Quercetin and other chemicals (including SP600125, PD98059 and LiCl), unless specified otherwise, were purchased from MilliporeSigma. Quercetin (to prepare the stock solution) was dissolved and diluted in DMSO. To the control wells, the maximum volume of DMSO used in the experiments was added, which was <1% per well and did not induce any cytotoxicity. Laboratory plasticware was obtained from Falcon (Corning Life Sciences). Mouse and rabbit monoclonal antibodies specific for caspase-3 (cat. no. 9661), caspase-7 (cat. no. 9491), poly(ADP-ribose) polymerase (PARP) (cat. no. 9542), phosphorylated (p)-JNK (cat. no. 9255), p-ERK1/2 (cat. no. 4377), p-p38 (cat. no. 9216), p-AMPKα (cat. no. 4188), p-GSK3-α/β (cat. no. 9331), cytochrome c (cat. no. 11940), Bcl-2 (cat. no. 15071), Bax (cat. no. 89477), Bak (cat. no. 12105), JNK-1 (cat. no. 3708), ERK1/2 (cat. no. 9102), p38 (cat. no. 8690), AMPKα (cat. no. 2532), GSK3-α/β (cat. no. 5676), β-actin (cat. no. 8457) and secondary antibodies [horseradish peroxidase (HRP)-conjugated anti-mouse IgG (cat. no. 7076) or anti-rabbit IgG (cat. no. 7074)] were purchased from Cell Signaling Technology, Inc.

The human tongue SCC-derived cell line SAS (JCRB0260) was purchased from Japanese Collection of Research Bioresources Cell Bank. SAS cells were cultured in a humidified chamber containing a 5% CO₂−95% air mixture at 37°C. All cells were maintained in culture medium containing 45% Dulbecco's modified Eagle's medium, 45% Ham's F12 medium and 10% fetal calf serum (all from Gibco; Thermo Fisher Scientific, Inc.). Cells were seeded to 6- or 24-well culture plates for each experiment and allowed to grow for 12–18 h (the recover overnight), and then treated with quercetin (10–300 µM) for different time intervals in the absence or presence of the inhibitors of SP600125 (20 µM), PD98059 (20 µM) or LiCl (100 µM) for 30 min at 37°C prior to treatment with quercetin.

The changes in cell morphology were detected according to a previous study (28). The cells were cultured on a glass slide at a density of 1×10⁶ cells/well at 37°C. After 24 h, a photomicrograph was obtained with a 20× objective lens using a cooled CCD camera attached to a Zeiss Axiovert 135-TV Inverted Fluorescence Phase Microscope (Zeiss AG).

An MTT assay was used to determine the effect of quercetin on the viability of SAS cells. The cells were cultured in a 24-well plate at a density of 2×10⁵ cells/well. After recovery overnight, the culture medium was removed and the cells were washed twice with PBS. Fresh medium with quercetin (10–300 µM) or cisplatin (10 µg/ml; as a positive control) was then added. After 24 h at 37°C, the cells were washed twice with PBS and fresh medium was added with 30 µl MTT (2 mg/ml; MilliporeSigma) at 37°C for 4 h. The medium was then removed and cells were washed twice with PBS. DMSO was added to dissolve the blue formazan crystals in cells and the absorbance of cells was detected at a wavelength of 570 nm to determine cell viability using an ELISA reader (model 550; Bio-Rad Laboratories, Inc.).

The externalization of phosphatidylserine residues on the outer plasma membrane of cells is an early event during apoptosis, which may be detected by annexin V (29). To assess quercetin-induced apoptosis and necrosis, flow cytometry was performed using the Annexin V-FITC-PI assay kit (BioVision, Inc.). SAS cells were seeded at 2×10⁵ cells/well in a 24-well plate and incubated with quercetin (10–100 µM) at 37°C for 24 h. Subsequently, the cells were incubated using 0.05% trypsin/EDTA for 1 min to detach the cells, which were then centrifuged (200 × g at 4°C for 5 min), re-suspended in 100 µl binding buffer, transferred to a 5-ml fluorescence-activated cell sorting tube and combined with 5 µl Annexin V-FITC and 10 µl PI (50 µg/ml). After incubation for 30 min at room temperature in the dark, 400 µl binding buffer was added to each tube and the samples were immediately analyzed using flow cytometry (FACScalibur; BD Biosciences) using CellQuest software (version 5.1; BD Biosciences). Four cell populations were identified as follows: the viable cell population was in the lower-left quadrant (low FITC and PI signals), the early apoptotic population was in the lower-right quadrant (high FITC and low PI signals), the late apoptotic population was in the upper-right quadrant (high FITC and PI signals), and the necrotic population was in the upper-left quadrant (low FITC and high PI signals).

