The tongue squamous cell line SCC15 was provided by Professor Shaohua Liu (Qilu Hospital of Shandong University, Jinan, China). SCC15 cells were cultured in DMEM (HyClone; Cytiva) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂.

Dioscin (purity ≥98%) was purchased from Beijing Solarbio Science & Technology Co., Ltd. Cells were treated with different concentrations of dioscin (0, 0.5, 1 or 2 µM) for 24 or 48 h, or with 1 µM dioscin for different durations (0, 2, 4 or 8 h). Nuclear Protein Extraction kit (cat. no. R0050) was purchased from Beijing Solarbio Science & Technology Co., Ltd and was used to extract nuclear proteins prior to western blotting. Primary antibodies targeted against GAPDH (cat. no. 60004-1-Ig) and histone H3 (cat. no. 17168-1-AP) were obtained from ProteinTech Group, Inc. Primary antibodies targeted against LATS1 (cat. no. 3477), p-LATS1 (Ser909; cat. no. 9157), p-MST (cat. no. 49332), MST2 (cat. no. 3952), YAP (cat. no. 4912), p-YAP (Ser397; cat. no. 13619), cyclin D1 (cat. no. 92G2) and BAX (cat. no. 5023) were purchased from Cell Signaling Technologies, Inc. The primary antibody targeted against BCL-2 (cat. no. WL01556) was purchased from Wanleibio Co., Ltd. The primary antibody targeted against RASSF1A (cat. no. ab91212) was purchased from Abcam. The goat anti-mouse or anti-rabbit HRP-conjugated secondary antibody (cat. nos. S0002 or S0001) was obtained from Affinity Biosciences.

After washing twice with ice-cold PBS, the total protein in cells were lysed using RIPA (cat. no. R0020; Beijing Solarbio Science & Technology Co., Ltd.) lysis buffer for 30 min on ice. Cells were harvested and centrifuged at 20,664 × g at 4°C for 15 min. In order to extract nuclear protein from the cells, cells were lysed using cytoplasmic protein extraction reagent lysis buffer for 30 min on ice. The proteins were collected from the supernatant after centrifugation at 20,664 × g at 4°C for 15 min, and the nuclear protein extraction reagent was added to the cell precipitation for centrifugation at 20,664 × g at 4°C for 15 min and collection of nuclear protein. Protein concentrations were determined using a BCA protein assay. Subsequently, proteins (20 µg) were separated via SDS-PAGE on 10% gels and transferred to PVDF membranes (EMD Millipore). After blocking with non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the appropriate primary antibodies (all 1:1,000). After washing three times with TBS-0.1% Tween-20 (TBST) for 15 min per wash, the membranes were incubated with a goat anti-mouse or anti-rabbit HRP-conjugated secondary antibody (1:20,000) for 1 h at room temperature. The membranes were washed three times with TBST and then protein bands were visualized using an enhanced chemiluminescence kit (EMD Millipore) under using an Imager 600 (Amersham; Cytiva). GAPDH and histone H3 were used as the loading controls. Protein expression levels were analyzed by ImageJ (1.8.0) software (National Institutes of Health).

Following treatment with dioscin for 24 h, lysates were prepared according to the manufacturer's instructions (Active Motif, Inc.). Following centrifugation at 4°C for 5 min at 137 × g, cells were suspended in 100 µl freshly prepared complete RIPA buffer and incubated for 30 min on a rotating platform at 4°C. Whole cell extract was combined with 5 µg IgG (cat. no. 3900) or anti-MST2 (cat. no. 3952) antibodies (both from Cell Signaling Technology, Inc.) in a final volume of 500 µl and incubated overnight at 4°C on a rotating platform. Subsequently, 25 µl protein G magnetic beads were added to the tube and incubated overnight at 4°C on a rotating platform. The protein G magnetic beads were washed carefully and the protein samples were obtained. The samples were resuspended in loading buffer (cat. no. P1040; Beijing Solarbio Science & Technology Co., Ltd.) and boiled for subsequent analysis via western blotting, according to the aforementioned protocol.

Cell proliferation was assessed by performing Cell Counting Kit-8 (CCK-8) assays (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol. SCC15 cells (3×10³ cells/well) were seeded into a 96-well plate. After treatment, cells were cultured for 1–4 days. Subsequently, 10 µl CCK-8 solution was added to each well with 100 µl DMEM and incubated for 2 h at 37°C. Optical density values were measured at a wavelength of 450 nm.

