Quercetin (cat. no. Q4951; ≥95%), propidium iodide (PI), Trypsin-EDTA, L-glutamine and penicillin-streptomycin were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fluo-3/AM, dihexyloxacarbocyanine iodide (DiOC₆) and dichloro-dihydro-fluorescein diacetate (H₂DCF-DA) were obtained by Invitrogen; Thermo Fisher Scientific, Inc.

Human oral cancer cells SAS cells were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). These cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine, and were cultured at 37°C in a humidified incubator in an atmosphere containing 5% CO₂ (26,27).

SAS cells (1×10⁵ cells/well) were placed in 12-well plates with DMEM for 24 h then quercetin (40 µM) or 1% dimethyl sulfoxide as a vehicle control was added to each well for 0, 12, 24 and 48 h. In order to examine morphological changes, cells in each well were examined and images were captured using contrast phase microscopy at a magnification, ×400. To measure the percentage of viable cells, cells were collected from each treatment well, counted and stained with PI (5 µg/ml) at room temperature in the dark then immediately analyzed using a Flow Cytometry system (BD Biosciences, San Jose, CA, USA) assay as previously described (26,28).

Cell apoptosis was measured using an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences) as described previously (29,30). Briefly, SAS cells (5×10⁴ cells/ml) in 12-well culture plates were treated with quercetin (40 µM) for 24 and 48 h or 1% DMSO as a vehicle control. Cells were harvested and then re-suspended in Annexin V binding buffer, followed by incubation with Annexin V-FITC/PI in the dark for 15 min according to the manufacturer's protocol for labeling of apoptotic cells (29,30). In each experiment, 1×10⁴ cells were analyzed using Cell Quest^™ program (Version 5.2.1; BD Biosciences). Experiments were performed in triplicate.

Flow cytometry was used to measure the levels of ROS, Ca²⁺ and MMP in SAS cells following exposure to quercetin. SAS cells (1×10⁵ cells/well) were placed in 12-well plates and were treated with 40 µM quercetin or 1% DMSO as a vehicle control for various time periods (1, 3, 6, 9, 12, 24 and 48 h). Cells were isolated and re-suspended in 500 µl H₂DCF-DA (10 µM), then kept in darkness for 30 min at 37°C to measure the levels of ROS (H₂O₂); re-suspended in 500 µl DiOC₆ (4 µmol/l) and maintained in darkness for 30 min at 37°C to measure the levels of MMP; or re-suspended in 500 µl of Fluo-3/AM (2.5 µg/ml) and kept in darkness for 30 min at 37°C for intracellular Ca²⁺ concentrations measurement. After 30 min of incubation, all samples were analyzed using flow cytometry as described previously (30,31).

Flow cytometry was used to measure the activities of caspase-3, caspase-8 and caspase-9. SAS cells (1×10⁵ cells/well) were incubated with 40 µM quercetin or 1% DMSO as a vehicle control for 6, 24 and 48 h, harvested, washed with 1X PBS and re-suspended in 25 µl 10 µM substrate solution sourced from caspase activity assay kits (PhiPhiLux-G1D1 for caspase-3, CaspaLux8-L1D2 for caspase-8 and CaspaLux9-M1D2 for caspase-9, OncoImmunin, Inc., Gaithersburg, MD, USA) prior to incubation at 37°C for 60 min. Following incubation, cells were washed with PBS and were immediately analyzed using flow cytometry as described previously (29,30).

