Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1

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1 Carcinogenesis vol.28 no.9 pp , 2007 doi: /carcin/bgm110 Advance Access publication August 11, 2007 Myricetin is a novel natural inhibitor of neoplastic cell transformation and MEK1 Ki Won Lee 1,2,3, Nam Joo Kang 1,4, Evgeny A.Rogozin 1, Hong-Gyum Kim 1, Yong Yeon Cho 1, Ann M.Bode 1, Hyong Joo Lee 4, Young-Joon Surh 3, G.Tim Bowden 5 and Zigang Dong 1, 1 Hormel Institute, University of Minnesota, th Avenue NE, Austin, MN 55912, USA, 2 Department of Bioscience and Biotechnology and Institute of Biomedical Science & Technology, Konkuk University, Seoul , Republic of Korea, 3 College of Pharmacy and 4 School of Agricultural Biotechnology, Seoul National University, Seoul , Republic of Korea and 5 Arizona Cancer Center, The University of Arizona, AZ , USA To whom correspondence should be addressed. Tel: þ ; Fax: þ ; zgdong@hi.umn.edu Evidence suggests that mitogen-activated protein kinase kinase (MEK) plays a role in cell transformation and tumor development and might be a significant target for chemoprevention. 3,5,4#-Trihydroxy-trans-stilbene (resveratrol), a non-flavonoid polyphenol found in various foods and beverages, including red wines, is reported to be a natural chemopreventive agent. However, the concentrations required to exert these effects might be difficult to achieve by drinking only one or two glasses of red wine a day. On the other hand, the flavonol content of red wine is 30 times higher than that of resveratrol. Here we demonstrated that 3,3#,4#,5,5#,7-hexahydroxyflavone (myricetin), one of the major flavonols in red wine, is a novel inhibitor of MEK1 activity and transformation of JB6 Pþ mouse epidermal cells. Myricetin (10 mm) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF)-induced cell transformation by 76 or 72%, respectively, compared with respective reductions of 26 or 19% by resveratrol (20 mm). A combination of myricetin and resveratrol exerted additive but not synergistic effects on either TPA- or EGF-induced transformation. Myricetin, but not resveratrol, attenuated tumor promoter-induced activation of c-fos or activator protein-1. Myricetin strongly inhibited MEK1 kinase activity and suppressed TPA- or EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) or p90 ribosomal S6 kinase, downstream targets of MEK. Moreover, myricetin inhibited H-Ras-induced cell transformation more effectively than either PD098059, a MEK inhibitor, or resveratrol. Myricetin directly bound with glutathione S-transferase-MEK1 but did not compete with ATP. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines. Introduction Abbreviations: AP-1, activator protein-1; DTT, dithiothreitol; EDTA, ethylenediamine tetraacetic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; MAP, mitogen-activated protein; MEM, Eagle s minimum essential medium; TPA, 12-O-tetradecanoylphorbol-13-acetate. Mitogen-activated protein kinase kinase (MEK)1 and MEK2 exhibit 79% amino acid homology and are equally effective at phosphorylating extracellular signal-regulated kinase (ERK) substrates (1). No substrates for MEK1 have been identified other than ERK1 and ERK2, and activated MEK1 catalyzes the phosphorylation of ERK at Thr183 and Tyr185 (1). This selectivity, coupled with a unique ability to phosphorylate both tyrosine and threonine residues, indicates that MEK is essential for integrating signals into the mitogen-activated protein (MAP) kinase pathway. MEK generally plays a critical role in transmitting signals initiated by tumor promoters, including 12-Otetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF) or platelet-derived growth factor (2,3). The constitutive activation of MEK1 results in cellular transformation, while a small molecular inhibitor of MEK inhibits transformation and tumor growth in both cell culture and mouse models (2,4). Additionally, a mutant H-Ras gene perpetually activates the MEK/ERK-signaling pathway and drives cells to develop a more aggressive cancer-like phenotype, such as anchorage-independent growth (5 7). Activator protein-1 (AP-1) is an inducible transcription factor comprising proteins encoded by the fos and jun oncogene families, and it plays an important role in pre-neoplastic to neoplastic transformation, tumor progression and metastasis (8,9). In JB6 mouse epidermal cell lines, TPA and EGF are known to induce AP-1 transcriptional activity in promotion-sensitive (Pþ) phenotypes but not in promotion-resistant (P ) phenotypes (8,9). On the other hand, blocking AP-1 induction causes Pþ cells to revert to the P phenotype, indicating the unique requirement for AP-1 activity in cell transformation (8,9). The MAP kinase signaling pathways are critical for AP-1 activation in response to a wide variety of extracellular stimuli including TPA, growth factors, cytokines, arsenic or ultraviolet irradiation (3). The first members of this family characterized were the ERK proteins, and blocking ERK activity by a dominant-negative ERK2 or an MEK1 inhibitor, PD098059, blocked TPA- or EGF-induced AP-1 transactivation and cell transformation (10). The MEK/ERK/AP-1-signaling pathway therefore represents a promising target for pharmacological interventions in carcinogenesis (5 7). Evidence suggests that 3,5,4#-trihydroxy-trans-stilbene (resveratrol, Figure 1A), a non-flavonoid phenolic phytochemical, might be a key substance in the cancer-preventive activity of red wines (11,12). However, even though resveratrol is noted for its striking inhibitory effects on diverse cellular events associated with carcinogenesis, the resveratrol content of red wine is only mg/l in French red wines and mg/l in various white wines (13). Previous studies suggest that the effective dose of resveratrol for cancer prevention ranges from 40 to 200 lm, whereas the achievable tissue concentration of resveratrol is in the low micromolar range (14). Research data suggest that