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Carcinogenesis vol.23 no.10 pp.1677–1684, 2002 Tangeretin induces cell-cycle G1 arrest through inhibiting cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells Min-Hsiung Pan1,4, Wei-Jen Chen1, Shoei-Yn Lin-Shiau2, Chi-Tang Ho3 and Jen-Kun Lin1* Institutes of 1Biochemistry and 2Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan, 3Department of Food Science, Cook College, Rutgers University, New Brunswick, NJ 08901, USA and 4Department of Marine Food Science, National Kaohsiung Institute of Marine Technology, Kaohsiung, Taiwan Tangeretin (5,6,7,8,4’-pentamethoxyflavone) is concentrated in the peel of citrus fruits. DNA flow cytometric analysis indicated that tangeretin blocked cell cycle progression at G1 phase in colorectal carcinoma COLO 205 cells. Over a 24 h exposure to tangeretin, the degree of phosphorylation of Rb was decreased after 12 h and G1 arrest developed. The protein expression of cyclins A, D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that tangeretin inhibited the activities of cyclin-dependent kinases 2 (Cdk2) and 4 (Cdk4) in a dosedependent manner in the cell-free system. As the cells were exposed to tangeretin (50 µM) over 48 h a gradual loss of both Cdk2 and 4 kinase activities occurred. Tangeretin also increased the content of the Cdk inhibitor p21 protein and this effect correlated with the elevation in p53 levels. In addition, tangeretin also increased the level of the Cdk inhibitor p27 protein within 18 h. These results suggest that tangeretin either exerts its growth-inhibitory effects through modulation of the activities of several key G1 regulatory proteins, such as Cdk2 and Cdk4, or mediates the increase of Cdk inhibitors p21 and p27. Introduction Flavonoids are widespread in fruit and vegetables. They are intensively studied for their role in human health, including cancer prevention. Citrus flavonoids have a broad spectrum of biological activity, including anticarcinogenic and antitumor activities (1). It is suggested that cancer induction can be prevented by ingestion of certain food ingredients (2), and flavonoids, in citrus fruits and juices are among the most prominent cancer-preventing agents (3–5). The biological effects of flavonoids seem to occur mainly through their interaction with protein tyrosine kinases (6) and cyclooxygenase (7). Polymethoxylated flavonoids, such as tangeretin and nobiletin, are more potent inhibitors of tumor cell growth than free hydroxylated flavonoids. They also possess potent anti-invasive and anti-metastatic activities (8,9). Tangeretin is a polymethoxylated flavone, 5,6,7,8,4’-pentamethoxyflavone, which is concentrated in the peel of citrus Abbreviations: CAK, Cdk-activating kinase; Cdk, cyclin-dependent kinase; CKI, Cdk inhibitor; EGCG, (-)-epigallocatechin-3-gallate; IP, immunoprecipitation; PCNA, proliferating cell nuclear antigen. © Oxford University Press 1677 Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 *To whom correspondence should be sent at Institute of Biochemistry, College of Medicine, National Taiwan University, No 1, Section 1, Jen-Ai Road, Taipei, Taiwan Email: [email protected] fruits (10) (Figure 1), and probably acts as a natural resistance factor against pathogenic fungi (11). Grapefruit and orange juice have been shown to inhibit human breast cancer cell proliferation (12) and to interact with drug administration (13). Several biological activities have been shown for tangeretin itself, including the ability to enhance gap junctional intercellular communication (14), to counteract tumor promoterinduced inhibition of intercellular communication (15) and to inhibit cancer cell proliferation (16). Previous studies have shown that many flavonoids exhibit potent antitumor activity against several rodent and human cancer cell lines (17,18). The antitumor properties of some flavonoids have been studied with respect to apoptosis and cell cycle arrest. Quercetin has been shown to impair the G1 to the S phase transition in a human gastric cancer cell line and also to cause apoptosis in several cell lines (19). The molecular mechanisms of cell cycle arrest by flavonoids remain largely unclear, but appear to involve modulation of multiple cell cycle regulatory proteins. The eukaryotic cell cycle is regulated through the sequential activation and inactivation of cyclin-dependent kinases (Cdks) that drive cell cycle progression through the phosphorylation (20) and