β-catenin signaling pathway

Biochemical Pharmacology 84 (2012) 1143–1153

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The b-catenin/TCF complex as a novel target of resveratrol in the Wnt/b-catenin signaling pathway Hui-Jye Chen a, Le-Shiang Hsu b, Yu-Ting Shia a, Meng-Wei Lin a, Chung-Ming Lin b,* a b

Graduate Institute of Molecular Systems Biomedicine, China Medical University, Taichung 404, Taiwan Department of Biotechnology, Ming Chuan University, Taoyuan 333, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 June 2012 Accepted 13 August 2012 Available online 19 August 2012

Wnts are secreted glycolipoproteins that play important roles in the regulation of embryonic development and tissue homeostasis. Binding of Wnt to receptors and co-receptors causes inactivation of the b-catenin destruction complex, which leads to the stabilization and nuclear translocation of bcatenin to initiate Wnt-responsive gene expression after associating with TCF in the nucleus. As its deregulation results in serious human diseases, especially cancers, the Wnt signaling pathway serves as a promising platform for screening anti-cancer drugs. Resveratrol was selected based on its ability to inhibit the b-catenin/TCF-mediated transcriptional activity. Resveratrol, a natural phytoalexin found in a variety of plants, possesses health-promoting properties including anti-aging, anti-inflammatory, antioxidant, anti-cancer, cardioprotective and neuroprotective activities. We found that resveratrol indeed exhibited dose-dependent suppression of Wnt signaling, reduced the expression of Wnt target genes such as cyclin D1 and conductin, and inhibited the growth of Wnt-stimulated cells and Wnt-driven colorectal cancer cells. Further studies indicated that resveratrol functions downstream of GSK3b. Treatment with resveratrol did not alter the amount of b-catenin and its distribution in the cytoplasm and nucleus, suggesting that resveratrol did not affect the accumulation and nuclear targeting of bcatenin. In contrast, co-immunoprecipitation and in vitro binding analyses substantiated that resveratrol was capable of disrupting the binding between b-catenin and TCF4, contributing to the decreased Wnt signaling. Our discoveries not only reveal a novel target of resveratrol in the Wnt signaling pathway but also show the potential of therapy with harmless resveratrol in colorectal cancer and other Wnt-related diseases. Crown Copyright ß 2012 Published by Elsevier Inc. All rights reserved.

Keywords: Resveratrol Wnt b-Catenin TCF Colorectal cancer

1. Introduction Wnts are a family of secreted glycolipoproteins that initiate a signaling cascade to regulate embryonic development at different stages and maintain homeostasis of adult tissues by binding to its cognate receptors. Aberrant Wnt signaling leads to a series of human diseases such as neurodegenerative diseases, familial exudative vitreorectinopathy (FEVR), osteoporosis, osteoarthritis, polycystic kidney disease, leukemia, schizophrenia, pulmonary fibrosis and various cancers [1–4]. Therefore, the Wnt signal transduction pathway has become a paradigm for pharmacological intervention in these human diseases [5,6]. In the absence of Wnt, the scaffolding protein Axin functions as a platform for its binding to adenomatous polyposis coli (APC), glycogen synthase kinase-3b (GSK3b), casein kinase 1 (CK1),

* Corresponding author at: Department of Biotechnology, Ming Chuan University, 5 De Ming Rd., Gui Shan District, Taoyuan 333, Taiwan. Tel.: +886 3 350 7001x3558; fax: +886 3 359 3878. E-mail address: [email protected] (C.-M. Lin).

microtubule actin crosslinking factor 1 (MACF1) and b-catenin to form the so-called ‘‘destruction complex’’ in the cytoplasm. In this complex, phosphorylation of b-catenin on residue Ser45 by CK1 primes the phosphorylation of residues Ser33, Ser37, and Thr41 by GSK3b, and these phosphorylation events create a docking site for E3 ubiquitin ligase beta-transducin repeat-containing protein (bTrcp) to poly-ubiquitinate b-catenin, and subsequently the ubiquitinated b-catenin is targeted to proteasome for degradation to maintain its very low level in the cytoplasm. Meanwhile, the TCF/LEF family of transcriptional factors associates with the repressor proteins Groucho and CtBP to repress the expression of Wnt target genes inside the nucleus. When the Wnt ligand binds to its receptor Frizzled (Fz) and co-receptor low-density lipoprotein receptor-related protein 5/6 (LRP5/6), the C terminal PPPSPxS motifs of LRP5/6 are phosphorylated by CK1 and GSK3b. The Axin complex will be translocated to the cell membrane from the cytoplasm via the assistance of MACF1 [7], and Axin docks on the phosphorylated LRP5/6, finally being degraded. b-Catenin is stabilized and accumulates in the cytoplasm, and subsequently mobilizes into the nucleus to complex with TCF/LEF as well as coactivators Bcl9, Pygopus, and CBP/p300 to activate the expression

0006-2952/$ – see front matter . Crown Copyright ß 2012 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.08.011

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of Wnt target genes such as cyclin D1, c-Myc, Axin2/conductin, endothelin-1 (ET-1) and brachyury/T [8]. Colorectal cancer (CRC) is the second leading cause of cancer death in many countries. Although there has been progress in the prevention, screening and chemotherapy of CRCs, patients with late-stage CRCs have a poor prognosis and a 40% overall mortality rate [9,10], indicating an urgent need to develop new drugs for the prevention and therapy of CRC. Familial adenomatous polyposis (FAP) and more than 80% of sporadic colorectal cancers have mutations in the APC gene that result in truncation of the protein [11,12]. Mutations in the N-terminal conserved phosphorylation sites of b-catenin were also observed in CRC patients [13,14]. These mutations prohibit the degradation of b-catenin by the proteasome, contributing to the stabilization and accumulation of b-catenin in the cytoplasm. The accumulated b-catenin is then translocated into the nucleus and activates the expression of Wnt target genes such as c-Myc and cyclin D1, leading to uncontrolled cell proliferation and eventually the development of CRC. As a result, disruption of b-catenin-mediated nuclear signaling would be a promising way to treat CRCs. Resveratrol (3,40 ,5-trihydroxystilbene) is a natural phytoalexin with the stilbene structure that can be found in many plants such as grapes, berries and peanuts [15]. Resveratrol displays pleiotropic health benefits, including anti-aging, anti-inflammatory, antioxidant, cardioprotective and neuroprotective activities, and can hence be regarded as a potential chemopreventive and therapeutic agent for cardiovascular disease, diabetes and neurodegenerative disorder. Besides, resveratrol exhibits anti-cancer activity by inhibiting cell proliferation and inducing apoptosis of many solid tumors and hematologic malignancies [16–18]. Mutations in the APC gene or serine/threonine phosphorylation sites within exon 3 of the b-catenin gene that result in increased b-catenin-mediated transcriptional activity are observed in many tumor cells, including hepatocellular carcinoma [19], gastric carcinoma [20], melanoma, endometrial cancer, and prostate cancer [21,22], in addition to CRCs [14], suggesting that aberrant b-catenin-mediated nuclear signaling underpins these tumors, and disruption of this signaling would suppress the growth of these tumors. To this purpose, we have undertaken drug screening and obtained the plant bioflavonoid resveratrol, which can inhibit b-catenin-mediated transcriptional activity. Further studies showed that resveratrol acts to disrupt the interaction of b-catenin with TCF4 and inhibits the growth of Wnt-stimulated cells as well as Wnt-driven CRC cells, and thus resveratrol is promising for the therapy of Wnt-related cancers.