achieving physiologically effective levels of resveratrol might be difficult and thus other phytochemicals could be responsible for the observed cancer-preventive activities of red wine. The levels of total flavonols are close to 53 mg/l, but may reach 200 mg/l in more expensive wines (15). Compared with resveratrol, the flavonol content of red wines is 30 times higher and the two major flavonols in red wine are 3,3#,4#,5,5#,7- hexahydroxyflavone (myricetin, Figure 1B) and 3,3#,4#,5,7-pentahydroxyflavone (quercetin) (16). Red wines contain sizable amounts of these compounds or typically about 20 50% of the total flavonol content (16). Myricetin also is a major flavonoid found in several foods including onions, berries and grapes as well as red wine (17 19). Research data suggest that one of the major chemopreventive actions of myricetin could be related to its activity as an antioxidant (20), but it also can act as a pro-oxidant (21). Reports indicate that myricetin does not cause tumor formation in mice and attenuates the number of diol-epoxide-induced pulmonary tumors per mouse (22). It inhibits polycyclic aromatic hydrocarbon metabolism and subsequent polycyclic aromatic hydrocarbon DNA adduct formation in mouse epidermis and lung (23). Myricetin exerts protective effects against two-stage skin tumorigenesis (24) and inhibits the growth of A549 lung cancer cells by suppressing thioredoxin reductase activity (25). It has also been reported to suppress invasion and both protein expression and enzyme activity of matrix metalloproteinase-2 in colorectal carcinoma cells (26). These accumulated data provide evidence that myricetin is a potent chemopreventive agent against carcinogenesis, but the underlying molecular mechanisms and molecular targets remain unclear. In the present study, we demonstrate that myricetin is a potent inhibitor of MEK1, which results in the subsequent inhibition of AP-1 transactivation and cell transformation. Ó The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1918

2 MEK1 is involved in myricetin-inhibited cell transformation Fig. 1. Chemical structures of (A) resveratrol, (B) myricetin and (C) PD and cytotoxic effects of (D) myricetin or (E) resveratrol on JB6 Pþ cells. JB6 Pþ cells were treated with myricetin (5 40 lm), resveratrol (5 40 lm) or the vehicle dimethyl sulfoxide (,0.1%), as a negative control and cultured in 5% FBS MEM for the indicated times. The viability of cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2Htetrazolium assay as described in Materials and Methods. Results are expressed as cell viability relative to untreated control, as determined from three independent experiments. Data are represented as means ± SDs. The asterisk indicates a significant difference compared with the untreated control (P, 0.05). Materials and methods Chemicals Eagle s minimum essential medium (MEM), basal medium Eagle, gentamicin, L-glutamine and EGF were purchased from Gibco BRL (Carlsbad, CA); fetal bovine serum (FBS) was from Gemini Bio-Products (Calabasas, CA) and TPA was obtained from Sigma Chemical (St Louis, MO). PD was purchased from Calbiochem (San Diego, CA). The antibodies against phosphorylated ERK (Tyr202/Tyr204), total ERK, phosphorylated p90rsk (Thr359/Ser363), total p90rsk, phosphorylated c-jun N-terminal kinase (JNK) (Thr183/Tyr185) and total JNK were from Cell Signal Biotechnology (Beverly, MA). MEK1 and ERK2 proteins were obtained from Upstate Biotechnology (Lake Placid, NY). CNBr-Sepharose 4B, glutathione Sepharose 4B and [c- 32 P]ATP were purchased from Amersham Pharmacia Biotech (Piscataway, NJ), and a protein assay kit was from Bio-Rad Laboratories (Hercules, CA). G418, the Cell- Titer96 Aqueous One Solution Cell Proliferation Assay Kit, and the luciferase assay substrate were obtained from Promega (Madison, WI). Cell culture JB6 Pþ (27,28) and the H-Ras-transformed JB6 Pþ mouse epidermal cell lines (H-Ras JB6 cells) were cultured in monolayers at 37 C in a 5% CO 2 incubator in 5% FBS MEM, 2 mm L-glutamine and 25 lg/ml gentamicin. The JB6 Pþ mouse epidermal cell line was stably transfected with the AP-1 luciferase reporter plasmid and maintained in 5% FBS MEM containing 200 lg/ml G418. Cell proliferation assay To estimate cell proliferation, JB6 Pþ cells ( ) were seeded into 96-well plates and cultured for 12 h. The cells were treated with different concentrations of myricetin (0 40 lm) or resveratrol (0 40 lm), and viability was determined using the CellTiter96Ò Aqueous One Solution detection kit (Promega) and by reading absorbance (OD 492 ) at 24 h intervals up to 72 h. Anchorage-independent cell transformation assay The effects of myricetin, resveratrol or a combination of both on TPA- or EGFinduced cell transformation were investigated in JB6 Pþ cells. Cells ( per ml) were exposed to TPA or EGF and myricetin (0 20 lm), resveratrol (0 20 lm) or myricetin (5 lm) combined with resveratrol (0 20 lm) in 1 ml of 0.33% basal medium Eagle agar containing 10% FBS or in 3.5 ml of 0.5% basal medium Eagle agar containing 10% FBS. Similarly, H-Ras JB6 cells ( per ml) were incubated with myricetin (0 20 lm), resveratrol (0 20 lm) or PD (0 20 lm) under the same conditions. All cultures were maintained at 37 C in a 5% CO 2 incubator for 14 days, and cell colonies were counted under a microscope with the aid of the Image-Pro Plus software program (Media Cybernetics, Silver Spring, MD) as described by Colburn et al. (28). Luciferase assay for AP-1 transactivation Confluent monolayers of JB6 Pþ cells stably transfected with the AP-1 luciferase plasmid were harvested, and viable cells suspended in 100 ll of 5% FBS MEM were added to each well of a 96-well plate. Plates were incubated at 37 C in5%co 2. When cells reached 80 90% confluence, they were starved by culturing in 0.1% FBS MEM for another 24 h. The cells were then treated 1 h with myricetin (0 20 lm) or resveratrol (0 20 lm), and then exposed to 20 ng/ml TPA or 10 ng/ml