The human CCA cell line QBC939 was obtained from American Type Culture Collection (Manassas, VA, USA). Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). All primary antibodies [anti-β-actin (cat. no. 4970), anti-CyclinE (cat. no. 20808), anti-Bax (cat. no. 5023), anti-Bcl-2 (cat. no. 4223), anti-Wnt3a (cat. no. 2721), anti-Snail1 (cat. no. 3879), anti-c-myc (cat. no. 13987) and anti-β-catenin (cat. no. 8480)] were acquired from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Human CCA QBC939 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were incubated in a humidified incubator at 37°C with 5% CO₂. Propofol was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. QBC939 cells were treated with various concentrations of propofol (0, 1, 5 or 10 µg/ml) for 24, 48 or 72 h at 37°C (19,20). Then, the cells were subjected to assays as described below.

Cells were collected at logarithmic phase, inoculated in a 96-well plate with 1×10⁴ cells/well and incubated at 37°C with 5% CO₂ for 12 h. Then, the cells were treated with various concentrations of propofol (0, 1, 5 or 10 µg/ml) for 24, 48 or 72 h at 37°C. Subsequently, MTT (20 µl; 5 mg/ml) was added to each well, and the cells were incubated for a further 4 h. The formazan crystals were dissolved in 150 µl DMSO and stirred slowly for 10 min. The optical density (OD) of each sample was determined at the wavelength of 570 mm with an immunoassay analyzer. The cell inhibition rate=(1-OD value of treatment group/OD value of control) ×100%. All experiments were performed in triplicate and repeated three times.

QBC939 cells were treated with various concentrations of propofol (0, 1, 5 or 10 µg/ml) for 48 h at 37°C. Then the cells were washed with PBS three times and lysed on ice in radioimmunoprecipitation buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Nanjing, China) with 1 mM PMSF for 30 min at 4°C. Protein was collected and stored −20°C. Bicinchoninic protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) was used to detect protein concentration. Equal amounts of protein (30 µg/lane) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Then, the membrane was blocked at room temperature for 2 h with 5% skimmed milk in PBS with 0.1% Tween-20 (PBST) and incubated with primary antibodies (β-actin, CyclinE, Bcl-2, Bax, Wnt3a, β-catenin, Snail1 and c-myc; all 1:1,000; Cell Signaling Technology, Inc.) overnight at 4°C. The following day, the membrane was washed four times in 1X PBST (10 min/wash) and incubated with anti-rabbit immunoglobulin G horseradish peroxidase-coupled secondary antibodies (cat. no. 7074; 1:1,000; Cell Signaling Technology, Inc.) for 2 h at room temperature. Proteins were detected using SignalFire^™ Plus ECL Reagent (cat. no. 12630; Cell Signaling Technology, Inc.) and imaged. β-actin was used as an internal control.

To investigate the effects of propofol on QBC939 cell migration and invasion, a 24-well Transwell plate (8-µm pore size) was used. The chamber inserts were coated with or without 200 mg/ml of BD Matrigel^™ Matrix (BD Biosciences, Franklin Lakes, NJ, USA) for the invasion and migration assay, respectively. Logarithmic phase QBC939 cells were inoculated into 6-well plates (1×10⁴ cells/well) and placed in a constant temperature incubator for routine culture. When the cells reached 70–80% confluence, they were treated with various concentrations of propofol for 48 h. Then, 100 µl DMEM containing 10% FBS was added to the upper chamber for 1 h. Subsequently, the cells were digested with 0.25% trypsin and resuspended in DMEM to prepare a single cell suspension. The cell density was adjusted to 10⁶ cells/ml. DMEM (0.5 ml) containing 10% FBS was added to the lower chamber, and 100 µl cell suspension was added to the upper chamber of each insert. The plates were cultured at 37°C with 5% CO₂ for 24 h. Then, cells that had not migrated or invaded from the upper chamber to the lower chamber were gently wiped away with a clean cotton swab. The cells on the lower chamber were stained with 0.5 ml 0.1% crystal violet at room temperature for 20 min. Five fields of view were observed for each chamber by a light microscope and the mean value was calculated.