Plasmid pET-TCF4 was constructed by cloning the BamHI–XhoI PCR fragments of human TCF4 cDNA into pET-23a (+) vector (Novagen, Madison, WI, USA). 2.2. Cell culture P19 cells were cultured in a-MEM medium containing 7.5% bovine calf serum (Hyclone, Thermo Scientific, Logan, UT, USA), 2.5% fetal bovine serum (Hyclone), 1% penicillin–streptomycin, and sodium pyruvate. COS-7, HCT 116, and WiDr cells were cultured in DMEM medium with 10% fetal bovine serum, 1% penicillin–streptomycin, and sodium pyruvate. SW480 cells were cultured in RPMI medium with 10% fetal bovine serum, 1% penicillin–streptomycin, and sodium pyruvate. All cells were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and incubated at 37 8C in a humidified chamber with 5% CO2. L cells and L-Wnt-3a cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Culture of L cells and L-Wnt-3a cells and the preparation of control-conditioned medium and Wnt-3a-conditioned medium were performed as described previously [7]. Briefly, L cells and L-Wnt-3a cells were split 1:10 into 10 ml of medium and cultured for 4 days. The culture medium was then collected. 10 ml of medium was added to the cells and cultured for an additional 3 days, and the medium was collected. The first batch and second batch of media were mixed in a 1:1 ratio and used as the controlconditioned medium and Wnt-3a-conditioned medium, respectively. COS-7 and P19 cells were treated with conditioned media for about 20 h and 16 h before harvesting. 2.3. Cell viability assay The cell viability was evaluated by a methylthiazol tetrazolium (MTT) assay. P19, HCT 116, SW480 and WiDr cells were seeded onto a 96-well plate at a density of 2  103/well. On the next day, the culture medium was replaced with fresh medium (for HCT 116, SW480 and WiDr cells) or Wnt-3a-conditioned medium (for P19 cells) containing different concentrations of resveratrol, and cultured for an additional 4 days. The medium was removed, and the cells were incubated with 50 ml of MTT (2 mg/ml), then 100 ml of DMSO was added to dissolve the formazan. The plate was read at a wavelength of 570 nm by an ELISA reader (Multiskan Fc, Thermo Scientific, Rockford, IL, USA). Each test was performed in five replicates. The reduction in viability of the drug-treated cells was expressed as a percentage compared to those without drug treatment, which was set as 100%.

2. Materials and methods

2.4. Immunoprecipitation and Western blotting

2.1. Reagents

Immunoprecipitation and Western blotting analyses were performed as described previously [7]. In brief, cells were lysed in protein lysis buffer (1% [v/v] Triton X-100, 150 mM NaCl, 50 mM Tris–Cl, pH 8.0, 5 mM EDTA, and protease inhibitors) and the cell lysate was incubated with primary antibody and protein A or G beads (GE Healthcare, Uppsala, Sweden) at 4 8C on a rotator overnight. The beads were washed, boiled in Laemmli buffer, and processed for Western blotting. The blots were then soaked in blocking solution (5% nonfat milk in TBS, 25 mM Tris, 150 mM NaCl, pH 7.4) for 1 h, washed and incubated with primary antibody for 2 h at room temperature or 4 8C overnight. Blots were then washed and incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), and finally the protein signals were visualized by ECL (Millipore, Billerica, MA, USA). Quantification of data and subsequent statistical analyses were performed using Image J (NIH) and the two-tailed Student t test, respectively.

Primary antibodies for b-tubulin, brachyury (T), Axin2 (Conductin), c-Myc, GAPDH, lamin A, TCF4, His (Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-catenin (BD Transduction Laboratories, San Jose, CA, USA), and cyclin D1 (Cell Signaling, Danvers, MA, USA) were obtained from the indicated vendors. Protease inhibitor cocktail tablets were obtained from Roche (Mannheim, Germany). Trans-resveratrol, MTT (3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), and lithium chloride (LiCl) were purchased from Sigma–Aldrich (St. Louis, MO, USA). DAPI (40 ,6-diamidino-2-phenylindole, dihydrochloride) was purchased from Invitrogen (Camarillo, CA, USA). pTOPFLASH was obtained from Dr. Hsiu-Ming Shih (Academia Sinica, Taiwan). pGEX-Bcatfl was a gift of Dr. David Rimm (Yale University; Addgene No. 24193). pGL3-OT was obtained from Dr. Bert Vogelstein (The Johns Hopkins University; Addgene No. 16558).

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Nuclear and cytosolic fractions were isolated from treated cells according to the manual for NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).

performed by mixing 200 ng of GST-b-catenin proteins with 125 ng of His-TCF4 proteins, incubating with different concentrations of resveratrol, and subjecting the mixture to co-immunoprecipitation analysis with His antibody. Detection was then performed using b-catenin antibody in Western blotting analysis.