EGF for 24 h. Cells were disrupted with 100 ll of lysis buffer [0.1 M potassium phosphate buffer (ph 7.8), 1% Triton X-100, 1 mm dithiothreitol (DTT) and 2 mm ethylenediamine tetraacetic acid (EDTA)], and luciferase activity was measured using a luminometer (Luminoskan Ascent, Thermo Electron, Helsinki, Finland). Reporter gene assay The firefly luciferase reporter gene assay was performed using lysates prepared from transfected cells. In addition, the reporter gene vector prl-sv40 (Promega) was co-transfected into each cell line and transfection efficiencies normalized to Renilla luciferase activity generated by this vector. Cell lysates were prepared by first washing the transfected JB6 Pþ cells (grown in 60 mm diameter dishes) once in phosphate-buffered saline at 37 C. After removing the phosphate-buffered saline completely, 500 ll of passive lysis buffer (Dual Luciferase Reporter Assay System, Promega) was added, and the cells were incubated for 1 h with gentle shaking. The lysate was then transferred to a reaction tube and the cellular debris was removed by centrifugation, and firefly and Renilla luciferase activities of the supernatant fraction were measured. Cell lysates (20 ll each) were mixed with 100 ll of Luciferase Assay II reagent, and the emitted firefly luciferase light was measured. Subsequently, coelenterazine reagent (100 ll) containing the substrate for the emission of Renilla luciferase light was mixed to normalize the firefly luciferase data. The c-fos promoter luciferase (pfos-wt GL3) constructs were kindly provided by Dr. Ron Prywes (Columbia University, New York, NY). In vitro MEK1 and ERK2 kinase assays The in vitro kinase assays were performed in accordance with the instructions provided by Upstate Biotechnology. In brief, every reaction contained 20 llof assay dilution buffer [20 mm 3-(N-morpholino) propanesulfonic acid (MOPS) (ph 7.2), 25 mm b-glycerol phosphate, 5 mm ethylene glycol tetraacetic acid 1919

3 K.W.Lee et al. (EGTA), 1 mm sodium orthovanadate and 1 mm DTT] and a magnesium ATP cocktail buffer. For MEK1, 1 lg of inactive ERK2 substrate peptide was included. A 4 ll aliquot was removed after incubating the reaction mixture at 30 C for 30 min, to which 20 lg of myelin basic protein substrate peptide and 10 ll of diluted [c- 32 P]ATP solution were added. For ERK2, 0.33 mg/ml of myelin basic protein substrate peptide was included. A 4 ll aliquot was removed after incubating the reaction mixture at 30 C for 30 min, to which 10 ll of diluted [c- 32 P]ATP solution was added. This mixture was incubated for 10 min at 30 C, and then 25 ll aliquots were transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min per wash and once with acetone for 2 min. The radioactive incorporation was determined using a scintillation counter. The effects of myricetin (0 20 lm) or resveratrol (0 20 lm) were evaluated by separately incubating each compound with the reaction mixtures at 30 C for 30 min. Each experiment was performed three times. Ex vivo MEK1 immunoprecipitation and kinase assay JB6 Pþ cells were cultured to 80% confluence and then serum starved in 0.1% FBS MEM for 24 h at 37 C. Cells were either treated or not treated with myricetin (0 20 lm) or resveratrol (0 20 lm) for 1 h, then treated with 20 ng/ml TPA for 30 min, disrupted with lysis buffer [20 mm Tris HCl (ph 7.4), 1 mm EDTA, 150 mm NaCl, 1 mm EGTA, 1% Triton X-100, 1mMb-glycerophosphate, 1 mg/ml leupeptin, 1 mm Na 3 VO 4 and 1 mm phenylmethylsulfonyl fluoride] and centrifuged at r.p.m. for 10 min in a microcentrifuge. The lysates containing 500 lg of protein were used for immunoprecipitation with an antibody against MEK1 and incubated at 4 C overnight. Protein A/G plus agarose beads were then added and the mixture was continuously rotated for another 3 h at 4 C. The beads were washed three times with kinase buffer [20 mm MOPS (ph 7.2), 25 mm b-glycerol phosphate, 5 mm EGTA, 1 mm sodium orthovanadate and 1 mm DTT], resuspended in 20 llof1kinase buffer supplemented with 1 lg of inactive ERK2 and incubated for an additional 30 min at 30 C. Then 20 lg ofmyelinbasic protein and 10 ll of diluted [c- 32 P]ATP solution were added and the mixture was incubated for 10 min at 30 C. A 20 ll aliquot was transferred onto p81 paper and washed three times with 0.75% phosphoric acid for 5 min per wash and then once with acetone for 5 min. The radioactive incorporation was determined using a scintillation counter. Each experiment was performed three times. Western blotting Cells ( ) were cultured in a 10 cm dish for 48 h, and then starved in serum-free medium for 24 h to eliminate the FBS activation of MAP kinases. The cells were then treated with myricetin (0 20 lm) for 1 h and then exposed to 20 ng/ml TPA or 10 ng/ml EGF for different times. The harvested cells were disrupted and the supernatant fractions were boiled for 5 min. The protein concentration was determined using a dye-binding protein assay kit (Bio- Rad Laboratories) as described in the manufacturer s manual. Lysate protein (20 lg) was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). After blotting, the membrane was incubated with the specific primary antibody at 4 C overnight. Protein bands were visualized by a chemiluminescence detection kit (Amersham Pharmacia Biotech) after hybridization with the horseradish peroxidase-conjugated secondary antibody. The relative amounts of proteins associated with specific antibodies were quantified using Scion Image (NIH, Bethesda, MD). Preparation of myricetin Sepharose 4B Freeze-dried powder (0.3 g) was suspended in 1 mm HCl and the couple solution [0.1 M NaHCO 3 (ph 8.3) and 0.5 M NaCl] was mixed. The mixture was rotated end over end at 4 C overnight. The medium was transferred to 0.1 M Tris HCl buffer (ph 8.0) and rotated end over end at 4 C overnight. The medium was washed three times with 0.1 M acetate buffer (ph 4.0) containing 0.5 M NaCl followed by a wash with 0.1 M Tris HCl (ph 8.0) containing 