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Quercetin induces growth arrest through activation of FOXO1
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transcription factor in EGFR-overexpressing oral cancer cells
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Chun-Yin Huang a, Chien-Yi Chan a,†, I-Tai Chou a,†, Chia-Hsien Lien a, Hsiao-Chi Hung a,
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Ming-Fen Lee b,*
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a
Department of Nutrition, China Medical University, Taichung, Taiwan, ROC
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b
Department of Nutrition and Health Sciences, Chang Jung Christian University, Tainan,
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Taiwan, ROC
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Correspondence: Dr. Ming-Fen Lee, Department of Nutrition and Health Sciences, Chang
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Jung Christian University, 396 Sec. 1 Changrong Rd., Gueiren Dist., Tainan, Taiwan. Tel:
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+886 6 2785123 x3308; Fax: +886 6 2785926, E-mail: leemf@mail.cjcu.edu.tw
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†
These authors contributed equally.
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Running title: Quercetin, FOXO1, EGFR, and oral cancer
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Keywords: EGFR; FOXO1; oral cancer; quercetin
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Funding sources: This work was supported by the grants, CMU97-303 (China Medical
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University) and NSC98-2320-B-039-021-MY3 (National Science Council) to Chun-Yin
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Huang, and NSC98-2320-B-309-002-MY3 and NSC101-2320-B-309-001 (National Science
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Council) to Ming-Fen Lee.
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Abstract
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The squamous cell carcinomas of the head and neck (SCCHN) with aberrant epidermal
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growth factor receptor (EGFR) signaling are often associated with poor prognosis and low
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survival. Therefore, efficient inhibition of the EGFR signaling could intervene with the
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development of malignancy. Quercetin appears anti-tumorigenesis, but the underlying
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mechanism remains unclear in oral cancer. Fork-head box O (FOXO) transcription factors,
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Akt downstream effectors, are important regulators of cell growth. Here, we hypothesized
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that FOXO1 might be crucial in quercetin-induced growth inhibition in
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EGFR-overexpressing oral cancer. Quercetin treatment suppressed cell growth by inducing
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G2 arrest and apoptosis in EGFR-overexpressing HSC-3 and TW206 oral cancer cells.
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Quercetin inhibited EGFR/Akt activation with a concomitant induction of FOXO1 activation.
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FOXO1-knockdown attenuated quercetin-induced p21 and FasL expression and subsequent
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G2 arrest and apoptosis, respectively. Likewise, quercetin suppressed tumor growth in HSC-3
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xenograft mice. Taken together, our data indicate that quercetin is an effective anti-cancer
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agent and that FOXO1 is crucial in quercetin-induced growth suppression in
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EGFR-overexpressing oral cancer.
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3
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1. Introduction
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The invasive squamous cell carcinomas of the head and neck (SCCHN) frequently
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overexpress epidermal growth factor receptor (EGFR) [1], leading to unfavorable clinical
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outcome–high recurrence and low survival rates [2, 3]. Aberrant EGFR/PI3K/Akt signaling
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pathway is often responsible for the malignant phenotype [4]. Although several
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EGFR-targeting drugs are clinically available [5-7], their wide application is limited by the
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resistance to the drugs and mutations in the EGFR downstream effectors in some patients [8].
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Therefore, to improve therapeutic efficacy, new anti-cancer agent should be considered
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accordingly.
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Quercetin (3,3’,4’,5,7-pentahydroxyflavone) is one of the major dietary flavonoids
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found in a wide range of fruits, vegetables, and beverages [9]. Among those, onion, apple,
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and red wine are good sources of quercetin. The antioxidant, anti-inflammatory, as well as
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anti-proliferative and apoptosis aspects of quercetin have been widely investigated [9]. A
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phase I clinical trial indicated that quercetin can be safely administered and its plasma levels
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are sufficient to inhibit lymphocyte tyrosine kinase activity [10]. Consumption of quercetin
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from onions and apples was inversely associated with lung cancer risk, supporting the
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chemopreventive effect of quercetin [11]. The chemotherapeutic efficacy of quercetin has
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also been demonstrated in many types of cancer [12-18]. Quercetin exerts its anti-cancer
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property mainly through induction of growth arrest in G1 or G2 [14, 15, 17, 19], apoptosis
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[15, 16, 20], and inhibition of angiogenesis [13] as evidenced by down-regulating the
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expression of oncogenes (HER2 [21], H-ras, K-ras, c-myc [22], or COX-2 [13] ) and mutant
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p53 [23], or up-regulating cell cycle control proteins (p21WAF1 and p27KIP1) [18, 19, 24].
