7,3′,4′-Trihydroxyisoflavone Inhibits Epidermal Growth Factor-induced Proliferation and Transformation of JB6 P+ Mouse Epidermal Cells by Suppressing Cyclin-dependent Kinases and Phosphatidylinositol 3-Kinase*

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3′,4′-Trihydroxyisoflavone (7,3′,4′-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3′,4′-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G1 phase. As shown by Western blot, 7,3′,4′-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G1 phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3′,4′-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3β/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3′,4′-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3′,4′-THIF provide a molecular basis for the possible development of new chemoprotective agents.

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3,4-Trihydroxyisoflavone (7,3,4-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3,4-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P؉ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G 1 phase. As shown by Western blot, 7,3,4-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G 1 phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3,4-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3␤/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3,4-THIF in the suppression of EGFinduced cell proliferation. These insights into the biological actions of 7,3,4-THIF provide a molecular basis for the possible development of new chemoprotective agents.
The complex process of carcinogenesis is believed to be comprised of three stages that include initiation, promotion, and progression. The reversible and lengthy stage of tumor promotion is a potential target for prevention strategies. Interventions at this stage appear more likely to be successful than interventions at the tumor initiation stage, which is irreversible and brief (1). Aberrant regulation of growth signaling is implicated in the abnormal biological process of tumorigenesis, and epidermal growth factor (EGF) 4 might be one of the tumor promoters needed for the induction of aberrant cell growth (2). Indeed, growth factors, including EGF, are expressed at high levels in a variety of human cancer cells, including those derived from skin, breast, colon, lung, and prostate. These growth factors stimulate cell proliferation, invasiveness, and angiogenesis (3,4).
The JB6 mouse epidermal cell system of clonal genetic variants, which are promotion-sensitive (Pϩ) or promotion-resistant (PϪ), provides an ideal model for investigations of the molecular mechanisms involved in neoplastic transformation, promotion, and progression (1). In JB6 Pϩ cells, EGF induces cell cycle progression and the formation of large, tumorigenic, anchorage-independent colonies in soft agar.
Uncontrolled cell growth is considered a major event in carcinogenesis. Multiple lines of evidence suggest the existence of a strong link between cell cycle deregulation and carcinogenesis (5). In particular, dysregulation of the cyclin-dependent kinases (CDKs) causes increased cell proliferation. CDK activity requires binding of regulatory subunits known as cyclins. The D-type cyclins are initially activated in response to mitogenic signals and preferentially bind to and activate CDK4 during the early G 1 stage of the cell cycle (6). Activation of these complexes leads to phosphorylation of retinoblastoma protein (Rb). The phosphorylated Rb (pRb) then binds to transcription factors, including E2Fs, and contributes to the expression of S-phase genes. The association of CDK2 with cyclin E and then with cyclin A is believed to coordinate events as cells progress from G 1 through S phase (7). pRb is initially phosphorylated on multiple sites by CDK4 and then additionally phosphorylated by cyclin E-bound CDK2. Therefore, cell cycle progression from G 1 to S phase is tightly controlled by interactions among pRb and the cyclin D1-CDK4 and cyclin E-CDK2 complexes.
The isoflavones daidzein and genistein are present at high concentrations in soybean-based foods (13,14). Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against EGF receptor-positive malignancies of breast, colon, skin, and prostate (15)(16)(17)(18). Genistein has been the subject of intensive study as a potential anti-carcinogenic compound, whereas daidzein and its metabolites have not received as much attention. Here, we investigated the chemopreventive effects of 7,3Ј,4Ј-trihydroxyisoflavone (7,3Ј,4Ј-THIF (see Fig. 1)), a daidzein metabolite, on EGF-stimulated cell proliferation and transformation. We report that 7,3Ј,4Ј-THIF is a potent inhibitor of CDKs and PI3K and induces cell cycle arrest at G 1 phase and suppresses proliferation and transformation of JB6 Pϩ mouse epidermal cells. The molecular mechanism suggested by this investigation might underlie the chemopreventive activity of 7,3Ј,4Ј-THIF and thus could at least partially account for the chemopreventive effect of soy foods.