0.5 M NaCl. In vitro pull-down assay Recombinant MEK1 (2 lg) or a JB6 Pþ supernatant fraction (500 lg) was incubated with myricetin Sepharose 4B (or Sepharose 4B alone as a control) beads (100 ll, 50% slurry) in reaction buffer [50 mm Tris (ph 7.5), 5 mm EDTA, 150 mm NaCl, 1 mm DTT, 0.01% Nonidet P-40, 2 lg/ml bovine serum albumin, 0.02 mm phenylmethylsulfonyl and 1 lg protease inhibitor mixture]. After incubation with gentle rocking overnight at 4 C, the beads were washed five times with buffer [50 mm Tris (ph 7.5), 5 mm EDTA, 150 mm NaCl, 1 mm DTT, 0.01% Nonidet P-40 and 0.02 mm phenylmethylsulfonyl], and proteins bound to the beads were analyzed by immunoblotting. ATP and myricetin competition assay 0.2 lg of active MEK1 was incubated with 100 ll myricetin Sepharose 4B or Sepharose 4B in reaction buffer (see previous section) for 12 h at 4 C, and ATP 1920 was added at different concentrations (0, 1, 10 or 100 lm) to a final volume of 500 ll and incubated for 30 min. The samples were washed and proteins were detected by western blotting. Expression and purification of recombinant MEK mutants For the expression of full-length and deletion mutants of MEK1, the appropriate plasmids (pgex-mek1 or deletion mutants for GST-MEK1) were expressed in Escherichia coli BL21. Single clones were selected after culturing in Luria Bertani medium at 37 C for 16 h with vigorous agitation. When liquid cultures exhibited an absorbance of 0.8 at 600 nm, they were diluted 100-fold with fresh Luria Bertani medium. Isopropyl-D-thiogalactoside was added to these cultures at a final concentration of 0.1 mm and the cultures were then continuously agitated for another 3 h at 25 C. The bacteria were collected by centrifugation, and the pellets were disrupted by sonication. The lysate was centrifuged, and a 50% slurry (250 ll) of glutathione Sepharose 4B beads was added to each supernatant fraction and then mixed gently for 1 h at room temperature to purify GST proteins. The molecular mass and relative protein expression were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Coomassie Blue staining. Statistical analysis Data are expressed as means ± SDs, and the Student s t-test was used in statistical analysis for single comparison. A probability value of P, 0.05 was used as the criterion for statistical significance. Results Effects of myricetin and resveratrol on the proliferation of JB6 Pþ cells We evaluated the effects of resveratrol or myricetin on the proliferation of JB6 Pþ cells using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfonyl)-2h-tetrazolium assay. Results showed that myricetin (Figure 1D) or resveratrol (Figure 1E) had no effect on cell growth at a concentration range of 5 20 lm at 1 or 3 days after treatment. The highest concentration of myricetin or resveratrol (40 lm) suppressed growth of JB6 Pþ cells by 25 and 13%, respectively (Figure 1D and E). Myricetin strongly inhibits TPA- or EGF-induced neoplastic transformation of JB6 Pþ cells We next examined the effect of myricetin, resveratrol or a combination of both on TPA- or EGF-induced neoplastic transformation of JB6 Pþ cells. Treatment with myricetin (10 lm), but not resveratrol (10 lm), markedly inhibited TPA-promoted neoplastic transformation of JB6 Pþ cells (Figure 2A). Furthermore, myricetin at 10 lm inhibited TPA-induced cell transformation by 76%, whereas resveratrol at 20 lm suppressed transformation by only 26% (Figure 2B). Similar results were observed for EGF-induced cell transformation (Figure 2C), and again myricetin at 10 lm suppressed EGF-induced cell transformation by 72% compared with a 19% inhibition by resveratrol at 20 lm (Figure 2D). The inhibition of TPA- or EGF-induced neoplastic cell transformation by myricetin was dose dependent (Figure 2B and D). These results indicated that myricetin is more potent than resveratrol at suppressing TPA- or EGF-induced neoplastic transformation of JB6 Pþ cells. Because both myricetin and resveratrol are constituents of red wine, we next evaluated potential synergistic effects of these polyphenols on neoplastic transformation. JB6 Pþ cells were treated with TPA (or EGF) and myricetin at 5 lm plus increasing doses of resveratrol (5 20 lm). However, the inhibitory effect on cell transformation appeared to be additive rather than synergistic (data not shown). Myricetin attenuates TPA-induced AP-1 and c-fos activation in JB6 Pþ cells Our previous studies demonstrated that resveratrol markedly induces transactivation of p53 and p53 protein expression in JB6 Pþ cells, which may contribute to its inhibition of tumor promotion (29 31). In contrast, myricetin at a concentration up to 40 lm had no effect on p53 transcriptional activation in JB6 Pþ cells (data not shown). This result suggested that a different mechanism is involved in the

4 MEK1 is involved in myricetin-inhibited cell transformation Fig. 2. Comparison of the effect of myricetin or resveratrol on TPA- or EGF-induced neoplastic transformation of JB6 Pþ cells. JB6 Pþ cells were treated as described in Materials and Methods and colonies were counted 14 days later. (A) The effect of myricetin or resveratrol on TPA-induced cell transformation comparing untreated control cells (a) and cells treated with TPA alone (b), TPA and 10 lm myricetin (c) or TPA and 10 lm resveratrol (d). The cell colonies were counted under a microscope with the aid of the Image-Pro Plus program. (B) The efficiency of inhibition by myricetin or resveratrol on TPA-induced cell transformation is expressed as a percentage of the transformation frequency compared with cells treated with TPA alone. (C) The effect of myricetin or resveratrol on EGF-induced cell transformation comparing untreated control cells (a) and cells treated with EGF alone (b), EGF and 10 lm myricetin (c) or EGF and 10 lm resveratrol (d). The cell colonies were counted under a microscope with the aid of the Image-Pro Plus program. (D) The efficiency of inhibition by