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Resveratrol inhibits STAT3 signaling pathway through the induction of SOCS-1: role in apoptosis induction and radiosensitization in head and neck tumor cells.

ABSTRACT

Background: Signal transducer and activator of transcription 3 (STAT3) is persistently activated in squamous cell carcinoma of the head and neck (SCCHN) and can cause uncontrolled cellular proliferation and division.

Hypothesis: Thus, its targeted abrogation could be an effective strategy to reduce the risk of SCCHN. Resveratrol is known for its anti-cancer efficacy in a variety of cancer models.

Study design: The effect resveratrol on STAT3 activation, associated protein kinases, phosphatases, cellular proliferation and apoptosis was investigated.

Methods: We evaluated the effect of resveratrol on STAT3 signaling cascade and its regulated functional responses in SCCHN cells.

Results: We found that HN3 and FaDu cells expressed strongly phosphorylated STAT3 on both tyrosine 705 and serine 727 residues as compared to other SCCHN cells. The phosphorylation was completely suppressed by resveratrol in FaDu cells, but not substantially in HN3 cells. STAT3 suppression was mediated through the inhibition of activation of upstream JAK2, but not of JAK1 and Src kinases. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the resveratrol-induced downregulation of STAT3, thereby indicating a critical role for a PTP. We also found that resveratrol induced the expression of the SOCS-1 protein and mRNA. Further, deletion of SOCS-1 gene by siRNA suppressed the induction of SOCS-1, and reversed the inhibition of STAT3 activation. Resveratrol down-regulated various STAT3-regulated gene products, inhibited proliferation, invasion, as well as induced the cell accumulation in the sub-Gl phase and caused apoptosis. Beside, this phytoalexin also exhibited the enhancement of apoptosis when combined with ionizing radiation treatment.

Conclusion: Our results suggest that resveratrol blocks STAT3 signaling pathway through induction of SOCS-1, thus attenuating STAT3 phosphorylation and proliferation in SCCHN cells.

Keywords:

Resveratrol

STAT3

Squamous cell carcinoma of the head and neck

SOCS-1

Introduction

Head and neck cancer is a malignancy that starts in the lip, oral cavity (mouth), nasal cavity (inside the nose), paranasal sinuses, pharynx, and larynx. 90% of head and neck cancers are squamous cell carcinomas, so they are often referred to as head and neck squamous cell carcinomas (HNSCC). It is the sixth most common cancer worldwide and eighth leading cause of mortality (Parfenov et al. 2014), with approximately 600,000 new cases diagnosed each year (Jemal et al. 2009). Although cancer treatment regimens have been advanced, up to 50% of HNSCC patients will experience recurrence or residual disease even after therapy and the median survival rate is less than 1 year (Cooper et al. 2004). Approximately 80% of HNSCC also exhibit up-regulation of persistent STAT3 expression, which can mediate radioresistance and chemoresistance. The hyperactivation of STAT3 in response to the aberrant activation of upstream receptor signals is frequently observed in a variety of human cancers, including head and neck cancer (Leong et al. 2003; Yin et al. 2010).

Signal transducer and activator of transcription 3 (STAT3) is an oncogenic transcription factor that transmits signals from cytokines and growth-factor receptors to the nucleus (Yu and Jove 2004). The aberrant STAT3 activation promotes the growth and survival of tumor cells through the modulation of cell cycle regulators (e.g., cyclin D1/D2 and c-Myc), upregulation of anti-apoptotic proteins (e.g., Mcl-1, Bcl-xL, and Survivin), downregulation of the tumor suppressor p53, and induction of angiogenesis by vascular endothelial growth factor (VEGF); these oncogenic overexpression eventually lead to tumor progression and resistance to chemotherapy and radiotherapy (Song and Grandis 2000; Yu and Jove 2004).

The suppressor of cytokine signaling (SOCS) proteins make up a family of intracellular proteins (Kubo et al. 2003; Yoshimura et al. 2007). There are seven SOCS family proteins: SOCS-1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, SOCS-6, and SOCS-7, each of which has a central SH2 domain, an amino-terminal domain of variable length and sequence, and a carboxy-terminal 40-amino-acid module known as the SOCS box (Tamiya et al. 2011). SOCS has been reported to inhibit STAT phosphorylation by binding and inhibiting JAKs or competing with STATs for phosphotyrosine binding sites on cytokine receptors (Krebs and Hilton 2001). Specifically, the SH2 domain of SOCS-1 directly binds to the activation loop of JAKs (Yasukawa et al. 1999). The SH2 domains of SOCS-2 and SOCS-3 bind to phosphorylated tyrosine residues on activated cytokine receptors (Kubo et al. 2003). SOCS-3 binds to gp130-related cytokine receptors, including the phosphorylated tyrosine 757 (Tyr757) residue of gp130, the Tyr800 residue of IL-12 receptor [beta]2, and Tyr985 of the leptin receptor (Lehmann et al. 2003; Sasaki et al. 2000). Additionally, both SOCS-1 and SOCS-3 can block JAK tyrosine kinase activity directly via their kinase inhibitory regions (Yasukawa et al. 1999).