Cells (1×10⁴/well) were seeded into a 48-well plate and cultured for 48 h. The EdU Apollo DNA in vitro Kit (Guangzhou RiboBio Co., Ltd.) was used to assess cell proliferation. Cells were cultured with 200 µl EdU for 2 h and were then fixed with 4% paraformaldehyde for 30 min at room temperature. Subsequently, cells were treated for 10 min with 2 mg/ml glycine and 0.5% Triton X-100 at room temperature. Then, DAPI working solution was added to each well and incubated for 30 min in the dark at room temperature. Quantification of EdU⁺ cells was expressed as a percentage of DAPI⁺ cells. The percentage of EdU⁺ cells was determined by fluorescence microscopy (Olympus Corporation).

cells (1×10⁵/well) were seeded into a 6-well plate and cultured for 24 h. At ~70% confluence, SCC15 cells were transduced with LV-homo-YAP particles [overexpression (OE) YAP group] or LV-5-negative control (NC; empty vector; OE NC group) for 12 h. Similarly, SCC15 cells were transduced with LV-homo-YAP-short hairpin (sh)RNA LV particles (sh YAP group) or a corresponding empty LV vector (sh NC group) for 12 h. The MOI for transduction was 20. All LV particles were purchased from Shanghai Genepharma Co., Ltd. After transduction for 48 h, cells were treated with 1 µg/ml puromycin for 2 weeks. Transduction efficiency was assessed via western blotting.

Cells (1×10⁶) were harvested, fixed in 75% ethanol overnight at 4°C, washed with cold PBS and incubated with 0.5 ml propidium iodide/RNase (Tianjin Sungene Biotech Co., Ltd.) working solution for 30 min at room temperature. Subsequently, the cell cycle distribution was determined using an Accuri C6 flow cytometer (Becton, Dickinson and Company) and analyzed using FlowJo software (version 10; FlowJo LLC).

The Annexin V-PI staining kit was used to assess apoptosis [Multi Sciences (Lianke) Biotech Co., Ltd.]. Cells were harvested, washed with cold PBS and suspended in 500 µl binding buffer. Subsequently, cells were incubated with 5 µl Annexin-V allophycocyanin working solution for 10 min in the dark at room temperature. Then, cells were incubated with 5 µl 7-aminoactinomycin D working solution for 5 min in the dark at room temperature. Within 1 h, early and late cell apoptosis was determined using an Accuri C6 flow cytometer (Becton, Dickinson and Company) and analyzed using FlowJo software (version 10; FlowJo, LLC).

Data are presented as the mean ± SD of three independent experiments. Statistical analyses were performed using GraphPad Prism software (version 6; GraphPad Software, Inc.). Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effects of dioscin on proliferation, SCC15 cells were treated with different concentrations of dioscin for 24 or 48 h, and cell proliferation was determined by performing CCK-8 assays. Compared with the control group (1.014±0.026), dioscin treatment significantly inhibited cell proliferation, even at 0.5 µM (0.820±0.004) (Fig. 1A). Similar results were obtained following treatment for 48 h (Fig. 1B). Moreover, the EdU incorporation assay results indicated that dioscin significantly decreased the ratio of EdU⁺ cells in a concentration-dependent manner (31.150±0.382, 22.92±0.352 and 18.100±0.311% at 0.5, 1 and 2 µM, respectively) compared with the control group (41.410±0.813%) (Fig. 1C and D). The results indicated that dioscin inhibited SCC15 cell proliferation in a dose-dependent manner.

To explore the cellular mechanism underlying the antiproliferative effect of dioscin on SCC15 cells, flow cytometry was performed to detect alterations in the cell cycle and apoptosis. Following treatment with 0.5, 1 or 2 µM dioscin, the number of cells in the G₂/M phase was significantly decreased (5.663±0.350, 1.816±0.304 and 0.5667±0.173%, respectively) compared with the control group (7.283±0.238%) (Fig. 2A and B). Moreover, following treatment with 1 or 2 µM dioscin, the number of cells in the G₀/G₁ phase was significantly increased (69.740±1.3960 and 74.200±0.305%, respectively) compared with the control group (65.830±0.644%). Subsequently, the effect of dioscin on cell apoptosis was evaluated. Compared with the control group (early apoptosis, 2.464±0.110%; late apoptosis, 6.254±0.078%), dioscin (0.5, 1 or 2 µM) significantly increased the population of early (3.100±0.112, 5.624±0.248 and 7.790±0.330%, respectively) and late (8.142±0.072, 15.100±0.353 and 21.020±0.213%, respectively) apoptotic cells (Fig. 2C and D). The apoptosis-inducing effect of dioscin was enhanced with increasing concentrations.

Previous research has demonstrated that RASSF1 supports the phosphorylation of MST2 and SAV, both of which are upstream molecules of YAP (22). A previous study reported that dioscin induced RASSF1 demethylation and inhibited the proliferative activity of bladder cancer cell lines (21). Therefore, the present study aimed to investigate the role of the Hippo signaling pathway in dioscin-treated cells. SCC15 cells were treated with different concentrations of dioscin (0, 0.5, 1 or 2 µM) for 24 h or with 1 µM dioscin for different durations (0, 2, 4 or 8 h). The ratios of p-LATS (Ser909)/LATS, p-MST/MST2 and p-YAP (Ser397)/YAP were significantly increased by dioscin treatment in a concentration- and time-dependent manner compared with the control group (Fig. 3A-D). Compared with the control group, dioscin did not notably alter the expression levels of total LATS1 and MST2, but markedly decreased the expression levels of total YAP (Fig. 3A-D). In addition, the expression levels of cell cycle- and apoptosis-related proteins were assessed. Cyclin D1 and BCL-2 expression levels were markedly decreased by dioscin in a concentration- and time-dependent manner compared with the control group (Fig. 3E and F). However, BAX expression levels displayed the opposite pattern. To investigate whether dioscin treatment altered the localization of YAP, nuclear and cytoplasmic proteins were extracted using the Nuclear Protein Extraction kit. As dioscin concentrations increased, nuclear YAP expression levels were markedly decreased, whereas YAP cytoplasmic accumulation was notably increased compared with the control group (Fig. 3G).

