Quercetin was bought from Sigma (US). 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Amresco (US). Antibody against Foxo3a, JNK, p-JNK, p-ERK, p-p38 and Lamin B1 were from Cell Signaling Technology (Boston, US). Anti-β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, US). SP600125 was from Tocris (Avonmouth, UK). SB203580 and PD98059 were bought from Calbiochem (USA).

Human breast cancer cell line MDA-MB-231 (ATCC, HTB-26) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were maintained at 37℃ and 5% CO₂ in a humidified atmosphere. Cells were starved in serum-free media for 12 h prior to quercetin treatment.

Cells were plated at a density of 1×10⁴ in 96 well-plates. Different concentrations of quercetin were used to treat cells. After incubation time, media were replaced with fresh media containing MTT at final concentration of 0.5 mg/ml. After 2 h incubation at 37℃, supernatant was carefully removed, the insoluble formazan crystal was dissolved in 200 µl of dimethyl sulfoxide (DMSO) and optical density (OD) was measured at 570 nm wave lengths.

RNAs was extracted from cells cultured in 6 well-plates using Trizol reagent (Invitrogen, Carlsbad, CA, US). For RT-PCR, SuperScript II Reverse Transcriptase (Invitrogen) was used to convert RNAs (2 µg) to cDNA. One µl of cDNA (1:20 dilutions) were used for real-time PCR. qPCR products were detected with SYBR Green (2xGreen star qPCR MasterMix, Bioneer, Korea) on Rotor-Gene Q (Qiagen, Hilden, Germany). qPCR procedure was denaturation at 95℃ for 10 mins, 40 cycles of denaturation at 95℃ for 20 s, annealing at 60℃for 20 s and extension at 72℃ for 25 s. Gapdh gene was used as internal control. Relative expression was analyzed using ΔΔCT method. PCR primers were used as followed: Gapdh, 5'-TTCAGCTCAGGGATGACCTT-3' (forward) and 5'-ACCCAGAAGACTGTGGATGG-3' (reverse); Foxo3a, 5'-ACTCACTTAGCCACAGCGAT-3'(forward) and 5'-TGACCAAACTTCCCTGGTTA-3' (reverse); FasL, 5'-AACCAAGTGGACCTTGAGAC-3'(forwa rd) and 5'-TTCACATGGCAGCCCAGAGT-3' (reverse).

Cells were washed with cold PBS and lysed using RIPA buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1% Triton X-100, 1 mM dithiothreitol) containing protease inhibitor cocktail (Roche) and phosphatase inhibitors. Protein concentration was determined using Protein assay kit (Biorad) following the manufacture's protocol. Total protein was separated on SDS-polyacryramide gel electrophoresis and transferred to PVDF membrane. The membrane was then blocked in 5% skimmed milk, incubated with indicated primary antibodies (all antibodies were diluted 1:1000 in 3% BSA) for 2 h at room temperature and incubated with respective horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using the enhanced chemiluminescence reagents (Bionote, Korea) and signals were detected and quantified using Fusion-Fx system (Vilber Lourmat, Eberhardzell, Germany).

For examine nuclear translocation of Foxo3a, cytoplasmic and nuclear fractions were prepared with the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific). After treatment with quercetin, MDA-MB-231 cells (2×10⁶) were harvested. According to the manufacturers' instructions, the nuclear and cytoplasmic fractions were collected, followed by western blot analysis. β-actin and Lamin B1 were used as loading controls of cytoplasmic and nuclear fractions, respectively.

For knockdown using siRNA, cells were seeded in 6-well plates and transfected with 100 nM siFoxo3a or scrambled control siRNA using Mission siRNA transfection agent (Sigma). SiFoxo3a were chemically synthesized by Bioneer (Daejon, South Korea), sequence of siFoxo3a as followed: 5'-GACGAUGAUGCGCCUCUCU dTdT-3' (sense) and 5'-AGAGAGGCGCAUCAUCGUC dTdT-3' (antisense). Scrambled control siRNA (sc-37007) were from Santa Cruz Biotechnology.

For knockdown using shRNA, cells were transfected with the shFoxo3a (Openbiosystem) or co-transfected with shFoxo3a and reporter constructs, using Genefectine transfection reagent (Genetrone Biotech, Korea). Scramble shRNA (Addgene) was used as control.

Cells were seeded in 48-well plates at density of 5×10⁴ cells/well and transfection was carried out with Genefectine. Foxo3a reporter (FHRE-luc), p21 promoter reporter (WWP-luc), p53 reporter (pG13-Luc) or GADD45 promoter reporter (Gadd45-Luc) (Addgene) (200 ng/well) and control reporter (pRL-TK, Promega) (20 ng/well) were used for co-transfection. After 24 h, cells were treated with 20 µM quercetin. Luciferase activities were measured after 24 h in luminometer (Glomax, Promega, CA, US) using a Dual-Luciferase assay kit (Promega), according to the manufacture's recommendations. Value of luciferase activity was normalized to Renilla luciferase activity.