Caspase-3 activity was assessed as previously described by Lee et al (28) and a Caspase-3 Activity Assay Kit (cat. no. 5723) was used (Cell Signaling Technology, Inc.). The cells were cultured in a 24-well plate at a density of 2×10⁵ cells/well. After treatment with quercetin (10, 30 and 50 µM) or cisplatin (10 µg/ml; as a positive control) at 37°C for 24 h, the cells were lysed and the caspase-3/CPP32 substrate (Ac-DEVD-AMC; 10 µM) was added for 1 h at 37°C. The fluorescence of cleaved substrates was determined using a spectrofluorometer (Spectramax; Molecular Devices, LLC) at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.

The alteration of MMP was monitored by flow cytometry as previously described by Chen et al (30). The cells were harvested and washed twice with PBS. All samples were stained with DiOC₆ (40 nM) for 30 min at 37°C and the fluorescence of DiOC₆ was analyzed by FACSscan flow cytometry (BD Biosciences), using CellQuest software (version 5.1; BD Biosciences).

Caspase 3/7 is widely accepted as a reliable indicator of apoptosis (31). A FLICA DEVD-FMK Caspase 3/7 Assay Kit (cat. no. 94; ImmunoChemistry Technologies, LLC) was used to determine apoptosis by flow cytometry. SAS cells were seeded at 2×10⁵ cells/well in a 24-well plate and incubated with quercetin (50 µM) in the absence or presence of SP600125 (20 µM), PD98059 (20 µM), or LiCl (100 µM) at 37°C for 24 h. Subsequently, the cells were collected in 1.5-ml Eppendorf tubes, centrifuged at 200 × g for 5 min at 4°C, washed twice with PBS and stained with fluorescent probes for 10 min in a dark environment at room temperature. Caspase 3/7 activity was determined based on the fluorescence intensity in cells using flow cytometry (FACScalibur; BD Biosciences) using CellQuest software (version 5.1; BD Biosciences).

Western blot analysis was performed according to a previously described protocol (28). In brief, the cells were lysed using Protein Extraction Solution (cat. no. 17081; iNtRON Biotechnology, Inc.) and the protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (50 µg) were resolved by SDS-PAGE (a 13.5% gel for caspase-3, −7, cytochrome c, Bcl-2, Bax, Bak; a 9% gel for p-JNK, JNK-1. P-ERK1/2, ERK1/2, p-p38, p38, p-AMPKα, AMPKα, p-GSK3-α/β, GSK3-α/β and β-actin) and transferred to polyvinylidene difluoride membranes. PBS-0.05% Tween-20 (PBST) containing 5% nonfat dry milk was used to block the membranes for 1 h at room temperature. The blots were then probed with primary antibodies (1:1,000 dilution) for 12–16 h at 4°C. The blots were washed twice with 0.1% PBST and were then probed with HRP-conjugated secondary antibodies (1:5,000 dilution) for 1 h at 4°C. The antibody-reactive bands were revealed using an Immobilon^® Western Chemiluminescent HRP substrate kit (MilliporeSigma) and analyzed using a luminescent image analyzer (ImageQuant™ LAS-4000; GE Healthcare Bio-Sciences). The bands underwent densitometric analysis using ImageJ version 1.50d software (National Institutes of Health). For the detection of cytosolic cytochrome c expression levels, the cells were detached, washed twice with PBS, and then homogenized with a pestle and mortar in the extraction buffer [0.4 M mannitol, 25 mM MOPS (pH 7.8), 1 mM EGTA, 8 mM cysteine, and 0.1% (w/v) bovine serum albumin]. The cell debris was removed via centrifugation at 6,000 × g for 2 min. The supernatant was centrifuged at 12,000 × g for 15 min to pellet the mitochondria. The cytochrome c levels in the supernatants (cytosolic fraction) were detected using western blot analysis. For the detection of the phosphorylated proteins and total proteins, the membrane was first probed with the p-protein antibody and analyzed. Subsequently, the same membrane was washed and stripped with western blot stripping buffer (cat. no. ab270550; Abcam) according to the manufacturer's instructions. After stripping, the membrane was probed with the total protein antibody and re-analyzed.