Resveratrol(3,5,4'-trihydroxy-trans-stilbene), a stilbenoid, is a phytoalexin produced naturally by several plants in response to injury or when the plant is constantly under attack by pathogens such as bacteria or fungi (Fremont 2000). Resveratrol is found in the skin of grapes, blueberries, raspberries, and mulberries. Resveratrol has been reported to exhibit a variety of biological activities including related to the inhibition of lipid peroxidation (Berrougui et al. 2009), free radical scavenging (Leonard et al. 2003), and the suppression of platelet aggregation (Stef et al. 2006). It can also exert anti-inflammatory effects (Bognar et al. 2013), vasorelaxing activity (Fitzpatrick et al. 1993), estrogenic activity (Klinge et al. 2003), and antineoplastic activity (Aggarwal et al. 2004) against a wide variety of disease states. Wung et al. first showed that resveratrol suppressed STAT3 phosphorylated at Tyr705 residue in IL-6-treated endothelial cells (Wung et al. 2005). Also, resveratrol exerted pro-apoptotic effects via suppression of STAT3 signaling against human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src) (Kotha et al. 2006), human multiple myeloma U266 cells (Bhardwaj et al. 2007), medulloblastoma cells (Yu et al. 2008), leukemia cells (Li et al. 2010a), malignant natural killer cells (Quoc Trung et al. 2013), human and murine melanoma cells (Habibie et al. 2014), and ovarian cancer cells (Zhong et al. 2015). Moreover, the effect of resveratrol in combination with irradiation and chemotherapy in Merkel cell carcinoma has been reported (Heiduschka et al. 2014). In addition, the chemopreventive potential of resveratrol in head and neck squamous cell carcinoma has also been observed (Shrotriya et al. 2015). Although resveratrol has been found to mitigate aberrant activation of STAT3 in various cell lines, our study is the first one to explore the effects of resveratrol both on STAT3 signaling cascades and on the negative regulators of phosphorylated STAT3 (SOCS-1) in HNSCC cells.

Along with standard treatments (such as surgery and chemotherapy), radiation therapy is one of the most important modalities for the treatment of head and neck cancer because the treatment modality generally consists of surgery with postoperative radiation therapy in patients (Vokes 1997). Unfortunately, the use of radiotherapy is largely limited by intrinsic or acquired resistance to ionizing radiation (1R). In an effort to overcome the radioresistance of cancer cells to improve the therapeutic efficacy of radiotherapy, a variety of phytochemicals have been examined for their potential radiosensitizing effects. For instance, curcumin (Chendil et al. 2004; Qian et al. 2015), resveratrol (Fang et al. 2012; Zoberi et al. 2002), genistein (Liu et al. 2013), luteolin (Cho et al. 2015), shikonin (Kwak et al. 2014), and rosmarinic acid (Alcaraz et al. 2014) have been reported to possess radiosensitizing effects on a variety of tumor cells. A significant amount of scientific evidence indicates that the blockage of STAT3 enhances radiation sensitivity in various tumor cells (Choe et al. 2015; Gao et al. 2010; Li et al. 2010b). To the best of our knowledge, the effect of resveratrol on radiosensitization of head and neck cancer cells has not yet been reported. We found that resveratrol suppressed constitutive and STAT3 activation through induction of SOCS-1; down-regulated STAT3-regulated gene products; and potentiated IR-induced apoptotic effects in the SCCHN cell line FaDu.

Materials and methods

Reagents

Resveratrol, with a purity greater than 99%, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tris base, glycine, NaCl, sodium dodecylsulfate (SDS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 and fetal bovine serum (FBS) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA). Annexin V was purchased from BD Biosciences (Palo Alto, CA). Anti-pSTAT3 (Tyr705 and Ser727), anti-p-JAK2, anti-p-JAKl, anti-JAK2, anti-JAKl, anti-p-Src, anti-Src, anti-Cydin Dl, anti-cleaved caspase3, anti-caspase9, and anti-cleaved caspase-9 were purchased from Cell Signaling Technology (Beverly, MA). Anti-STAT3, anti-Lamin B, anti-SOCS-1, anti-Bcl-2, anti-Bcl-xL, anti-Survivin, anti-IAP-1, anti-VEGF, anti-MMP-9, anti-MMP-2, anti-caspase-3, anti-PARP, anti-[beta]-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell lines

The human head and neck squamous cell carcinoma (HNSCC) SCC4 and FaDu cells were obtained from the American Type Culture Collection (Manassas, VA), YD-8 and YD-10B cells were obtained from Korean Cell Line Bank (Seoul, Korea), HN3, HN9, LN686, and J17AR cells were provided by Dr. Sang-wook Lee (ASAN Medical Center, Korea), immortal human keratinocyte (HaCaT) cells were provided by Cell Lines Service (Eppelheim, Germany). SCC4 cells were cultured in DMEM containing 10% FBS, vitamin solution, non-essential amino acid, penicillin (100 units/ml), and streptomycin (100 [micro]g/ml). YD-8, YD-10B, HN3, HN9, FaDu, LN686, and J17AR cells were cultured in MEM containing 10% FBS, penicillin (100 units/ml), and streptomycin (100 [micro]g/ml). HaCaT cells were cultured in DMEM containing 10% FBS, penicillin (100 units/ml), and streptomycin (100 [micro]g/ml).