dephosphorylation of several regulatory proteins. In normal cells, Cdks exist predominantly in quaternary complexes consisting of a Cdk, a cyclin, a proliferating cell nuclear antigen (PCNA) and a 21 kDa protein (p21) (21). Cdk activation requires cyclin binding and phosphorylation of conserved threonine residue by Cdk-activating kinase (CAK). The activated Cdk–cyclin complexes can be changed to an inactive state by phosphorylation of a conserved threonine– tyrosine pair or binding to Cdk inhibitory subunits (CKIs). Progression from G1 to S phase in mammalian cells is regulated by the accumulation of cyclins D, E and A, which bind to and activate different Cdk catalytic subunits. The activation of Cdk4–cyclin D and/or Cdk6–cyclin D complex is necessary for transition from early to mid G1 phase. Transition through mid G1 to S phase is regulated by activation of the Cdk2– cyclin E complex. Progression through late G1 to S phase also requires the presence of Cdk2–cyclin A complex (22). The retinoblastoma tumor suppressor protein (Rb) is a critical target protein that is phosphorylated via these Cdk–cyclin complexes (23). Rb controled gene expression is mediated by a family of heterodimeric transcriptional regulators, collectively termed the E2F, which can transactivate genes whose products are important for transition from G1 to S phase (24). Phosphorylation of Rb frees these regulators, enabling them to transactivate the target genes. Therefore, the hypophosphorylated forms of Rb are predominantly found in G0–G1 phase, but the hyperphosphorylated forms of Rb are required during S and G2–M phase (23). Recent studies show that Cdk regulation involves a diverse family of proteins, termed the CKIs (Cdk inhibitors), that bind and inactivate Cdk–cyclin complexes. In mammalian cells CKIs fall into two classes: (i) p21 (Cip1/Waf1/Cap20/Sdi1/ M.-H. Pan et al. system and in cultured cells. The effects of these flavonoids on the p53 protein levels were also investigated. Materials and methods Cell culture The cell line COLO 205 (CCL-222; American Type Culture Collection) was developed from a poorly differentiated human colon adenocarcinoma. Cell lines were maintained in RPMI-1640 supplemented with 10% FCS (Gibco BRL, Grand Island, NY), 1% penicillin/streptomycin–1% glutamate and kept at 37°C in a humidified atmosphere of 5% CO2 in air. Determination of cell growth curve Human colon cancer (5⫻104) cells were plated in 35 mm petri dishes. The next day, the medium was changed and various flavonoids were added. Control cells were treated with DMSO to a final concentration of 0.05% (v/v). At the end of incubation, cells were harvested for cell count using a hemocytometer (17). Flow cytometric cell analysis Cell cycle distribution was analyzed by flow cytometry as described previously (35). Briefly, cells were trypsinized, washed once with PBS, and fixed in 100% ethanol for 1 h at –20°C. Fixed cells were washed with PBS, incubated with 0.5 ml PBS containing 0.05% RNase and 0.5% Triton X-100 for 30 min at 37°C and stained with propidium iodide. The stained cells were analyzed using a FAScan laser flow cytometer (Becton Dickinson, San Jose, CA). Fig. 1. Structures of the different flavonoids. Pic1), p27 (Kip1), and p57 (Kip2) are related proteins with a preference for Cdk2– and Cdk4–cyclin complexes; (ii) p16INK4A, p15INK4B, p18INK4C and p19INK4D are closely related CKIs specific for Cdk4– and Cdk6–cyclin complexes (25). The majority of in vivo studies suggest that the inhibitory effect of p21 is largely exerted during the G1 phase of the cell cycle, with preferential binding to Cdk4- and Cdk2- containing complexes, and that it either inhibits their kinase activities or prevents their activation by CAK (26). In addition, the regulation of p21 is largely dependent on the presence of functional p53, a transcriptional regulator that mediates cell cycle arrest following DNA damage (27) and in senescence (28). However, the expression of p21 in a variety of tissues from p53 null mice suggests that it is also regulated by a p53-independent mechanism (29). These effects probably play a role in the ability of p53 to act as a tumor suppressor. In addition, a number of chemopreventive agents have been shown to exert their anti-tumorigenic activity through p53-dependent mechanisms (30–33). In this study, we examined the effects of tangeretin and other flavonoids on the growth of colorectal carcinoma COLO 205 cells, expression of G1 cyclins, phosphorylation