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In addition, quercetin inhibits several tyrosine and serine–threonine kinases, whose activities
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are linked to cell surface receptors-transduced survival pathways (PI3K/Akt/PKB) [25-27].
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Nonetheless, the effect of quercetin on oral cancers remains uncertain. Quercetin seems to
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induce apoptosis in some human oral cancer cell lines [28-30]; however, the underlying
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mechanisms are still elusive.
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Fork-head box O (FOXO) transcription factors share a conserved “fork-head box”
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DNA-binding domain, which comprise four members in mammals: FOXO1, FOXO3a,
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FOXO4, and FOXO6 [31, 32]. FOXO factors are involved in a wide range of biological
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processes, including cell cycle arrest, apoptosis, DNA repair, glucose metabolism, oxidative
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stress resistance, and longevity [32]. The biological activity of FOXO factors is highly
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dependent on their intracellular trafficking, mediated by post-translational modifications in
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phosphorylation, acetylation, or ubiquitination [33]. In addition, FOXO factors share highly
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conserved sites within and nearby their DNA binding domains for phosphorylation by the
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survival PI3K/Akt signaling pathway. The growth factors-mediated Akt phosphorylation on
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these conserved residues negatively regulates FOXO expression and activity [34]. In this
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regard, FOXO factors are implicated in pathogenesis including aging, diabetes, immune
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disease, infertility, neurodegeneration, and cancer [35]. Among FOXO factors, Increased
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p-FOXO1 or decreased FOXO1 expression is often associated with tumorigenesis [36].
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FOXO1 may exert its tumor suppression function through its transcription-dependent
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expression on growth arrest and apoptosis-related genes, including p15, p19, NOXA, FasL,
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TRAIL, and Bim [37-39]. The promoters of these genes comprise the consensus sequence
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T/C-G/A-A-A-A-C-A-A recognized by FOXO1 [35]. Alternatively, FOXO1 can mediate
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cancer cell death through induction of autophagy which is independent of its transcriptional
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activity [40]. Similarly, FOXO1 inhibits Runx2-mediated cell migration and invasion
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independent of its transcriptional activity [41]. Together, FOXO1 appears to be a potential
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therapeutic target of anti-cancer reagents [42-46].
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Aberrant EGFR expression and PI3K mutation are often observed in invasive SCCHN;
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however, the role of transcription factor FOXO1 as a downstream target of activated
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EGFR/PI3K/Akt signaling axis in oral cancer still remains unclear. Therefore, in the present
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study, we aimed to investigate the anti-cancer efficacy of quercetin in human oral cancer cell
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lines with elevated EGFR expression, and to further identify the crucial role of FOXO1 factor
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in quercetin-mediated growth suppression.
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2. Materials and methods
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2.1 Reagents, antibodies, and plasmids
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All chemicals including quercetin were purchased from Sigma (St. Louis, MO) and
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antibodies were purchased from Cell Signaling Technology (Beverly, MA), respectively,
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unless specified otherwise. PVDF membrane and ECL detection reagents were from Perkin
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Elmer Life Sciences, Inc. The annexin V-FITC apoptosis detection kit was purchased from
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BD Biosciences (San Jose, CA). Antibody against cyclin D1 was purchased from Santa Cruz
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Biotechnology (Santa Cruz, CA) and anti-α-tubulin antibody was purchased from Abcam
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(Cambridge, MA). FOXO1-specific siRNA and the Lipofectamine RNAiMAX reagent were
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purchased from Invitrogen (Grand Island, NY). The pGL3-FHRE-luc plasmid was obtained
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from Addgene (Cambridge, USA; Addgene database plasmid 1789) and the PolyJet In Vitro
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DNA Transfection Reagent was purchased from SignaGen Laboratories (Ijamsville, MD).
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The NE-PER Nuclear and Cytoplasmic Extraction Reagents were purchased from Thermo
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Fisher Scientific (Rockford, IL).
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2.2 Cell culture and treatment
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HSC-3 and TW206 human oral squamous carcinoma and HGF human gingival fibroblasts
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cells were kind gifts of Dr. Hsin-Ling Yang at China Medical University (Taichung, Taiwan).