To detect Hippo signaling pathway activation following dioscin treatment, the expression levels of RASSF1A, a protein consistently linked to tumor onset and poor cancer prognosis (23), were detected. The western blotting results indicated that RASSF1A expression levels were markedly increased by dioscin treatment in a concentration- and time-dependent manner compared with the control group (Fig. 3A and B). However, the mechanism underlying dioscin-induced inactivation of the Hippo signaling pathway is not completely understood. RASSF1A has been reported to form a complex with MST2 and SAV, which phosphorylates LATS1, leading to YAP phosphorylation (24). The aforementioned results indicated that dioscin treatment resulted in RASSF1A accumulation (Fig. 3A and B), and endogenous RASSF1Aor LATS1 were coimmunoprecipitated with the MST2protein (Fig. 3H). As shown in Fig. 3H, RASSF1A and LATS1 were detected in the anti-MST2 precipitate, but not in the IgG precipitate.

The aforementioned results indicated that the RASSF1A/MST2/YAP axis was involved in dioscin-induced inhibition of cell proliferation. To evaluate the hypothesis, SCC15 cells were transduced with OE YAP or OE NC and then treated with 1 µM dioscin. The flow cytometry results demonstrated that, compared with the OE NC group (3.450±0.886%), dioscin treatment significantly decreased the number of cells in the G₂/M phase (1.097±0.112%), whereas the number of cells in the proliferative phases was significantly increased in the OE YAP group (7.887±0.206%) compared with the OE NC group. (Fig. 4A and B). Meanwhile, compared with the OE NC + dioscin group (1.097±0.112%), the increase of proliferation activity of OE YAP + dioscin group (6.890±0.237%), indicated that overexpression of YAP gene abrogated dioscin-induced inhibition of cell proliferation. (Fig. 4B). The apoptosis assay results demonstrated that dioscin treatment significantly increased the number of apoptotic cells (39.010±0.464%), whereas in the OE YAP group this increase was attenuated (19.150±0.831%) compared with the OE NC group (24.770±0.382%) (Fig. 4C and D). The EdU incorporation (Fig. 4E and F) and CCK-8 (Fig. 4G) assay results demonstrated that, compared with the OE NC group or OE YAP group, dioscin treatment significantly suppressed SCC15 cell proliferation in the OE NC + dioscin and OE YAP + dioscin groups, whereas YAP overexpression in the OE YAP or OE YAP + dioscin groups enhanced cell proliferation compared with the OE NC or OE NC + dioscin groups, respectively. The dioscin-induced suppression of proliferation was inhibited in the OE YAP group. The protein expression levels of cell cycle- and apoptosis-related proteins were measured via western blotting. The western blotting results further verified the alterations in the cell cycle and apoptosis (Fig. 4H).

To further understand the role of YAP in mediating the suppressive effects of dioscin on SCC15 cells, SCC15 cells were transduced with sh YAP or sh NC and then treated with 1 µM dioscin. Flow cytometry was performed to assess alterations in cell cycle distribution and apoptosis. Both YAP knockdown and dioscin treatment led to a decrease of cells in G₂/M phase, and the combination of these interventions augmented this effect (G₂/M phase cells: sh NC, 11.770±0.0880%; sh NC + dioscin, 11.000±0.152%; sh YAP, 10.300±0.152%; and sh YAP + dioscin, 6.263±0.489%) (Fig. 5A and B). Moreover, compared with the sh NC group (19.860±0.474%), the proportion of apoptotic cells was increased following dioscin treatment (21.600±0.462%) (not statistically significant) or YAP knockdown (22.010±0.353%), and the combination of these two interventions was synergistic (27.020±0.277%) (Fig. 5C and D). Subsequently, SCC15 cell proliferation was evaluated by performing EdU incorporation (Fig. 5E and F) and CCK-8 (Fig. 5G) assays. Compared with the sh NC group, both dioscin and YAP knockdown significantly decreased cell proliferation. In addition, the combination of dioscin treatment and YAP knockdown resulted in further inhibition of cell proliferation compared with either intervention alone, as determined by flow cytometry, EdU staining, western blotting and CCK-8 assay at 24 h.

Subsequently, the expression levels of YAP, cyclin D1, BCL-2 and BAX were measured. Similarly, the combination of dioscin treatment and YAP knockdown led to more notable suppressive effects on protein expression compared with either intervention alone. However, the expression levels of the proapoptotic protein BAX displayed the opposite trends (Fig. 5H).