Phosphatidylinositol 3-kinase, a novel target molecule for the inhibitory effects of kaempferol on neoplastic cell transformation

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1 Carcinogenesis vol.31 no.8 pp , 2010 doi: /carcin/bgq102 Advance Access publication June 8, 2010 Phosphatidylinositol 3-kinase, a novel target molecule for the inhibitory effects of kaempferol on neoplastic cell transformation Kyung Mi Lee 1,2, Dong Eun Lee 1,2, Sang Kwon Seo 1, Mun Kyung Hwang 1,2, Yong-Seok Heo 3, Ki Won Lee 2, and Hyong Joo Lee 1 1 Major in Biomodulation, Department of Agricultural Biotechnology, Seoul National University, Seoul , Republic of Korea, 2 Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center and 3 Department of Chemistry, Konkuk University, Seoul , Republic of Korea To whom correspondence should be addressed. Tel: þ ; Fax: þ ; kiwon@konkuk.ac.kr Correspondence may also be addressed to Hyong Joo Lee. Tel: þ ; Fax: þ ; leehyjo@snu.ac.kr Kaempferol (KF), which is a natural dietary flavonoid, has potential beneficial effects as a chemopreventive agent for critical health conditions, such as cancer. However, the molecular mechanisms underlying the activity of KF remain unknown. We report on the inhibition of neoplastic cell transformation by KF through the suppression of phosphatidylinositol 3-kinase (PI3K) activity. Epidermal growth factor (EGF)-induced neoplastic transformation of mouse epidermal JB6 P1 cells was inhibited by 40 mmkf. The activation of activator protein-1 and nuclear factor-kb induced by EGF was also attenuated by KF. The EGF-induced phosphorylation of Akt (protein kinase B) was completely suppressed by KF, although extracellular signal-regulated kinase, p38, c-jun N-terminal kinase and p90 ribosomal S6 kinase were unaffected by KF. Kinase assay data revealed that KF bound directly to PI3K, which is upstream of Akt, and suppressed its activity. Furthermore, KF inhibited ultraviolet B (UVB)-induced PI3K activity and attenuated UVB-induced phosphorylation of Akt. Our results suggest that KF docks at the adenosine triphosphate-binding site of PI3K, which is located between the N-lobe and C-lobe of the kinase domain. Inhibition by KF of PI3K, which is an important factor in carcinogenesis, and its downstream effects may explain the chemopreventive action of KF. Phosphatidylinositol 3-kinase (PI3K) is a major signaling component downstream of many growth factor receptor tyrosine kinases (1). PI3K is a heterodimeric protein composed of a catalytic subunit (p110a/b/c/d) and a regulatory subunit (p85a/b) that participates in multiple cellular processes, including transformation, proliferation and differentiation (2). Activated PI3K is recruited to the plasma membrane, where it catalyzes the conversion of membrane phosphoinositide 4,5-biphosphate in the D3 position, to generate phosphoinositide 3,4,5-triphosphate. Akt (also known as protein kinase B), which is a well-characterized downstream target of PI3K, is activated by growth factors and oncogenes. Uncontrolled activation of Akt is common in tumor cells with PI3K activation and is thought to play a critical role in maintaining proliferation, thereby preventing apoptosis (3). Previous studies have demonstrated activation of the PI3K/Akt signaling pathway in epidermal growth factor (EGF)-induced cell transformations (4,5). Therefore, this pathway represents a significant target for pharmacologic interventions in carcinogenesis. The PI3K/Akt pathway controls several growth-regulatory transcription factors, such as activator protein 1 (AP-1) and transcription factor nuclear factor-jb (NF-jB), leading to the expression of several target genes. (6). AP-1 activity is linked to tumor promotion and the progression of various types of cancers. Previous study demonstrated that PI3K inhibitor attenuated EGF-induced AP-1 activation (4,5). AP-1 plays important roles in preneoplastic-to-neoplastic transformation and proliferation (7). The active form of NF-jB is located in the nuclei of many different cancer cells (8). NF-jB has a key function in the expression of target genes that are involved in the initiation or acceleration of tumorigenesis (9,10). NF-jB is the downstream of PI3K/Akt cascade, using dominant negative-akt stable transfectants in JB6 Pþ cells (11). Therefore, AP-1 and NF-jB are believed to be crucial for cell