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HSC-3 and HGF cells were maintained in DMEM-F12 and DMEM (Invitrogen), respectively,
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supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antibimycotic (Gibco).
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All cells were cultured with 5% CO2 at 37°C.
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2.3 Cell viability assay
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Cell viability was measured by trypan blue method. Cells were allowed to grow in 6-well
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culture plates and then treated with various concentrations of quercetin (0-50 μM) for
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indicated hours. Then, cells were trypsinized and viable cells were stained with trypan blue
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for hemocytometric counting.
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2.4 Western blotting
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Cells were washed with cold PBS and lysed in RIPA buffer containing 150 mM NaCl, 10
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mM Tris (pH 7.2), 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, and
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protease/phosphatase inhibitors. Protein concentration was determined by BCA protein assay,
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and denatured proteins were separated in 8% to 15% SDS-PAGE, and transferred onto PVDF
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membranes. Nonspecific binding was blocked with 5% milk in TBST buffer (20 mM Tris
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base, 140 mM NaCl, pH 7.6, 0.1% Tween-20) for 1 h, followed by incubation with primary
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antibodies at 4°C overnight and secondary antibodies at room temperature for 1 h. Blots were
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visualized by ECL detection reagents.
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2.5 Cell cycle analysis
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HSC-3 and TW206 cells were seeded onto 6-well plates and serum-starved for 18 h followed
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by quercetin treatment in growth media for 24 h. Cells were then trypsinized, washed with
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PBS, and fixed in cold 70% ethanol at -20 °C. The fixed cells were collected and stained in a
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solution containing 0.5 ml of 4 μg/ml of PI, 0.5 mg/ml of RNase, and 1% Triton X-100 for 30
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min at 4 °C. DNA content of these cells was analyzed using a FACScan flow cytometer
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(Becton Dickinson).
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2.6 Colony formation assay
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After incubation with the indicated concentration of quercetin, cells were washed with PBS,
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fixed with 10% formalin (Mallinckrodt Chemicals) for 10 min, and stained with 0.05%
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crystal violet (Panreac Quimica S.A.U.) for 30 min. Dishes were subjected to colony
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counting and optical density measurement at 540 nm.
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2.7 Apoptosis analysis
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Cells were seeded onto 6-well plates in growth media and treated with quercetin for 24 h.
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Cells were then trypsinized, washed with PBS, and resuspended in binding buffer (10 mM
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Hepes/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2) at a concentration of 1×106
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cells/ml. One hundred microliters of the lysis solution were transferred to a polystyrene tube,
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followed by 5 μl of annexin V-FITC and 5 μl of PI. The mixtures were sheltered from light
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and incubated for 15 min at room temperature. Finally, 400 μl of 1× binding buffer was
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added to each tube and cells were analyzed using a FACScan flow cytometer (Becton
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Dickinson).
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2.8 Reporter assay
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The pGL3-FHRE-luc and β-galactosidase plasmids with or without FOXO1 siRNA were
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co-transfected into HSC-3 cells for 24 h, followed by another 24 h of quercetin treatment.
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Cell lysates were collected in lysis solution (Applied Biosystems, Carlsbad, CA) according to
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the manufacturer’s protocol. The Dual Light® System (Applied Biosystems) was used to
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quantify luciferase and β-galactosidase activities.
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2.9 Xenograft mice
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Seven-week-old male nude mice were purchased from National Laboratory Animal Center
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(Taipei, Taiwan). The animal study protocol, IACUC 98-71-N, was reviewed and approved
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by the Institutional Animal Care and Use Committee (IACUC) of the China Medical
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University. The animals were given free access to autoclaved food and water during the study.
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After 2-week of acclimation, animals were implanted subcutaneously with HSC-3 cells (3 X
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106) suspended with liquid Matrigel to a final volume of 100 μl into the flank regions and
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randomly assigned into the control or quercetin group. Tumor size was recorded every other
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day for 40 days. Two weeks after the implantation, quercetin (3 mg/kg/day) or PBS was
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given via gavage feeding. Tumor volume = ½(length × width2).
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2.10 Statistical analysis
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Data are expressed as mean ± SD from at least three independent experiments. Statistical
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significance was analyzed using Student’s t test or Duncan’s multiple range tests. Results
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were considered significantly different at p < 0.05.