myricetin or resveratrol on EGF-induced cell transformation is expressed as a percentage of the transformation frequency compared with cells treated with EGF alone. For (B and D), data are represented as the means ± SDs of transformation frequency calculated from three independent experiments. The asterisk indicates a significant difference between the group treated with TPA (or EGF) and myricetin (or resveratrol) and the group treated with TPA (or EGF) alone (P, 0.05). suppression of cell transformation by myricetin. Because previous studies suggested that AP-1 transactivation is strongly involved in TPA-induced neoplastic transformation of JB6 Pþ cells (8,9), we investigated whether AP-1 activation was involved in the inhibition of cell transformation by myricetin. Results indicated that myricetin blocked TPA- or EGF-induced transactivation of AP-1 in a dosedependent manner. Myricetin at 10 lm inhibited TPA- or EGF-induced AP-1 luciferase activity by 70 or 85%, respectively, whereas resveratrol at the same concentration had little effect (Figure 3A and B). Because TPA induces c-fos expression mainly through the ERKsignaling pathway resulting in AP-1 transactivation, we next investigated whether myricetin could inhibit c-fos promoter activation. This assay was performed using a reporter plasmid carrying the luc gene under the control of the c-fos promoter. Results indicated that TPA- or EGF-induced c-fos promoter activity was also suppressed by myricetin in a dose-dependent manner (Figure 3C and D). The inhibitory effects of myricetin on AP-1 and c-fos activity are consistent with this compound s suppression of cell transformation and indicate that myricetin is substantially more effective than resveratrol in blocking AP-1 transactivation in JB6 Pþ cells. Effects of myricetin and resveratrol on MEK1 and ERK2 kinase activities Our previous studies have shown that the MEK/ERK-signaling pathway is clearly involved in TPA- or EGF-induced cell transformation and AP-1 activation in JB6 Pþ cells (8,9). Notably, the chemical structure of myricetin is very similar to that of PD (Figure 1C), a well-known inhibitor of MEK. Based on this similarity, we next investigated the effects of myricetin on MEK1 kinase activity in vitro and ex vivo to determine whether MEK1 might be a target of myricetin. In vitro kinase assay results indicated that resveratrol had no effect, whereas myricetin at 1 or 5 lm strongly suppressed MEK1 kinase activity by 29 and 88%, respectively (Figure 3E). In addition, myricetin also inhibited ERK2 kinase activity but to a lesser degree. The highest concentration of myricetin (20 lm) caused a reduction in ERK2 kinase activity of only 41% (Figure 3F). Furthermore, we found that myricetin inhibited TPA-induced MEK1 kinase activity in a dose-dependent manner ex vivo (Figure 3G). In contrast, resveratrol had no effect on MEK1 or ERK2 under the same experimental conditions. Overall, the results indicate that MEK1 appears to be a more important target molecule of myricetin than ERK for the inhibition of TPA- or EGF-induced cell transformation and AP-1 activation. Effects of myricetin on TPA- or EGF-induced phosphorylation of ERK, p90rsk and JNK in JB6 Pþ cells We next examined the ability of myricetin or resveratrol to modulate phosphorylation of MEK1 downstream signaling proteins, ERK and p90rsk. Results showed that myricetin blocked TPA- or EGFinduced phosphorylation of ERK (Figure 4A and B) or p90rsk (Figure 4C and D) in JB6 Pþ cells. Unlike myricetin, resveratrol (up to 20 lm) had no effect on TPA- or EGF-induced phosphorylation of ERK or p90rsk (data not shown). Because JNKs are also critical in mediating AP-1 transactivation and malignant transformation (32), we evaluated the ability of myricetin or resveratrol to modulate phosphorylation of JNK. Results showed that myricetin blocked 1921

5 K.W.Lee et al. Fig. 3. Comparison of the effect of myricetin or resveratrol on TPA- or EGF-induced AP-1, c-fos, MEK1 or ERK2 activation. (A and B) Myricetin, but not resveratrol, inhibits (A) TPA- or(b) EGF-induced AP-1 transactivation. For the luciferase assay, stably transfected JB6 AP-1 luciferase reporter cells were cultured as described in the Materials and Methods. The cells were starved in 0.1% FBS MEM and then treated or not treated with myricetin or resveratrol at the indicated concentrations (0, 5, 10 or 20 lm) for 1 h before being exposed to 20 ng/ml TPA or 10 ng/ml EGF for 24 h. Luciferase activity was assayed and AP-1 activity is expressed as percent inhibition compared with TPA- or EGF-treated cells. Data are represented as means ± SDs of the AP-1 luciferase activity calculated from three separate experiments. (C and D) Myricetin, but not resveratrol, suppresses (C) TPA- or(d) EGF-induced c-fos promoter activity. For the reporter gene assay, JB6 Pþ cells were transfected with a plasmid mixture containing the c-fos-luciferase reporter gene (0.5 lg) and the prl-sv40 gene (0.5 lg). At 24 h after transfection, cells were incubated in 0.1% FBS MEM for 24 h at 37 C in a 5% CO 2 atmosphere. Cells were then treated or not treated with myricetin or resveratrol at the indicated concentrations (5, 10 or 20 lm) for 1 h before being exposed to 20 ng/ml TPA or 10 ng/ml EGF for 12 h. The firefly luciferase activity was determined in cell lysates and normalized against Renilla luciferase activity. The c-fos-luciferase activity is expressed as percent inhibition compared with TPA- or EGF-treated cells. Data are represented as means ± SDs of c-fos-luciferase activity calculated from three separate experiments. For (A D), the asterisk indicates a significant difference between groups treated with TPA (or EGF) and myricetin (or resveratrol) compared with the respective group treated with TPA (or EGF) alone (P, 0.05). (E) Myricetin, but not resveratrol, inhibited MEK1 kinase activity in vitro. An in vitro MEK1 kinase assay was performed as described in Materials and Methods and the