Cell Cycle Analysis-Cell cycle progression was analyzed by a published method (19) with slight modifications. JB6 Pϩ cells were seeded in 60-mm dishes (1.5 ϫ 10 5 cells/dish), cultured for 24 h, and then serum-starved in 0.1% FBS-containing MEM (0.1% FBS-MEM) for 36 h to synchronize the cells at G 1 phase (19). The cells were then treated with 7,3Ј,4Ј-THIF at various concentrations. After 1 h, EGF (final concentration, 10 ng/ml) or FBS (final concentration, 5%) was added to stimulate cell growth. The cells were trypsinized 24 h later, washed with icecold Dulbecco's phosphate-buffered saline (36 mg/liter sodium pyruvate, 50 mg/liter streptomycin sulfate, 100 mg/liter kanamycin monosulfate, 1000 mg/liter glucose, calcium chloride, and magnesium chloride), and fixed in ice-cold 70% ethanol at Ϫ20°C overnight. The cells were then washed twice with Dulbecco's phosphate-buffered saline and incubated with 20 g/ml RNase A and 200 g/ml propidium iodide in Dulbecco's phosphate-buffered saline at room temperature for 30 min in the dark. The cell cycle phase was determined using a FACSCalibur flow cytometer (BD Biosciences). The data were gathered using ModFit LT software (Verity Software House, Inc., Topsham, ME).
Western Blot Analysis-Cells were cultured in 10-cm dishes (4 ϫ 10 5 cells/dish) for 24 h and then starved in 0.1% FBS-MEM for an additional 36 h. After treatment with 7,3Ј,4Ј-THIF for 1 h, they were treated with EGF for 10 min or 24 h and harvested. The harvested cells were disrupted, and the supernatant fractions were boiled for 5 min. The protein concentration in the lysates was determined using a dye-binding protein assay kit according to the manufacturer's protocol. Lysate protein (30 g) was subjected to 10% SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane. After blotting, the membrane was incubated with a primary antibody against cyclin D1, cyclin E, CDK4, CDK2, phosphorylated Akt, GSK-3␤, or Rb at 4°C overnight. Protein bands were visualized using a chemiluminescence detection kit after hybridization with a horseradish peroxidase-conjugated secondary antibody.

7,3,4-THIF Inhibits Proliferation and Cell Transformation
of 200 mM ATP, and 1 g/l Rb-C fusion protein were added, and the reaction mixture was incubated at 30°C for an additional 30 min. Phosphorylation of the Rb-C fusion protein was detected by Western blotting with a pRb (Ser-807/Ser-811) polyclonal antibody.
To determine PI3K activity, an active PI3K protein (100 ng/l) was incubated with 7,3Ј,4Ј-THIF (0, 10, 20, or 40 m) for 10 min at 30°C. Then, 20 l of 0.5 mg/ml phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) was added, and the mixture was incubated for 5 min at room temperature. Reaction buffer (100 mM HEPES, pH 7.6, 50 mM MgCl 2 , 250 M ATP) containing 10 Ci of [␥-32 P]ATP was added, and the reaction was incubated for an additional 10 min at 30°C. The reaction was stopped by the addition of 15 l of 4 N HCl and 130 l of chloroform:methanol (1:1) and vortexing. The lower chloroform phase (30 l) was spotted onto a 1% potassium oxalatecoated silica-gel plate (previously activated for 1 h at 110°C) and subjected to thin-layer chromatography and autoradiography to visualize the 32 P-labeled phosphatidylinositol 3-phosphate product.
Luciferase Assay for AP-1 Transactivation-Confluent monolayers of JB6 Pϩ cells stably transfected with an AP-1 luciferase reporter plasmid were trypsinized, and 8 ϫ 10 3 viable cells were suspended in 100 l of 5% FBS-MEM and added to each well of a 96-well plate. The plates were incubated at 37°C under a humidified 5% CO 2 atmosphere. When the cells reached 80 -90% confluence, they were serum-starved by culturing in 0.1% FBS-MEM for an additional 24 h. The cells were then treated for 1 h with 7,3Ј,4Ј-THIF (0, 10, 20, or 40 M) and exposed to 10 ng/ml EGF for 24 h. After treatment, cells were disrupted with 100 l of lysis buffer (0.1 M potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mM dithiothreitol, 2 mM EDTA), and luciferase activity was measured using a luminometer (Luminoskan Ascent, Thermo Electron, Helsinki, Finland).
Molecular Modeling-Insight II (Accelrys Inc., San Diego, CA) was used for the docking study and structure analysis with the crystal coordinates of PI3K in complex with ATP or myricetin (accession codes 1E8X or 1E90) available in the Protein Data Bank (www.rcsb.org/pdb/).
Statistical Analysis-When applicable, data are expressed as means Ϯ S.D. values, and the Student's t test was used for single statistical comparisons. A probability value of p Ͻ 0.05 was used as the criterion for statistical significance.