Western blot analysis

After the cells were treated with the indicated concentrations of resveratrol, the cells were lysed and the total protein concentrations were determined by Bradford reagent (Bio-Rad, Hercules, CA). Equal amounts of lysates resolved on sodium dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a nitrocellulose membrane, and the membrane was blocked with 1 x TBS containing 0.1% Tween 20 and 5% skim milk at room temperature. After the blocking, the membranes were incubated overnight at 4[degrees]C with the respective primary antibodies. The membranes were washed three times and incubated with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) for 1 h at room temperature. After four times washing, the membranes were detected using an enhanced chemiluminescence (ECL) kit (Millipore, Bedford, MA).

EMSA for STAT3-DNA binding

STAT3-DNA binding was analyzed by EMSA using a 5'-biotinylated STAT3 oligonucleotide (5'-GATCCTTCTGGGAATTCCTAG ATC-3' and 3'-CTAGGAAGACCCTTAAGGATCTAG-5'). Briefly, nuclear extracts were prepared from resveratrol-treated cells and incubated with the 5'-biotinylated STAT3 oligonucleotide probe. The DNA-protein complex formed was separated from free oligonucleotide on 5% native polyacrylamide gels and transferred to a positively charged nylon membrane. The membrane was detected following manufacturer instructions using LightShift[R] Chemiluminescent EMSA kit (Thermo Fisher Scientific Inc., Waltham, MA).

Immunocytochemistry for STAT3 localization

After 2 h of resveratrol (150 [micro]M) treatment, the cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature and then washed three times in PBS. The cells were permeabilized with 0.2% Triton X-100 in PBS for 20 min, washed three times in PBS, and then blocked with 5% BSA in PBS for 1 h at room temperature. After that the cells were incubated with rabbit polyclonal anti-human STAT3 antibody at a 1:100 dilution. After overnight incubation at 4[degrees]C, the slides were washed, incubated with FITC-conjugated secondary antibodies at a 1:200 dilution (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. Next, the cells were stained with a 1 [micro]g/ml DAPI solution and mounted onglass slides using Fluorescent Mounting Medium (GBI Laboratories, Manchester, UK). Using a FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan), DAPI and FITC fluorescence were excited (Ex: 405 nm and 488 nm) and detected (Em: 461 nm and 519 nm) with 2.1% laser transmissivity and 5.0% laser transmissivity, respectively.

RNA analysis and reverse transcription polymerase chain reaction

Total RNA was extracted using a Trizol reagent according to the manufacturer's instructions (Invitrogen, Life Technologies). Total RNA of 1 [micro]g was converted to cDNA by M-MLV reverse transcriptase (Promega, Fitchburg, WI) and then amplified by a Taq polymerase using reverse transcription polymerase chain reaction (RT-PCR) (TAKARA, Tokyo, Japan).The relative expression of SOCS-1, Cyclin D1, and MMP-9 were analyzed by quantitative RT-PCR with glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as an internal control. The following pairs of forward and reverse primer sets were used SOCS-1 (5'ACGCAGGATTAACGTGGATG-3' and 5'-CCCTGGTTTGTGCAAAGATACT3'), Cyclin D1, (5'-ACAAGCTCTGCATCTACACCGACA-3' and 5'-AGAAGCTGTGCATCTACACCGACA-3'). and MMP-9 (5'-TTGAGGAGCGGCTCTCCAAG-3, and 5'-CGGTCCTGGCAGAAATAGGC-3'). The reaction of SOCS-1 was done at 94[degrees]C for 5 min, and 30 cycles of 94[degrees]C for 30 s, 55[degrees]C for 30 s, and 72[degrees]C for 30 s, with extension at 72[degrees]C for 5 min, the reaction of Cyclin D1 was done at 94[degrees]C for 5 min, and 30 cycles of 94[degrees]C for 30 s, 58 CC for 30 s, and 72[degrees]C for 30 s, with extension at 72[degrees]C for 5 min, and the reaction of MMP-9 was done at 94[degrees]C for 5 min, and 30 cycles of 94[degrees]C for 30 s, 62[degrees]C for 30 s, and 72[degrees]C for 30 s, with extension at 72[degrees]C for 5 min. PCR products were run on 1% agarose gel and then stained with Dyne Loading STAR (DyneBio, Korea). Stained bands were visualized under a UV light and photographed.

Transfection with SOCS-1 siRNA

FaDu cells were transfected with 50 nM of SOCS-1 siRNA or scrambled siRNA using serum-free Lipofectamine2000 (Invitrogen) according to the manufacturer's protocols and treated 24 h post-transfection.