effect of myricetin or resveratrol on MEK1 kinase activity is expressed as the percent inhibition relative to untreated control MEK1 kinase activity. (F) Myricetin, but not resveratrol, inhibited ERK2 kinase activity in vitro. An in vitro ERK2 kinase assay was performed as described in Materials and Methods and the effect of myricetin or resveratrol on ERK2 kinase activity is expressed as the percent inhibition relative to untreated control ERK2 kinase activity. (G) Myricetin inhibits MEK1 kinase activity ex vivo. Cells were pretreated with myricetin or resveratrol at the indicated concentrations (0, 1, 5, 10 and 20 lm) for 1 h and then stimulated with 20 ng/ml TPA for 30 min. Cells were harvested followed by immunoprecipitation and an in vivo MEK1 kinase assay as described in Materials and Methods. The effect of myricetin or resveratrol on MEK1 kinase activity is expressed as the percent inhibition relative to cells treated with TPA only. For (E G), the mean 32 P count was determined from three independent experiments and data are expressed as means ± SDs. The asterisk indicates a significant difference between groups treated with TPA and myricetin (or resveratrol) and the group treated with TPA alone (P, 0.05). TPA-induced phosphorylation of JNK (Figure 4E), but not EGFinduced phosphorylation of JNK (Figure 4F). Overall results suggest that myricetin appears to exert a stronger inhibitory activity on TPAor EGF-induced phosphorylation of ERK or p90rsk, compared with its effect on JNK. Resveratrol (up to 20 lm) had no effect on TPA- or EGF-induced phosphorylation of JNK (data not shown). Therefore, this finding indicated that myricetin, but not resveratrol, is a markedly effective suppressor of TPA- or EGF-induced phosphorylation of ERK and p90rsk, an effect that is most probably due to myricetin s inhibition of upstream MEK1 kinase activity. Myricetin strongly inhibits H-Ras-induced neoplastic transformation in JB6 cells Various human cancers are known to exhibit mutations and overexpression of ras genes; and one of the most frequent events in carcinogenesis is the uncontrolled activation of the Ras-signaling pathway (33). H-Ras is also known to induce cell transformation mainly through the MEK/ERK/AP-1-signaling pathway. We previously showed that H-Ras activates the growth signal pathway involving MEK and ERK protein kinases, leading to anchorage-independent 1922 growth and elevated AP-1 activity in JB6 Pþ cells (6,7). In the present study, we compared the inhibitory effect of myricetin, PD and resveratrol on H-Ras-induced cell transformation. Based on the numbers of cell colonies, 10 lm myricetin inhibited neoplastic transformation of H-Ras JB6 cells by 94%, compared with a 53 or 20% inhibition by 10 lm PD or resveratrol, respectively (Figure 5A and B). We also found that myricetin was a more effective inhibitor than PD in TPA- or EGF-induced anchorage-independent neoplastic transformation in JB6 Pþ cells (data not shown). These results indicated that myricetin is a much stronger suppressor of cell transformation than either PD or resveratrol and acts by targeting the MEK1/ERK/ AP-1-signaling pathway. Myricetin specifically binds with MEK1 but does not compete with ATP for binding with MEK1 The results above indicated that the inhibition of cell transformation by myricetin is associated with the suppression of MEK1 kinase activity and its downstream signaling pathway. To further confirm whether myricetin directly interacts with MEK1, we first used an in vitro myricetin pull-down assay. Our results revealed that MEK1

6 MEK1 is involved in myricetin-inhibited cell transformation Fig. 4. Effect of myricetin on TPA- or EGF-induced phosphorylation of ERK, p90rsk or JNK in JB6 Pþ cells. Cells were pretreated with myricetin at the indicated concentrations (0, 5, 10 or 20 lm) for 1 h, stimulated with 20 ng/ml TPA and then harvested 15 min later (A and C) and 6 h later (E). Cells were pretreated with myricetin at the indicated concentrations (0, 5, 10 or 20 lm) for 1 h, stimulated with 10 ng/ml EGF and then harvested 15 min later (B and D) and 6 h later (F). The level of phosphorylated and total ERK (A and B), p90rsk (C and D) or JNK (E and F) proteins was then determined by western blot analysis as described in the Materials and Methods using specific antibodies against the corresponding phosphorylated or total proteins. Data are representative of two independent experiments. bound to myricetin Sepharose 4B beads (Figure 6A, left panel, lane 3), but not to Sepharose 4B beads alone (Figure 6A, left panel, lane 2). We also observed ex vivo binding of myricetin and MEK1 in JB6 Pþ cell lysates (Figure 6A, right panel, lane 3). These results indicated that MEK1 could directly bind with myricetin. Furthermore, ATP did not compete with myricetin for binding with MEK1, as indicated by data showing that the binding of myricetin with MEK1 was not changed with increasing amounts of ATP (Figure 6B). Likewise, the binding of ATP with MEK1 also did not change with increasing concentrations of myricetin (data not shown). These results suggested that myricetin is non-competitive with ATP for suppressing MEK1 kinase activity. Previous studies have demonstrated that, like myricetin, MEK1- selective inhibitors such as PD098059, PD184352, PD or U0126 also do not compete with ATP in the binding of MEK1, but the direct binding sites of other inhibitors, except for PD184352, remain unclear (34 36). Thus, to determine the region of binding between myricetin and MEK1, we constructed three MEK1 deletion mutants from full-length GST-MEK1 one containing only the N- terminal fragment (GST-MEK1 1 67); one containing the N-terminal fragment and the binding region of PD (GST-MEK1, 1 206); and the other containing the N-terminal fragment, the binding region of PD and a phosphorylation site of MEK1 (GST-MEK ) (Figure 6C). We then performed the bacterial expression and purification of GST-MEK wild-type and mutant proteins as described in Materials and Methods. The binding affinity of the four expressed GST-MEKs was assessed using the myricetin Sepharose 4B pull-down assay. Results indicated that GST-MEK interacted efficiently with myricetin, whereas GST-MEK was not detected (Figure 6D). These results suggested that myricetin is a potent inhibitor of MEK1 without competing with ATP and that the region where myricetin binds with MEK1 may be similar to that of the MEK1 inhibitor, PD

