at different times (12, 24, 48, and 72 h), and a 10 µL CCK8 reagent was added (APExBIO, K1018, USA) to each well. After incubation at 37°C for 1 h, the optical density (OD) was measured at 450 nm. IC50 was calculated using GraphPad Prism 7.0 software.
2.5 Plate cloning
Both cell lines were cultured in a well plate at a density of 300 cells per well. The cells were gently rotated to disperse the cells evenly. After 6 h, the Cholangiocarcinoma cells were treated with Tan-IIA (0, 5, 10, 20, and 30 µg/mL) and incubated in a cell culture incubator at 37ºC with 5% CO2 for two weeks. Next, the cells were washed with PBS three times. Subsequently, 4% paraformaldehyde was used to fix the cells for 15 min. The cells were then stained with crystal violet for 10 min. Subsequently, the staining solution was washed off with PBS. The six well plates were then inverted and an overlay of transparency sheet with a grid was performed and the clones was manually counted directly with the naked eye: clone formation rate = (number of clones/number of inoculated cells) × 100%.
2.6 Scratch-induced wound healing assay
Plated in six well culture dishes were 4 × 105 of Cholangiocarcinoma cells. After 24 h, a 200 μL tip was used to wound confluent cells. The detached cells were washed with PBS three times, and the cells were incubated with Tan-IIA (HuCCT-1: 27µg/mL, RBE: 49µg/mL) for 24 h. Cell migration images were recorded using an inverted microscope. The results of the scratch experiment were obtained using the formula wound closure rate = post-healing area/initial wound area. All experiments were repeated three times.
2.7 Transwell invasion assay
Cell invasion was detected in a 24-well Transwell chamber. The upper chamber was precoated with 50 µL Matrigel. Cholangiocarcinoma cells (5 x 104) were suspended in 100 µL serum-free medium supplemented with or without Tan-IIA (HuCCT-1: 27µg/mL, RBE: 49µg/mL) and were seeded into the upper chamber. At the same time, 600 µL DMEM containing 10% FBS was placed into the lower chamber. After 24 h incubation, Cholangiocarcinoma cells on the upper side of the membrane were wiped with a clean swab. Then, the cells on the underside of the membrane were fixed with methanol and stained with crystalline violet. Nine regions were randomly selected to count the number of invading cells using an inverted microscope.
2.8 Apoptosis determined by the Annexin-V-FITC/PI
Cholangiocarcinoma cells were cultured in six well plates at a density of 2 × 105 cells per well. The cells were then treated with or without Tan-IIA (HuCCT-1: 27µg/mL, RBE: 49µg/mL) for 24 h. Then, cells were digested by trypsin without EDTA and harvested and then washed twice with cold PBS. Subsequently, the cells were suspended in binding buffer and stained with 5 μL Annexin V-FITC for 30 min. Next, PI (10 µL) was added for 15 min at room temperature in darkness. Finally, the samples were analyzed by Accuri C6 flow cytometry (BD Biosciences, CA, USA).
2.9 Identification of common targets for Tan-IIA and Cholangiocarcinoma
The targets of Tan-IIA were identified using the TCMSP database (https://tcmspw.com/tcmsp.php), and then gene annotation of the TCM targets was conducted on the Uniprot website (https://www.uniprot.org/). From OMIM (https://omim.org/), GeneCards (https://www.genecards.org/), PharmGKB (https://www.pharmgkb.org/), and TTD (http://db.idrblab.net/ttd/) databases were used to obtain relevant targets of Cholangiocarcinoma, and R software was used to analyze the common target genes of both.
2.10 Protein–protein interaction (PPI) network
For building a protein–protein interaction (PPI) network, the common targets of Tan-IIA and Cholangiocarcinoma were entered into the string website (https://string-db.org/) to construct PPI plots, and then Cytoscape software was used to construct network pharmacograms.
2.11 GO and KEGG pathway analysis
Gene Ontology (GO) is an international standard classification system for gene function, consisting of three major components: cellular components, molecular function, and biological processes 12. To investigate the gene functions involved in common targets, we ran GO functional enrichment using R software. The analysis of common target-associated pathways between Tan-IIA and Cholangiocarcinoma was performed using Kyoto Encyclopedia of Genes and Genome (KEGG) 13.
2.12 Western blotting
Cholangiocarcinoma cells were lysed in RIPA for 30 min at 4°C and total protein was extracted by centrifugation for 20 min. Then, BCA protein analysis reagent was used to determine protein concentrations. Protein extracts were boiled for 5 min. After resolution using SDS/PAGE (10%), proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA). At room temperature, the membranes were sealed with 5% bovine serum albumin (BSA) for 2 h. Diluted primary antibody: Akt (1: 1000), phospho (p)-Akt (1: 2000), PI3K (1:1000), mTOR (1: 1000), phospho (p)-mTOR (1: 2000) (CST, CA, USA), and β-actin(1:5000). Subsequently, the target membranes were incubated with the specific primary antibodies overnight at 4°C. Membranes were then washed with TBST three times and then diluted HRP-coupled secondary antibodies were added and incubated for 2 h at room temperature. Finally, immunocomplexes were detected using an ECL detection reagent (Millipore, MA, USA). The measurement dates were obtained from three separate experiments. The intensity of each band was determined using ImageJ software.
2.13 In vivo experiments
NOD-SCID (NOD CB17-Prkdcscid/NcrCrl, male, 5 weeks of age) mice were provided by Beijing Vital River Lab-oratory Animal Technology Co., Ltd (Beijing, China). All mice were placed in a 12-h light/dark cycle at 25+-1℃ and 56% humidity with free access to food and water. The initial body weights of these mice ranged from 20 to 23 grams. Following subcutaneous injection of 2 × 106 HuCCT-1 Cholangiocarcinoma cells into the back of 15 NOD-SCID mice, the mice were divided into three groups: the control group (n=5), the Tan-IIA (50 mg/kg) treatment group (n=5), and the Tan-IIA (50 mg/kg) combined with 740y-p (10 mg/kg) treatment group. Tan-IIA was diluted with DMSO: Methanol: Hydroxypropyl-β-cydodextrin (HP-β-CD) = 1: 1: 1. 740y-p was dissolved in the same way. Seven days after the injection of HuCCT-1 Cholangiocarcinoma cells, drugs were injected intraperitoneally into the experimental groups of mice on every other day, and normal saline was given to the control group. Mice were killed at day 21 of inoculation with tumor cells. All mice were executed by dislocation of the cervical vertebrae. Tumor volumes were measured every 3 days before execution.
2.14 Statistical analysis
The statistical software GraphPad 7 was used for data analysis. The experimental results listed in the article represent the dates of at least three separate replicate experiments. Dates are shown as mean + standard deviation. Student’s t-test was used for differences between two groups. Multiple group comparisons were made using one-way ANOVA. Differences were considered statistically significant at P values of less than .05.