transformation and tumor promotion (12,13). Cancer chemoprevention, which can be defined as the use of substances to prevent the process of carcinogenesis, is one of the major strategies currently being developed for cancer control. Natural flavonoids, which may have the ability to halt or retard carcinogenesis, are important phytonutrient components found in a large variety of fruits, vegetables and beverages (14). Kaempferol (KF), which has a single -OH moiety on the B-ring (Figure 1A), is one of the most commonly found dietary flavonoids. Several studies have indicated that KF acts as an antioxidant and chemopreventive agent (15 17). KF or KF glycosides may be selective inhibitors of ribosomal S6 kinase (RSK), which is a downstream effector of mitogen-activated protein kinase (MAPK) (18 20). Mouse epidermal JB6 Pþ cells are well suited to studies of tumor promotion owing to their sensitivity to tumor promoter-mediated cell transformation and promotion (21). In the present study, we demonstrate that KF is a potent antitumor-promoting agent that inhibits PI3K activity, AP-1/NF-jB transactivation and the transformation of JB6 Pþ cells. Materials and methods Introduction Abbreviations: AP-1, activator protein 1; ATP, adenosine triphosphate; DTT, dithiothreitol; EGF, epidermal growth factor; ERK, extracellular signalregulated kinase; FBS, fetal bovine serum; JNK, c-jun N-terminal kinase; KF, kaempferol; MEM, minimum essential medium; MAPK, mitogenactivated protein kinase; NF-jB, nuclear factor-jb; PI3K, phosphatidylinositol 3-kinase; RSK, ribosomal S6 kinase; UVB, ultraviolet B. Materials Eagle s minimum essential medium (MEM), Eagle s basal medium, gentamicin, L-glutamine and EGF were purchased from Gibco BRL (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Calabasas, CA). EGF and KF were obtained from Sigma Chemical Company (St Louis, MO). The antibodies directed against total Akt, phospho-akt (Ser473), total extracellular signal-regulated kinase (ERK), phospho-erk (Tyr202/Tyr204), total p38, phospho-p38 (Tyr180/Tyr182), total p90rsk, phospho-p90rsk (Thr359/ Ser363), total c-jun N-terminal kinase (JNK) and phospho-jnk (Thr183/ Tyr185) were purchased from Cell Signaling Technology (Beverly, MA). The antibody against PI3K p85 and recombinant human active PI3K was obtained from Upstate Biotechnology (Lake Placid, NY). CNBr-Sepharose 4B, glutathione-sepharose 4B and [c- 32 P] adenosine triphosphate (ATP) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). A dye-binding protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA). The aminoglycoside antibiotic G418 and the Luciferase Assay Substrate were obtained from Promega (Madison, WI). Cell culture The JB6 Pþ mouse epidermal cell line was cultured in monolayers at 37 C in a5%co 2 incubator in MEM that contained 5% FBS, 2 mm L-glutamine and 25 lg/ml gentamicin. The cell line was stably transfected with the AP-1 and NF-jB luciferase reporter plasmid and maintained in MEM medium that was supplemented with 5% FBS and 200 lg/ml G418. Anchorage-independent cell transformation assay The effects of KF on EGF-induced cell transformation were investigated in JB6 Pþ cells. Cells ( /ml) were exposed to EGF with or without KF (0 40 lm) in 1 ml of 0.33% basal medium eagle agar that contained 10% Ó The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org 1338

2 Inhibition of cell transformation and PI3K by KF after hybridization with the horseradish peroxidase-conjugated secondary antibody. The relative amounts of proteins associated with specific antibodies were quantified using the Sicon Image software (National Institutes of Health, Bethesda, MD). In vitro PI3K assay Active PI3K protein (100 ng) was incubated with KF for 10 min at 30 C. The mixture was then incubated with 20 ll of 0.5 mg/ml phosphatidylinositol (Avanti Polar Lipids, Alablaster, AC) for 5 min at room temperature and incubated in reaction buffer [100 mm N-2-hydroxyethylpiperazine-N#- 2-ethanesulfonic acid (ph 7.6), 50 mm MgCl 2, 250 lm ATP containing 10 lci of [c- 32 P]ATP] for an additional 10 min at 30 C. The reaction was stopped by adding 15 ll of 4 N HCl and 130 ll of chloroform:methanol (1:1). After vortexing, 30 ll of the lower chloroform phase were spotted onto 1% potassium oxalate-coated silica gel plate, which was previously activated for 1hat110 C. The resulting 32 