EGCG (E8120) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Simvastatin (S6196) was purchased from Merck KGaA (Sigma-Aldrich; Darmstadt, Germany) and dissolved in DMSO to make stock solutions. Primary rabbit monoclonal anti-human antibodies against TAZ (cat. no., 70148), phosphorylated (p)-TAZ (Ser89) (cat. no., 59971), large tumor suppressor 1 (LATS1; cat. no., 3477), MOB kinase activator 1 (MOB1; cat. no., 13730), mammalian sterile 20-like 1 (MST1; cat. no., 14946), salvador 1 (SAV1; cat. no., 13301), c-Jun N-terminal kinase (JNK; cat. no., 9252), p-JNK (Thr183/Tyr185) (cat. no., 4668), extracellular regulated protein kinases (Erk; cat. no., 4695), p-Erk (Thr202/Tyr204) (cat. no., 4370), protein kinase B (Akt) (cat. no., 4691), p-Akt (Ser473) (cat. no., 4060), B cell lymphoma-2 (Bcl-2; cat. no., 2872), Bcl-2 associated X protein (Bax; cat. no., 5023), poly ADP-ribose polymerase (PARP; cat. no., 9532), cleaved PARP (cat. no., 5625), vimentin (cat. no., 5741), and E-cadherin (cat. no., 3195), GAPDH (cat. no., 5174) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were obtained from Affinity Biosciences (cat. no., S0001; Cincinnati, OH, USA).

The TSCC cell lines, CAL27 and SCC15, were obtained from the American Type Culture Collection (Manassas, VA, USA). They were identified using short tandem repeats. CAL27 cells were cultured in Dulbecco's modified Eagle's medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), while SCC15 cells were incubated in minimum essential medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

Cell proliferation was measured by a Cell-Counting Kit-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were cultured in 96-well tissue culture plates (4.0×10³ cells/well) with 10% FBS for 24 h. Then, the cells were exposed to different concentrations of EGCG (0, 40, 80, 120, 160 and 200 µM) for different durations (0, 24, 48, 72, 96 and 120 h). Following incubation, 10 µl CCK-8 solution was added to each well, and the plates were incubated for an additional 2 h. The absorbance was measured using a spectrometer at a wavelength of 450 nm.

Cell proliferation was also assessed using an EdU Apollo DNA in vitro kit (Guangzhou RiboBio Co., Ltd., Guangzhou, China) following the manufacturer's protocol. CAL27 cells were seeded at a density of 1.0×10⁵ cells/cm² in 24-well plates and incubated for 24 h in normal growth medium. Cells were treated with 200 µl EdU for 2 h and then fixed with 4% paraformaldehyde for 15-20 min at room temperature. The cells were then incubated with 2 mg/ml glycine at room temperature for 10 min, followed by washing with PBS. Then, the cells were permeabilized with 100 µl/well permeabilization buffer (0.5% Triton X-100 in PBS) at room temperature for 30 min and incubated with 200 µl 1X Apollo solution for 30 min at room temperature in the dark. Subsequently, the cells were incubated with 200 µl 1X Hoechst 33342 solution for 30 min at room temperature in the dark. The percentage of EdU-positive cells was determined by fluorescence microscopy (magnification, ×100) and was calculated using Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA).

Cell apoptosis was analyzed by flow cytometry. Cells with green fluorescent probes and empty vector were stained using the Annexin V-APC staining kit (Sungene Biotech Co., Ltd., Tianjin, China). Briefly, following washing with cold PBS, the cells were suspended in 500 µl 1X binding buffer, and then incubated with 5 µl Annexin V-fluorescein APC at room temperature in the dark for 10 min. Finally, 5 µl 7-AAD solution was added and cells were incubated for 5 min at room temperature in the dark. Untreated control cells without transfection were measured using the Annexin V-FITC/PI kit [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd., Hangzhou, China] following the manufacturer's protocol. The percentage of apoptotic cells was measured using a FACSCalibur (BD Biosciences, San Jose, CA, USA), and analyzed using CellQuest software version 5.1 (BD Biosciences).

Cells (8.0×10⁵/well) were seeded in 6-well plates, and allowed to attach and reach 80% confluence. Cell monolayers were wounded by scratching with 200-µl pipette tips and then washed twice with PBS to remove floating cells. Cells in each well were subsequently exposed to FBS-free medium with or without EGCG for up to 48 h. Cells were imaged at ×100 magnification with a phase-contrast microscope at each time point. The wound healing areas at different time points were measured using ImageJ software 14.8 for Windows (National Institutes of Health, Bethesda, MD, USA).

