PD (>98%, PubChem CID:132399081) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. It was dissolved in PBS to obtain a stock solution (100 µmol/l), then stored at −20°C. MTT and Hoechst 33342 were purchased from Sigma-Aldrich; Merck KGaA. FITC Annexin V Apoptosis Detection kit I was purchased from BD Biosciences. Primary antibodies against cleaved caspase-9 (1:1,000; cat. no. 9505), cleaved caspase-3 (1:1,000; cat. no. 9661), Bax (1:1,000; cat. no. 5023), Bcl-2 (1:1,000; cat. no. 4223), SAPK (stress-activated protein kinase)/JNK (1:1,000; cat. no. 9252), phosphorylated-SAPK/JNK (p-JNK; 1:1,000; cat. no. 9251), matrix metalloproteinase (MMP)-2 (1:1,000; cat. no. 87809), MMP-9 (1:1,000; cat. no. 3852), cyclin B1 (1:1,000; cat. no. 4138), cytochrome c (1:1,000; cat. no. 4272) and β-tubulin (1:1,000; cat. no. 2146), and all secondary antibodies (1:1,000; cat. no. 7074) were purchased from Cell Signaling Technology, Inc. The same secondary antibody was used for all primary antibodies. The antibody against cyclin-dependent kinase 1 (CDK1; 1:1,000; cat. no. ab133327) was purchased from Abcam.

The human GBC NOZ, GBC-SD and SGC-996 cell lines were obtained from the The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were maintained in DMEM supplemented with 10% FBS (both Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin. All cells were cultured at 37°C in a humidified atmosphere with 5% CO₂.

GBC cells were added into 96-well plates at a density of 2×10³ cells/well and cultured overnight at 37°C and 5% CO₂. Subsequently, different concentrations of PD (0, 5, 10, 15, 20 and 25 µmol/l) were added to each well, and the cells were cultured for 24, 48 or 72 h, separately. MTT (5 mg/ml) solution was added to the wells (10 µl/well) and incubated at 37°C for 4 h. The culture medium was then replaced with DMSO (100 µl/well) to dissolve the purple formazan and a microplate reader (BioTek Instruments, Inc.) was used to measure the absorbance at 490 nm.

NOZ and GBC-SD cells were collected and counted manually. A total of 600 cells/well were added into 6-well plates (Corning Inc.). Subsequently, PD at different concentrations (0, 5, 10 and 15 µmol/l) was used to treat the cells. The cells were treated for ~14 days. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for 15 min at room temperature. All colonies with >50 cells were recorded manually with a fluorescence microscope (magnification ×40; Leica Microsystems GmbH).

NOZ and GBC-SD cells were cultured with PD at various concentrations (0, 5, 10 and 15 µmol/l) for 48 h at 37°C and 5% CO₂. After culturing, the cells were collected and washed with PBS. Next, the cells were diluted to the appropriate density (10⁶ cells/ml) using a Annexin V binding buffer (BD Biosciences). The cell suspension (200 µl) was gently mixed with Annexin V-FITC (5 µl) (BD Biosciences) and PI (5 µl) (BD Biosciences) and incubated for 15 min in the dark at room temperature, these were a part of the kit mentioned earlier and were used according to the manufacturer's protocol. Subsequently, 300 µl of the binding buffer was added. Flow cytometry using a BD FACSCanto II (BD Biosciences) was used to analyze the sample within 1 h and BD FACSDiva Software v6.1.3 (BD Biosciences) was used to analyze the results.

NOZ and GBC-SD cells were added into 12-well plates and incubated overnight at 37°C and 5% CO₂. Subsequently, PD at 0, 5, 10 and 15 µmol/l was added to the wells, and the plates were incubated for 48 h at 37°C and 5% CO₂. After treatment, the cells were stained with Hoechst 33342 for 30 min in the dark at 37°C and then washed with PBS. The cells were observed using a fluorescence microscope (magnification, ×200; Leica Microsystems GmbH).