The mRNA expression levels of Bcl-2, Bax, Bak, caspase-3, caspase-7, caspase-9 and PARP were analyzed using RT-qPCR as described in a previous study (32). Cells were cultured in a 10-cm² dish and incubated with quercetin (50 µM) in the absence or presence of SP600125 (20 µM), PD98059 (20 µM) or LiCl (100 µM) at 37°C for different time intervals (2–16 h). Subsequently, total intracellular RNA was extracted using an RNeasy kit (Qiagen GmbH) according to the manufacturer's protocol. To eliminate genomic DNA (gDNA), the sample was added to DNase I. Subsequently, the sample was incubated at room temperature for 15 min and heated to 70°C for 10 min to denature DNase I, and then rapidly placed on ice. To reverse transcribe RNA into cDNA, 5 µg RNA was added to a reaction buffer: 2.5 mM deoxynucleotide mix, 40 U/µl RNAase inhibitor (Promega Corporation), 100 nmol random hexamer primers, 1X reverse transcriptase (RTase) buffer (which was supplied with the RTase enzyme) and 30 units AMV RTase enzyme, to which nuclease-free water was added to reach a final volume of 20 µl. The temperature steps of the RT reaction consisted of 10 min at room temperature, 15 min at 42°C and 5 min at 95°C and then the sample was placed on ice. The real-time SYBR Green primers for human caspase-3, caspase-7, caspase-9, PARP, Bcl-2, Bax, Bak and β-actin used in the present study are shown in Table I. Each sample (2 µl) was assessed using Real-Time SYBR Green PCR reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and transgene-specific primers in a 25-µl reaction volume, and amplification was performed using an ABI StepOnePlus™ Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The cycling conditions consisted of 2 min at 50°C, 10 min at 95°C, 40 cycles at 95°C for 30 sec and 60°C for 1 min. Real-time fluorescence detection was performed during the 60°C annealing/extension step of each cycle. Melt-curve analysis was performed on each primer set to ensure that no primer dimers or nonspecific amplifications were present under the optimized cycling conditions. After 40 cycles, data analysis was performed using StepOne™ software (version 2.1; Applied Biosystems; Thermo Fisher Scientific, Inc.). The fold differences in mRNA expression between the treatment and control groups were determined using the relative quantification method, which utilizes qPCR efficiencies and normalizes them to a housekeeping gene (β-actin was used in the present study), thus comparing relative Cq changes (ΔCq) between the control and experimental samples. The fold change values were calculated using the expression 2^−ΔΔCq, where ΔΔC represents ΔCq_{condition of interest}-ΔCq_control (33). Prior to conducting the statistical analyses, the fold change from the mean of the control group was calculated for each individual sample (including the individual control samples to assess the variability in this group).

Data are presented as the mean ± standard deviation of at least four independent experiments. All data analyses were performed using SPSS software version 12.0 (SPSS, Inc.). Significant differences among the groups were assessed by one-way analysis of variance followed by Tukey's post-hoc test to identify group differences. P<0.05 was considered to indicate a statistically significant difference.

The present study first investigated whether quercetin had an apoptotic effect on tongue SCC cells. Cells were treated with quercetin (10–300 µM) for 24 h. As shown in Fig. 1A, quercetin (30 and 50 µM) markedly induced morphological changes (as indicated by the arrows), including cell shrinkage, as observed by an inverted phase-contrast microscope. Furthermore, cell viability was significantly decreased following treatment with quercetin (10–300 µM; Fig. 1B). The 50% lethal concentration (LC₅₀) in SAS cells was ~50 µM quercetin (Fig. 1B). As determined by flow cytometry, the fluorescence intensity of Annexin V-FITC (Fig. 1C-a and b) and the percentage of apoptotic cells (as indicated by high Annexin V-FITC + low PI plus high Annexin V-FITC + high PI signals; Fig. 1C-c) were significantly increased in quercetin-treated cells in a dose-dependent manner. However, quercetin did not induce cell necrosis in quercetin-treated ASA cells (Fig. 1C). Furthermore, caspase-3 activity was also significantly and dose-dependently increased in cells treated with quercetin (Fig. 1D). Cisplatin (10 µg/ml), as a positive control, was used to induce cytotoxicity (Fig. 1B) and apoptosis (Fig. 1D) in SAS cells. These results indicated that quercetin may be capable of inducing SAS cell apoptosis and death.