state of Rb, and kinase activities of Cdk2 and Cdk4 in a cell-free 1678 Western blot analysis Equal amounts of total cellular proteins (50 µg) were resolved by SDS– polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham, Arlington, IL), and then probed with primary antibody followed by secondary antibody conjugated with horseradish peroxidase or alkaline phosphatase. The immunocomplexes were visualized using enhanced chemiluminescence kits (α-tubulin, Rb, cyclin A, cyclin E, Cdk2, Cdk4, p21, p27, p53 and cyclin D1). In vitro and cell culture Cdks kinase assay For in vitro Cdks kinase assay, exponentially growing COLO 205 cells were washed with cold PBS and lysed with Gold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM EGTA, 5 mM EDTA, 10 mM NaF, 1 mM sodium pyrophosphate, 20 mM Tris–HCl pH 7.9, 100 µM β-glycerophosphate, 137 mM NaCl, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin] for 30 min at 4°C. The cell lysate was clarified by centrifugation at 12 000 g for 10 min at 4°C. 4 mg of protein were incubated with anti-Cdk2 or anti-Cdk4 antibody and protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 18 h at 4°C. The immunoprecipitate was washed three times with immunoprecipitate buffer [1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, 0.5% NP-40] and three times with kinase buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 2.5 mM EDTA, 10 mM β-glycerophosphate, 1 mM NaF, 1 mM DTT for Cdk4; 50 mM Hepes, pH 7.4, 10 mM MgCl2, 2.5 mM EDTA, 1 mM DTT for Cdk2) another three times and separated into 6–8 tubes. The kinase reactions were carried out in a final volume of 40 µl containing 2 µg histone H1 (Calbiochem, CA) or 1 µg Gst-Rb (Santa Cruz), 20 µM cold ATP, 5 µCi [γ-32P]ATP (5000 Ci/mmol, Amersham) and incubated for 20 min at 25°C. Each sample was mixed with 10 µl of 5⫻Laemmli’s loading buffer to stop the reaction, heated for 10 min at 100°C, and subjected to SDS–PAGE. The gels were dried, visualized by Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 Materials Apigenin, kaempferol, myricetin, quercetin, rutin, luteolin and all protease inhibitors were purchased from Sigma (St Louis, MO). Tangeretin and nobiletin were isolated from orange peel extract rich in polymethoxyflavones which was provided by Florida Flavors Inc. (Lakeland, FL). The extract (5 g) was subjected to silica gel column chromatography. The column was eluted with chloroform containing increasing amounts of ethyl acetate followed by ethyl acetate and methanol. Tangeretin and nobiletin were isolated as two major products at 72 mg and 52 mg, respectively. The purity of these two compounds were ⬎⬎98% as judged by HPLC. Tangeretin and nobiletin were identified by comparison of 1H and 13C NMR spectra with literature values (34). The antibodies of p27, cyclins A, D1, E and Cdk2 were obtained from the Santa Cruz Biotechnology (Santa Cruz, CA); anti-p21 monoclonal antibody was obtained from Transduction Laboratory (Lexington, KY); anti-α-tubulin monoclonal antibody from Oncogene Science (Cambridge, UK); anti-Rb monoclonal antibody and Cdk4 from Upstate Biotechnology (Lake Placid, NY); p53 from Oncogene Research Products (Cambridge, MA); anti-pRb antibody from Cell Signaling (Beverly, MA). It is important to be precise about which antibodies are used, especially with respect to anti-phospho-Rb. Tangeretin induces G1 arrest in human colorectal carcinoma cells Table I. Effect of different flavonoids on the growth of COLO 205 cells Compounds Apigenin Kaemperol Myricetin Qucercetin Rutin Luteolin Tangeretin Nobiletin IC50 (µM) ⬎100 ⬎100 ⬎100 ⬎100 ⬎100 47.6 ⫾ 0.15 37.5 ⫾ 0.12 66.2 ⫾ 1.25 Cells were treated with various concentrations of flavonoids for 24 h. The numbers of viable cells were determined by counting the trypan blueexcluding cells in a hemocytometer. Each experiment was independently performed three times and expressed as mean ⫾ SE. Results Several flavonoids inhibit cell proliferation Previous studies have shown that flavonoids are potent antiproliferation (17–18) and anticancer agents (36). Here we investigated the anti-proliferation of eight structurally related flavonoids: apigenin, luteolin, quercetin, rutin, tangeretin, nobiletin, myricetin and kaempferol. The structures of these flavonoids are illustrated in Figure. 