The present study chose human EBV positive NPC cell (C666-1) and EBV negative cells (HK1) (18). C666-1 and HK1 cells were purchased from the National Infrastructure of Cell Line Resource, Peking Union Medical College (Beijing, China). C666-1 and HK1 cells were cultured in Dulbecco's modified Eagle medium (HyClone; Cytiva) containing 10% fetal bovine serum (HyClone; Cytiva). All cells were then incubated at 37°C in a humidified atmosphere of 5% CO_2.

Ophiopogonin B (OP-B; purity of ≥97%; Shanghai Tauto Biotech Co., Ltd.) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA). For treatment of cells, OP-B was diluted in culture medium to a final concentration of 5, 10 and 20 µM (0.01% DMSO) at 25°C for 30 min.

Cells were seeded (5×10⁴ cells/well) in 96-well plates and then OP-B (5, 10 and 20 µM) was added for 12, 24, 48 or 72 h. MTT (20 µl; 5 mg/ml; Sigma-Aldrich; Merck KGaA) was added and incubated for another 4 h at 37°C. Afterwards, 150 µl dimethyl sulfoxide was added at room temperature for 10 min. Absorbance was assessed at 490 nm using a microplate reader (BioTek Instruments, Inc.).

Cell proliferation was detected using the BeyoClick™ EdU-594 detection kit (cat. no. C0078S; Beyotime Institute of Biotechnology). Briefly, following treatment with OP-B, cells were incubated with 50 mM EdU for 2 h at 37°C and incubated with 4′,6-diamidino-2-phenylindole for 30 min at 37°C. After staining, images were photographed under a fluorescence microscope (Olympus).

The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was used to detect cell apoptosis. Briefly, following treatment with OP-B, cells were collected. 5 µl Annexin V-FITC and 5 µl PI were then added, mixed and incubated for 15 min at room temperature. Apoptosis was evaluated using a flow cytometer (BD Accuri C6 Plus; BD Biosciences) and FlowJo software (v10.6.2; FlowJo, LLC). The apoptotic rate was calculated as the percentage of early + late apoptotic cells.

MMP was examined using the fluorescent probe 5,5′,6,6-tetrachloro-1, 1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyotime Institute of Biotechnology). Briefly, following treatment with OP-B, cells were incubated with JC-1 staining solution at 37°C for 20 min. MMP was detected using flow cytometry as aforementioned (BD Biosciences).

Intracellular ROS were detected using an ROS assay kit (Nanjing Jiancheng Bioengineering Institute). In brief, following treatment with OP-B, 10 µM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was added to the cells at 37°C for 20 min. The median fluorescence intensity of ROS was measured using a flow cytometer as aforementioned (BD Biosciences).

MDA and SOD activity were assessed using MDA assay kit and SOD assay kit (Nanjing Jiancheng Bioengineering Institute).

The yes-associated protein (YAP) siRNA (sense: 5′-ACUUUUCGCUGCAAGUUGCUA-3′; antisense: 3′-GCAACUUGCAGCGAAAAGUUU-5′), control siRNA (sense: 5′-UUCUCCGAACGUGUCACGUTT-3′; antisense: 3′-ACGUGACACGUUCGGAGAATT-5′) were synthesized by Shanghai GenePharma Co., Ltd. C666-1 and HK1 cells (10⁶ cells/well) were seeded in six-well plates. The cells were transfected with 100 nM of YAP siRNA or control siRNA using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C, and the medium was changed for fresh after 6 h. After 48 h, the C666-1 and HK1 cells were harvested and exposed to OP-B (5 µM) for 24 h at 37°C.