P-labeled phosphatidylinositol-3-phosphate was separated by thin-layer chromatography, and the radiolabeled spots were visualized by autoradiography. Fig. 1. (A) Chemical structure of KF and (B) the inhibitory effects of KF on EGF-induced neoplastic transformation of JB6 Pþ cells. KF inhibits EGFinduced cell transformation. JB6 Pþ cells were treated as described in Materials and Methods and colonies were counted after 14 days. Cell colonies were counted under a microscope using the Image-Pro Plus (ver. 4) software. The average colony number was calculated and photographed from three independent experiments. Significant differences were evaluated using the Student s t-test ( P, 0.01). A significant decrease is observed for EGFinduced transformation of KF-treated cells as compared with the positive control cells. FBS or in 3.5 ml of 0.5% basal medium eagle agar that contained 10% FBS. The cultures were maintained at 37 C in a 5% CO 2 incubator for 14 days, and the cell colonies were counted under a microscope using the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD), as described by Colburn et al. (22). Luciferase assay of AP-1 and NF-jB transactivation Confluent monolayers of JB6 Pþ cells that were stably transfected with the AP-1 and NF-jB luciferase plasmid were trypsinized, and viable cells suspended in 100 ll of 5% FBS MEM were added to each well of a 96-well plate. The plates were incubated at 37 C in a humidified atmosphere of 5% CO 2.When the cultures reached 80 90% confluence, they were starved in 0.1% FBS MEM for an additional 24 h. The cells were treated for 30 min with KF (0 40 lm) and then exposed to 10 ng/ml EGF for 24 h. Thereafter, the cells were disrupted with 100 ll of lysis buffer [0.1 M potassium phosphate (ph 7.8), 1% Triton X-100, 1 mm dithiothreitol (DTT), 2 mm ethylenediaminetetraacetic acid], and luciferase activity was measured using a luminometer (Luminoskan Ascent; Labsystems, Franklin, MD). Western blotting Following culture ( cells) in a 10 cm dish for 48 h, the cells were starved in 0.1% FBS MEM for an additional 24 h to reduce the influence of FBS on the activation of MAPKs. The cells were then treated with KF (0 40 lm) for 30 min, before being exposed to 10 ng/ml EGF or irradiation with ultraviolet B (UVB) (0.05 J/cm 2 ) for different time periods. The harvested cells were disrupted and the supernatant fractions boiled for 5 min. The protein concentrations were determined using a dye-binding protein assay kit (Bio-Rad Laboratories). Lysate protein samples (20 lg) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride 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 the chemiluminescence detection kit (Amersham, Piscataway, NJ) Cell lysates PI3K immunoprecipitation and kinase assay PI3K activity was assayed as described previously (23). In brief, the cells were treated with 10 ng/ml of EGF or irradiated with UVB (0.05 J/cm 2 ) alone or together with KF (0 40 lm)for30minandthenlysedin400ll of lysis buffer [20 mm Tris HCl (ph 7.4), 137 mm NaCl, 1 mm MgCl 2,10% glycerol, 1% NP-40, 1 mm DTT, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 10 lg/ml aprotinin and 10 lg/ml leupeptin]. Proteins in the cell lysates (500 lg) were immunoprecipitated with 2 lg of a monoclonal anti-phosphotyrosine antibody (py99), followed by incubation with protein A/G-Sepharose beads. The immunoprecipitates were washed twice with each of the following buffers: (i) phosphate-buffered saline that contained 1% NP-40, 1 mm DTT and 0.1 mm sodium orthovanadate; (ii) 100 mm Tris HCl (ph 7.6), 0.5 M LiCl, 1 mm DTT and 0.1 mm sodium orthovanadate and (iii) 10 mm Tris HCl (ph 7.6), 0.1 M NaCl, 1 mm DTT and 0.1 mm sodium orthovanadate. After the final wash, the immunoprecipitates were resuspended in 20 ll ofbuffer3onicefor5min, and 20 ll of 0.5 mg/ml phosphatidylinositol (Avanti Polar Lipids) were added. After 5 min at room temperature, the immunoprecipitates were incubated with reaction buffer [100 mm N-2-hydroxyethylpiperazine-N#- 2-ethanesulfonic acid (ph 7.6), 50 mm MgCl 2, 250 lm ATP containing 10 lci of [c- 32 P]ATP] for an additional 10 min at 30 C. The reaction was stopped by adding 15 ll of 4 N HCl and 130 ll of chloroform:methanol (1:1). After vortexing, 30 ll of the lower chloroform phase were spotted onto a 1% potassium oxalate-coated silica gel plate, which was previously