Cells (1.0×10⁴ cells/well) were seeded in the upper chamber of Transwell inserts (8-µm pore size) pre-coated with Matrigel (both from Corning Incorporated, Corning, NY, USA) and exposed to FBS-free medium with or without different concentrations of EGCG. Medium containing 10% FBS was placed in the lower chamber, and cells for each treatment were incubated for 24 h at 37°C in a humidified environment with 5% CO₂. Then, non-invaded cells in the upper chamber were removed with a cotton swab, and invaded cells were washed with PBS, fixed with 4% para-formaldehyde for 30 min and stained with 0.1% crystal violet (Solarbio, Shanghai, China) for 10 min. Images were captured with a light microscope at ×200 magnification, and the number of cells that had penetrated the membrane was counted using Image-Pro Plus 6.0 software.

A total of 1×10⁶ CAL27 cells were seeded and transfected at ~80% confluence with culture medium with 8 µg/ml Polybrene and 100 µl overexpression TAZ lentivirus particle LV5-homo-TAZ (NM_000116.4) (TAZ-overexpression group). Similarly, CAL27 cells at ~80% confluence were transfected with the culture medium with 8 µg/ml Polybrene and 100 µl empty vector lentivirus LV5-NC (NM_000116.4) (negative control group). After 6-8 h, the medium was changed to basal medium (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS, and the cells were cultured for further analyses. The efficiency of TAZ overexpression was determined using western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays.

Cells were collected and lysed in ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), and the concentration of each protein sample was quantified using a bicinchoninic acid assay. Subsequently, 20 µg of protein from each sample was separated using 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking in 5% fat-free milk for 1 h at room temperature, the membranes were probed with primary antibodies (dilution 1:1,000) at 4°C overnight. Next, they were washed with Tris-based saline-Tween-20 (20 mmol/l Tris-HCl, 150 mmol/l NaCl and 0.05% Tween-20) at room temperature three times for 10 min each. Then, the membranes were incubated with HRP-conjugated secondary antibodies (dilution 1:20,000) at room temperature for 1 h. Finally, protein bands were detected using the Immobilon Western chemiluminescent HRP substrate kit (EMD Millipore). Quantitative analysis of protein bands was calculated using ImageJ with 64-bit Java 1.6.0-24 program for Windows.

Total RNA was extracted from cells at 80% confluence using TRIzol^® reagent (Gibco; Thermo Fisher Scientific, Inc.) and then reverse transcribed into cDNA using the PrimeScript™ RT II reagent kit (Takara Bio, Inc., Otsu, Japan) according to manufacturer's protocol. The generated cDNA was used as template for Quantitative real-time PCR (RT-qPCR) using SYBR Premix Ex Taq™ kit (Takara, Tokyo, Japan) following the manufacturer's protocol. Amplification conditions were as follows: Initial step of 95°C for 30 sec; 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. RT-qPCR was performed using a LightCycler^® 480 II and changes in gene expression were calculated using the 2^−ΔΔCq method (21). The expression levels of GAPDH and TAZ were analyzed and the primers used in the present study were as follows: TAZ forward, 5′-GCT GCT TCT GGA CCA AGT ACA-3′ and reverse, 5′-AGA TGT GGC GGA GTT TCA GG-3′; GAPDH forward, 5′-GCT TGT CAT CAA CGG GAA G-3′ and reverse, 5′-GAT GTT AGT GGG GTC TCG-3′.

All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Differences between multiple groups were examined using one-way analysis of variance followed by Tukey's post hoc test and a two-tailed t-test was used for the comparison between two groups. All results are expressed as the mean ± standard deviation from at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of EGCG on proliferation of CAL27 cells, cells were treated with different concentrations of EGCG and the changes in proliferation were measured using a CCK-8 assay. Cell proliferation was significantly decreased in response to EGCG treatment in time- and dose-dependent manners (Fig. 1A and B). Following incubation for 120 h, the cell numbers in the 160 µM EGCG group were significantly lower compared with those in the control groups. Eventually, 80 µM was established as the optional drug concentration. Additionally, cell apoptosis was measured by flow cytometry (Fig. 1C). The apoptosis rates, total and early apoptosis, of the 80 µM EGCG group were significantly higher compared with those of the group without EGCG. In addition, the 160 µM EGCG group had a higher proportion of cells in late apoptosis compared with the 80 µM EGCG group. EGCG also significantly inhibited cell invasion in a concentration-dependent manner as evaluated using a Transwell assay (Fig. 1D). Similarly, as revealed using the scratch assay, EGCG significantly reduced the time of wound healing (Fig. 1E). Therefore, the results of the present study indicated that EGCG inhibited proliferation, migration and invasion, and promoted apoptosis in CAL27 cells. In order to confirm these findings, the aforementioned experiments were repeated in SCC15 cells, whereby similar changes were observed (Fig. 2)

Western blot assays were performed to confirm whether EGCG treatment induces changes in the expression of proteins associated with proliferation, apoptosis, migration and invasion (Fig. 3). Akt is known as a key molecule of the PI3K/Akt/MTOR signaling pathway (22), and Erk-1/2 is a component of the mitogen-activated protein kinase (MAPK) signaling pathway (23), which affects the expression of proliferation-associated proteins. According to the results of the present study, EGCG significantly reduced the expression of proliferation-associated proteins compared with normal (0 µM) cells, including p-Akt, while no differences were observed in total Akt expression. Although the level of total Erk was unaffected, changes in p-Erk levels indicate a reduction in proliferative abilities.

