The TSCC cell line, CAL27, was obtained from Shanghai Ninth People's Hospital (Shanghai, China) and cultured in a high-glucose Dulbecco's modified Eagle's medium (DMEM; HyClone Laboratories; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 5% CO₂ at 37°C. For morin treatment, tumor cells were cultured in a complete cell culture medium supplemented with different doses of morin (0, 50, 100 and 150 µM; Yuanye Chemicals Co., Ltd., Shanghai, China), while dimethyl sulfoxide (DMSO; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the solvent, was used as the control treatment for up to 14 days.

Tumor cell viability was assayed following morin treatment using a cell viability Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Briefly, Cal27 cells were seeded into 96-well plates at a density of 3,000 cells per well and grown overnight in a complete cell culture medium (see above) and then refreshed with morin-containing medium for 24 to 48 h. At the end of each experiment, cell growth medium was added to 10 µl of CCK-8 working solution and cells were further cultured for 90 min at 37°C. The optical density value (OD) of the plates was then measured using a microplate reader at 450 nm. The experiment was performed in triplicate and repeated at least three times.

Cal27 cells were plated into 6-well plates (1,000 cells/well) and grown overnight. Following this incubation, the cell culture medium was refreshed with morin-containing medium (0, 50, 100 and 150 µM) every 2 days for 14 days. At the end of the experiments, cells were washed with ice-cold phosphate-buffered saline (PBS), fixed using freshly made 4% polyformaldehyde/PBS solution and stained with 0.1% crystal violet. Cell colonies with more than 50 cells were counted under an inverted microscope (Olympus Corp., Tokyo, Japan). The experiment was performed in triplicate and repeated at least three times.

We performed the EdU incorporation assay using an EdU kit from Guangzhou RiboBio Co., Ltd. (Guangzhou, China) according to the manufacturer's instructions. Specifically, Cal27 cells were plated into 24-well plates at a density of 50,000 cells/well and grown overnight. The cells were then treated with different concentrations of morin (0, 50, 100 and 150 µM) for 24 h. Following this, cell culture was added to 50 µl of the EdU working solution and further cultured for 2 h. At the end of the experiment, cells were washed with ice-cold PBS and fixed in 4% paraformaldehyde for 15 min. The cells were then treated with 2 mg/ml glycine at room temperature for 5 min and stained with Apollo^® 567 and Hoechst working solution, in the dark, for 30 min. Finally, the cell images were obtained using fluorescence microscopy (Olympus Corp.).

Cal27 cells were grown and treated with various concentrations of morin (0, 50, 100 and 150 µM) for 24 h. For the tumor cell wound-healing assay, the cells were tripsinized and reseeded into 6-well plates at a density of 1×10⁶ cells/well and grown overnight to reach ~90% confluency. The cell monolayer was then scratched with a sterile 200-µl pipette tip across the plates, washed with DMEM twice and cultured for up to 24 h in DMEM/0.1% FBS as well as different the concentrations of morin. At each time-point (0, 6 or 24 h), the cell monolayers were photographed using an inverted microscope (Olympus Corp.). The wound healing areas were measured using ImageJ software 14.8 for Windows (National Institutes of Health, Bethesda, MD, USA). The experiment was performed in triplicate and repeated at least three times.

Cal27 cells were first plated into 6-well plates (1×10⁶ cells/well) and incubated overnight at 37°C. Cells were then treated with different doses of morin (0, 50, 100 and 150 µM) for up to 48 h. At the end of each experiment, both detached and attached cells were collected and incubated with the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (eBioscience, Vienna, Austria) according to the manufacturer's protocol. Specifically, ~20,000 cells were suspended in the binding buffer and 5 µl of Annexin V-FITC was added. Cells were incubated for 10 min, followed by a further 5-min incubation after the addition of 5 µl of the PI staining solution at room temperature in the dark. Finally, the apoptosis level was measured using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).

Cal27 cells were plated into 6-well plates (1×10⁶ cells/well) and incubated overnight. Cells were then treated with different doses of morin (0, 50, 100 and 150 µM) for 24 h. The cells were then harvested and washed with ice-cold PBS once and fixed in 70% ethanol at 4°C for 12 h. On the next day, the cells were washed again with PBS and then stained with PI working solution at room temperature for 30 min in the dark. The cell cycle distribution of the cells was analyzed using flow cytometry (FACSCalibur; BD Biosciences).

Morin-treated Cal27 cells were lysed in radioimmunoprecipitation assay buffer (RIPA buffer; Beyotime Institute of Biotechnology, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology) for 30 min on ice. Both cytoplasmic and nuclear extracts were then extracted using a nuclear fractionation protocol, following the manufacturer instructions (Beijing Solarbio Science & Technology Co., Ltd.). The concentration of each protein sample was assayed using the bicinchoninic acid assay kit (Beijing Solarbio Science & Technology) and equal amounts (20 µg of each) of protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel (Beyotime Institute of Biotechnology) electrophoresis and electrically transferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen; Thermo Fisher Scientific). For western blotting, the membranes were first incubated in 5% non-fat milk at room temperature for 1 h and then incubated with a rabbit monoclonal anti-human YAP antibody (cat. no. 14074), a rabbit monoclonal anti-human phospho-YAP antibody (cat. no. 13619), a rabbit polyclonal anti-human MST1 antibody (cat. no. 3682), a rabbit polyclonal anti-human phospho-MST1 antibody (cat. no. 3681), a rabbit monoclonal anti-human MOB1 antibody (cat. no. 13730), and a rabbit monoclonal anti-human phospho-MOB1 antibody (cat. no. 8699) (all from Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. The next day, the membranes were washed with Tris-based saline-Tween-20 (TBS-T; 20 mmol/l Tris-HCl, 150 mmol/l NaCl and 0.05% Tween-20) three times for 10 min each time and then incubated in 1:5,000 HRP-conjugated goat anti-rabbit IgG (cat. no. 7074S; Cell Signaling Technology, Inc.). The protein bands were visualized using the Chemiluminescent HRP Substrate kit (EMD Millipore, Billerica, MA, USA). The level of protein expression was then normalized to that of GAPDH (cytoplasmic protein) and Histone H3 (nuclear protein).