SAS cells (1.5×10⁶ cells/dish) were incubated in a 10-cm dish for 24 h at 37°C, then incubated with 40 µM quercetin for 6, 12, 24 and 48 h or 1% DMSO as a vehicle control. Cells were collected then lysed and denatured using ice-cold lysis buffer for 1 h (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 0.3 mM PMSF, 0.2 mM sodium orthovanadate, 0.1% SDS, 1 mM EDTA, 1% NP-40, 10 mg/ml leupeptin and 10 mg/ml aprotinin). Total protein quantity was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as described previously (29). Equal amounts of total protein (40 µg) were separated using SDS-PAGE (12% v/v) and then transferred onto polyvinylidene difluoride membranes. Following blocking with 5% non-fat dried milk in PBS with Tween-20 for 1 h at room temperature, membranes were immunoblotted with specific primary antibodies [Apoptosis-inducing factor (AIF; cat. no. sc-13116), activating transcription factor-6α (ATF-6α; cat. no. sc-22799), ATF-6β (cat. no. sc-30597), cytochrome c (Cyto c; cat. no. sc-13560), endonuclease G (Endo G; cat. no. sc-365359), gastrin-releasing peptide-78 (GRP-78; cat. no. sc-13968), iron-responsive element-1α (IRE-1α; cat. no. sc-20790), X-box binding protein 1 (XBP-1; cat. no. sc-7160), TNF-related apoptosis-inducing ligand (TRAIL; cat. no. sc-80393) antibodies were supplied by Santa-Cruz Biotechnology, Inc. (Dallas, TX, USA; all dilution, 1:1,000); apoptotic protease activating factor 1 (Apaf-1; cat. no. 8723), Bcl-2-associated death promoter (Bad; cat. no. 9239), Bcl-2 homologous antagonist killer (Bak; cat. no. 12105), B-cell lymphoma 2 (Bcl-2; cat. no. 2870), Caspase-3 (cat. no. 9665s), Caspase-8 (cat. no. 9746), Caspase-9 (cat. no. 9508) antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA; all dilution, 1:1,000); Caspase-6 (cat. no. AB10512; dilution, 1:2,000), BH3 interacting-domain death antagonist (Bid; cat. no. AB1730; dilution, 1:500), Fas-associated protein with death domain (FADD; cat. no. 05-486; dilution, 1:1,000), Fas ligand (Fas-L; cat. no. 05-571; dilution, 1:2,000) antibodies were supplies by EMD Millipore (Billerica, MA, USA); Caspase-4 (cat. no. 556459; dilution, 1:500) and Fas (cat. no. 610198, dilution, 1:5,000) antibodies were obtained from BD Biosciences (Bedford, MA, USA); poly(ADP-ribose) polymerase (PARP; cat. no. ab6079-1; dilution, 1:400) and Caspase-7 (cat. no. ab181579; dilution, 1:5,000) antibodies were obtained from Abcam (Cambridge, MA, USA); Bcl-extra large (Bcl-x; cat. no. B9304; dilution, 1:2,000), Caspase-2 (cat. no. C7349; dilution, 1:200) and β-actin (cat. no. A5316; dilution, 1:10,000) antibodies were supplied by Sigma-Aldrich; Merck KGaA] for 1 h at room temperature (25°C). Subsequently, the membranes were incubated with secondary antibodies [horseradish peroxidase (HRP)-conjugated mouse immunoglobulin G (IgG; cat. no. GTX213112) and rabbit HRP-conjugated IgG secondary antibodies (cat. no. GTX213110) at dilution, 1:5,000 (GeneTex, Inc., Irvine, CA, USA)] for 1 h at room temperature. Chemiluminescence signals were enhanced using enhanced chemiluminescence reagent (EMD Millipore) (27,29,30), and images were captured using the MultiGel-21 Image system (TOP BIO CO., Taipei, Taiwan).

SAS cells (1×10⁵ cells/well) were maintained on 6-well chamber slides with or without 40 µM quercetin or 1% DMSO as a vehicle control for 48 h and then fixed in 4% formaldehyde in PBS for 15 min at room temperature. Triton X-100 in PBS (0.1%) was added to the cells for 15 min at room temperature, followed by 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature to block non-specific binding sites. Cells were then incubated overnight with primary antibodies, including anti-Cyto c (cat. no. sc-13560; 1:250 dilution), AIF (cat. no. sc-13116; 1:250 dilution) and anti-Endo G (cat. no. sc-365359; 1:250 dilution) (all in green fluorescence) at 4°C, and then followed by incubation with a secondary antibody (FITC-conjugated goat anti-mouse IgG; cat. no. 115-545-003; 1:100 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) for 1 h at room temperature, and PI (red fluorescence) staining for examination as described previously (30). Slides were mounted, examined with oil immersion and images (magnification, ×630) were captured using a Leica TCS SP2 Confocal Spectral Microscope (Leica Microsystems GmbH, Mannheim, Germany).