The apoptotic pathway is tightly regulated by pro- and anti-apoptotic members of the Bcl-2 protein family (24). Bcl-2 is an anti-apoptotic protein of the Bcl-2 family, whereas Bcl-2 associated X apoptosis regulator is a pro-apoptotic protein of the same family. PARP is a zinc-finger DNA-binding enzyme that is activated by binding to DNA breaks. When it is activated, PARP is cleaved (25). It has been reported that EMT affects tumor metastasis (26), and the expression of proteins associated with EMT in the present study changed in response to EGCG treatment, indicating that EGCG serve a role in tumor metastasis. The expression of E-cadherin, an epithelial protein, significantly increased with EGCG treatment compared with untreated control cells. In contrast, the expression of vimentin, a mesenchymal protein, significantly decreased in response to EGCG treatment.

In previous studies, researchers have demonstrated that TAZ is associated with tumorigenesis-associated processes in TSCC, including cell proliferation, survival and migration in vitro (19,20). Therefore, whether the expression of TAZ and its upstream signaling molecules were affected by EGCG treatment were investigated in the present study. As expected, the protein levels of total TAZ and p-TAZ were significantly decreased in response to different doses of EGCG for 24 h of CAL27 cells (Fig. 4A). To investigate whether EGCG altered the nuclear localization of TAZ, proteins were extracted from the nucleus and cytoplasm of cells, and the relevant protein levels of TAZ were decreased in parallel with total TAZ levels (Fig. 4B). Next, the effect of EGCG on the upstream signaling pathway was determined. As determined by the western blot assay, EGCG treatment significantly decreased LATS1 and MOB1 protein levels compared with untreated control cells. However, EGCG had minimal or no effect on MST1 and SAV1 expression (Fig. 4A). Next, total JNK and p-JNK protein levels were examined. p-JNK levels were significantly decreased while total JNK level remained constant (Fig. 4A) compared with untreated control cells, consistent with the results of a previous study (10). In order to confirm these findings, all the aforementioned experiments were performed on SCC15 cells, whereby Hippo-associated proteins and p-JNK protein exhibited the same trend as CAL27 cells, indicating that EGCG inhibited the biological behaviors of TSCC through the Hippo-TAZ signaling pathway (Fig. 5).

To investigate whether the expression level of TAZ influenced EGCG-treated cells, CAL27 cells were treated with TAZ overexpression lentiviral particles or control lentiviral particles for another 24-48 h. Western blot analysis and RT-qPCR assays indicated the successful overexpression of TAZ (Fig. 6A and B). Treatment with EGCG significantly inhibited proliferation, migration and invasion and promoted apoptosis in control cells. However, TAZ overexpression alleviated the effects of EGCG compared with that in EGCG-treated control cells (Fig. 6C-G). No significant differences were observed in the protein expression levels between the empty vector group and non-transfected cell group, which were all stimulated with EGCG, indicating that the transfection of control lentiviral particles exhibited little influence on CAL27 cells. The protein levels of TAZ, Bcl-2, p-Akt and vimentin significantly increased in TAZ-overexpression cells, and E-cadherin protein significantly decreased compared with the non-transfected cells and the empty vector groups, which was in parallel with the morphological features observed (Fig. 7). Taken together, these results revealed that TAZ overexpression, at least in part, abolished the effects of EGCG, further demonstrating that the Hippo-TAZ pathway serves an important role in regulating CAL27 cell proliferation and migration in response to EGCG stimulation.

Simvastatin, which has been identified as the inhibitor of TAZ, was used to treat CAL27 cells stimulated with EGCG (20). To further determine the additive effects of EGCG and simvastatin, CCK-8 assays, flow cytometry, scratch and Transwell assays were used to examine the corresponding changes in the proliferative, apoptotic, migrative and invasive abilities of CAL27 cells (Fig. 8). Simvastatin decreased the TAZ protein level in a dose manner, particularly at 20 µM, thus 20 µM was selected as the optional drug concentration (Fig. 8A). It was demonstrated that 80 µM EGCG and 20 µM simvastatin treatment separately inhibited the proliferation, migration and invasion of CAL27 cells, but more significant suppressive changes were detected when cells were treated with a combination of EGCG and simvastatin (Fig. 8B, C and E). In addition, 80 µM EGCG and 20 µM simvastatin promoted cell apoptosis to varying degrees, the simulative effect was more significant when cells were exposed to both drugs compared with single treatment (Fig. 8D).