7,3,4-THIF Inhibits Proliferation and Cell Transformation
increased the percentage of cells in S phase, and pretreatment with 7,3Ј,4Ј-THIF before EGF treatment suppressed EGF-stimulated cell cycle progression at S phase. Because the flow cytometry data revealed that the 24-h pretreatment with 7,3Ј,4Ј-THIF did not affect the number of quiescent JB6 Pϩ cells in sub G 1 (data not shown), the inhibition caused by 7,3Ј,4Ј-THIF was not attributable to cytotoxicity or apoptosis. 7,3Ј,4Ј-THIF also induced cell cycle arrest of FBS-stimulated JB6 Pϩ cells at G 1 phase (Fig. 2B).
7,3Ј,4Ј-THIF Inhibits the Expression of G 1 Phase-regulatory Proteins-To determine the mechanism responsible for 7,3Ј,4Ј-THIF-induced cell cycle arrest at G 1 phase, we examined the expression of cyclins and CDKs involved in the G 1 /S-phase progression. Western blotting results showed that 7,3Ј,4Ј-THIF inhibited EGF-induced expression of the early G 1 regulators cyclin D1 and CDK4 and of the late G 1 regulators cyclin E and CDK2 (Fig. 3A).
Because phosphorylation of Rb plays a central role in the G 1 to S transition, we determined the effect of 7,3Ј,4Ј-THIF on Rb phosphorylation at Ser-807/Ser-811 and Ser-795, the sites at which pRb is reportedly phosphorylated by CDK4 (Fig. 3B). Pretreatment of cells with 7,3Ј,4Ј-THIF (10, 20, or 40 m) before EGF stimulation significantly inhibited Rb phosphorylation at these sites (Fig. 3B). These results suggest that 7,3Ј,4Ј-THIF-induced inhibition of the expression of G 1 regulatory proteins promotes blockage of the G 1 /S transition of EGF-stimulated JB6 Pϩ cells.
7,3Ј,4Ј-THIF Suppresses the Kinase Activity of Cyclin D1-CDK4 and Cyclin E-CDK2 Complexes-Rb phosphorylation is initiated by the cyclin D1-CDK4 complex and enhances complete pRb phosphorylation by the cyclin E-CDK2 complex, forming a positive feedback loop (11). Therefore, both the cyclin D1-CDK4 and cyclin E-CDK2 complexes are important G 1 phase-regulating proteins.
Because CDK4 must form a complex with cyclin D1 for activation, we next examined the effect of 7,3Ј,4Ј-THIF on the activity of the cyclin D1-CDK4 complex. Treatment with 7,3Ј,4Ј-THIF completely suppressed the kinase activity of the cyclin D1-CDK4 complex at all concentrations tested (Fig. 4A, left  panel). Treatment with 7,3Ј,4Ј-THIF also partially inhibited the activity of the cyclin E-CDK2 complex (Fig. 4A, right panel), but its inhibitory effect on cyclin E-CDK2 appeared weaker than that on the cyclin D1-CDK4 complex.

7,3,4-THIF Inhibits Proliferation and Cell Transformation
Akt/GSK-3␤ pathway plays a key regulatory role in cell cycle progression from G 1 to S phase. Therefore, we next evaluated the effect of 7,3Ј,4Ј-THIF on the Akt/GSK-3␤ signaling pathway by Western blot. Treatment with 7,3Ј,4Ј-THIF inhibited the phosphorylation of both Akt and GSK-3␤ (Fig. 5A) but had no effect on the phosphorylation of ERKs or p38 (data not shown).

7,3Ј,4Ј-THIF Inhibits EGF-induced AP-1 Transactivation in and Neoplastic Transformation of JB6
Pϩ Cells-The promoter region of the cyclin D1 gene contains two binding sites for AP-1, a positive regulator of cyclin transcription (21). Thus, AP-1 activation is thought to play an important role in tumor promoter-induced cell proliferation and neoplastic transformation.
To determine whether the repression of proliferation and cyclin D1 expression by 7,3Ј,4Ј-THIF involves inhibition of AP-1, we measured AP-1 transactivation using JB6 cell lines stably transfected with an AP-1 luciferase reporter plasmid. Results showed that 7,3Ј,4Ј-THIF inhibited EGF-induced transactivation of AP-1 in a dose-dependent manner (Fig. 6A), suggesting that the inhibition of cell proliferation and cyclin D1 expression by 7,3Ј,4Ј-THIF occurs through the suppression of AP-1 activity.