2.6. b-Catenin/TCF-mediated luciferase activity assay

3. Results

COS-7 (6  104 cells/well), P19, HCT 116, SW480 or WiDr (1  105 cells/well) cells were seeded onto a 24-well plate overnight and transfected with pGL3-OT (for all cells except WiDr cells, for which pTOPFLASH was used) and pTK-Renilla vectors, and either treated with fresh medium or fresh medium containing resveratrol or treated with control-conditioned medium or different concentrations of resveratrol in Wnt-3a-conditioned medium before dual luciferase activity assay using the DualLuciferase Reporter Assay System (Promega, Madison, WI, USA). Some Wnt-reporter-transfected cells were either treated with fresh medium, LiCl in fresh medium or resveratrol with LiCl in fresh medium before the activity assay. Each test was performed in triplicate. Firefly luciferase activity was calibrated by Renilla luciferase activity.

3.1. Resveratrol inhibits Wnt/b-catenin signaling

2.5. Isolation of nuclear and cytosolic fractions

Total RNAs were isolated using TRIZOL Reagent (Invitrogen) and cDNAs were synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche) and amplified using the Platinum PCR SuperMix (Invitrogen) according to the providers’ brochures. The primers used were: mouse conductin (50 -GTTAGTGACTCTCCTTCCAGATCC-30 and 50 -GAGTGTAAAGACTTGGTCCACCTG-30 , amplicon 237 bp), human conductin (50 -GCCTGGCTCCAGAAGATCACAAAG30 and 50 -CCAGATCTCCTCAAACACCGCTCC-30 , amplicon 245 bp), mouse endothelin-1 (ET-1) (50 -CCAAGGAGCTCCAGAAACAGC-30 and 50 -GCCCTGAGTTCTTTTCCTGC-30 , amplicon 351 bp), human and mouse c-Myc (50 -AGCCGCCGCTCCGGGCTCTGCTC-30 and 50 GCTGGAGGTGGACAGACGCTGTG-30 , amplicon 336 bp and 339 bp, respectively), human cyclin D1 (50 -GCCAACCTCCTCAACGACCGGGTGC-30 and 50 -CAACGAAGGTCTGCGCGTGTTTGCGG-30 , amplicon 502 bp) and human and mouse GAPDH (50 CGTATTGGGCGCCTGGTCACCAGG-30 and 50 -GAGGGGCCATCCACAGTCTTCTGG-30 , amplicon 539 bp). All primers were obtained from either Invitrogen or MB MISSION biotech (Taipei, Taiwan).

Wnts are secreted glycolipoproteins that function as signaling molecules to regulate embryonic development and determine the cell fate. Deregulation in Wnt signaling results in a series of human diseases, notably cancers. Therefore, the Wnt/b-catenin signaling pathway would be an ideal target for the screening of anti-cancer drugs. In searching for drugs for the therapy of Wnt-related cancers, we set up a drug screening platform based on the bcatenin/TCF-mediated transcriptional activity. A couple of potential drugs that inhibit Wnt/b-catenin signaling were obtained, one of which is resveratrol. To further confirm the inhibitory effects of resveratrol on Wnt/b-catenin signaling, cells were transfected with Wnt reporter plasmids and treated with either the controlconditioned medium or Wnt-3a-conditioned medium or different concentrations of resveratrol in the Wnt-3a-conditioned medium, and the resulting lysate was assayed for the b-catenin/TCFmediated luciferase activity. Two mammalian cells, COS-7 and P19 cells, were chosen in this study as they have good response to the Wnt ligand. As shown in Fig. 1A, treatment of COS-7 cells with resveratrol decreased the Wnt/b-catenin signaling in a concentration-dependent fashion. Resveratrol also abolished the b-catenin/ TCF-mediated transcriptional activity at all tested concentrations in P19 cells (Fig. 1B). These results suggested that this inhibition is a general effect for Wnt-stimulated cells. To address whether resveratrol inhibits the expression of endogenous Wnt target genes at the mRNA and protein levels, we first treated cells with resveratrol for RT-PCR analyses, and the result showed that resveratrol treatment reduced the expressions of Wnt target genes such as ET-1, conductin (Axin2), and c-Myc (Fig. 1C). Western blotting analyses also indicated that treatment of cells with increasing concentrations of resveratrol reduced the protein level of Wnt target genes such as T (brachyury), conductin, and cyclin D1 (Fig. 1D). These data suggested that resveratrol can inhibit Wnt/bcatenin signaling.

2.8. Immunofluorescent staining

3.2. Resveratrol acts downstream of GSK3b

Cells were seeded onto coverslips the day before staining. On the next day, cells were fixed with 4% paraformaldehyde (Electron Microscope Sciences, Hatfield, PA, USA) for 15 min at room temperature, blocked with 10% normal goat serum (Invitrogen) for 30 min, and probed with primary antibody at 4 8C overnight. Then, cells were stained with Alexa 488 or 568-conjugated goat anti-mouse or anti-rabbit secondary antibodies (Invitrogen/ Molecular Probes) for one hour, stained with DAPI for 5–10 min, washed and mounted with Shandan mounting medium (Thermo Fisher, Rockford, IL, USA). Images were captured using a Zeiss AX10 Fluorescence microscope equipped with an imaging system, AxioCam MRm (Carl Zeiss, Germany).

To pinpoint on which step of Wnt/b-catenin signaling resveratrol acts, we treated cells with resveratrol in the presence or absence of LiCl. LiCl is a chemical that can inhibit the kinase activity of GSK3b, an important enzyme that phosphorylates bcatenin, and phosphorylated b-catenin is subjected to proteasomal degradation. Treatment of cells with LiCl causes the accumulation of b-catenin in the cytoplasm and an increase in b-cateninmediated nuclear signaling. As shown in Fig. 2A, LiCl treatment resulted in an increase of b-catenin/TCF-mediated transcriptional activity, while LiCl treatment with resveratrol decreased the activity to the basal level in COS-7 cells. We also observed the same phenomenon in P19 cells (Fig. 2B). However, as compared with treatment with control medium, a lower transcriptional activity was observed following treatment with resveratrol and LiCl, which is very likely due to the suppression of both the LiCl-induced and intrinsic Wnt activities by resveratrol in P19 cells. The data presented in Fig. 2A and B showed that resveratrol seems to inhibit Wnt/b-catenin signaling downstream of GSK3b. However, we cannot rule out the possibility that resveratrol targets GSK3b per se to increase its activity, leading to a reduced amount