NOZ and GBC-SD cells were treated with different concentrations (0, 5, 10 and 15 µmol/l) of PD for 48 h at 37°C and 5% CO₂. Subsequently, the cells were stained with Mito-Tracker green (Beyotime Institute of Biotechnology) at 37°C for 30 min in the dark. The cells were observed using a fluorescence microscope (magnification ×100; Leica Microsystems GmbH).

Transwell plates with 24 wells (Corning Inc.) were used to perform cell migration and invasion assays. The upper chambers with or without Matrigel^® (1 mg/ml) were dried at 37°C for 30 min. NOZ and GBC-SD cells treated with PD (0, 5, 10 and 15 µmol/l) for 48 h were collected and diluted in serum-free DMEM at a density of 2×10⁵ cells/ml. Subsequently, 100 µl of the cell suspension was added to the upper chambers. Simultaneously, 500 µl of DMEM supplemented with 10% FBS was added to the lower chambers. After 24 h, the cells on top of the membrane were removed using a cotton swab. The cells on the lower membrane were fixed with 4% paraformaldehyde for 15 min at room temperature, stained with crystal violet for 15 min at room temperature, and observed using a phase-contrast microscope (magnification, ×100; Olympus Corporation). Five fields of vision were randomly selected per well.

For cell cycle analysis, the Cell Cycle and Apoptosis Analysis kit was used (Beyotime Institute of Biotechnology). NOZ and GBC-SD cells were seeded into 6-well plates and incubated overnight at 37°C and 5% CO₂. The supernatant was replaced with different concentrations (0, 5, 10 and 15 µmol/l) of PD. After 48 h of incubation at 37°C and 5% CO₂, the cell layer was digested using trypsin, washed with cold PBS and fixed with 70% ethanol at 4°C for at least 2 h before storing at 4°C overnight. Subsequently, the cells were washed with cold PBS and resuspended in staining buffer. Next, 0.1 mg/ml RNase A and 0.1 mg/ml PI were added to the cell suspension. The staining buffer, RNase A and PI were part of the Cell Cycle and Apoptosis Analysis kit (Beyotime Institute of Biotechnology) and were used according to the manufacturer's instructions. The cells were analyzed using flow cytometry after incubation at 37°C in the dark for 30 min. BD FACSDiva Software v6.1.3 (BD Biosciences) was used to analyze the distribution of the cell cycle phases.

NOZ and GBC-SD cells treated with PD (0, 5, 10 and 15 µmol/l) for 48 h were collected and washed with PBS. Total protein was extracted from the cells using RIPA buffer (Beyotime Institute of Biotechnology). After sonication (15–25 KHz, 25 sec on ice), the mixture was centrifuged at 14,000 × g for 20 min at 4°C. Protein concentration was quantified using a bicinchoninic acid assay kit (Beyotime Institute of Biotechnology). Subsequently, the proteins (30 µg/lane) were separated via 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with the corresponding aforementioned primary antibodies at 4°C overnight. The membranes were washed three times with TBS supplemented with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.) for 1 h at room temperature. Subsequently, the membranes were washed again with TBST three times and Millipore Western Blot Chemiluminescence HRP Substrate ECL Luminescent solution was added (EMD Millipore). The membranes were observed using the Gel Doc 2000 (Bio-Rad Laboratories, Inc.) and ImageJ software version 1.48 (National Institutes of Health) was used to quantify the densitometric values of the detected bands.

Each experiment was performed three times. The data was expressed as the mean ± SD. Shapiro-Wilk normality tests were used to assess normality of quantitative variable distributions. If the variables followed a normal distribution, one-way ANOVAs followed by Dunnett's multiple comparisons test was performed using GraphPad Prism version 7.0 (GraphPad Software, Inc.) to analyze the results. Alternatively, Kruskal-Wallis test followed by Dunn's multiple comparisons test was used in cases where the samples were not normally distributed. P<0.05 was considered to indicate a statistically significant difference.