activated for 1 h at 110 C. The resulting phosphatidylinositol-3-phosphate was separated by thin-layer chromatography, and the radiolabeled spots were visualized by autoradiography. Pull-down assays JB6 Pþ cellular supernatant fractions (500 lg protein) with recombinant PI3K (2 lg), which were treated with 10 ng/ml EGF or irradiated with UVB (0.05 J/ cm 2 ), were incubated with KF-Sepharose 4B (or Sepharose 4B as a control) beads (100 ll, 50% slurry) in reaction buffer [50 mm Tris HCl (ph 7.5), 5 mm ethylenediaminetetraacetic acid, 150 mm NaCl, 1 mm DTT, 0.01% NP-40, 2 lg/ml bovine serum albumin, 0.02 mm phenylmethylsulfonyl fluoride and 1 protease inhibitor mixture]. After incubation with gentle rocking overnight at 4 C, the beads were washed five times with buffer [50 mm Tris HCl (ph 7.5), 5 mm ethylenediaminetetraacetic acid, 150 mm NaCl, 1 mm DTT, 0.01% NP-40 and 0.02 mm phenylmethylsulfonyl fluoride], and the proteins bound to the beads were analyzed by immunoblotting. ATP and KF competition assay Briefly, 0.2 lg of active PI3K was incubated with 100 ll of KF-Sepharose 4B or 100 ll of Sepharose 4B in the reaction buffer used in the in vitro pull-down assay for 12 h at 4 C, and ATP was added at different concentrations (10 and 100 lm) to a final volume of 500 ll for 30 h. The samples were washed, and the proteins were detected by western blotting. Molecular modeling Insight II (Accelrys, San Diego, CA) was used for docking and structure analyses with the crystal coordinates of PI3K complexed with ATP or quercetin (accession numbers 1E8X and 1E8W in the Protein Data Bank; http: // Statistical analysis The data are expressed as means ± standard deviations. The Student s t-test was used to perform statistical analysis of single comparisons. The probability value of P, 0.05 was used as the criterion for statistical significance. 1339

3 K.M.Lee et al. Results KF strongly inhibits EGF-induced neoplastic transformation The mouse epidermal JB6 Pþ cell system is a well-developed model for studying the molecular mechanisms of tumor promotion and antitumor agents. Initially, we examined the inhibitory activities of KF on EGF-induced neoplastic JB6 Pþ cell transformation. Treatment with 40 lm KF significantly inhibited by 58% neoplastic transformation as compared with EGF-induced transformation, indicating that KF is a potent inhibitor (Figure 1B). KF attenuates EGF-induced AP-1 and NF-jB activation The regulation of cell proliferation processes by AP-1 and NF-jB activation is believed to be of crucial importance for the multistage development of tumors (6). To determine whether the transformation suppressed by KF involves the inhibition of AP-1 and NF-jB activities, we measured AP-1 and NF-jB transactivation using JB6 Pþ cells that were stably transfected with an AP-1 and an NF-jB luciferase reporter plasmid. Pretreatment with KF significantly reduced EGF-induced transactivation of AP-1 (Figure 2A) and NF-jB (Figure 2B). These results indicate that KF suppresses AP-1 and NF-jB, which may be related to its antitumor activity. KF blocks EGF-induced activation of Akt but not MAPKs Previous studies have indicated that the MAPKs and Akt signaling pathway are involved in EGF-induced JB6 Pþ cell transformation (4). Therefore, we investigated the influence of KF on EGF-induced activation of Akt, ERK, p38 and JNK kinase. EGF-induced phosphorylation of Akt was completely inhibited by 20 lm or40lm KF (Figure 3), whereas EGF-induced phosphorylation of p38, JNK and ERK was not inhibited. Moreover, KF had no effect on EGF-induced phosphorylation of RSK, a downstream kinase of ERK (Figure 3). These results suggest that the inhibition of the Akt signal pathway by KF leads to a decrease in EGF-induced cell transformation. KF binding inhibits EGF-induced PI3K activity Since KF strongly inhibited EGF-induced phosphorylation of Akt, we studied the effect of KF on the kinase activity of PI3K. The results of the in vitro kinase assay showed that KF strongly inhibited PI3K (Figure 4A). In addition, we measured the effect of KF on EGFinduced PI3K activity in JB6 Pþ cells. The increase in PI3K activity induced by EGF treatment for 10 min was attenuated by KF (Figure 4B). These results suggest that PI3K is a molecular target of KF and that KF inhibits downstream signaling by blocking PI3K activity. To investigate the mechanism