2.7. Reverse transcription-polymerase chain reaction analysis

2.9. Protein purification and in vitro binding assay pGEX-Bcatfl and pET-TCF4 were transformed into bacteria BL21 (DE3). GST-tagged b-catenin and His-tagged TCF4 proteins were purified using glutathione-Sepharose 4B beads (GE Healthcare) and HisBind Resin (Novagen) respectively according to the manufacturers’ brochures. The in vitro binding assay was

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Fig. 1. Resveratrol inhibits Wnt/b-catenin signaling in Wnt-stimulated cells. (A) Resveratrol inhibits b-catenin/TCF-mediated transcriptional activity in COS-7 cells. Cells were transfected with pGL3-OT and pTK-Renilla vectors, treated with control-conditioned medium (CTL), Wnt-3a-conditioned medium (Wnt) or different concentrations of resveratrol (Res) in Wnt-3a-conditioned medium for 20 h, and then assayed for b-catenin/TCF-mediated transcriptional activity. Each bar is the mean  S.E.M. Each experiment was performed in triplicate. (B) Resveratrol inhibits b-catenin/TCF-mediated transcriptional activity in P19 cells. Cells were treated and analyzed as in (A), except that cells were treated with 0–20 mM of resveratrol for 16 h. (C) The effects of resveratrol on the expression of Wnt target genes in P19 cells. Cells were treated with control-conditioned medium (CTL), Wnt-3a-conditioned medium (Wnt), or 20 mM of resveratrol in Wnt-3a-conditioned medium (Res; 20 mM) for 16 h, and the expressions of Wnt responsive genes were analyzed by RT-PCR for ET-1, conductin, and c-Myc genes. GAPDH served as an internal control. The signal intensities from Wnt target genes were normalized by that of GAPDH. Data from the treatments with control-conditioned medium were set to 1, and the relative gene expressions of cells treated with Wnt-3a-conditioned medium and resveratrol in Wnt-3a-conditioned medium were then calculated. (D) The effects of resveratrol on the protein expression of Wnt target genes in P19 cells. Cells were treated with controlconditioned medium (CTL), Wnt-3a-conditioned medium (Wnt), or 20–30 mM of resveratrol (Res) in Wnt-3a-conditioned medium for 16 h and assayed for Western blotting analyses with the indicated proteins. GAPDH served as a loading control. The data were quantified and analyzed as in (C). *P < 0.05 for cells treated with resveratrol in the presence of Wnt versus Wnt3a-stimulated cells.

of b-catenin and decreased b-catenin/TCF-mediated transcriptional activity in the presence of LiCl. As GSK3b activity is required for b-catenin turnover, the increase in GSK3b activity by resveratrol would be expected to result in a decrease in the amount of b-catenin. To address this possibility, we treated cells with resveratrol in the presence of LiCl and checked the amount of b-catenin by Western blotting analysis. As shown in Fig. 2C, LiCl

treatment resulted in the accumulation of b-catenin and treatment with resveratrol and LiCl did not reduce the level of b-catenin in P19 cells. Moreover, as shown in Fig. 3A, Wnt treatment resulted in the accumulation of b-catenin, and treatment with resveratrol under Wnt did not reduce the level of b-catenin in P19 cells. The data presented in Figs. 2A–C and 3A suggested that resveratrol appears not to interfere with GSK3b activity, and the inhibitory

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Fig. 2. Resveratrol acts downstream of GSK3b. (A and B) Resveratrol alleviated the LiCl-elicited b-catenin/TCF-mediated transcriptional activity in COS-7 (A) and P19 (B) cells. Cells were transfected with pGL3-OT and pTK-Renilla vectors, treated with either fresh medium or 25 mM of LiCl in fresh medium or 25 mM of LiCl with 20 mM of resveratrol in fresh medium for (A) 20 h or (B) 16 h, and assayed for b-catenin/TCF-mediated transcriptional activity. Each bar is the mean  S.E.M. Each experiment was performed in triplicate. (C) Resveratrol does not reduce the amount of b-catenin in the presence of LiCl in P19 cells. P19 cells were treated as in (B) and cells were collected for Western blotting analysis with anti-b-catenin. GAPDH served as the loading control. (D) Resveratrol reduced the LiCl-elicited b-catenin/TCF-mediated transcriptional activity in HCT 116 cells. Cells were transfected with pGL3-OT and pTK-Renilla vectors, and treated with either fresh medium or 10 mM of LiCl in fresh medium or LiCl with 20, 30, 50 mM of resveratrol in fresh medium for 22 h, and assayed for b-catenin/TCF-mediated transcriptional activity. Each bar is the mean  S.E.M. Each experiment was performed in triplicate. (E) Resveratrol reduced the LiCl-elicited b-catenin/TCF-mediated transcriptional activity in SW480 cells. Cells were transfected with pGL3-OT and pTK-Renilla vectors, and treated with either fresh medium or 20 mM of LiCl in fresh medium or LiCl with 20, 30, 50 mM of resveratrol in fresh medium for 9 h, and assayed for b-catenin/TCF-mediated transcriptional activity. (F) Resveratrol reduced the LiCl-elicited b-catenin/TCF-mediated transcriptional activity in WiDr cells. Cells were transfected with pTOPFLASH and pTK-Renilla vectors, treated with either fresh medium or 5 mM of LiCl in fresh medium or LiCl with 20, 30, 50, and 75 mM of resveratrol in fresh medium for 22 h, and assayed for b-catenin/TCF-mediated transcriptional activity.

effect is on other steps downstream of GSK3b in the Wnt/bcatenin signaling pathway.