Real-time cell proliferation analysis

FaDu cells (5000 cells/well) were seeded onto 16-well E-plates, integrated with gold microelectrode arrays, and incubated in realtime cell analysis (RTCA) was carried out with the xCELLigence System (Roche, Mannheim, Germany). Application of a low-voltage alternating current signal generates an electric field between the electrodes, which is modulated by the cells covering the electrodes. Cell proliferation in the wells results in changes in the impedance readout, obtained from each well with the RTCA DP Instrument. The generated signal is displayed in arbitrary units, referred to as the cell index. After a 20 h initial incubation on the E-plates, FaDu cells were treated with the resveratrol (50 or 150 [micro]M). Non-treated samples were used as controls. The cell index was monitored for 48 h, with measurements every 15 min.

MTT assay

Cell viability was measured by an MTT assay to detect NADH-dependent dehydrogenase activity. Thirty microliters of MTT solution (2 mg/ml) in 1 x phosphate-buffered saline (PBS) was directly added to the cells, which were then incubated for 2h to allow MTT to metabolize to formazan. Absorbance was measured with an automated spectrophotometric plate reader at a wavelength of 570 nm. Cell viability was normalized as relative percentages in comparison with untreated controls.

Cell cycle analysis

To determine apoptosis, cell cycle analysis was performed using PL After treatment with resveratrol, the cells were collected, washed with cold PBS, fixed with 70% ethanol, and incubated for 30 min at 37[degrees]C with 0.1% RNase A in PBS. Cells were then washed, resuspended, and stained in PBS containing 10 [micro]g/ml of PI for 10 min at room temperature. Cell distribution across the cell cycle was analyzed with a flow cytometry (Becton-Dickinson, Heidelberg, Germany).

Annexin V assay

One of the early indicators of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cell's cytoplasmic interface to the extracellular surface. This loss of membrane asymmetry can be detected using the binding properties of Annexin V. To detect apoptosis, we used Annexin V antibody conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). FaDu cells were treated with the indicated concentration of resveratrol and then subjected to Annexin V and PI staining. The cells were washed and observed accordingly with a flow cytometry (Becton-Dickinson, Heidelberg, Germany). Acquisition and analysis of the data were performed using Cell Quest 3.0 software.

Wound healing assay

The FaDu cells were plated in triplicate and treated with resveratrol (15 [micro]M) for 48 h. Before plating the cells, two parallel lines were drawn at the underside of the wells to serve as fiducially marks demarcating the wound areas to be analyzed. Before inflicting the wound, the cells should be fully confluent. The growth medium was aspirated off and replaced by calcium-free PBS to prevent killing of the cells at the edge of the wound by exposure to high calcium concentrations before two parallel scratch wounds were made perpendicular to the marker lines with a sterile 200-[micro]l autoclaved pipette tip. Thereafter, the calcium-free medium was then changed to serum-free medium with or without resveratrol. After incubation for 48 h, the wounds were observed using bright field microscopy and multiple images were taken at areas flanking the intersections of the wound and the marker lines at the start and end of the experiment. Gap distance of the wound was measured at three different wells using Photoshop software, and the data were normalized to the average of the control. Graphs were plotted against the percentage of migration distance the cells moved before and after treatment, normalized to control.

Invasion assay

We employed the Roche xCELLigence Real-Time Cell Analyzer (RTCA) DP instrument (Roche Diagnostics GmbH, Germany) to measure cellular invasion. The RTCA DP instrument uses the cellular invasion/migration (CIM)-Plate 16, which features microelectronic sensors integrated onto the underside of the microporous polyethylene terephthalate membrane of a Boyden-like chamber. For invasion experiments, the top chamber of the CIM-Plate 16 was coated with Matrigel (BD Biosciences, San Diego, CA) before addition of the medium to the bottom chamber. The CIM-Plate 16 was assembled by placing the top chamber onto the bottom chamber and snapping the two together. Serum-free medium was placed in the top chamber to hydrate and preincubate the membrane for 1 h in the C[O.sub.2] incubator at 37[degrees]C before obtaining a background measurement. Cells were lightly trypsinized, pelleted, and resuspended at the indicated cell densities in serum-free medium. Once the CIM-Plate 16 has been equilibrated, it was placed in the RTCA DP station, and the background cell-index values were measured. The CIM-Plate 16 was then removed from the RTCA DP station, and the cells are added to the top chamber at the desired density.

Real-time quantitative PCR

Total RNA was extracted using a Trizol reagent according to the manufacturer's instructions (Invitrogen, Life Technologies). Total RNA of 1 [micro]g was converted to cDNA by M-MLV reverse transcriptase (Promega, Fitchburg, WI). The expression levels of Bd-2 and Bcl-xL were determined with a Real-Time quantitative PCR as described in the manufacturer's protocol (Applied Biosystems, MA). [2.sup.[DELTA][DELTA]Ct] value compared to the NT samples was determined with StepOne software (Applied Biosystems, MA).The following pairs of forward and reverse primer sets were used Bcl-2 (5'-TCCCTCGCTGCACAAATACTC-3' and 5'-GACGACCCGATGGCCATA-3'), and Bcl-xL (5'-TACCAGCCTGACCAATATGGC-3' and 5'-TGGGTTCAAGTGATTCTCCTG-3'), and GAPDH (5'-ACCTGACCTGCCGTCTAGAAAA-3' and S'-ACGCCTGCTTCACCACCTT-3') was used as a house keeping gene and endogenous control.