1. To assess the inhibitory effects of selected flavonoids on the growth of colon cancer cells, we first determined the growth rates of COLO 205 colon cancer cells. Exponentially growing cultures of COLO 205 cells were continuously cultured in the absence or presence of different concentrations of flavonoids. After 48 h of treatment, the cell growth rates were determined by trypan blue exclusion. As shown in Table I, luteolin and nobiletin induced a dosedependent inhibited cell proliferation, assuming an IC50 value of ~47.6 µM and 66.2 µM, but the potencies of their inhibition were lower than that by tangeretin (IC50, 37.5 µM). Tangeretin strongly inhibited COLO 205 cell growth (Table I). Exponentially growing COLO 205 cultures rapidly underwent growth inhibition with the addition of various concentrations of tangeretin, as evidenced by a decrease of cell proliferation over the experimental period (Figure 2). Tangeretin induces G0/G1 cell cycle arrest in COLO 205 cells In this study, we demonstrated that tangeretin induced significant growth inhibition of human colon cancer cells (Figure 2). In order to determine whether tangeretin has a cell cycle arrest effect in human COLO 205 colon cancer cells, the cells treated with DMSO or tangeretin for 12 and 24 h were subjected to flow cytometric analysis after staining their DNA. Histograms of flow cytometric data are shown in Figure 3. Control cells (DMSO) progressed through the cell cycle well. In contrast, tangeretin-treated COLO 205 cells were blocked in the G0/G1 phase (over 90%) after 24 h treatment. Effects of tangeretin on pRb phosphorylation, cyclins and Cdks 2 and 4 protein expression The phosphorylation mediated by both cyclin D/Cdk4 and cyclin E/Cdk2 of the Rb protein is required for the cells to Fig. 2. Effects of tangeretin on the growth of COLO 205 cells. Cells were treated without (d) or with various concentrations of tangeretin and cell number was determined every 12 h thereafter, the numbers of viable cells were determined by counting the trypan blue-excluding cells in a hemocytometer. Data represent means ⫾ SE. Fig. 3. Tangeretin caused cell cycle arrest at G0/G1 phase. Cells were treated with DMSO (0.05%) or tangeretin (50 µM) for indicated time, and cell cycle analysis was measured as described in Materials and methods. Data shown are representative of at least three independent experiments. progress from G1 into S phase in those cells possessing a functional pRb. To elucidate the arrest point of tangeretintreated COLO 205 cells in the G1 phase we analyzed the phosphorylation state of pRb and the expression of G1 cyclin protein and Cdks 2 and 4. As shown in Figure 4A, the degree of phosphorylation of Rb was decreased after 12 h of 50 µM 1679 Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 autoradiography, and quantitated by densitometry (IS-1000 Digital Imaging System). For cell culture Cdks kinase assay, cells (2⫻105/dish) were precultured in 100 mm plastic dishes for 2 days and treated for various periods. Cell lysis and immunoprecipitation were performed as described above. Kinase assay was carried out in 50 µl of kinase buffer with Cdk2 or Cdk4-immunoprecipitate from 250 µg of protein of the lysate. The kinase reaction was performed as described above. M.-H. Pan et al. tangeretin treatment compared with total Rb protein. Exposure to 50 µM tangeretin, the levels of cyclins A, D1 and E were analyzed by immunoblotting over a 24 h period. As shown in Figure 4B and C, cyclin A, D1 and E levels did not change with increasing time of 0.05% DMSO exposure, while tangeretin appeared to have a suppressing effect on the levels of cyclin A, cyclin D1 and E. By contrast, there was no change in the protein expression of Cdks 2 and 4, which are associated with Rb phosphorylation. However, small changes in the levels of cyclins were not sufficient to explain the reduction of the 1680 phosphorylation of Rb. Therefore, we subsequently investigated the effects of tangeretin on the activities of Cdks 2 and 4 in a cell-free system and in cultured cells, as well as its effects on the expression of Cdk inhibitory subunits (CKIs). Effects of tangeretin on the activities of Cdks 2 and 4 in a cell-free system and in cultured cells To examine whether tangeretin would directly inhibit Cdks in COLO 205 cells, we first evaluated the effect of tangeretin on the kinase activities of Cdks 2 and 4 in cell-free systems. The kinase activities of Cdks 2 and 4 were measured after immunoprecipitation from exponentially growing COLO 205 cells using anti-Cdk2 or anti-Cdk4 antibodies, then tangeretin was added and histone H1 (for Cdk2) or Gst–Rb fusion protein (for Cdk4) as substrates. As