DISCUSSION
Because chemopreventive agents are likely to be used continually by essentially healthy people, naturally occurring substances are ideal candidates, presuming safety during long term use. Isoflavones are a class of naturally occurring bioactive phytochemicals found in many plants (22). Numerous experimental studies have shown that soy isoflavones such as genistein and daidzein have anti-carcinogenic effects, and soy foods are generally recognized as safe. Several in vitro and in vivo studies have demonstrated that soy extracts and isoflavones have an inhibitory effect on proliferation of EGF-overexpressing cancer cells (17,23,24).
A substantial percentage of dietary daidzein is converted to biologically active metabolites by intestinal microflora or liver microsomes (25,26). We previously showed that equol is one of the most biologically active metabolites of daidzein (27). We to synchronize cells at G 0 and then treated with 7,3Ј,4Ј-THIF at the indicated concentrations for 1 h followed by stimulation with 10 ng/ml EGF for 10 min, and then harvested. The levels of phosphorylated and total Akt and GSK-3␤ proteins were then determined by Western blot analysis using specific antibodies against the corresponding phosphorylated or total proteins as described under "Experimental Procedures." The data shown are representative of three independent experiments. B, 7,3Ј,4Ј-THIF inhibits PI3K activity. Kinase assays were performed as described under "Experimental Procedures." The 32 P-labeled phosphatidylinositol 3-phosphate product was resolved by TLC and visualized by autoradiography. The data shown are representative of three independent experiments. C, 7,3Ј,4Ј-THIF specifically binds to PI3K. Binding of 7,3Ј,4Ј-THIF to PI3K was assessed by immunoblotting using a specific p110 antibody. Lane 1, PI3K protein standard (input control); lanes 2 and 3, PI3K pulldown experiments were performed using Sepharose 4B beads (control; lane 2) or 7,3Ј,4Ј-THIF-Sepharose 4B beads (lane 3), as described under "Experimental Procedures." D, 7,3Ј,4Ј-THIF competes with ATP for binding to PI3K. Active PI3K (2 g) was incubated with ATP (0, 10, or 100 m) and 100 l of 7,3Ј,4Ј-THIF-Sepharose 4B beads or 100 l of Sepharose 4B (as a negative control) in reaction buffer in a total volume of 500 l. The mixtures were incubated at 4°C overnight with shaking. After washing, the pulled down proteins were detected by Western blotting. Lane 1, PI3K protein standard (input control); lane 2, negative control: PI3K did not bind to Sepharose 4B; lane 3, positive control: PI3K bound to 7,3Ј,4Ј-THIF-Sepharose 4B; lanes 4 and 5, as the ATP concentration increased, 7,3Ј,4Ј-THIF binding to PI3K decreased. also suggested that equol, but not daidzein, is a potent inhibitor of mitogen-activated protein kinase activity and that equol has chemopreventive activity in JB6 Pϩ cells, suppressing 12-Otetradecanoylphorbol 13-acetate-induced neoplastic transformation (27).
In rats (28) and humans (25), isoflavones are subject to oxidative biotransformation through hepatic metabolism. One of the major products of the hepatic metabolism of daidzein is 7,3Ј,4Ј-THIF (29). However, no reports on the possible chemopreventive effects or molecular mechanism of action of this hepatic metabolite have been published. In the present study, we found that 7,3Ј,4Ј-THIF prevented the transition of JB6 Pϩ cells from G 1 to S phase, significantly blocking EGF-stimulated progression of the cell cycle at G 1 . It also effectively suppressed EGF-induced neoplastic transformation of JB6 Pϩ cells, whereas we previously found daidzein itself had no effect (27). This study thus demonstrates that 7,3Ј,4Ј-THIF is a potent chemopreventive agent against EGF-induced proliferation of JB6 Pϩ cells. Our findings also provide information about the molecular mechanisms and targets of this activity.
Defective CDK4 inhibition and deregulated CDK4 activity have been observed in many cancers. Phosphorylation of Rb by CDK4 contributes to the release and activation of E2F target genes, including those encoding E-and A-type cyclins, which facilitate progression through G 1 phase. In particular, Ser-795 and Ser-807/Ser-811 in Rb are specific sites for phosphorylation by CDK4 and are additionally phosphorylated by cyclin E-bound CDK2 (30,31). The G 1 /S-phase transition is tightly regulated by the interactions of the CDK4-cyclin D and CDK2cyclin E complexes with Rb. Therefore, CDK inhibitors have been considered relevant candidates for anticancer therapeutic agents owing to their potential ability to restore control of the cell cycle (5,32). The development of a natural inhibitor of CDK4 or CDK2 activity is a promising approach in this chemopreventive strategy. Our results show that 7,3Ј,4Ј-THIF completely suppresses CDK4 activity and partially inhibits CDK2 activity by binding directly to these proteins. Thus, 7,3Ј,4Ј-THIF exerts its potent chemopreventive effect by suppressing CDK activities.