The present study next examined whether the mitochondrial pathway was involved in quercetin-induced SAS cell apoptosis. Cells were treated with quercetin (50 µM) for 6 or 24 h. As shown in Fig. 2A and B, the MMP was significantly decreased and cytosolic cytochrome c protein expression was effectively increased in quercetin-treated cells. Furthermore, the expression levels of mitochondrial apoptosis-related proteins were also detected. Both Bax and Bak protein expression levels were time-dependently increased, whereas Bcl-2 protein expression was time-dependently decreased in response to quercetin (Fig. 2C-a). Cisplatin (10 µg/ml), as a positive control, was used to induce apoptosis-related protein expression in SAS cells (Fig. 2C-a and D-a). In addition, the protein expression levels of cleaved caspase-3, caspase-7 and PARP were time-dependently increased in SAS cells following quercetin exposure (Fig. 2D-a). Moreover, quercetin decreased Bcl-2, and increased Bax, Bak, caspase-3, caspase-7 and PARP mRNA expression levels in SAS cells (Fig. 2C-b and D-b). These results suggested that the mitochondrial pathway may be involved in quercetin-induced apoptosis in human tongue SCC SAS cell cells.

Quercetin induces apoptosis through the JNK, ERK1/2 and GSK3-α/β signaling pathways in human tongue SCC SAS cells. The present study also investigated the upstream signals for quercetin-induced mitochondrial apoptosis in SAS cells. Cells were treated with quercetin (50 µM) for 30–120 min. As shown in Fig. 3, quercetin significantly and time-dependently increased the protein expression levels of p-ERK1/2, p-JNK1/2 and p-GSK3-α/β, but not those of p-p38 and p-AMPKα in SAS cells. Furthermore, the roles of JNK and ERK in quercetin-induced apoptosis were investigated (Fig. 4). Cells were treated with or without a JNK inhibitor (SP600125, 20 µM) or an ERK inhibitor (PD98059, 20 µM) for 30 min, and were then treated with quercetin (50 µM) for 24 h. As shown in Fig. 4A, B and D, SP600125 and PD98059 effectively inhibited the quercetin-induced increase in protein and mRNA expression levels of caspase-3, caspase-7, caspase-9, PARP and Bax, and reversed the quercetin-induced decrease in the protein and mRNA expression levels of Bcl-2 in SAS cells. Furthermore, the role of GSK3 in quercetin-induced apoptosis was assessed. The cells were treated with or without the GSK3 inhibitor LiCl (100 µM) for 30 min and were then treated with quercetin (50 µM) for 16 or 24 h. As shown in Fig. 4C and D, LiCl effectively inhibited the increased protein and mRNA expression levels of caspase-3, caspase-7, caspase-9, PARP and Bax, and decreased the protein and mRNA expression levels of Bcl-2 in quercetin-treated cells. These inhibitors (SP600125, PD98059, and LiCl) could also significantly reverse the decreased MMP (Fig. 4E) and the increased caspase-3/7 activity (Fig. 4F) in quercetin-treated cells. These results indicated that quercetin-induced mitochondrial apoptosis may be regulated by JNK1/2, ERK1/2 and GSK3 signaling pathways in SAS cells.

The present study examined the relationship between JNK, ERK and GSK3-α/β signals in SAS cells in the presence or absence of quercetin. As shown in Fig. 5A, SP600125 effectively reduced the quercetin-increased protein expression levels of p-JNK1/2, p-ERK1/2 and p-GSK3-α/β. Moreover, PD98059 treatment effectively reduced the quercetin-increased protein expression levels of p-ERK/12 and p-GSK3-α/β but not p-JNK1/2 (Fig. 5B). Similarly, LiCl treatment effectively reduced the quercetin-increased protein expression levels of p-GSK3-α/β and p-ERK1/2 but not p-JNK1/2 (Fig. 5C). These results suggested that JNK1/2 activation downstream-regulated ERK1/2- and GSK3-α/β-mediated apoptotic signaling pathways may have important roles in quercetin-induced tongue SCC SAS cell apoptosis.