C666-1 and HK1 cells (1×10⁶) were harvested and lysed them with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology). Total protein was quantified by using an Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Total protein (50 µg) was loaded on 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Subsequently, the gel was transferred to a polyvinylidene fluoride membrane (Millipore). The membrane was blocked by using 5% non-fat milk for 1 h at 25°C. After blocking, the membrane was incubated with anti-YAP (1:1,000; cat. no. 4912), anti-phosphorylated (p-)YAP (S127; 1:1,000; cat. no. 4911), anti-mammalian sterile 20-like kinase 1 (MST1; 1:1,000; cat. no. 3682), anti-large tumor suppressor 1 (LATS1; 1:1,000; cat. no. 3477), anti-transcriptional enhanced associate domain 1 (TEAD1; 1:1,000; cat. no. 12292), anti-Bcl-2 (1:1,000; cat. no. 3498), anti-Bax (1:1,000; cat. no. 2774), anti-caspase-3 (1:1,000; cat. no. 9662), anti-cleaved caspase-3 (1:1,000; cat. no. 9661), anti-poly(ADP-ribose) polymerase (PARP; 1:1,000; cat. no. 9542), anti-cleaved-PARP (1:1,000; cat. no. 9545), anti-forkhead box transcription factor O1 (FOXO1; 1:1,000; cat. no. 2880), anti-p-FOXO1 (1:1,000; cat. no. 2486; all from Cell Signaling Technology, Inc.) and anti-glyceraldehyde-3-phosphate dehydrogenase (1:5,000; cat. no. P30008; Abmart Pharmaceutical Technology Co., Ltd.) overnight at 4°C. Membranes were incubated with secondary antibody (1:5,000; cat. no. M21002, Abmart Pharmaceutical Technology Co., Ltd.) at room temperature for 2 h. Protein bands were detected using a chemiluminescence kit and a gel imaging system (Tanon 2500; Tanon Science and Technology Co., Ltd.).

Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software, Inc.). The data represented mean ± standard deviation from three independent experiments. One-way ANOVA and Tukey's post hoc test were conducted to evaluate changes among groups. P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of OP-B is shown in Fig. 1A. The effect of OP-B on cell proliferation was investigated in C666-1 and HK1 cell lines. The MTT assay demonstrated that OP-B inhibited C666-1 and HK1 cells proliferation in a dose and time-dependent manner (Fig. 1B). As shown in Fig. 1C, OP-B effectively inhibited the proliferation of C666-1 and HK1 cells.

Next, whether OP-B induced apoptosis was investigated. Flow cytometry assays were used to confirm that OP-B activated apoptosis in C666-1 and HK1 cells (Fig. 2A). Furthermore, OP-B increased the expression of Bax, cleaved-PARP and cleaved-caspase-3, whereas the expression of Bcl-2, PARP and caspase-3 was decreased by OP-B in C666-1 and HK1 cells (Fig. 2B). OP-B induced a concentration-dependent decrease in red/green fluorescence ratios in C666-1 and HK1 cells (Fig. 2C).

ROS from mitochondria are related to cell apoptosis (19). Following treatment with OP-B, intracellular ROS levels were increased in C666-1 and HK1 cells (Fig. 3A). As shown in Fig. 3B and C, OP-B increased the MDA content and decreased SOD activity.

OP-B treatment decreased the ratio of p-FOXO1 vs. total FOXO1 in C666-1 and HK1 cells (Fig. 4A). In addition, FOXO1 protein level decreased in cytosolic fraction and increased in the nuclear fraction following treatment with OP-B (Fig. 4B).

OP-B significantly increased the expression of MST1 and LATS1, increased the ratio of p-YAP vs. total YAP and decreased TEAD protein levels (Fig. 5). Therefore, the results suggested that OP-B inhibited NPC cells tumorigenesis through the regulation of the Hippo signaling pathway.

siYAP was transfected into C666-1 and HK1 cells and the cell proliferation and apoptosis ability of these two NPC cell lines detected. The expression of YAP decreased rapidly due to OP-B treatment, YAP knockdown significantly reduced YAP expression (Fig. 6A). YAP knockdown promoted the inhibitory effect of OP-B on the proliferation of C666-1 and HK1 cells (Fig. 6B). In addition, YAP knockdown induced the enhancing effect of OP-B on apoptosis in C666-1 and HK1 cells (Fig. 6C).