underlying this effect, we performed a KF pull-down assay. PI3K bound to the KF-Sepharose 4B beads, but not to the Sepharose 4B beads alone (Figure 4C). The PI3K protein was loaded as a control. Using cell lysates pull-down assays, we also investigated whether KF binding to PI3K occurs in cell lysates. After treatment with EGF for 10 min, cell lysates were collected and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. PI3K was observed only in association with the KF-Sepharose 4B beads (Figure 4D). We confirmed the specificity of PI3K binding to KF by the competition assay using the free KF. PI3K was found in the KF-Sepharose 4B beads, but not in free KF. Fig. 2. (A and B) KF inhibits EGF-induced AP-1 and NF-jB transactivation. For the luciferase assay, JB6 cells were stably transfected with an AP-1 and NF-jB luciferase reporter and cultured as described in Materials and Methods. Cells were starved in 0.1% FBS/MEM and left untreated or treated with KF at 10, 20 or 40 lm for 30 min before being exposed to 10 ng/ml EGF for 24 h. Luciferase activity was assayed; the AP-1 and NF-jB activities are expressed relative to the values obtained for the control cells (without EGF). Data shown are the mean (± SD) AP-1 and NF-jB luciferase activities from three independent experiments Fig. 3. Effects of KF on EGF-induced phosphorylation of Akt, ERK, p38, JNK and RSK. KF blocks Akt phosphorylation in the presence of EGF, but not ERK, p38, JNK or RSK phosphorylation. Cells were treated with KF at 10, 20 or 40 lm for 30 min before incubation with 10 ng/ml EGF for 15 min. Lysates were prepared, and western blotting was performed for phosphorylated and total protein levels of Akt, ERK, p38, JNK and RSK, as described in Materials and Methods. Data shown are representative of three independent experiments that generated similar results.

4 Inhibition of cell transformation and PI3K by KF Fig. 4. Effects of KF on EGF-induced PI3K activity both in vitro and cell lysates. (A) KF inhibits PI3K activity in vitro. PI3K protein was pre-incubated with KF at the indicated concentrations for 10 min at 30 C and then incubated with phosphatidylinositol substrate and [c- 32 P]ATP for an additional 10 min at 30 C. The resulting 32 P-labeled phosphatidylinositol-3-phosphate was measured as described in Materials and Methods. (B) KF inhibits EGF-induced PI3K activity in JB6 Pþ cells. JB6 Pþ cells were pretreated with KF at 10, 20 and 40 lm for 30 min and stimulated with 10 ng/ml EGF for an additional 10 min. The cells were lyzed, and PI3K activity was determined. (C) KF specifically binds to the p85 subunit of PI3K in vitro as confirmed by immunoblotting with an antibody directed against the p85 subunit. Lane 1, PI3K protein standard (input control); lane 2, Sepharose 4B used to pull down PI3K (control) and lane 3, PI3K pulled down using the KF- Sepharose 4B affinity beads. (D) PI3K KF binding in JB6 Pþ cells is confirmed by immunoblotting with an antibody against the p85 subunit of PI3K. Lane 1, whole cell lysates (input control); lane 2, lysates precipitated with the Sepharose 4B beads (control) and lane 3, whole-cell lysates precipitated by the KF- Sepharose 4B affinity beads. The binding ability of KF-Sepharose 4B with PI3K altered in free KF (supplementary Figure is available at Carcinogenesis Online). These data support that free KF can bind with PI3K. To confirm the specific inhibition of PI3K by KF, we examined the activities of other kinases; however, KF had no effect on ERK, p38 or JNK kinase activity (data not shown). These data indicate that KF binds to PI3K and subsequently inhibits PI3K activation and downstream signaling. KF-binding to PI3K inhibits UVB-induced activity To confirm the specificity of the inhibition of PI3K activities by KF, we assayed UVB-induced PI3K kinase activity. JB6 Pþ cells were treated with various concentrations of KF for 30 min and then exposed to UVB irradiation. The increased activity of PI3K stimulated by UVB treatment subsequently decreased in the presence of KF (Figure 5A). KF strongly decreased UVB-induced phosphorylation of Akt (Figure 5B). Furthermore, we examined KF binding to PI3K in cell lysates using pull-down assays. After treatment with UVB for 15 min, the cell lysates were collected and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. PI3K was observed to be