MDA-MB-231 cells were prepared in 6 well-plates. After exposure to 20 µM quercetin for 24 h or 48 h, cells were collected and subjected to Annexin V and propidium iodide (PI) staining using FITC Annexin V Apoptosis Detection kit I (BD Pharmingen), following the protocol provided by the manufacturer. Apoptotic cells were then analyzed by flow cytometry (FACSCalibur, BD Biosciences, San Diego, CA, USA). Post-compensation and analysis of FACS data were done using Flowing 2.5.1 version software.

After treatment with 20 µM quercetin for 48 h, cells were washed twice with PBS and fixed with 70% ethanol for 1 h at 4℃, and then resuspended with PBS containing 20 µg/ml PI (Sigma) and 50 µg/ml RNase A. Cells were incubated at room temperature in the dark for 30 mins and cell cycle was analyzed by flow cytometry. The data was analyzed using Cell Quest Pro 6.0 (BD Biosciences) and ModFit LT software (BD Biosciences).

Cells grown on coverslips were fixed in 4% paraformandehyde in PBS for 10 mins at 4℃, permeablized in 0.25% Triton X-100 in PBS for 10 mins, and blocked in 5% donkey serum (Chemicon) and 0.3% Triton X-100 in PBS for 30 mins at RT. Next, cells were incubated with rabbit polyclonal anti-Foxo3a antibody (Cell signaling) (1:200) in 1% BSA and 0.3% Trition X-100 in PBS for overnight at 4℃. After that, incubation of cells with Alexa 488-conjugated goat anti-rabbit IgG (Molecular Probes) (1:1000) in 1% BSA and 0.3% Trition in PBS was carried out in RT. After deeply washing the cells with PBS, DAPI (1 µg/ml in PBS) was added to stain nuclei. Cells were mounted with Fluoromount-G (Southern biotech) and images were taken by a fluorescence microscope.

Statistical differences were analyzed by Graphpad Prism 5.0 (San Diego, CA) and evaluated by two-tailed Student's t-test. All data was presented as mean±SEM of at least three independent experiments. p<0.05 was considered statistically significant.

To determine the effect of quercetin on TNBC cells, we treated MDA-MB-231 cells with various quercetin concentrations (2.5~80 µM) for 24 h, 48 h and 72 h and cell viability was determined by MTT assay. As shown in Fig. 1, quercetin demonstrated dose- and time-dependent effects on reduction of the cell viability in MDA-MB-231 cells. Treatment with either 40 µM or 80 µM of quercetin for 24 h reduced cell viability about 20% and 35%, respectively, while treatment with lower concentrations (2.5~20 µM) just caused a minor reduction in cell viability (about 5~10%) in comparison with control. However, the reduction in cell viability increased steadily from 24 h to 72 h in quercetin treatment with all doses.

Further, to study whether the reduced viability was due to cell apoptosis triggered by quercetin treatment, FACS analysis was used to detect apoptotic cells followed by treatment of 20 µM quercetin at 24 h and 48 h. Results showed an increase of 15% apoptotic cells (cells with Annexin V positive) after 48 h of quercetin treatment. However, no significant difference for cell death was observed after 24 h of quercetin treatment, compared with control (Fig. 2A). Obtained results are consistent with MTT results as treatment with 20 µM of quercetin for 24 h did not affect cell viability and apoptosis significantly. However, after 48 h of quercetin treatment, cell viability was reduced and population of apoptotic cells was increased significantly. The increase of apoptotic cell death (about 15%) was less than the decrease of cell viability observed by MTT assay (about 30%). It may be further explained by growth arrest. Therefore, we analyzed changes in cell cycle after 48 h of stimulation with quercetin (20 µM) using flow cytometry. As shown in Fig. 2B, quercetin caused cell cycle arrest at S and G2/M phase. Namely, treatment with quercetin resulted in 3 fold increase in cell population of S phase and 2 fold increase in cell population of G2/M phase, along with 30% reduction in cell population of G0/G1 phase. Fas ligand (FasL) is a pro-apoptotic factor that can activate Fas receptor leading to apoptosis. Here we observed that expression of FasL mRNA was enhanced by quercetin after 24 h treatment (Fig. 2C). p53 is known as a cell cycle regulator that activates and inhibit genes involved in cell cycle. GADD45, a p53-regulated and DNA damage-inducible protein have been shown to induce G2-M cell cycle arrest [2829]. p21 (p21^WAF1/Cip1), a protein transcriptionally regulated by p53 has been reported to inhibit CDK2 activity, leading to G1/S arrest. [30]. Therefore, signaling activities of p53, p21 and GADD45 were analyzed after quercetin treatment. Fig. 2D shows that quercetin treatment induced signaling activity of p53 (about 3 folds) and promoter activities of p21 (4 folds) and GADD45 (3 folds), suggesting a possibility that increased activities of p53, p21 and GADD45 might lead to cell cycle arrest triggered by quercetin.