shown in Figures 5A and 6A, the in vitro inhibition of Cdks 2 and 4 by tangeretin was concentration-dependent, assuming an IC50 of ~26.5 µM and Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 Fig. 4. Effects of tangeretin on the Rb protein phosphorylation and the protein expression of G1 cyclins as well as Cdk2 and Cdk4. (A) COLO 205 cells were treated with 50 µM of tangeretin for the time indicated (top). The phosphorylated Rb and total Rb protein were detected with specific antibodies. (B) COLO 205 cells were treated with 50 µM of tangeretin or (C) DMSO [final concentration of 0.05% (v/v)] for different time periods. The cell lysis and Western blotting were performed as Materials and methods indicate. Western blot data presented are representative of those obtained in at least three separate experiments. Fig. 5. Effects of tangeretin on the activities of Cdk2 kinase in cell-free system and cultured cell. (A) Cdk2 immuno complex was prepared from growing COLO 205 cells and reacted with [γ-32P]ATP, various concentrations of tangeretin and substrates (histone H1) for 20 min at room temperature as described under Materials and methods. Quantification of the phosphorylated-histone H1 was performed by densitometric analysis (IS-1000 Digital System). Data represent the means ⫾ SE of three samples. (B) COLO 205 cells were treated with 50 µM of tangeretin for increasing periods. (C) Cells were treated with different concentrations of tangeretin for 24 h. Total cell lysates were used for immunoprecipitation, the kinase activities were assayed with histone H1 as substrates. The experiments were performed as described under Materials and methods. Data shown are representative of at least three independent experiments. Tangeretin induces G1 arrest in human colorectal carcinoma cells 19.2 µM for Cdk2 and Cdk4 respectively. We next examined the activities of Cdks 2 and 4 from tangeretin-treated COLO 205 cells. The cells were treated with 50 µM tangeretin for increasing periods, and the kinase activity was then determined by the immunoprecipitation method described above. As shown in Figure 5B and 6B, the kinase activities of both Cdks 2 and 4 were inhibited in a time-dependent manner, and ⬎90% of the activities were inhibited compared with untreated control cells (100%) after 12 h treatment. On the other hand, the inhibition of Cdks 2 and 4 activities by tangeretin were also concentration-dependent in cultured COLO 205 cells (Figure 5C and 6C). These findings were consistent with the lack of Cdks 2 and 4 activities in tangeretin-treated cells, the underphosphorylated form of Rb, and the failure of these cells to progress from G1 to S phase. Effects of tangeretin on protein expression of Cdk inhibitors p27Kip1, p21Cip1and p53 in colon cancer cells To examine further whether tangeretin could induce other members of the Cdk inhibitor protein family, we investigated the effect of tangeretin on the expression of p27 and p21 proteins COLO 205 cells. The expression of p27 protein was significantly increased within 18 h after exposure to 50 µM tangeretin (Figure 7A). As with p27 protein, tangeretin treatment induced a significant increase of p21 protein. We studied further the effect of tangeretin on the levels of p27 and p21 proteins. As shown in Figure 7B, tangeretin markedly up-regulated the level of p27 and p21 in a concentration-dependent manner. The increase of p21 is reported to be regulated by either a p53dependent or p53-independent mechanism. To determine whether the growth-inhibitory response to tangeretin was dependent on the p53 status, additional experiments were performed as described below, and it was found that p53 protein was also induced by tangeretin in COLO 205 cells. Effects of tangeretin and related compounds on the expression of p53 in COLO 205 colon cancer cell line The p53 tumor suppressor has been shown to be a key regulator in interpreting the extrinsic signals that induce cell cycle arrest (37). Flavonoids such as apigenin have been reported to induce the expression of p53 protein in mouse fibroblasts (38). To 1681 Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 Fig. 6. Effects of tangeretin on the activities of Cdk4 kinase in cell-free system and cultured cell. (A) Cdk4 immuno complex was prepared from growing COLO 205 cells and reacted with [γ-32P]ATP, various concentrations of tangeretin and substrates (GST-Rb) for 20 min at room temperature as described under Materials and methods. Quantification