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3
Results
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3.1 Quercetin inhibits cell growth in human oral cancer cells
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The growth-inhibitory effect of quercetin has been shown in various types of cancer; however,
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its efficacy on oral squamous cancer cells remains elusive. To examine the effect of quercetin
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on oral cancer, we exposed two human tongue squamous carcinoma cell lines, HSC-3 and
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TW206 [47], and a control, human gingival fibroblast HGF cells, to various concentrations of
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quercetin. Quercetin potently inhibited HSC-3 cell growth as compared with TW206 and
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HGF cells (Fig. 1A). The IC50 of quercetin in HSC-3 cells was around 20 μM, much less
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than that of TW206 (45 μM). These results indicate that the metastatic HSC-3 cell line is
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more susceptible to quercetin than the other two cell lines tested. Indeed, quercetin
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supplementation for 7 days significantly suppressed HSC-3 colony formation in a
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dose-dependent manner with an IC50 of 7 μM (Fig. 1B). These data support quercetin as a
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potential anti-cancer reagent in oral squamous carcinomas.
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Cell cycle arrest and apoptosis are two main factors responsible for growth suppression.
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Quercetin has been shown to cause growth arrest in various cancer cells [14, 15, 17, 19]. To
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investigate the effect of quercetin on cell proliferation in oral cancer, we performed flow
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cytometric analysis for cell cycle distribution upon quercetin addition. In both HSC-3 and
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TW206 cells, quercetin (20 μM) treatment for 24 h resulted in a significant increase of cell
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numbers in G2/M phase (Fig. 2A), with concomitant reductions of G2/M phase cyclins,
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cyclin A and B1 [48], whereas quercetin showed no suppressive effect on G1 phase cyclin,
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cyclin D1 (Fig. 2B). In addition, quercetin induced the expression of p21 but not negative G1
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regulator, p15 [49] (Fig. 2B). These results demonstrate that quercetin is a potent inducer of
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growth arrest in human oral squamous carcinoma cells.
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Next, we evaluated the apoptotic efficacy of quercetin in oral cancer cells. Upon
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quercetin treatment (0-30 μM), both HSC-3 and TW206 cells displayed an accumulation of
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apoptotic cells in a dose-dependent manner (Fig. 2C). The apoptosis-inducing effect was
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further confirmed by the observations of the condensed chromatin staining (Fig. 2D) and the
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increased FasL and cleaved caspase 3 (Fig. 2E) upon quercetin treatment in HSC-3 cells.
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However, quercetin had little effect on the expression of Bax, a pro-apoptotic member of the
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Bcl-2 family (Fig. 2E). These results suggest the potential involvement of FasL-mediated
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extrinsic apoptotic pathway in quercetin-mediated cell death of HSC-3. Taken together, both
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growth arrest and apoptosis are responsible for the anti-cancer effect of quercetin in oral
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squamous carcinoma cells.
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3.2 Quercetin modulates the EGFR/Akt signaling axis
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Aberrant EGFR activation is the key cause of oral cancer malignancy. Therefore, we studied
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the effect of quercetin on EGFR signaling. In EGFR-overexpressing HSC-3 cells (Fig. 3A),
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the protein levels of p-Y1086-EGFR and its downstream effector, p-Akt, were decreased
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upon quercetin treatment in both time- (Fig. 3B) and dose-(Fig. 3C) dependent patterns,
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whereas the p-Y1045-EGFR levels were gradually increased (Fig. 3B). It is noteworthy that
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the phosphorylation at tyrosine 1045 is responsible for c-Cbl-mediated ubiquitination and
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degradation of EGFR protein [50]. These results suggest quercetin as an important modulator
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of the EGFR/Akt pathway.
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3.3 Quercetin regulates FOXO1 activation
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FOXO transcription factors play a role in the regulation of cell growth and apoptosis. The
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activity of FOXO factors can be regulated by Akt-mediated nucleus exclusion and subsequent
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degradation [51]. Therefore, we examined the effect of quercetin on FOXOs. While HSC-3
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cells displayed increased FOXO1 protein levels in both dose- and time-dependent manners
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(Fig. 4A-B) upon quercetin treatment, no change of FOXO3a expression was observed (Fig.