bound to the KF-Sepharose 4B beads, but not to the Sepharose 4B beads alone (Figure 5C). These results strongly support the notion that KF specifically binds to PI3K. Discussion KF is a natural flavonoid that has been isolated from broccoli, tea, propolis, grapefruit and other plant sources (24,25). Accumulated evidence suggests that KF has chemopreventive or chemotherapeutic effects on cancer (26,27). In the present study, we found that KF inhibited EGF-induced JB6 Pþ cell transformation, which strongly supports its potential as an inhibitor of cell transformation. AP-1 and NF-jB are extremely important transcription factors for neoplastic transformation (6). 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. Blocking AP-1 activation specifically prevents neoplastic transformation (7). It has been reported that NF-jB rapidly induces stressresponsive transcription factor functions to amplify the transcription of various genes (28). NF-jB signal transduction pathways are also important in cell transformation (29). We performed a luciferase assay to detect AP-1 and NF-jB transcriptional activities. Our results show that KF blocks EGF-induced activation of AP-1 or NF-jB in JB6 Pþ cells. Thus, it appears that KF targets the AP-1 or NF-jB-signaling Fig. 5. Effect of KF on UVB-induced PI3K activity. (A) KF inhibits UVBinduced PI3K activity. JB6 Pþ cells were pretreated with KF at 10, 20 and 40 lm for 30 min, irradiated with UVB (0.05 J/cm 2 ) and harvested 10 min later. The cells were lyzed, and PI3K activity was determined, as described in Materials and Methods. (B) KF inhibits UVB-induced Akt phosphorylation. Cells were pretreated with KF at 10, 20 or 40 lm for 30 min, irradiated with UVB (0.05 J/cm 2 ) and harvested 15 min later. The cells were lyzed, and Akt phosphorylation levels were analyzed by western blotting. Data shown are representative of three independent experiments that generated similar results. (C) KF directly binds to the p85 subunit of PI3K, as confirmed by immunoblotting with an antibody against the p85 subunit. Lane 1, whole-cell lysates (input control); lane 2, lysates precipitated with the Sepharose 4B beads (control) and lane 3, whole-cell lysates precipitated by the KF-Sepharose 4B affinity beads. pathway to exert its antitumor effect. We performed that the AP-1 or NF-jB DNA-binding activity was analyzed by gel shift assay. However, KF cannot inhibit binding of AP-1 or NF-jB to the promoter regions in our experimental condition. We thought that KF suppresses 1341

5 K.M.Lee et al. AP-1 or NF-jB promoter activity instead of protein-binding affinity of AP-1 or NF-jB sites. Further research need to know more about the role of KF on transcriptional factor. The activities of NF-kB and AP-1 are regulated by multiple signaling pathways. Among these pathways, the PI3K/Akt- and MAPKmediated signaling pathways play critical roles. Therefore, we investigated whether the inhibition of MAPK phosphorylation by KF suppresses EGF-induced cell transformation. As shown in Figure 3, KF suppressed EGF-induced Akt activation. In contrast, none of the MAPK proteins were directly inhibited by KF. We hypothesize that the molecular target of KF that limits EGF-induced cell transformation is a kinase upstream of Akt. It has been shown that the survival signal PI3K controls multiple cell signaling pathways that lead to tumor development (30) and can form complexes with cellular oncoproteins, which possess transforming activities (31). The transforming effect of PI3K is associated with the activation of its downstream effector kinases, including Akt (32). Previous studies have demonstrated that the PI3K inhibitor LY blocks EGF-induced transformation of JB6 Pþ cells (5). These findings suggest that PI3K is an important regulatory protein for tumor promotion. Our results clearly show that KF significantly inhibits PI3K activity and binds directly to PI3K (Figure 4). These results indicate that KF inhibits EGF-induced cell transformation and subsequently attenuates EGF-induced phosphorylation of Akt by regulating PI3K activity. UVB exposure, which is the most important risk factor for skin cancer, also induces PI3K activation (33). We elucidated the inhibition of UVB-induced PI3K/Akt signaling pathway by KF in JB6 Pþ cells. KF not only suppressed UVB-induced phosphorylation of Akt but also blocked