Radiotherapy

Radiotherapy was administered at room temperature with a Linear Accelerator (Clinac IX, Varian Associates, Inc., CA) operating at 6 MV and with a dose rate of 1, 5, and 10 Gy by Dr. Dong-Oh Shin (Kyung Hee University Medical Center, Korea).

Statistical analysis

All numeric values are represented as the mean [+ or -] SD. Statistical significance of the data compared with the untreated control was determined using the Student's unpaired t-test. Significance was set at p < 0.05.

Results

Resveratrol inhibited constitutive STAT3 phosphorylation in FaDu cells

We investigated the effect of resveratrol on STAT3 activation in HNSCC cells. The structure of resveratrol is shown in Fig. 1A. As the first step in the present study, we examined constitutive STAT3 activation in a wide variety of HNSCC cells. As shown in Fig. 1B, HN3 and FaDu cells relatively overexpressed constitutively active STAT3 as compared to other tumor cells. So we set out to determine whether resveratrol could inhibit STAT3 activation in HN3 and FaDu cells. The results showed that resveratrol inhibited the constitutive activation of STAT3 in FaDu cells in a dose-and time-dependent manner (Fig. 1C and D, right panels). In contrast to FaDu cells, resveratrol slightly down-regulated constitutive activation of STAT3 in HN3 cells (Fig. 1C and D, left panels). Therefore, we decided to investigate the in depth molecular mechanism(s) of resveratrol-induced inhibition of STAT3 signaling pathway in only FaDu cells.

Resveratrol modulated constitutive activation of JAK2

STAT3 has been reported to be activated by tyrosine kinases of the Janus family (JAKs) and the Src kinase families (Ihle 1996; Schreiner et al. 2002). Hence, we investigated whether resveratrol suppresses constitutive phosphorylation of JAK1, JAK2, and Src in FaDu cells. As shown in Fig. IE, resveratrol inhibited the constitutive activation of JAK2 in FaDu cells, whereas it did not have any effect on the phosphorylation of both JAK1 and Src kinases.

Resveratrol inhibited binding of STAT3 to the DNA

Because tyrosine phosphorylation causes the dimerization of STAT3 and their translocation to the nucleus (Yu et al. 1995), where they bind to DNA and regulate gene transcription, we determined whether resveratrol suppresses the DNA binding activities of STAT3. EMSA analysis of nuclear extracts prepared from FaDu cells showed that resveratrol inhibited STAT3-DNA binding activities in a dose-and time-dependent manner (Fig. 2A). Oct-1 was used as a loading control. These results show that resveratrol abrogated the DNA binding ability of STAT3.

Resveratrol suppressed the nuclear translocation of STAT3 in FaDu cells

Because the active dimer of STAT3 is capable of translocating to the nucleus and inducing transcription of specific target genes (Bowman et al. 2000), we next investigated whether resveratrol suppresses the nuclear translocation of STAT3. Immunocytochemistry (Fig. 2B) and Western blot analysis (Fig. 2C) of nuclear extracts clearly demonstrated that resveratrol blocked the translocation of STAT3 into the nucleus in FaDu cells. Thus, resveratrol inhibited the binding of STAT3 to DNA could result from the decreased nuclear translocation of STAT3.

Tyrosine phosphatases are involved in resveratrol-induced inhibition of STAT3 activation

Because protein tyrosine phosphatases (PTPs) have also been reported to play a critical role in STAT3 activation (Han et al. 2006), we determined whether resveratrol-induced inhibition of STAT3 tyrosine phosphorylation could be due to activation of a PTP. Treatment of FaDu cells with the broad-acting tyrosine phosphatase inhibitor, sodium pervanadate reversed resveratrol-induced suppression of STAT3activation in a dose-dependent manner (Fig. 2D).This result indicates that tyrosine phosphatases may play an important part in resveratrol-induced inhibition of STAT3 activation in FaDu cells.

Resveratrol induced the expression of SOCS-1 and SOCS-1 mRNA

The suppressor of cytokine signaling 1 (SOCS-1) is also a negative regulator of STAT3 (Rottapel et al. 2002). We examined whether resveratrol can modulate the expression of SOCS-1 in FaDu cells. We found that resveratrol-induced the expression of SOCS-1 protein in a time-dependent manner (Fig. 2E, upper panels).We also examined the effect of resveratrol on the transcription of SOCS-1.For this, cells were incubated with resveratrol at different intervals and total RNA was examined for SOCS-1 mRNA expressions by RT-PCR analysis. As shown in (Fig. 2E, lower panels'), resveratrol clearly induced the expression of SOCS-1 mRNA in a time-dependent manner.