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4B). In addition, quercetin reduced the phosphorylation of cytoplasmic FOXO1 at serine 256
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(Fig. 4C), and induced the translocation of FOXO1 from cytoplasm to nucleus as shown by
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Western blotting (Fig. 4C) and immunofluorescence (Fig. 4D) in HSC-3 cells. These results
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suggest that quercetin induces the nuclear translocation of FOXO1 and, thereby,
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transactivates FOXO1. Indeed, quercetin activated a luciferase reporter gene driven by three
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fork-head responsive elements (FHREs) in HSC-3 cells, whereas such activation was
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abolished upon FOXO1 knockdown (Fig. 4E). Since these FHREs were originally identified
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in FasL promoter [52], our data indicate that quercetin activates FOXO1 and induces FasL
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expression. Furthermore, as in the treatment of quercetin (Fig. 3A), the administration of
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LY294002, a PI3K inhibitor, increased FOXO1 expression in a time-dependent manner in
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HSC-3 cells (Fig. 4F). Taken together, FOXO1 is a downstream transducer of quercetin.
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3.4 Quercetin mediates growth inhibition through FOXO1 activation
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To verify if FOXO1 plays a key role in quercetin-mediated growth inhibition in oral cancer
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cells, we suppressed FOXO1 expression by FOXO1-specific siRNA in the presence of
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quercetin. FOXO1 knockdown relieved the suppression effect of quercetin on cell growth
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(Fig. 5A) and colony formation (Fig. 5B). In particular, FOXO1 suppression abolished
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quercetin-mediated G2/M arrest in both HSC-3 and TW206 cells (Fig. 5C). FOXO1
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knockdown reduced quercetin-induced p21 expression (Fig. 5D); ectopic expression of
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FOXO1 resulted in increased p21 expression (Fig. 5E). These results support the role of
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FOXO1 in quercetin-mediated growth inhibition where the subsequent p21 expression might
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be responsible for the G2 arrest.
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We next examined the role of FOXO1 in quercetin-mediated apoptosis. FOXO1
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knockdown alleviated quercetin-induced apoptosis (Fig. 6A) and reduced the expression
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levels of FasL and cleaved caspase 3 (Fig. 6B) in HSC-3 cells. These results further suggest
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that quercetin-induced cell death might, in part, through FOXO1-mediated FasL expression.
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3.5 Quercetin inhibits tumor growth with concomitant FOXO1 induction in vivo
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The anti-growth effect of quercetin on HSC-3 cells was further tested with xenograft mice.
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Both tumor size and weight of HSC-3-engineered xenografts were significantly suppressed
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upon quercetin administration as compared with the control (Fig. 7A-B). In addition,
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quercetin-fed xenograft mice also exhibited higher FOXO1 expression than those of control
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animals (Fig. 7C). Therefore, in consistent with the in vitro results, the in vivo data indicate
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that quercetin inhibits tumor growth and induces FOXO1 expression.
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4
Discussion
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Aberrant activation of the EGFR/PI3K/Akt signaling pathway is often associated with cancer
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malignancy [53]. Accordingly, this deregulated signaling pathway becomes a conceivable
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therapeutic target in anti-cancer strategy. HSC-3 cell line, a cervical lymph node metastasis
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originated from a tongue primary tumor, displays activated EGFR/PI3K/Akt signaling by
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expressing augmented EGFR levels and a constitutively active PI3K mutant [53]. Quercetin
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has been reported as a potent PI3K inhibitor [25], and the efficacy of quercetin inhibition on
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tyrosine kinase may continue for 16 h [10]. In the current study, we observed
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down-regulation of EGFR and Akt phosphorylation upon quercetin treatment for 12 h in
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HSC-3 cells. It is possible that quercetin might compete with ATP for the binding to the
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catalytic site of the PI3K [25]. Our data potentiate quercetin as an alternative regimen for oral
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cancer patients carrying an aberrant EGFR/PI3K/Akt signaling axis.
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Oral squamous cancer cells with EGFR overexpression appear susceptible to
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anti-proliferation effect of quercetin. The concentration of quercetin to inhibit SCC-1483 cell
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viability by half is about 40 μM [29], in contrast with 20 μM for EGFR-overexpressing
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HSC-3 oral cancer cell line. In the current study, quercetin-induced G2 arrest is associated
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with the induction of FOXO1-dependent p21 expression in HSC-3 human oral cancer cells.
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Similar induction effect on p21 has also been reported in quercetin-treated breast and prostate
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cancer cells [17, 18]. However, the concentration of quercetin to induce p21 expression in
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PC-3 cells was much higher (50-100 μM) than that of HSC-3 cells.