We then hypothesized that the molecular target of 7,3Ј,4Ј-THIF in the inhibition of EGF-induced cyclin D1 expression might be a kinase upstream of Akt. We found that 7,3Ј,4Ј-THIF effectively and directly inhibits PI3K activity, resulting in the down-regulation of Akt and GSK-3␤. Furthermore, 7,3Ј,4Ј-THIF competes with ATP for PI3K binding, which might explain the reduced kinase activity of 7,3Ј,4Ј-THIF-bound PI3K. Collectively, these results suggest that the inhibition of cyclin D1 expression by 7,3Ј,4Ј-THIF is primarily the result of direct suppression of PI3K activity.
To investigate the molecular mechanism of the PI3K inhibition by 7,3Ј,4Ј-THIF, we carried out a modeling study using the crystal structure of PI3K in complex with ATP or myricetin (37,38). PI3K consists of four domains: a Ras-binding domain, a C2 domain, a helical domain, and a catalytic domain. The catalytic domain of PI3K consists of an N-lobe and a C-lobe with a fold similar to other protein kinases, and this structural similarity is also conserved in the ATP binding site that is flanked by these two lobes. Consequently, ATP binds between these lobes in a manner similar to ATP binding in many protein kinases. The N-and C-lobes are linked through a loop, which is called the "hinge region." The backbone of this loop interacts with the adenine moiety of ATP by hydrogen bonding. Considering the experimental result showing that 7,3Ј,4Ј-THIF is an ATPcompetitive inhibitor of PI3K, we docked the compound to the ATP binding site of PI3K (Fig. 7A). The hydroxyl groups at the 3Ј and 4Ј positions of 7,3Ј,4Ј-THIF could make hydrogen bonds with the backbone atoms of Val-887 in the hinge region of PI3K. The hydroxyl group at the 7 position could also form hydrogen bonds with the side chains of Asp-836. In addition, 7,3Ј,4Ј-THIF would be sandwiched by the side chains of the hydrophobic residues in the ATP binding site, including Met-804, Trp-812, Ile-831, Tyr-867, and Ile-879 from the N-lobe and Ala-885, Met-953, Phe-961, and Ile-963 from the C-lobe.
The potent inhibitory activity of 7,3Ј,4Ј-THIF against PI3K would be due to these hydrogen bonds and hydrophobic interactions. Because 7,3Ј,4Ј-THIF was shown to be an ATP-noncompetitive inhibitor of CDKs, a docking study was not possible due to the lack of available structure data for the binding of ATP-noncompetitive inhibitors with CDKs.
From the experimental result showing that 40 M 7,3Ј,4Ј-THIF does not affect other protein kinases such as EGF receptor, ERKs, MSK1, Akt, MKK3/6, or p38 kinases (data not shown) and inhibits CDKs in an ATP-noncompetitive manner, we can hypothesize that the selective binding of the compound to the ATP binding site of PI3K is due to a similar, but distinct structure of the ATP binding site of PI3K compared with those of protein kinases such as the CDKs. Binding of 7,3Ј,4Ј-THIF to CDKs might induce a structural rearrangement of CDKs into an inactive conformation or weaken the binding affinity of cyclins to CDKs. Further studies with x-ray crystallography to determine the inhibitor complex structures would elucidate the exact binding modes of 7,3Ј,4Ј-THIF to PI3K and CDK2/4.
A recently adopted strategy in anticancer therapeutics development has been to develop agents that target a variety of different kinases. The hope is that these agents will be more efficacious than are the highly selective kinase inhibitors administered as single agents. Moreover, a multitargeted kinase inhibitor might have broad-range antitumor efficacy. Two such multitargeted kinase inhibitors, sunitinib and sorafenib, have proven to be effective in clinical testing (39).
Although CDK inhibitors are thought to be relevant drug candidates for cancer therapy, the first-generation CDK inhibitors, such as flavopiridol and UCN-01, have not exhibited significant clinical advantages. A possible explanation for this disappointing result is that different kinds of tumors might have different sensitivities to different types of CDK inhibition, depending on their pathogenic spectrum of mutations (32). Therefore, naturally occurring dietary substances capable of regulating different kinases, rather than or in addition to chemotherapy, might be ideal agents for chemoprevention.