Deletion of SOCS-1 expression by siRNA reversed the inhibition of STAT3activation by resveratrol

We determined whether the suppression of SOCS-1 expression by siRNA would abrogate the inhibitory effect of resveratrol on STAT3 activation. Western blot analysis showed that resveratrol-induced SOCS-1 expression was effectively abolished in the cells treated with SOCS-1 siRNA, whereas treatment with scrambled siRNA had no effect (Fig. 2F, upper panels). We also found that resveratrol failed to suppressSTAT3 activation in cells treated with SOCS-1 siRNA (Fig. 2F, lower panels). These siRNA results corroborate our earlier evidence on the critical role of SOCS-1 in the suppression of STAT3 phosphorylation by resveratrol.

Resveratrol suppressed the proliferation of FaDu cells

To specifically examine the anti-tumor activity of resveratrol in FaDu cells, the cells were treated with resveratrol (50 and 150 [micro]M) for indicated time intervals, and then cell viability was analyzed by RTCA. As shown in Fig. 3A, resveratrol significantly suppressed cell proliferation of FaDu cells. Next, FaDu cells and HaCaT cells were treated with resveratrol (100 and 150 [micro]M) for 24 h, and then cell viability was measured by MTT assay. Fig. 3B shows that resveratrol significantly exerted cytotoxicity in FaDu cells, but not in HaCaT cells, indicating that resveratrol is relatively non-toxic to normal cells.

Resveratrol caused the accumulation of the cells in the sub-G1 phase of the cell cycle and increased Annexin V positive cells

We set out to determine the effect of resveratrol on cell cycle phase distribution. After treatment for 24 h, resveratrol (150 [micro]M) significantly increased the sub-G1 DNA contents 17.4% at resveratrol, compared to non-treated cells (5.0%) (Fig. 3C). Next, FaDu cells were treated with resveratrol (150 [micro]M) for 24 h, and the cells were incubated with Annexin-V/FITC and PI and then analyzed by a flow cytometer. Fig. 3D showed that the Annexin V positive cells were clearly increased in resveratrol-treated cells as compared with the non-treated cells.

Resveratrol suppressed FaDu cells invasion activity

Next, we investigated whether resveratrol can modulate FaDu cells invasion activity. Wound healing assay (Fig. 3E) and invasion assay (Fig. 3F) indicated that resveratrol significantly suppressed tumor cell invasion activity.

Resveratrol suppressed expression of various proteins involved in apoptosis, proliferation, metastasis, and angiogenesis

STAT3 activation has been shown to regulate the expression of various gene products involved in proliferation, anti-apoptosis, invasion, and angiogenesis (Aggarwal et al. 2006). We found that resveratrol down-regulated the expression of Bcl-2, Bcl-xL, Survivin, IAP-1, Cydin Dl, VEGF, MMP-9, and MMP-2 all of which have been reported to be regulated by STAT3. The expression of these oncogenic proteins decreased in a dose-dependent manner (Fig. 4A and B). We also found that mRNA expression of Bcl-2, Bcl-xL (Fig. 4C), cyclin D1, and MMP-9 (Fig. 4D) were down-modulated by resveratrol treatment in a dose-dependent manner.

Resveratrol activated caspase-3/9and caused PARP cleavage

Whether suppression of constitutively active STAT3 in FaDu cells by resveratrol leads to apoptosis was also investigated. In FaDu cells treated with resveratrol, there was a dose-dependent activation of pro-caspase-3 and pro-caspase-9 (Fig. 4E). The activation of downstream caspases led to the cleavage of a 118 kDa PARP protein into an 85 kDa fragment (Fig. 4F). These results clearly suggest that resveratrol induced caspase-dependent apoptosis in FaDu cells.

Resveratrol sensitized FaDu cells to radiotherapy

The principal aim of this study was to test whether resveratrol can enhance the therapeutic effect of radiotherapy on tumor growth in FaDu cells. As the first step, FaDu cells were treated with radiotherapy (1, 5 or 10 Gy) after 2 h of pretreatment with resveratrol (50 or 100 [micro]M). As shown in Fig. 5A, cell viability of FaDu cells treated with radiotherapy (1, 5 or 10 Gy) was 110%, 116%, and 103%, respectively, compared to untreated controls, radiotherapy alone had no effect on the cell viability of FaDu cells. But cell viability of FaDu cells treated with combination of 100 [micro]M of resveratrol and 10 Gy of radiotherapy was 57%, whereas cell viability treated with 100 [micro]M of resveratrol alone was 75%. This result shows that resveratrol caused radiosensitization of FaDu cells.

Resveratrol potentiated the therapeutic effect of radiotherapy for FaDu cells

To specifically examine the effect of combination of resveratrol and radiotherapy, FaDu cells were treated with radiotherapy (10 Gy) after 2h of pretreatment with resveratrol (100 [micro]M) and incubated for 2h. Fig. 5B shows that combination of resveratrol (100 [micro]M) and radiotherapy (10 Gy) treatment inhibited the phosphorylation of STAT3 and JAK2. Next, FaDu cells were treated with radiotherapy (10 Gy) after 2h of pretreatment with resveratrol (100 [micro]M) and incubated for 24 h. Fig. 5C shows that radiotherapy (10 Gy) had minimal effect or increased the expression of Bcl-2 and Survivin proteins. However, combination of resveratrol and radiotherapy significantly down-regulated the expression of these proteins.