3.4. Disruption of the b-catenin/TCF complex by resveratrol in Wnt/ b-catenin signaling

3.3. Resveratrol does not interfere with the mobilization of b-catenin into the nucleus during Wnt/b-catenin signaling

In canonical Wnt signaling, once inside the nucleus, b-catenin associates with TCF/LEF transcription factor, and then the complex binds to TCF/LEF binding site(s) on the promoter of Wntresponsive genes to activate the gene expression. To assess whether resveratrol has any effect on the binding of b-catenin to TCF/LEF, we obtained cell lysate from cells treated with controlconditioned medium, Wnt-3a-conditioned medium or resveratrol under Wnt stimulation and performed co-immunoprecipitation experiments. As shown in Fig. 4A, Wnt treatment enhanced the binding between b-catenin and TCF4 as compared with treatment with control-conditioned medium (compare lane 2 to lane 1), while treatment with resveratrol in the presence of Wnt substantially inhibited the association of b-catenin with TCF4 (compare lane 3 to lane 2). This inhibitory effect was not due to unequal amounts of b-catenin being precipitated (IPed b-catenin, lanes 1–3 in the lower panel of Fig. 4A) or the reduced expression of TCF4 and b-catenin upon resveratrol treatment in the presence of Wnt (lanes 5 and 6 in the right panel of Fig. 4A). To address whether resveratrol can directly disrupt the b-catenin/TCF association, we expressed and purified GST-b-catenin and His-TCF4 proteins from bacteria for an in vitro binding assay. As shown in Fig. 4B,

As resveratrol did not affect the accumulation of b-catenin in the cells during Wnt signaling (Figs. 2C and 3A), we further checked whether the inhibitory effect is on the mobilization of bcatenin into the nucleus. For this purpose, we isolated cytoplasmic and nuclear fractions from control-conditioned medium, Wnt-3aconditioned medium, or both Wnt-3a and resveratrol-treated cells and examined the b-catenin amount in both fractions by Western blotting. As shown in Fig. 3B, treatment of COS-7 cells with Wnt increased the b-catenin amount in both the cytoplasm and nucleus as compared with cells treated with control-conditioned medium. However, treatment of cells with resveratrol under Wnt did not change the distribution of b-catenin in the nucleus and cytoplasm. We also obtained the same conclusion from P19 cells (Fig. 3C). Furthermore, immunofluorescent staining showed that b-catenin mobilization into the nucleus is not disturbed by resveratrol treatment under Wnt stimulation (Fig. 3D). These data suggested that Wnt-induced nuclear targeting of b-catenin is not affected by resveratrol.

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Fig. 3. Resveratrol does not interfere with the translocation of b-catenin into the nucleus upon Wnt stimulation. (A) The protein level of b-catenin was not affected by resveratrol under Wnt stimulation in P19 cells. Cells were treated with control-conditioned medium (CTL), Wnt-3a-conditioned medium (Wnt) or 20 mM of resveratrol (Res) in Wnt-3a-conditioned medium for 16 h and the cell lysate was subjected to Western blotting analysis for b-catenin and GAPDH. (B and C) The effect of resveratrol on the distribution of b-catenin in the cytoplasm and nucleus of COS-7 (B) and P19 (C) cells. Cells were treated as in (A) except COS-7 cells, which were treated for 20 h. Cells were then fractionated into cytoplasmic and nuclear portions for Western blotting analyses as indicated. b-tubulin (indicated by an arrow) and lamin A served as the cytosolic and nuclear markers, respectively. C, cytosol. N, nucleus. Asterisk, a non-specific cross-reacting signal in the nuclear fraction with anti-b-tubulin. (D) Resveratrol does not interfere with the nuclear translocation of b-catenin. COS-7 cells were treated as in (B), fixed and processed for immunofluorescent staining with anti-b-catenin (colored in green) and with DAPI for the nuclei (colored in blue). Merge, the merged picture. The images originally photographed at 400 magnification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

resveratrol directly disrupted the binding between b-catenin and TCF4 in a concentration-dependent manner. These results revealed that resveratrol could directly disturb the binding of b-catenin to TCF, leading to the inhibition of Wnt/b-catenin signaling. 3.5. Resveratrol inhibits the survival of Wnt-stimulated cells and colon cancer cells Wnt/b-catenin signaling is essential for cell growth. As resveratrol inhibits Wnt/b-catenin signaling in P19 cells, we then

tested the effect of resveratrol on the survival of Wnt-treated P19 cells. As shown in Fig. 5A, resveratrol inhibited the survival of Wntstimulated P19 cells in a dose-dependent manner, with IC50 of 17.86  0.79 mM. Many colorectal cancer cells are Wnt-driven cells. The oncogenecity originates from aberrant Wnt signaling in these cells due to mutations in Wnt signaling components. For example, HCT 116 cells harbor a b-catenin mutation [11] that forbids its degradation by the proteasomal system. SW480 and WiDr cells both have different APC mutations [11], contributing to b-catenin

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Fig. 4. Resveratrol disrupts the interaction of b-catenin with TCF4. (A) Resveratrol interrupts the association of b-catenin with TCF4 in Wnt-challenged P19 cells. Cells were treated with control-conditioned medium (CTL), Wnt-3a-conditioned medium (Wnt) or 25 mM of resveratrol (Res) in Wnt-3a-conditioned medium for 16 h and the cell lysate was used for co-immunoprecipitation analyses with anti-bcatenin, the resulting blot being detected with anti-TCF4. Western blotting analyses of TCF4, b-catenin, GAPDH (as the loading control) and precipitated b-catenin (IPed b-catenin) are also shown. Total, total cell lysate. (B) Resveratrol directly disrupts the interaction of b-catenin with TCF4. GST-b-catenin proteins and His-TCF4 proteins were expressed and purified from bacteria for the in vitro binding assays. Equal molar amounts of GST-b-catenin proteins (GST-b-cat; 200 ng) and His-TCF4 proteins (125 ng) were mixed, incubated with different concentrations of resveratrol (Res), and the mixture was subjected to co-immunoprecipitation (IP) with His antibody and Western blotting analysis with b-catenin antibody. Western blotting controls (WB) for GST-b-catenin and His-TCF4 proteins are shown on the right side of the figure. The precipitated His-TCF4 is also shown (IPed His-TCF4).

accumulation. As resveratrol can restrain the growth of Wnttreated P19 cells, we then examined whether resveratrol is able to inhibit the growth of colorectal cancer cells such as HCT 116, WiDr, and SW480 through inhibiting the b-catenin-mediated nuclear signaling of the cells. As shown in Fig. 5B–D, resveratrol inhibited the survival of HCT 116, WiDr and SW480 cells in a dosedependent manner, although a higher concentration of resveratrol was needed for the inhibition of WiDr (IC50 = 104.13  0.77 mM) and SW480 (IC50 = 73.18  0.22 mM) than that of HCT 116 (IC50 = 33.27  1.03 mM) cells. These data indicated that resveratrol can repress the viability of these colorectal cancer cells as well as Wnt-stimulated cells. 3.6. Resveratrol inhibits Wnt signaling in colorectal cancer cells by disrupting the binding of b-catenin to TCF4 Given that resveratrol can inhibit the cell growth of CRC cells, we suspected that resveratrol also could inhibit their intrinsic Wnt signaling. As shown in Fig. 6A, resveratrol was able to suppress the b-catenin/TCF-mediated transcriptional activity in HCT 116 cells