7 K.W.Lee et al. Fig. 5. Comparison of the effect of myricetin, resveratrol or PD on H-Ras-induced cell transformation. H-Ras-induced cell transformations were performed as described in Materials and Methods and colonies were counted 14 days later. (A) The effect of myricetin, resveratrol or PD on H-Ras-induced cell transformation comparing untreated control cells (a) and cells treated with (b) 10 lm myricetin, (c) 10 lm resveratrol or (d) 10 lm PD The cell colonies were counted under a microscope with the aid of the Image-Pro Plus program. (B) The efficiency of inhibition by myricetin, resveratrol or PD on cell transformation is expressed as a percentage of the transformation frequency compared with untreated H-Ras control cells. Data are represented as the mean ± SD of transformation frequency calculated from three independent experiments. The asterisk indicates a significant difference between the group treated with myricetin, resveratrol or PD compared with the untreated H-Ras control group (P, 0.05). Discussion 1924 Epidemiological studies demonstrated that, in spite of a highcholesterol diet, the French population appears to have lower coronary disease compared with others (37). Resveratrol, a non-flavonoid polyphenolic compound found in grape skins and red wine, was suggested as an explanation for the so-called French paradox. This suggestion is based on data indicating that it may inhibit platelet aggregation, increase high-density lipoprotein cholesterol and act as an antioxidant, thereby serving as a cardioprotective agent (38). Accumulating research evidence also suggested that resveratrol may be used alone or in combination with traditional chemotherapeutic agents to prevent or treat cancer (11,12). Previous studies showed that resveratrol effectively inhibited TPA-induced tumorigenesis in a mouse skin cancer model (39) and TPA-induced anchorage-independent neoplastic transformation of mouse epidermal JB6 Pþ cells (30,40). However, even though these preclinical studies have produced promising results, the concentrations required to exert these effects may be difficult to achieve by drinking only one or two glasses of red wine a day. The content of flavonols such as myricetin and quercetin is much higher than resveratol in red wines. The present study demonstrated that myricetin exhibited a much stronger inhibitory effect on TPA- or EGF-induced neoplastic cell transformation compared with resveratrol (Figure 2) and that myricetin and resveratrol exerted additive, but not synergistic, effects (data not shown). These results suggested that myricetin might play a major role in the cancer-preventive activity of red wine. Others and we have reported that the chemopreventive activities of resveratrol and its derivatives are related to its ability to trigger apoptosis in diverse cell systems (11). Resveratrol suppressed tumor promoter-induced cell transformation and strongly induced apoptosis, transactivation of p53 and increased expression of the p53 protein in the same cell line and at the same dose (30,40). Activation of p53 resulting in apoptosis can suppress tumor development in vivo, particularly in response to oncogenic signaling. Resveratrol-induced apoptosis in cells expressing wild-type p53 but not in p53-deficient cells, indicating that activation of p53 was required for resveratrol s effects. In the present study, myricetin did not affect p53 transcriptional activity indicating that other mechanisms must be involved in the antitumor-promoting activity of myricetin (data not shown). AP-1 plays a key role in pre-neoplastic to neoplastic transformation in both cell culture and animal models and is involved in tumor promotion, progression and metastasis (8,9); and blocking AP-1 activation prevents neoplastic transformation. Thus, AP-1 is a highly relevant target for chemopreventive agents. The present study demonstrated that myricetin (5 lm) blocked TPA- or EGF-induced AP-1 or c-fos activation in JB6 Pþ cells, whereas resveratrol had no effect (Figure 3). Thus, compared with the action of resveratrol, myricetin appears to act in a different manner, mainly by targeting the AP-1-signaling pathway to exert its antitumor-promoting effect. The MAP kinase signaling pathways are critical for AP-1 activation. As indicated earlier, TPA and EGF induce high levels of AP-1 activation and a high frequency of neoplastic transformation in JB6 Pþ cells but have no effect on P cells (8,9). The lack of response was shown to be directly attributable to a low level of both TPA- and EGFinduced phosphorylation of ERK and total ERK protein levels (8,9). MEK1 and MEK2 are dual-specificity protein kinases that phosphorylate the downstream target ERK at specific tyrosine and threonine residues. MEK functions as a key component of this evolutionarily conserved signaling module and is activated by phosphorylation of key serine residues in the catalytic domain by an upstream serine kinase, Raf. The experimental data indicate that the MEK-signaling pathway is an attractive target for pharmacological interventions in proliferative and inflammatory diseases. A key role for MEK in the development of tumors was described and a small molecular inhibitor of MEK was shown to be capable of inhibiting up to 80% of growth of human and murine colon carcinomas in mice (4). Our results demonstrated that low concentrations of myricetin inhibited MEK kinase activity both in vitro and ex vivo in a dose-dependent manner (Figure 3E and G) but was much less effective to inhibit ERK2 kinase even at the highest concentration used (Figure 3F). These results indicated that MEK is a more important target molecule of myricetin than ERK for inhibiting TPA- or EGF-induced cell transformation and AP-1 activation. Myricetin, but not resveratrol, also inhibited TPA- or EGF-induced phosphorylation of ERK and p90rsk (Figure 4A D) in JB6 Pþ cells. In contrast to this result, others did not detect an effect of myricetin on EGF-induced ERK phosphorylation (41). This discrepancy may be due to differences in experimental conditions (e.g. EGF or myricetin concentrations or time of detection). In contrast to its effect on ERK and p90rsk phosphorylation, higher amounts of myricetin were required to inhibit TPA-induced phosphorylation of JNK and it had no effect on EGF-induced JNK phosphorylation