Combination of resveratrol and radiotherapy produced enhanced effect on the induction of apoptosis in FaDu cells

We set out to determine the synergic effect on induction of apoptosis in FaDu cells. FaDucells were treated with radiotherapy (10 Gy) after 2h of pretreatment with resveratrol (100 [micro]M) and incubated for 24 h. As shown in Fig. 5D, combination of resveratrol and radiotherapy potentially induced PARP cleavage. To further confirm, Annexin V assay was performed in FaDu cells. The combination of resveratrol and radiotherapy increased the population of Annexin-V/FITC and PI positive cells (20%) compared to resveratrol (12%) or radiotherapy (4%) alone (Fig. 5E).

Discussion

Although resveratrol has been shown to exert anti-tumor effects in head and neck squamous cell carcinomas (HNSCC) (Hu et al. 2012; Lin et al. 2008; Tyagi et al. 2011), there are no prior reports elaborating the potential effect of resveratrol on proteins regulating JAK/STAT signaling pathway in HNSCC cells. The purpose of this study was to determine whether resveratrol exerts its anticancer effects through the modulation of the STAT3 signaling pathway in head and neck squamous cell carcinomas FaDu cells. We found that HN3 and FaDu cells clearly induced constitutive STAT3 activation and resveratrol suppressed STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 in only FaDu cells. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase 2 (JAK2). Interestingly, resveratrol triggered the expression of SOCS-1 protein, which is known as a negative regulator of STAT3 activation. Moreover, the knockdown of SOCS-1 using siRNA suppressed the induction of SOCS-1 and reversed the inhibition of STAT3 activation. Resveratrol down-regulated the expression of various STAT3-regulated gene products including Bcl-2, Bcl-xL, Survivin, IAP-1, Cyclin D1, VEGF, and MMP-9/2. It also caused the inhibition of proliferation, increased accumulation of cells in S phase, induced substantial apoptosis, suppressed invasive activity, and significantly potentiated IR-induced apoptotic effects in FaDu cells.

We found for the first time that resveratrol could suppress constitutive STAT3 phosphorylation both at Tyr705 and Ser727 residues in HN3 and FaDu cells. These phosphorylations were inhibited by resveratrol in FaDu cells, but not in HN3 cells. We also observed that resveratrol suppressed DNA-binding activity and nuclear translocation of STAT3 in FaDu cells. Next, how resveratrol affects STAT3signaling pathway was also investigated in detail. The activation of JAKs have been closely correlated with STAT3 activation (Ihle 1996) and we observed that resveratrol inhibited the activation of constitutively active JAK2 in FaDu cells, but not JAK1 and Src. JAKs are essential for the tyrosine phosphorylation of STAT3 in response to growth factors and cytokines (Toyonaga et al. 2003). Several reports show that constitutively activated STAT3 in HNSCC is a major regulator of proliferative and prosurvival signaling under both in vitro and in vivo settings (Kijima et al. 2002; Masuda et al. 2002). Thus. STAT3 decoy oligonucleotide has been found to significantly inhibit proliferation and STAT3-mediated gene expression in HNSCC cells (Leong et al. 2003).

We found evidence that resveratrol-induced inhibition of STAT3 activation involves a negative regulator of STAT3, namely SOCS-1. SOCS-1 has been shown to be involved in the negative regulation of IL-6R/JAK-mediated activation of STAT3 and the expression of SOCS-1 following demethylation or transient transfection can suppresses STAT3 activation in HNSCC cells (Lee et al. 2006). It has also been reported that the reduced SOCS1 expression is found in a variety of cancers, including prostate cancer, multiple myeloma, acute myeloid leukemia, and pancreatic cancer and lymphoma (Zhang et al. 2012). On the contrary, higher expression of S0CS1 mRNA has been linked with initial tumor stages and better clinical outcomes in breast cancer (Sasi et al. 2010). Our study demonstrates for the first time that the loss of SOCS-1 following transfection with SOCS-1 siRNA abolished the STAT3 inhibitory effects of resveratrol in HNSCC cells.

Several previous reports have shown that resveratrol is a potent inhibitor of nuclear factor-[kappa]B (NF-[kappa]B) activation through inhibition of IKK and the phosphorylation of [kappa]B[beta] and of p65 (Manna et al. 2000) and resveratrol can also suppress both the constitutive and the interleukin 6-induced activation of STAT3 in human multiple myeloma cells, thereby indicating that the abrogation of STAT3 activation by resveratrol may be linked with suppression of NF-[kappa]B (Bhardwaj et al. 2007). Surprisingly, the p65 subunit of NF-[kappa]B has been found to directly interact with STAT3 protein (Yu et al. 2002) and JAK2 kinase activation has been shown to be required for erythropoietin-induced NF-[kappa]B activation (Digicaylioglu and Lipton 2001). Thus, it may be possible that the suppression of JAK2 activation is the potential link for inhibition of both NF-[kappa]B and STAT3 activation by resveratrol.