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in a concentration-dependent manner, with a 75% or greater reduction after treatment with 20 mM of resveratrol. The mRNA and protein levels of Wnt target genes such as cyclin D1, c-Myc, and conductin (Axin2) were decreased with an increase in the concentration of resveratrol (Fig. 6B and C). To ascertain whether resveratrol acts downstream of GSK3b in colorectal cancer cells, as seen in COS-7 and P19 cells (Fig. 2A and B), we treated HCT 116, SW480 and WiDr cells with LiCl in the absence and presence of resveratrol and assayed for b-catenin/TCF-mediated transcriptional activity. The results showed that LiCl treatment resulted in an increase in b-catenin/TCF-mediated transcriptional activity, whereas the combined treatment of LiCl with resveratrol inhibited this activity in the three colorectal cancer cells (Fig. 2D–F). These data suggested that resveratrol seems to target downstream of GSK3b in these colon cancer cells. As the movement of b-catenin into the nucleus is important for transmitting the downstream Wnt signal, it is likely that resveratrol impeded Wnt signaling by inhibiting the movement of b-catenin from the cytoplasm to the nucleus in CRC cells. Treatment of HCT 116 cells with resveratrol did not change the distribution of b-catenin in the cytoplasm and nucleus and the amount of nuclear transcription factor TCF4 (Fig. 6D). Consistently, treatment of HCT 116 cells with resveratrol did not interfere with the translocation of b-catenin into the nucleus as compared with the control by immunofluorescent staining (Fig. 6E). The same phenomenon was observed in SW480 and WiDr cells (Fig. 7A and B). These data suggested that resveratrol has other targeting site(s) downstream of Wnt signaling. As can be seen from Fig. 4, resveratrol could disrupt the interaction of b-catenin with TCF4 in Wnt-stimulated P19 cells, and we infer that resveratrol could also inhibit Wnt signaling through the same mechanism in HCT 116 colorectal cancer cells. As shown in Fig. 6F, treatment of cells with resveratrol decreased the binding of b-catenin to TCF4 at all tested concentrations, and this inhibitory effect was not due to the decreased expression of bcatenin or TCF4, as the amounts of b-catenin and TCF4 were maintained at all tested concentrations of resveratrol (right panel of Fig. 6F). In conclusion, resveratrol can inhibit the growth of colorectal cancer cells by blocking the intrinsic Wnt signaling through interfering with the binding of b-catenin to TCF4, a promising drug-targeting site. 4. Discussion The Wnt signaling pathway is not only involved in various biological processes including cell proliferation, differentiation, motility, survival and/or apoptosis, but also plays pivotal roles in embryonic development and maintenance of homeostasis in mature tissues. De-regulation in Wnt signaling causes various human diseases. Thus, drugs that block Wnt signaling could have potential for the therapy of these diseases. The natural compound resveratrol is harmless to human subjects [23] and is able to inhibit the growth of cancer cells, to arrest the cell cycle progression and to elicit cell apoptosis [18,24–29]. It seems to be an ideal reagent for cancer prevention and chemotherapy. As up-regulation of Wnt/ b-catenin signaling is correlated with the formation of various cancers, especially colorectal cancers [1,4,5,10], it would be interesting to ascertain whether the anti-tumor activity of resveratrol is attributed to the inhibition of Wnt/b-catenin signaling. Current studies on the effects of resveratrol on Wnt signaling are perplexing. Studies have shown that resveratrol at 10–100 mM inhibits Wnt signaling [17,30]. However, it has been reported that resveratrol augments Wnt signaling to promote osteoblastogenesis and angiogenesis through the activation of ERKs to phosphorylate GSK3b, leading to b-catenin stabilization and its nuclear translocation [31,32]. In this paper, we found that resveratrol is able to inhibit Wnt signaling and cell survival based

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Fig. 5. Resveratrol inhibits the survival of Wnt-stimulated P19 cells and Wnt-driven colon cancer cells. (A) Resveratrol inhibits the survival of Wnt-stimulated P19 cells. P19 cells were treated with Wnt-3a-conditioned medium (Wnt) or different concentrations of resveratrol (Res, 2.5 to 160 mM) in Wnt-3a-conditioned medium and the cell viability was analyzed by MTT assay as described in Materials and Methods. B–D, colorectal cancer cells HCT 116 (B), WiDr (C) or SW480 (D) cells were treated with different concentrations of resveratrol (0–160 mM) and analyzed by MTT assay. The viability of cells without drug treatment was set as 100%. Each bar is the mean  S.E.M. Each treatment was performed in five replicates. The calculated IC50s of resveratrol to P19, HCT 116, WiDr and SW480 cells were 17.86  0.79 mM, 33.27  1.03 mM, 104.13  0.77 mM, and 73.18  0.22 mM, respectively.

on its ability to inhibit the b-catenin/TCF-mediated transcriptional activity and reduce the expression of Wnt target genes in both Wnt-stimulated cells and Wnt-driven colon cancer cells (Figs. 1, 5 and 6A–C). We further examined the underlying mechanism of resveratrol inhibition, and the data demonstrated that resveratrol appears to act downstream of GSK3b (Figs. 2 and 3A). However, resveratrol did not affect the accumulation, cellular distribution, and nuclear targeting of b-catenin in Wnt signaling (Figs. 2C, 3, 6D and E, and 7A and B). Furthermore, our co-immunoprecipitation and in vitro binding data confirmed that the interaction between b-catenin and TCF4 is substantially disrupted by resveratrol (Figs. 4 and 6F), contributing to the inhibition of Wnt signaling. Our findings about the molecular mechanism of resveratrol in Wnt signaling are not in line with previous reports showing that resveratrol inhibits the mobilization of b-catenin into the nucleus [17,30]. It has been reported that resveratrol decreased the expression of Wnt target genes and inhibited the nuclear translocation of b-catenin in cultured cells from a patient with Waldenstro¨m’s Macroglobulinemia, a low-grade lymphoproliferative disorder [17]. Another report also discovered that resveratrol reduced the nuclear translocation of b-catenin by immunofluorescent staining in Wnt-stimulated RKO cells, a colon cancer cell line that lacks intrinsic activation of the Wnt/b-catenin signaling pathway. They further proposed that the defect in nuclear translocation of b-catenin is likely due to the reduced expression of two b-catenin nuclear-targeting factors, Legless (Lgs) and Pygopus (Pygo) [30]. If this is the case, it seems that resveratrol targets some other cellular target(s) rather than directly targeting the components of the Wnt signaling pathway, leading to reduced expression of both nuclear targeting factors of b-catenin.