The MTT assay was used to detect the proliferative ability of PD-treated GBC cells. The results indicated that the proliferation of GBC cells decreased with an increased concentration of PD in a time-dependent manner. In NOZ and GBC-SD cells, the IC₅₀ of PD was ~10 µmol/l at 48 h (Fig. 2A). PD had a stronger inhibitory effect on NOZ and GBC-SD cells than on SGC-996 cells (Fig. S1); therefore, NOZ and GBC-SD cells were selected for subsequent experiments. A colony forming assay was performed to further investigate the colony forming ability of PD-treated GBC cells. The results indicated that PD had a negative effect on the ability of GBC cells to form colonies (Fig. 2B). Additionally, the PD-treated groups developed fewer clones than those in the control group (Fig. 2C). When treated with 5 µmol/l of PD, a significant decrease in colonies was observed in NOZ cells, while a significant decrease in colonies was observed in GBC-SD cells when the cells were treated with 10 µmol/l of PD (Fig. 2C).

To assess the potential mechanisms of action behind PD-mediated growth inhibition, PD-treated GBC cells were analyzed using flow cytometry (Fig. 3A). As the drug concentration increased, the proportion of apoptotic cells also increased, while the percentage of surviving cells was reduced (Fig. 3B). Subsequently, the morphology of the nuclei was analyzed using Hoechst 33342 staining. In the control group, the morphology of the cells was normal and the chromatin was uniformly distributed, while the chromatin in the PD-treated group were markedly aggregated and broken (Fig. 3C). Subsequently, Mito-Tracker green was used to stain the mitochondria. After treatment, bright green fluorescence was observed in the control group, while the fluorescence intensity of the PD-treated group was visibly reduced (Fig. 4A). Additionally, cytochrome c expression was higher in the PD-treated groups than that in the control group (Fig. 4B).

To explore the influence of PD on the migratory and invasive abilities of GBC cells, cell migration and invasion assays were performed. Compared with the control group, PD treatment (10 and 15 µmol/l) markedly suppressed the migration numbers of NOZ and GBC-SD cells (Fig. 5A) and the difference was statistically significant (P<0.01; Fig. 5B). In addition, in the cell invasion assay, PD treatment led to a significant anti-invasion effect in GBC cells and this effect was dose-dependent (Fig. 5C and D). Additionally, western blotting analysis confirmed that MMP-2 and MMP-9 had low expression in PD-treated GBC cells compared with the control group (Fig. 5E). These data indicated that PD inhibited the migration and invasion of GBC cells.

To further understand how PD affected the proliferation of GBC cells, flow cytometry was performed to measure the proportion of PD-treated GBC cells at different cell cycle stages (Fig. 6A). When cells were treated with 10 µmol/l PD, the proportion of cells in G₂/M phase increased significantly, while the proportion of cells in G₀/G₁ phase decreased in both cell lines (Fig. 6B). This suggested that PD inhibited the cell cycle progression of GBC cells. Next, the levels of proteins that are closely associated with the cell cycle, such as cyclin B1 and CDK1, were examined. The expression levels of these proteins in GBC cells were markedly decreased compared with the control group (Fig. 6C). Therefore, PD may induce G₂/M phase arrest in GBC cells to inhibit cell proliferation.

Caspase family and Bcl-2 family proteins are both important in the regulation of apoptosis (19). To further elucidate how PD induced apoptosis in GBC cells, the expression levels of apoptosis-associated proteins were investigated. It was observed that the expression levels of Bax, cytochrome c, cleaved caspase-9 and cleaved caspase-3 were increased with an increased concentration of PD, while Bcl-2 expression was decreased with an increased concentration of PD compared with the control group (Figs. 4B and 7A). According to a previous study, the Bax/Bcl-2 ratio is a key factor in the regulation of the apoptotic process (20). In the present study, PD decreased the expression levels of the anti-apoptotic protein Bcl-2 and increased those of the pro-apoptotic protein Bax, thereby decreasing the ratio of Bcl-2/Bax (Fig. 7B). Additionally, it was observed that p-JNK expression was upregulated, while JNK expression was not markedly changed. In summary, the present results suggested that PD may induce apoptosis by initiating mitochondrial destruction through the JNK signaling pathway in GBC cells.