of the phosphorylated-GST-Rb was performed by densitometetric analysis (IS-1000 Digital System). Data represent the means ⫾ SE of three samples. (B) COLO 205 cells were treated with 50 µM of tangeretin for increasing periods. (C) Cells were treated with different concentrations of tangeretin for 24 h. Total cell lysates were used for immunoprecipitation, the kinase activities were assayed with GST-Rb as substrates. The experiments were performed as described under Materials and methods. Data shown are representative of at least three independent experiments. Fig. 7. Effects of tangeretin on protein expression of p27, p21 and p53 in COLO 205 cells. (A) COLO 205 cells were treated with 50 µM of tangeretin for the time indicated. (B) Cells were treated with various concentrations of tangeretin for 24 h. Cell lysis and western blotting were performed as described under Materials and methods. Data shown are representative of at least three independent experiments. M.-H. Pan et al. investigate the structure–activity relations the effects of selected flavonoids on the expression of p53 were examined by western blotting. Among these compounds, tangeretin and nobiletin exhibited rather strong induced activities in the COLO 205 cells (Figure 8A). However, tangeretin was the most potent inducer of p53 and induced p53 protein expression in a dose-dependent manner (Figure 8B). These results suggest that p53 protein plays a critical role in the tangeretin-induced G0/G1 cell cycle arrest. Discussion Flavonoids are naturally occurring plant polyphenols found in abundance in diets rich in fruit, vegetable and plant-derived beverages such as tea. Epidemiological studies have shown that the intake of certain vegetables, fruits and tea in the daily diet provides effective cancer prevention (39). Polymethoxyflavonoids, including tangeretin, occur exclusively in citrus fruits, and the Dancy tangerine has the highest total, containing approximately five times the amount found in the peel of sweet orange varieties (40). Citrus flavonoids have a broad spectrum of biological activity including anticarcinogenic and antitumor activities. Polymethoxylated flavonoids, such as tangeretin and nobiletin, are more potent inhibitors of tumor cell growth than hydroxylated flavonoids (1). Tangeretin was reported to inhibit tumor invasion and metastasis (8) and to induce apoptosis in HL-60 cells (9). In addition, nobiletin was reported to induce differentiation of mouse myeloid leukemia cells (41), to exhibit anti-proliferative activity towards a human squamous cell carcinoma cell line (42), to exert antimutagenic activity (43), to suppress the induction of matrix metalloproteinase-9 (44), and to inhibit phorbol ester-induced skin inflammation, oxidative stress, and tumor promotion in mice (5). Our previous report demonstrated that (-)-epigallocatechin3-gallate (EGCG) could block cell cycle progression in the G1 phase in human breast carcinoma MCF-7 cells (34). In the present study, we showed that tangeretin also arrested 1682 Downloaded from http://carcin.oxfordjournals.org/ at Pennsylvania State University on March 5, 2014 Fig. 8. Effects of tangeretin and related compounds on protein expression of p53 in COLO 205 cells. (A) COLO 205 cells were treated with 50 µM of various compounds for 24 h (C, control; A, apigenin; K, kaempferol; M, myricetin; R, rutin; Q, quercetin; T, tangeretin; N, nobiletin; L, luteolin). (B) COLO 205 cells treated with increasing doses of tangeretin for 24 h. Cell lysis and western blotting were performed as described under Materials and methods. Data shown are representative of at least three independent experiments. cell cycle progression in the G1 phase in human colorectal carcinoma COLO 205 cells (Figure 3). As shown in Figure 2, among selected flavonoids, tangeretin was more potent to inhibit COLO 205 cell proliferation. Previous studies showed that many flavonoids, such as apigenin, genistein and quercetin, which have a different structure than tangeretin, induce G2/M arrest in several cell lines (45–47). On the other hand, many flavonoids also induce G1 arrest (47). These results suggest that arrest of the cell cycle by flavonoids or tangeretin is an effect which is dependent on the structural groups of these agents. Our previous studies have shown that EGCG exerts inhibitory activity against Cdk2 in cell-free systems (35). Tangeretin also exhibited a concentration-dependent inhibitory effect, not only on Cdk2 but also on