Total RNA was extracted from QBC939 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RNA concentration was detected using NanoDrop 2000 (Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNAs using the PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. qPCR was performed using QuantiFast SYBR Green PCR kit (Qiagen, Inc., Valencia, CA, USA) and a CFX Connect Real-Time system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Amplification conditions were as follows: 10 min at 95°C, followed by 35 cycles of 15 sec at 95°C and 40 sec at 55°C. The primer sequences used for RT-qPCR were listed in Table I. Relative gene expression was analyzed using the 2^−ΔΔCq method (21).

QBC939 cells were collected in logarithmic growth phase, and the cell suspension density was adjusted and inoculated into 6-well plates at 1×10⁵ cells/well. Subsequently, cells were treated with various concentrations of propofol for 48 h. Then, the cells were fixed with 70% methanol at −20°C overnight, washed with PBS twice, stained with propidium iodide (PI; cat. no. 4087; Cell Signaling Technology, Inc.) and incubated at 4°C for 30 min in the dark. Flow cytometry was performed to detect cell-cycle distribution. For detection of cell apoptosis, cells were stained with Annexin V and PI for 15 min at room temperature in the dark prior to flow cytometry. Data were analyzed using WinMDI (version 2.5; Purdue University Cytometry Laboratories, West Lafayette, IN, USA).

All quantitative data are presented as the mean ± standard deviation. All experiments were repeated three times. Differences between multiple groups were compared by one-way analysis of variance followed by Tukey's test, and differences between two groups were compared by Student's t-test. GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analysis. P<0.05 was considered to indicate a statistically significant difference.

In order to study the anti-tumor effect of propofol on human CCA cells, an MTT assay was performed. Following treatment with various concentrations of propofol for 24, 48 or 72 h, respectively, inhibition of proliferation appeared to increase as concentration of propofol in QBC939 cells and the duration of the incubation increased (Fig. 1).

The effects of propofol on migration and invasion of QBC939 cells were examined by Transwell assay. The results indicated that migration and invasion of cells decreased gradually with increased concentrations of propofol compared with the control (Fig. 2). Doses of 1, 5 and 10 µg/ml significantly decreased migration and invasion compared with the 0 µg/ml group.

To investigate whether propofol inhibited the proliferation of QBC939 cells via inducing apoptosis, flow cytometry was performed to detect cell apoptosis. Following treatment with various concentrations of propofol for 48 h, the apoptosis rate of QBC939 cells appeared to increase as concentration of propofol increased (Fig. 3A and B). Furthermore, RT-qPCR and western blot analysis were conducted to determine the expression levels of apoptosis-associated genes, Bax and Bcl-2. The results indicated that with an increase of propofol concentration, the Bax mRNA and protein level in QBC939 cells gradually increased, while the mRNA and protein level of Bcl-2 decreased (Fig. 3C-E).

To demonstrate the possible mechanism of propofol-induced cell growth inhibition in QBC939 cells, cell cycle progression was also analyzed. QBC939 cells were treated with different concentrations of propofol for 48 h and then the cell cycle distribution was analyzed using flow cytometry (data not shown). The results indicated that the percentage of G1 phase of QBC939 cells appeared to increase as concentration of propofol increased (Fig. 4A). In addition, the mRNA and protein expression of CyclinE, an important regulator of G1 and S phases in the cell cycle, was detected. It was identified that the expression of CyclinE was gradually decreased at the mRNA and protein level in QBC939 cells as propofol concentration increased compared with the control (Fig. 4B and C).

QBC939 cells were treated with various concentrations of propofol for 48 h, then the Wnt/β-catenin signaling pathway was analyzed. RT-qPCR and western blot assays indicated that the expression of Wnt3α, β-catenin, Snaill and c-myc was gradually decreased at both the protein (Fig. 5A) and mRNA (Fig. 5B-E) level in QBC939 cells as propofol concentration increased compared with the control.