The total cellular RNA was isolated from Cal27 cells that had been treated with morin for 24 h using a TRIzol^® reagent (Takara Bio, Inc., Otsu, Japan). The RNA was then reversely transcribed into cDNA using a Reverse Transcriptase kit (Takara Bio) according to the manufacturer's protocols. These cDNA samples were then amplified with qPCR in 20 µl of the reaction system, which contained the SYBR^® Primix Ex Taq™ (Takara Bio), cDNA, each primer and RNase-free H₂O, following the manufacturer's protocol. Amplification conditions were set to an initial step of 95°C for 30 sec and then 45 cycles of 95°C for 5 sec, 60°C for 35 sec, and 72°C for 60 sec and a final step at 40°C for 30 sec. The level of GAPDH mRNA was used as a control and the relative expression level of each RNA sample was calculated using the 2^−ΔΔCq method (30). All experiments were performed in triplicate and repeated at least once. The primer sequences were GAPDH, 5′-GCACCGTCAAGGCTGAGAAC-3′ and 5′-TGGTGAAGACGCCAGTGGA-3′; CTGF, 5′-TCTCCAACCTCTCCTACTAC-3′ and 5′-GCACGTAGTTTCGATCACT3′; CYR61, 5′-CCTTGTGGACAGCCAGTGTA-3′ and 5′-ACTTGGGCCGGTATTTCTTC-3′; and ANKRD, 5′-AGTAGAGGAACTGGTCACTGG-3′ and 5′-TGGGCTAGAAGTGTCTTCAGAT-3′.

All data are summarized as the mean ± standard error of the mean (SEM) of at least three replicates of each experiment and statistically analyzed using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) with the two-tailed Student's t-test for two groups of data or the one-way analysis of variance (ANOVA) plus Tukey's post hoc test for multiple groups of data. P<0.05 indicates statistical significance.

To evaluate the effects of morin on the regulation of TSCC cell proliferation, we performed cell viability (CCK-8), EdU incorporation and colony formation assays. Our CCK-8 assay data revealed that morin treatment significantly reduced tumor cell viability in a dose-dependent manner both at 24 and 48 h (Fig. 1). The results from the tumor cell colony formation assay also revealed that morin treatment significantly reduced the number of tumor cell colonies compared with the number of tumor cell colonies observed in the untreated control cells (Fig. 2). Moreover, morin treatment for 24 h led to a significantly lower percentage of EdU-positive cells than that observed in the control cells, in a dose-dependent manner (Fig. 3). These data indicated that morin inhibited TSCC cell proliferation.

We further investigated how morin treatment inhibits tumor cell proliferation using flow cytometric apoptosis and cell cycle assays. Our data showed that morin treatment, for 24 h, arrested tumor cells at the G1 phase of the cell cycle, and showed a significantly smaller percentage of S phase cells than this percentage in the control group (Fig. 4A and B). Moreover, morin-treated tumor cells underwent apoptosis. Both the percentages of early and late apoptotic cells were significantly higher at 48 h of morin treatment at 100 and 150 µM than in the control cells (Fig. 4C and D).

We then performed the tumor cell wound-healing assay to investigate the effect of morin on the migratory capacity of Cal27 cells. We found that, following morin treatment, cells showed a lower tumor cell wound healing capacity than the control cells, which showed quick closure of the wound (Fig. 5).

Thus far, we demonstrated the antitumor activity of morin in TSCC cells in vitro. To further this study, we aimed to assess the underlying molecular pathways responsible for the action of morin on TSCC cells. Our western blot analysis revealed that the total levels of MST1 and MOB1 proteins were not significantly altered after morin treatment of TSCC cells (there was no significant difference between the results of the treated cells and the control cells; Fig. 6A and B). In contrast, the expression levels of phospho-MST1 and phospho-MOB1 proteins were significantly higher in morin-treated TSCC cells than these levels in the control cells (Fig. 6C and D). Furthermore, the cytoplasmic level of YAP protein was significantly upregulated in morin-treated TSCC cells and the ratio of phosphorylated YAP was also greater in the morin-treated tumor cells (Fig. 6E and F). In contrast, TSCC cells treated with high concentrations of morin exhibited a lower level of nuclear YAP protein than that of the control cells (Fig. 6G and H). We further investigated whether the changes in YAP protein localization that were observed following morin treatment correlated with an alteration in YAP-targeting proteins, such as CTGF, CYR61 and ANKRD. Our data showed that the expression of CTGF, CYR61 and ANKRD genes was significantly lower in the morin-treated Cal27 cells than that in the control cells (Fig. 6I). Taken together, our current data demonstrated that morin treatment upregulated the activity of the Hippo pathway but suppressed YAP nuclear translocation and YAP-related transcriptional activity in Cal27 cells.