Moreover, Wang et al. found that treatment of endothelial cells with 5 mM of resveratrol increased the b-catenin/TCF-dependent transcriptional activity through the inactivation of GSK3b [31,32], while 5 mM of resveratrol inhibited the b-catenin/TCF-dependent transcriptional activity in our study (Fig. 1B). The observed differential resveratrol effects could be either due to different types of cells being used or the difference in activation by Wnt. However, we found that resveratrol does target a component of the Wnt pathway by disrupting the formation of the b-catenin/TCF complex, a result that provides a basis for the design of more effective, less toxic resveratrol derivatives that target the bcatenin/TCF complex through molecular docking. Scientists have been looking for chemicals that target the Wnt/ b-catenin signaling pathway for the therapy of Wnt-related diseases. Through high-throughput screening, chemicals that inhibit the upstream Wnt signaling have been obtained [33,34]. For example, chemicals that target tankyrase result in b-catenin degradation. However, these drugs are limited for use because they are inactive in diseases that have mutations in the downstream Wnt/b-catenin signaling pathway such as APC or b-catenin mutations in colorectal cancers. Drugs that inhibit the function of b-catenin in the nucleus are more efficacious for the therapy of these diseases. Possible mechanisms that lead to the inactivation of b-catenin include reduced mobilization into the nucleus, disruption of the formation of the transcription activation complex, binding of this complex to target DNA, and some other events after binding to target DNA. Our data showed that resveratrol inhibits the formation of the TCF/b-catenin complex (Figs. 4 and 6F), a target site on downstream of the Wnt/b-catenin signaling pathway. Therefore, resveratrol is not only suitable for the

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Fig. 6. Resveratrol represses Wnt signaling by disrupting the binding of b-catenin to TCF4 in colorectal cancer cells. (A) Resveratrol decreases the b-catenin/TCF-mediated transcriptional activities in HCT 116 cells. Cells were transfected with pGL3-OT and pTK-Renilla vectors, treated with resveratrol (Res, 0, 20, 30, 50 mM) for 22 h and assayed for dual luciferase activities. Each bar is the mean  S.E.M. Each experiment was performed in triplicate. (B) Resveratrol reduces the RNA expression of Wnt target genes in HCT 116 cells. Cells were treated with different concentrations of resveratrol (Res, 0, 20, 30, 50 mM) for 22 h, and cells were collected for RT-PCR analyses for cyclin D1, c-Myc, conductin, and GAPDH (as the internal control), respectively. (C) Resveratrol reduces the protein expression of Wnt target genes in HCT 116 cells. Cells were treated with different concentrations of resveratrol (Res, 0, 20, 30, 50 mM) for 22 h, and cell lysates were collected for Western blotting analyses for cyclin D1, c-Myc, conductin, and GAPDH (as the loading control), respectively. (D) Resveratrol does not interfere with the cellular distribution of b-catenin and the expression of TCF4. Cells were treated with 0, 20, 30, and 50 mM of resveratrol for 22 h and separated into cytosolic and nuclear fractions, and the cell lysate was then subjected to Western blotting analyses for b-catenin, TCF4, b-tubulin (a marker for cytosol) and lamin A (a marker for the nucleus). C, cytosol. N, nucleus. (E) Resveratrol does not influence the mobilization of b-catenin into the nucleus. HCT 116 cells were treated with 20 mM of resveratrol for 22 h, fixed and stained for b-catenin (green) and nuclei (DAPI, in blue). The images originally photographed at 400 magnification. (F) Resveratrol disrupts the interaction of b-catenin with TCF4 in HCT 116 cells. HCT 116 cells were treated with different concentrations of resveratrol (0, 20, 30, 40 mM) for 22 h and the cell lysates were used for co-immunoprecipitation analyses with anti-TCF4, the resulting blot being detected with anti-b-catenin. Western blotting analyses of b-catenin, TCF4, GAPDH (as the loading control) and precipitated TCF4 (IPed TCF4) are also shown. Total, total cell lysate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

treatment of diseases that are over-activated upstream of Wnt signaling, but is also promising for therapy of those diseases that have intrinsic downstream Wnt signaling. In this study, we tested three colorectal cancer cell lines, SW480, WiDr, and HCT 116, for their sensitivity to resveratrol by MTT assay, and their IC50s to resveratrol were found to be 73.18  0.22 mM, 104.13  0.77 mM, and 33.27  1.03 mM, respectively (Fig. 5). In SW480 and WiDr colorectal cancer cells, APC is mutated at codons 1338 and 1556, respectively [11,35]. These mutations occur on the b-catenin binding site of APC proteins [35],

which interferes with its binding to b-catenin and hence the regulation of b-catenin. In HCT 116 cells, APC is not mutated but b-catenin is mutated at codon 45 [11]. These mutations lead to bcatenin stabilization and cause abnormal b-catenin-mediated nuclear signaling in these cells. However, in SW480 and WiDr cells, p53 is also mutated at codon 273 on its central DNA binding domain [36]. The p53 mutation would be very likely to make SW480 and WiDr cells more resistant to apoptosis and hence more resistant to resveratrol. This may explain the observed higher IC50 for resveratrol in these cells. Therefore, resveratrol is effective in curing Wnt-driven