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The involvement of p21 in G2 arrest could result from either DNA damage or mitogen
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starvation [49]. Seoane et al. proposed that, in response to TGF-β, FOXO1 forms a complex
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with Smad 3/4, which further induces p21 expression through the binding of the Forkhead
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binding element within the p21 promoter [54]. The link between FOXO1 and p21 was further
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supported by the finding that FOXO1-mediated p21 expression is crucial in the antineoplastic
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effect of calorie restriction [55].
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Quercetin may exert apoptotic function in a FOXO1-dependent fashion. Here, we
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demonstrated that quercetin treatment caused apoptosis in both HSC-3 and TW206 cells (Fig.
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2C). This phenomenon was supported by increased levels of cleaved caspase 3 and FasL
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upon quercetin addition (Fig. 2E). In particular, FOXO1 suppression alleviated
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quercetin-mediated cell death and reduced the induction of FasL and cleaved caspase 3 (Fig.
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6). Mutant p53 could block apoptosis, whereas quercetin can down-regulate mutant p53
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protein levels [23]. Since HSC-3 carries a truncated p53 mutant [56], this could be an
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alternative mechanism how quercetin facilitates cell death in HSC-3 cells.
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In summary, our data support quercetin as an efficient anti-cancer agent in
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EGFR-overexpressing oral cancers; the schematic mechanism is illustrated in Fig. 8. By
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suppressing EGFR/Akt activation, quercetin stimulates FOXO1 activation and subsequently
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induces p21 and FasL which link to G2 arrest and apoptosis, respectively. The current study
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provides several lines of evidence both in vivo and in vitro to support quercetin as a potential
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therapeutic agent for a subset of oral squamous carcinomas. Specifically, the mechanism
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involving FOXO1 in the anti-cancer action of quercetin may be translated to the future
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application as clinical stratagem in cure of oral cancer.
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Conflict of interest
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The authors declare no conflict of interest.
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5
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Figure legends
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Fig. 1. Quercetin inhibits cell growth in human oral cancer cell lines. (A). HGF (control) and
498
two human oral cancer cell lines, TW206 and HSC-3 cells, were treated with quercetin (0-50
499
μM) dissolved in DMSO for 24 h. The final DMSO concentration in the culture media was no
500
more than 0.3 %. Viable cell numbers were counted (*, p < 0.05 as compared with control, 0
501
μM). The cell number of each quercetin-untreated group was set as 1. (B). Effect of quercetin
502
on colony formation. HSC-3 cells were incubated with the indicated concentration of
503
quercetin for 7 days. Cell colonies were stained with crystal violet and quantified. Bars with
504
different letters indicate statistically significant difference from each other (p<0.05).
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Fig. 2. Quercetin induces cell cycle arrest and apoptosis in human oral cancer cells. (A).
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Effect of quercetin on cell cycle progression. Both HSC-3 and TW206 cells were treated with
508
quercetin (20 μM) for 24 h and then undergone flow cytometric analysis for cell cycle
509
distribution (*, p<0.05). (B). Effect of quercetin on cell cycle-related genes. HSC-3 cells were
510
treated with quercetin (0-20 μM) for 24 h, and the protein lysates were analyzed by Western
511
blotting with the indicated antibodies where α-tubulin served as a loading control. (C-E).
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Effect of quercetin on apoptosis. Both HSC-3 and TW206 cells were subjected to annexin
513
V-FITC and PI staining after 24 h treatment of quercetin (0-30 μM). Annexin V-positive cells
29
514
were analyzed and quantified using a FACScan flow cytometer (*, p<0.05 as compared with
515
Q0, quercetin 0 μM) (C). HSC-3 cells after 24 h treatment of quercetin were stained with
516
DAPI (D) or analyzed for apoptosis-related gene expression by Western blotting with the
517
indicated antibodies where GAPDH served as a loading control (E).
518
519
Fig. 3.
520
HSC-3, TW206, and HGF cell lines were detected by Western blotting, where p38 served as
521
a loading control. Quercetin suppresses phosphorylation of EGFR and its downstream
522
effector, Akt, in time- (B) and dose-dependent (C) manners. HSC-3 cell lysates from each
523
respective treatment were extracted and analyzed by Western blotting with the indicated
524
antibodies. The experiments repeated three times with consistent results.
Effect of quercetin on EGFR/Akt signaling. (A) The endogenous levels of EGFR in
525
526
Fig. 4.