All data are expressed as the mean ± standard deviation. Differences between groups were analyzed using one-way analysis of variance followed by the Dunnett's method. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SigmaPlot for Windows (version 12.0; Systat Software, Inc., San Jose, CA).

SAS cells were treated with 40 µM of quercetin for 0, 12, 24 and 48 h. Cells were examined for morphological changes and the percentage of viable cells. The results indicated that quercetin induced cell morphological changes including cell shrinkage and cell floating, and significantly decreased the quantity of viable cells (Fig. 1). These effects were time-dependent. A 40 µM dose of quercetin decreased cell viability to ~50% when compared with the control group (0 h) that was selected for all experiments.

In order to investigate the effects of quercetin on apoptosis in SAS cells, Annexin V/PI-double staining was performed. The results of dot plots (Fig. 2A) indicated that quercetin induced apoptosis of cells, with a increase in the percentage of early apoptotic cells detected at 24 h (5.97%) and 48 h (21.06%). The percentage of apoptotic cells at different times are presented in Fig. 2B; the results indicate that quercetin significantly induced early-stage apoptosis in a time-dependent manner.

In order to investigate whether the cell apoptosis induced by quercetin in SAS cells involve the production of ROS and Ca²⁺ or dysfunction of mitochondrial, cells were treated with quercetin for various time periods and then analyzed using a flow cytometric assay. Fig. 3A and B identified that quercetin significantly increased ROS production following 3 h treatment. Fig. 3C and D showed that quercetin treatment significantly decreased the levels of ΔΨ_m following 24 h treatment. In addition, it was demonstrated that quercetin significantly promoted Ca²⁺ production following 24 h treatment (Fig. 3E and F).

It was hypothesized that quercetin was able to induce cell apoptosis in SAS cells through the activation of caspases. Following treatment of SAS cells with quercetin (40 µM) for various time periods, caspase-3, −9 and −8 activities were assayed using flow cytometric analysis. The results indicated that quercetin significantly increased the activities of caspase-3 (Fig. 4A and B), caspase-9 (Fig. 4C and D) and caspase-8 (Fig. 4E and F) in a time-dependent manner.

In order to understand whether quercetin induced the apoptosis of SAS cells through the effects of apoptosis-associated proteins, SAS cells were treated with quercetin (40 µM) for various time periods and then the levels of apoptosis-associated proteins were measured using western blotting. The results demonstrated that quercetin markedly increased the expression of caspase-2, Bak, Bid and Bad (Fig. 5A), Cyto c, Apaf-1, Endo G, AIF and PARP (Fig. 5B), active form of caspase-9, caspase-3, caspase-6 and −7 (Fig. 5C), TRAIL, Fas-L, Fas, FADD, and active form of caspase-8 (Fig. 5D), ATF-6α, ATF-6β, XBP-1, IRE-1α, caspase-4 and GRP-78 (Fig. 5E), but inhibited the expression of Bcl-2 and Bcl-x (Fig. 5A), pro-caspase-3 (Fig. 5C). These results suggested that quercetin induced apoptosis of SAS cells via cell surface receptor (Fas-L and Fas) and mitochondria-dependent pathways.

In order to investigate the effect of quercetin on the release and translocation of Cyto c, AIF, and Endo G during apoptosis of SAS cells, cells were treated with or without 40 µM of quercetin for 48 h, then stained by anti-Cyto c, -AIF and -Endo G. Images were captured using a confocal laser microscopic system. The results revealed that quercetin markedly increased cytochrome c (Fig. 6A), AIF (Fig. 6B) and Endo G (Fig. 6C) release from mitochondria to the cytoplasm in SAS cells compared with the corresponding control group.