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Fig. 7. Resveratrol does not influence the mobilization of b-catenin into the nucleus of SW480 and WiDr colorectal cancer cells, and a model was proposed for the novel role of resveratrol in Wnt signaling. (A and B) Resveratrol does not influence the mobilization of b-catenin into the nucleus of SW480 and WiDr cells. SW480 (A) or WiDr (B) cells were treated with 20 mM of resveratrol for 22 h, fixed and stained for b-catenin (green) and nuclei (DAPI, in blue). The images originally photographed at 400 magnification. (C) A model depicting the novel role of resveratrol in Wnt signaling. In the presence of Wnt, Wnt binds to receptor Frizzled and co-receptor LRP5/6, and Disheveled (DVL) binds to Frizzled. The Axin protein complex including b-catenin and GSK3b proceeds to the membrane and Axin binds to phosphorylated LRP5/6. b-catenin is released and accumulates in the cytoplasm, then enters the nucleus to associate with TCF and activate the transcription of Wnt target genes. Resveratrol acts to disrupt the interaction of bcatenin with TCF4, contributing to the decrease in Wnt signaling. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

colorectal cancer that without any p53 mutations, such as HCT 116 cancer cells. For those Wnt-driven cancers that harbor p53 mutations, combined therapy of resveratrol with other anti-cancer drugs or therapies could be considered. The theory that cancer stem cells are the initiating cells of tumors has been proposed [37]. Normal stem cells are endowed with the capabilities of self-renewal and differentiation into multiple cell lineages. Cancer stem cells are a small population of cells that are located on the tumor. Similar to normal stem cells, they are immortal and can undergo unlimited division and differentiation. These cells have the potential to develop into tumors. Meanwhile, they are regarded as being responsible for cancer metastasis, recurrence or resistance to chemotherapy and

radiotherapy [38–42]. Eradication of cancer stem cells would be an effective way to fight cancer and increase the survival rate of patients. It has been reported that the Wnt/b-catenin signaling pathway is required for the growth and maintenance of colorectal, mammary, epithelial, and haematopoietic normal stem cells [43– 45]. Furthermore, it is also important for the development of leukemia stem cells in acute myelogenous leukemia (AML) [46] as well as colorectal cancer stem cells [45]. Thus, drugs that block Wnt/b-catenin signaling would be able to stop or inhibit the growth of cancer stem cells. Our data showed that 20 mM of resveratrol is sufficient to inhibit Wnt/b-catenin signaling and the growth of P19 cells, a pluripotent stem cell of murine teratocarcinoma (Figs. 1B–D, 4A, and 5A), suggesting that resveratrol shows

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potential for the erasing of cancer stem cells in addition to cancer cells and may be beneficial for the treatment of drug resistance, metastasis and recurrence of cancers. Based on our studies, a model depicting the molecular mechanism of resveratrol in Wnt/b-catenin signaling is proposed (Fig. 7C). Upon binding of the Wnt ligand to receptor Frizzled and co-receptor LRP5/6, Dishelleved (DVL) will bind to Frizzled and the Axin protein complex will translocate from the cytoplasm to the cell membrane and bind to phosphorylated LRP5/6, resulting in the accumulation of b-catenin in the cytoplasm. Stabilized b-catenin then moves into the nucleus to complex with TCF and activates the expression of Wnt target genes. In the presence of resveratrol, the formation of the b-catenin/TCF complex is disrupted, contributing to the reduction in Wnt signaling and inhibition of cell growth. Consequently, we have in this study identified the b-catenin/TCF complex as a novel cellular target of resveratrol, and our data suggest that resveratrol holds promise not only for the therapy of CRC, but also for other Wnt-related diseases. Acknowledgments This work was sponsored by grants to H.J.C. from the National Science Council (NSC 98-2320-B-039-035-MY3) and China Medical University (CMU97-237), Taiwan. References [1] Clevers H. Wnt/beta-catenin signaling in development and disease. Cell 2006;127:469–80. [2] De Ferrari GV, Moon RT. The ups and downs of Wnt signaling in prevalent neurological disorders. Oncogene 2006;25:7545–53. [3] Johnson ML, Rajamannan N. Diseases of Wnt signaling. Rev Endocr Metab Disord 2006;7:41–9. [4] Moon RT, Kohn AD, De Ferrari GV, Kaykas A. WNT and beta-catenin signalling: diseases and therapies. Nat Rev Genet 2004;5:691–701. [5] Luo J, Chen J, Deng ZL, Luo X, Song WX, Sharff KA, et al. Wnt signaling and human diseases: what are the therapeutic implications. Lab Invest 2007;87: 97–103. [6] Verkaar F, Zaman GJ. New avenues to target Wnt/beta-catenin signaling. Drug Discov Today 2011;16:35–41. [7] Chen HJ, Lin CM, Lin CS, Perez-Olle R, Leung CL, Liem RK. The role of microtubule actin cross-linking factor 1 (MACF1) in the Wnt signaling pathway. Genes Dev 2006;20:1933–45. [8] MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009;17:9–26. [9] Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127:2893–917. [10] Markowitz SD, Bertagnolli MM. Molecular origins of cancer: molecular basis of colorectal cancer. N Engl J Med 2009;361:2449–60. [11] Rowan AJ, Lamlum H, Ilyas M, Wheeler J, Straub J, Papadopoulou A, et al. APC mutations in sporadic colorectal tumors: a mutational hotspot and interdependence of the two hits. Proc Natl Acad Sci USA 2000;97:3352–7. [12] Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67. [13] Polakis P. Wnt signaling and cancer. Genes Dev 2000;14:1837–51. [14] Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, Vogelstein B, et al. Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 1997;275:1787–90. [15] Shukla Y, Singh R. Resveratrol and cellular mechanisms of cancer prevention. Ann N Y Acad Sci 2011;1215:1–8. [16] Yu W, Fu YC, Wang W. Cellular and molecular effects of resveratrol in health and disease. J Cell Biochem 2012;113:752–9. [17] Roccaro AM, Leleu X, Sacco A, Moreau AS, Hatjiharissi E, Jia X, et al. Resveratrol exerts antiproliferative activity and induces apoptosis in Waldenstrom’s macroglobulinemia. Clin Cancer Res 2008;14:1849–58. [18] Liang YC, Tsai SH, Chen L, Lin-Shiau SY, Lin JK. Resveratrol-induced G2 arrest through the inhibition of CDK7 and p34CDC2 kinases in colon carcinoma HT29 cells. Biochem Pharmacol 2003;65:1053–60.

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