8 MEK1 is involved in myricetin-inhibited cell transformation Fig. 6. Myricetin specifically binds with MEK1 and does not compete with ATP. (A) Myricetin specifically binds with MEK1 in vitro and ex vivo. The MEK1 myricetin binding in vitro was confirmed by immunoblotting using an antibody against MEK1 (A, left panel): lane 1, MEK1 protein standard served as an input control; lane 2, as a negative control, Sepharose 4B was used to pull down MEK1 as described in Materials and Methods; lane 3, MEK1 was pulled down using myricetin Sepharose 4B affinity beads. The MEK1 myricetin binding ex vivo was confirmed by immunoblotting using an antibody against MEK1 (A, right panel): lane 1, whole-cell lysate from JB6 Pþ cells served as input control; lane 2, as a negative control, a lysate of JB6 Pþ cells was precipitated with Sepharose 4B beads as described in Materials and Methods; lane 3, whole-cell lysate from JB6 Pþ cells was precipitated by myricetin Sepharose 4B affinity beads as described in Materials and Methods. (B) Myricetin does not compete with ATP for binding with MEK1. Active MEK1 (2 lg) was incubated with ATP at different concentrations (0, 1, 10 or 100 lm) and 50 ll of myricetin Sepharose 4B or 50 ll of Sepharose 4B (as a negative control) in reaction buffer in a final volume of 500 ll. The mixtures were incubated at 4 C overnight with shaking. After washing, the pulled-down proteins were detected by western blotting: lane 2, negative control, MEK1 cannot bind with Sepharose 4B; lane 3, positive control, MEK1 binding with myricetin Sepharose 4B; lanes 4 6, increasing amounts of ATP have no effect on myricetin binding with MEK1. (C) GST deletion mutant constructs of MEK1. (D) In vitro interactions of myricetin with full-length (FL) GST-MEK1 (aa 1 363), GST-MEK1 (aa 1 225), GST-MEK1 (aa 1 206) or GST-MEK1 (aa 1 67). GST proteins were incubated with myricetin Sepharose 4B beads at 4 C overnight. Precipitates were analyzed by immunoblotting using a GST antibody (upper panel) and Coomassie Blue staining (lower panel). (Figure 4E and F). These results support our idea that MEK is an important molecular target of myricetin, which may account, at least in part, for its anticancer-promoting potential. The Ras/MEK/ERK-signaling pathway is responsible for the coordination and regulation of cell growth and differentiation in response to extracellular stimulation. MAP kinases, and in particular, ERK-mediated phosphorylation is important for the expression and post-translational modification of the AP-1 complex (10,42). Additional evidence suggests that Raf and/or Ras are constitutively activated in several tumor cell lines and that the transforming actions of several oncogenes are dependent on the MEK/ERK/AP-1 pathway (5 7). Myricetin exerted a stronger inhibitory activity compared with PD098059, an inhibitor of MEK, or resveratrol in H-Ras-induced cell transformation (Figure 5). These results suggested that compared with resveratrol and PD098059, myricetin is a more potent inhibitor of cell transformation and mainly targets the MEK-signaling pathway. Additionally, myricetin directly binds with MEK1 in vitro and ex vivo (Figure 6A) and does not compete with ATP for binding with MEK1 (Figure 6B). Previous studies suggested that, similar to myricetin, PD and PD bind with MEK1 and are ATP 1925

9 K.W.Lee et al. non-competitive inhibitors of MEK1 (34 36,43). Furthermore, the binding of PD may lock the enzyme into an inactive conformation, preventing catalysis but still allowing phosphorylation of Ser218 and Ser222 in the activation segment by Raf (43). Currently, only the direct binding site of PD is known (43). In the present study, we found that the deletion mutant GST-MEK (containing the N-terminal domain and binding region of PD184352) interacted strongly with myricetin, whereas GST-MEK (containing only the N-terminal domain) was not detected (Figure 6D). These results suggested that the binding region of myricetin with MEK1 is similar to that of the MEK1 inhibitor PD The careful analysis of these aspects in the further studies will help to define clearer molecular mechanisms responsible for the antitumor effect of myricetin. X-ray crystallography studies are also needed to elucidate the structure of the myricetin-mek1 complex. Large-scale animal studies are also needed to address the bioavailability, toxicity and side effects of myricetin. In summary, myricetin interacts with MEK1 to suppress its activity and downstream signaling to the ERK/p90RSK/AP-1 pathway, leading to cell transformation. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines. Funding Hormel Foundation, BioGreeen21 program, Rural Development Administration (nos and ), Republic of Korea, National Institutes of Health [CA27502 (note: Chemoprevention of Skin Cancer, Alberts/Bowden) CA120388, CA111536, CA88961, CA81064 to Z.D.]; National Research Laboratory, Ministry of Science and Technology (no. B050007), Republic of Korea to Y.-J.S. Postdoctoral fellowship for K.W.L. from the Korea Science and Engineering Foundation. 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