Cdk4 obtained from cycling COLO 205 cells, resulting in an IC50 of ~26.5 µM and 19.2 µM, respectively (Figure 5A and 6A). The kinase activities of Cdks 2 and 4 were inhibited by 50 µM tangeretin in a time-dependent (Figure 5B and 6B) and a dose-dependent (Figure 5C and 6C) manner in cultured cells. The Cdks 2 and 4 inhibition due to these direct and indirect actions inhibited phosphorylation of Rb by tangeretin (Figure 4A). However, Cdk activation and inhibition also requires phosphorylation or dephosphorylation at some conserved amino acid residues (20). It is therefore possible that tangeretin inhibits the Cdkactivating kinases (CAKs) or activates the Cdk-inactivating phosphatases, which are regulators for Cdks. Additional studies are needed to determine whether the inhibition of CAKs or activation of Cdk-inactivating phosphatases contributes to the inhibition of Cdks 2 and 4. In addition, Cdk6 might phosphorylate pRb in cells. Issues of whether or not tangeretin directly inhibits Cdk6 activity remain to be addressed. There was little change in the levels of cyclin D1 and E after 24 h of tangeretin exposure (Figure 4B). But, it seems that the protein amount of cyclins was not the main causative effect on the activities of Cdks in tangeretin-treated COLO 205 cells. However, we cannot rule out the possibility that tangeretin might block cyclin binding to Cdk, thereby inhibiting the kinase complex activities of Cdks. The tumor suppressor, p53, has been implicated in a variety of cellular processes (48). However, the undisputed roles of p53 are the induction of cell growth arrest and apoptosis (27). Among the transcriptional targets of p53, the Cdk inhibitor p21Cip1 plays a key role in mediating G1 arrest (49). Another CKI is p27Kip1, which mediates growth arrest and is thought to play a critical role in negative regulation of cell division in vivo (22,50). The outcome of CKIs induction in most cells is cessation of cell proliferation, differentiation, or even cell death. Since an inhibition of CKIs activity is one of the factors causing uncontrolled proliferation of tumor cells, one possible strategy to control cancer cell proliferation is to induce CKIs expression, which would lead to G0/G1 arrest and stop tumor cell growth. In this study, we observed that tangeretin can cease cell proliferation in COLO 205 cells which possess functional p53 and induce the increase of p53 protein in a time-dependent manner. G1 arrest produced by tangeretin occurred with an accompanying p21Cip1 and p27Kip1 accumulation (Figure 7). The p21Cip1 continued to show a markedly up-regulation from 18 h to 24 h after exposure to tangeretin. Inhibition of Cdk activity might also have occurred through up-regulation of p21Cip1 by tangeretin. These effects might be triggered by an increased expression of p53 by tangeretin. In this study, tangeretin also increased the p27 protein level in Tangeretin induces G1 arrest in human colorectal carcinoma cells Acknowledgments: This study was supported by the National Science Council NSC 90-2320-B002-163, NSC 90-2320-B-002-164, NSC 90-2313-B-022-004; by the National Health Research Institute, NHRI-EX918913BL; by the National Research Institute for Chinese Medicine, NRICM 90102; and by the Ministry of Education, ME 89-B-FA01-1. References 1. Attaway,J.A. (1994) Citrus juice flavonoids with anticarcinogenic and antitumor properties, In Food Pytochemicals for Cancer Prevention I. 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In summary, we have shown that the growth-inhibitory response to tangeretin, including the inhibition of Cdk2 and Cdk4 kinase activities, and the increase in the level of p21 and p27, is dependent on p53 activity. The p21 and/or p27 up-regulation by tangeretin might play an important role in its anti-proliferative activity in cultured cells. A predominance of polymethoxyflavonoids (tangeretin and nobiletin) over polyhydroxyflavonoids was demonstrated by the induced p53 levels (Figure 8). In previous reports polymethoxyflavonoids were more potent than polyhydroxyflavonoids in antitumor activities using a cell culture system (42). Differences in their activities may be derived from the relatively greater membrane uptake efficiencies of polymethoxyflavonoids than those of polyhydoxyflavonoids because methylation of the phenolic hydroxyl groups, in general, increases the molecular hydrophobicity that promotes the transportation rates (5). 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