527
time- (A) and dose-dependent (B) manners. HSC-3 cell lysates from each respective
528
treatment were extracted and analyzed by Western blotting with antibodies against FOXO1,
529
FOXO3a, or α-tubulin. (C-D). Effect of quercetin on subcellular localization of FOXO1. (C).
530
HSC-3 cells were treated with quercetin (0 or 10 μM) for 24 h and then undergone
531
cytoplasmic and nuclear extraction. Protein lysates were subjected to Western blotting
532
analysis of p-FOXO1 and FOXO1 expression with α-tubulin and TBP as loading controls for
Quercetin induces FOXO1 activation. Quercetin induces FOXO1 expression in
30
533
cytoplasmic and nuclear portions, respectively. (D). HSC-3 cells were seeded onto a chamber
534
slide, treated with quercetin for 24 h, and then fixed and stained with an antibody against
535
FOXO1 (green). Nuclei were counterstained with DAPI (blue). (E). Quercetin induces
536
FOXO1 transactivation. HSC-3 cells, in triplicate, were cotransfected with
537
pGL3-FHRE-luciferase (0.25 μg) and β-galactosidase (0.05 μg) plasmids, in the presence or
538
absence of FOXO1 siRNA, for 24 h, and then treated with quercetin (0, 20 μM) for another
539
24 h. The luciferase activity was normalized to β-galactosidase for transfection efficiency and
540
presented as -fold activation. Each bar represents the mean + S.D. (*, p<0.05). (F). Ly294002
541
(LY), a PI3K inhibitor, mimics quercetin’s effect on FOXO1. HSC-3 cells were treated with
542
LY at the indicated time points, and cell lysates were then analyzed by Western blotting for
543
p-Akt and FOXO1 expression.
544
545
Fig. 5.
546
transfected with either scramble siRNA (Control) or FOXO1 siRNA were treated with
547
quercetin as indicated for 24 h (A) or 7 days (B) for the measurement of viable cells and
548
colony numbers, respectively (*, p<0.05 vs. control). (C). Effect of FOXO1 knockdown on
549
quercetin-mediated cell cycle arrest. Both HSC-3 and TW206 cells with FOXO1 knockdown
550
were treated with quercetin for 24 h and then analyzed for cell cycle distribution by a
551
FACScan flow cytometer. (D). Effect of FOXO1 knockdown on quercetin-induced p21
Role of FOXO1 in quercetin-mediated growth suppression. (A-B). HSC-3 cells
31
552
expression. HSC-3 cells were transfected with either FOXO1 or scramble siRNA for 24 h
553
followed by quercetin treatment for another 24 h. Cell lysates were analyzed for FOXO1 and
554
p21 expression by Western blotting. (E). FOXO1 mimics quercetin’s effect on p21. HSC-3
555
cells were transiently transfected with a Flag-FOXO1 expression construct, and cells lysates
556
were analyzed by Western blotting with anti-flag and anti-p21 antibodies.
557
558
Fig. 6. Role of FOXO1 in quercetin-induced apoptosis. HSC-3 cells undergone FOXO1
559
siRNA and/or 24 h of quercetin treatment were stained with annexin V-FITC and PI, analyzed
560
by a FACScan flow cytometer (*, p<0.05) (A), and subjected to Western blotting analysis
561
with the indicated antibodies (B).
562
563
564
Fig. 7.
565
mice were established, with or without quercetin treatment, for 35 days. (A) Tumor size was
566
recorded every other day (*, p < 0.05 as compared with control). (B) At the end of the study,
567
tumors were excised and weighed. The picture represents the average tumor growth in either
568
group (n=6). A two-tailed, unpaired t test was used to assess the differences of tumor weight
569
between the two groups (*, p<0.05). (C). Quercetin induces FOXO1 expression in HSC-3
570
implanted xenograft mice. At the end of the experiment, tumors were excised and
Quercetin suppresses tumor growth in vivo. (A-B). HSC-3 implanted xenograft
32
571
homogenized for protein extraction. Protein lysates were then subjected to Western blotting
572
analysis for FOXO1 expression.
573
574
Fig. 8.
575
carcinomas. Quercetin efficiently inhibits cell growth by targeting EGFR/Akt signaling in
576
EGFR-overexpressing oral cancer. The resulting FOXO1 activation leads to elevated
577
expression of p21 and FasL, and, subsequently, growth arrest in G2 phase and apoptosis.
Role of FOXO1 in quercetin-mediated growth suppression of oral squamous
33