We further demonstrate that resveratrol suppressed the expression of various STAT3-regulated gene products, such as antiapoptotic (Bcl-xL, Bd-2, Survivin, and IAP1), proliferative (Cydin D1), metastatic (MMP-2and MMP-9), and angiogenetic (VEGF). Constitutive STAT3 activation has been known to induce resistance to apoptosis (Catlett-Falcone et al. 1999), possibly through upregulation of Bcl-2, Bcl-xL, IAP1, and Survivin expression (Nielsen et al. 1999). The down-modulation of Cyclin Dl expression by resveratrol has been found to be associated with the suppression in proliferation and accumulation of cells in the sub-G1 phase of the cell cycle, suggesting that this protein plays an important role in the observed anti-proliferative effects of resveratrol. We also found that resveratrol significantly inhibited tumor cell invasion activity in FaDu cells, which may be explained by its ability to negatively regulate the expression of MMP-9 and MMP-2 proteins. Bcl2, Bcl-xL, and survivin expressions are mainly regulated by STAT3 pathway, and these proteins are also overexpressed in HNSCC cells (Sharma et al. 2004). The downregulation of Bel- 2, Bcl-xL, and Survivin proteins could also account for resveratrol's ability to induce substantial apoptosis in FaDu cells.

We also noted that resveratrol can potentially improve 1R sensitivity in FaDu cells. Interestingly, resveratrol was found to significantly potentiate the cytotoxic effect upon co-treatment with radiotherapy. The combinational treatment potentiated IR-induced apoptosis through the downregulation of various STAT3-regulated gene products. Fang et al. reported that resveratrol can significantly enhance radiation sensitivity in prostate cancer by inhibiting cell proliferation and promoting cellular senescence and apoptosis (Fang et al. 2012). Also, resveratrol can enhance prostate cancer cell response to ionizing radiation through the modulation of the AMPK, Akt, and mTOR pathways (Rashid et al. 2011). Our data suggests that the potential of resveratrol as a novel radio-sensitizer for HNSCC treatment and warrants further investigation.

Overall, our results indicate for the first time that resveratrol can inhibit constitutive STAT3 signaling pathway through upregulation of SOCS-1 protein in HNSCC cells, as well as can enhance the effects of radiotherapy through the downregulation of gene products that mediate tumor cell survival, proliferation, invasion, and metastasis in HNSCC.

http://dx.doi.org/10.1016/j.phymed.2016.02.011

ARTICLE INFO

Article history:

Received 8 December 2015

Revised 12 February 2016

Accepted 15 February 2016

Abbreviations: ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; ICC, immunocytochemistry; JAK, Janus tyrosine kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PTPs, protein tyrosine phosphatases; RTCA, real-time cell analysis; SCCHN, squamous cell carcinoma of the head and neck; SDS-PAGE, sodium dodecyl-polyacrylamide gel electrophoresis; SOCS, suppressor of cytokine signaling; STAT3, signal transducer and activator of transcription 3.

Conflict of interest

The authors declare no competing financial interests.

Acknowledgments

This research was supported by the Convergence of Conventional Medicine and Traditional Korean Medicine R&D project funded by the Ministry of Health & Welfare through the Korea Health Industry Development Institute (KHIDI) (grant no. H114C1723). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2015R1A4A1042399). The authors would also like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through the Research Group Number (RG-1435-081).

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Seung Ho Baek (a), (1), Jeong-Hyeon Ko (a), (1), Hanwool Lee (a), Jinhong Jung (b), Moonkyoo Kong (b), Jung-woo Lee (c), Junhee Lee (a), Arunachalam Chinnathambi (d), ME Zayed (d), Sulaiman Ali Alharbi (d), Seok-Geun Lee (a), Bum Sang Shim (a), Gautam Sethi (d,e), Sung-Hoon Kim (a), Woong Mo Yang (a), Jae-Young Um (a), Kwang Seok Ahn (a), *

(a) College of Korean Medicine, Kyung Hee University, 24 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-701. Republic of Korea

(b) Department of Radiation Oncology, Kyung Hee University Medical Center, Kyung Hee University School of Medicine, 23 Kyungheedae-ro, Dongdaemoon-gu, Seoul 130-872, Republic of Korea

(c) Department of Oral & Maxillofacial Surgery, Kyung Hee University Dental Hospital, Kyung Hee University School of Dentistry, 23 Kyungheedae-ro, Dongdaemun-gu, Seoul 130-872, Republic of Korea

(d) Department of Botany and Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia

(e) Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 117600, Singapore

* Corresponding author. Tel.; +82 2 961 2316.

E-mail address: ksahn@khu.ac.kr (K.S. Ahn).

(1) These authors contributed equally to this study.
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Title Annotation:Signal transducer and activator of transcription 3
Author:Baek, Seung Ho; Ko, Jeong-Hyeon; Lee, Hanwool; Jung, Jinhong; Kong, Moonkyoo; Lee, Jung-woo; Lee, Ju
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9SING
Date:May 15, 2016
Words:8554
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