Above data showed that quercetin significantly induced apoptosis and cell cycle arrest in breast cancer cells. Identification of signaling molecules that are regulated by quercetin and can contribute to apoptosis and cell cycle arrest was required. It is known that Foxo3a signaling pathway is involved in cell apoptosis and cell cycle arrest by regulating transcription of genes involved in triggering apoptosis [31] and inhibiting cell cycle [32]. Therefore, any involvement of Foxo3a after quercetin treatment in MDA-MB-231 cells was investigated. Data showed that quercetin treatment for 24 h induced about 3 folds of Foxo3a activity in breast cancer cells, as detected by Luciferase assay (Fig. 3A). Further, to elucidate whether quercetin regulated Foxo3a activity was affecting at gene or protein level, we measured mRNA and protein levels of Foxo3a in quercetin-treated cancer cells. The result displayed that quercetin did not affect gene expression of Foxo3a after 12 h, 24 h and 48 h of treatment (Fig. 3B), however, quercetin triggered a steady increase in protein level of Foxo3a after 3~12 h of treatment, maximal increment being 5 folds after 12 h of treatment (Fig. 3C).

Since transcriptional activity of Foxo3a requires its nuclear translocation, we tried to examine the subcellular localization of Foxo3a in quercetin-treated MDA-MB-231 cells. Our immunofluorescence analysis shows increased nuclear Foxo3a of quercetin-treated cells compared to control (Fig. 3D). The result obtained was further confirmed by western blot with nuclear and cytoplasmic fractions. The data shows that quercetin-treated MDA-MB-231 cells exhibited 6~7 folds increases in nuclear Foxo3a protein levels at 6~24 h of treatment, in comparison with control at 0 h. However, no change of cytoplasmic protein levels of Foxo3a by quercetin treatment was observed (Fig. 3E). The results indicate that overall elevated Foxo3a protein in quercetin-treated MDA-MB-231 is associated with its increased levels in nuclei.

Since quercetin regulated Foxo3a activity, it may be essential to clarify upstream signaling pathways responsible for regulation of Foxo3a activity in quercetin treatment. Therefore, we investigated whether activation of several signaling pathways such as ERK, p38, JNK was induced by quercetin. Western blot analysis using antibodies against phosphorylated forms of ERK, p38 and JNK was carried out to determine activation of respective signaling pathways. As presented in Fig. 4A, a significant increase of JNK activation (p-JNK/JNK) was observed after 1~2 h of quercetin treatment. This activation of JNK might be responsible for increased Foxo3a protein level as observed after 3~12 h of quercetin treatment. However, the other signaling pathway activation (p-ERK and p-p38) showed no significant alteration by quercetin.

To confirm that JNK is one of main signaling pathway participating Foxo3a activity regulation, a JNK specific inhibitor (SP600125) was used. Results showed that pre-treatment with 1 µM of JNK inhibitor (SP600125) completely abolished the effect of quercetin on elevation of protein level and transcriptional activity of Foxo3a (Fig. 4A and 4B). Taken together, the data suggests that JNK signaling pathway participated in regulation of Foxo3a activity in quercetin-treated breast cancer cells.

Moreover, involvement of p38 and ERK signaling pathway in increased Foxo3a activity in response to quercetin treatment was further confirmed. The result shows that treatment with 20 µM of p38 inhibitor SB203580 or 50 µM of ERK inhibitor PD98059 [33] did not affect on quercetin-induced Foxo3a activity (data not shown), suggesting that p38 and ERK signaling pathways may be not necessary for the induction of Foxo3a in response to quercetin treatment.

To verify whether Foxo3a was involved in apoptosis and cell cycle arrest triggered by quercetin treatment, MDA-MB-231 cells were transiently transfected with Foxo3a-specific siRNA to deplete the endogenous expression of Foxo3a and the cells transfected with scramble siRNA was used as control. As shown in Fig. 5A, quercetin-induced apoptosis was reduced significantly in Foxo3a siRNA-transfected cells, compared to control. Moreover, Foxo3a knockdown reduced the effect of quercetin on increasing cell population in S and G2/M phases significantly (Fig. 5B). The result suggests that Foxo3a may regulate apoptosis and cell cycle arrest induced by quercetin.

Moreover, effect of Foxo3a knockdown on increased activities of p53, p21 and GADD45 signaling in quercetin treatment was analyzed. MDA-MB-231 cells were co-transfected with Foxo3a shRNA (or scramble shRNA for control) and a plasmid containing p21, p53 or GADD45 luciferase reporter gene prior to quercetin treatment for 24 h. As shown in Fig. 5C, the increase in the reporter activities of p53, p21 and GADD45 were abolished in Foxo3a knockdown cells compared with control. This data suggests that Foxo3a was possibly involved in regulation of p53, p21, and GADD45 signaling activities enhanced by quercetin.

As JNK signaling pathway was involved in regulation of Foxo3a in quercetin treatment, we also examined the involvement of JNK in induction of p21, p53 and GADD45 signaling activities. Similarly to Foxo3a knockdown treatment, JNK inhibitor also abolished the effect of quercetin on increasing reporter activities of p21, p53 and GADD45 in MDA-MB-231 cells (Fig. 5D).