UVB-induced PI3K activity by specifically binding to PI3K (Figure 5). Although previous studies have indicated that KF has a specific inhibitory effect on RSK2 (20), we were interested in determining whether KF could affect other kinase activities. Interestingly, we found that KF particularly targets the PI3K/Akt cell signaling pathway, thus mediating its anticancer effects. The binding of KF to PI3K was altered in a concentration-dependent manner in the presence of ATP (Figure 6A), which suggests that KF could act as an ATP-competitive inhibitor of PI3K. To determine the binding mode of KF to PI3K, we conducted a modeling study using the crystal structure of PI3K complexed with ATP or quercetin (34,35). PI3K consists of the: (i) Ras-binding domain; (ii) C2 domain; (iii) helical domain and (iv) catalytic domain. The catalytic domain consists of an N-lobe and a C-lobe with a fold similar to that seen in protein kinases, and this structural similarity is conserved in the ATPbinding site flanked by these two lobes. Consequently, ATP binds between these lobes in a manner similar to the ATP binding observed for protein kinases. The N-lobe and C-lobe are linked through a loop, which is termed the hinge region. The backbone of this loop interacts with the adenine moiety of ATP through hydrogen bonds. Based on the premise that KF is an ATP-competitive inhibitor, we docked the compound to the ATP-binding site (Figure 6B). The carbonyl group at position 4 and the hydroxyl groups at position 5 of KF formed hydrogen bonds with the backbone atoms of Val887 in the hinge region of PI3K. The hydroxyl groups at positions 7 and 4 formed hydrogen bonds with the side-chains of Lys890 and Asp841, respectively. In addition, KF would appear to be sandwiched by the side-chains of the hydrophobic residues in the ATP-binding site, i.e. Met804, Trp812, Ile831, Ile879 and Ile881 in the N-lobe, and Ala885, Phe961, Met953 and Ile963 in the C-lobe. The high-level inhibitory activity of KF may be attributable to these hydrogen bonds and hydrophobic interactions. Further studies to elucidate the inhibitor s complex structure, using techniques such as X-ray crystallography, may determine the precise mode of KF PI3K binding. Fig. 6. Hypothetical models of PI3K complexed with KF. (A) KF binds to PI3K in an ATP-competitive manner. Active PI3K (2 lg) was incubated with ATP at different concentrations (10 or 100 lm), and 50 ll of KF-Sepharose 4B or 50 ll of Sepharose 4B (as a negative control) in reaction buffer was added to give 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 1, input control; lane 2, PI3K unbound to Sepharose 4B (negative control); lane 3, PI3K bound to KF-Sepharose 4B (positive control) and lanes 4 and 5, increasing concentrations of ATP alter KF binding with PI3K. Three replicates were performed. (B) Hypothetical model of the PI3K complex with KF, including an in-depth view. The Ras-binding domain, C2 domain and helical domain of PI3K are colored in gray. The N-lobe, C-lobe and hinge region of the catalytic domain are colored in yellow, purple and cyan, respectively. KF (atomic color) binds to the ATP-binding site in the catalytic domain of PI3K. The residues in gray ellipses are the hydrophobic residues that interact with KF. The hydrogen bonds are depicted as white lines. 1342

6 Inhibition of cell transformation and PI3K by KF In summary, KF is effective in inhibiting neoplastic transformation induced by EGF in JB6 Pþ cells. This inhibition is mediated primarily through the blocking of the PI3K/Akt signaling pathways and the subsequent suppression of AP-1 and NF-jB activities. KF inhibits PI3K activity by directly binding with PI3K. Furthermore, KF directly blocks UVB-induced PI3K activation. Given the critical role of PI3K in carcinogenesis, these results suggest that PI3K is a potent molecular target of KF and may provide the molecular basis for the development of new chemopreventive agents. Supplementary material Supplementary Figure can be found at Funding WCU Program (R ); World Class Institute Program; Priority Research Centers Program ( ); Postdoctorate Fellowship Program (NRF F00027); National Research Foundation; BioGreen 21 Program ( and 027); Rural Development Administration. Acknowledgements Conflict of Interest Statement: None declared. 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