Black tea polyphenols inhibit IGF-I-induced signaling through Akt in normal prostate epithelial cells and Du145 prostate carcinoma cells
Russell D. Klein,1 and
Susan M. Fischer
The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, PO Box 389, Smithville, TX 78957, USA
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Abstract
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Tea polyphenols have been proposed as potential chemopreventive agents against prostate cancer, primarily because of their high intake by populations with reduced cancer incidence and their reported ability to inhibit proliferation and increase apoptosis in prostate cancer cells in culture. Insulin-like growth factor-I (IGF-I) has been implicated as a risk factor for the development of prostate cancer by epidemiological studies and has been shown to be causative in animal models. One of the primary signal transduction pathways activated by IGF-I binding to its receptor is the Akt pathway. We determined that phosphorylated Akt levels are very low in serum-starved human normal prostate epithelial cells (PrEC) and Du145 prostate carcinoma cells, and that treatment of these cells with IGF-I results in a rapid and sustained phosphorylation of Akt. Pre-treatment of PrEC and Du145 cells with doses as low as 20 µg/ml of a mixture of black tea polyphenols (BTP) substantially reduced IGF-I-mediated Akt phosphorylation. This effect of BTP appears to be due partially to the reduced autophosphorylation of IGF-I receptor-1 in BTP-treated cells. BTP pre-treatment also decreased downstream effects of Akt activation including phosphorylation of glycerol synthase kinase-3, increased cyclin D1 protein levels and increased DNA synthesis. Our results indicate that polyphenols from black tea inhibit the IGF-I signal transduction pathway, which has been linked to increased prostate cancer incidence in human populations and, therefore, provide further support for the potential of BTP to prevent prostate cancer.
Abbreviations: BTP, black tea polyphenol; DMEM, Dubellco's modified medium; EGCG, epigallocatechin gallate; GSK-3 glycerol synthase kinase-3; IGF-I, insulin-like growth factor-I; IGFR-1, insulin-like growth factor receptor-1; PrEC, prostate epithelial cells.
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Introduction
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Currently, almost 10% of men in the US develop clinically detectable prostate cancer in their lifetime (1). The incidence of clinically relevant prostate cancer is much lower in Asian countries, although the incidence of microscopic or latent prostate carcinoma does not differ substantially (2). These observations have led to the hypothesis that environmental factors such as diet are capable of altering the progression of this disease. Understanding the molecular mechanisms involved in the progression of prostate cancer from a latent to a clinically relevant form, and discovering compounds that inhibit those mechanisms, will enhance our ability to prevent the progression of prostate cancers. Recent epidemiological evidence has linked elevated serum levels of insulin-like growth factor-I (IGF-I) with the development of prostate cancer (3), and expression of an IGF-I transgene in mice has been demonstrated to cause the development of prostate cancers (4). IGF-I is capable of both enhancing proliferation and inhibiting apoptosis in normal and malignant prostate epithelial cells in culture (5,6). IGF-I-related signal transduction might, therefore, be an important factor in the development of prostate cancer. In many cell types, the binding of IGF-I to the IGF-I receptor causes rapid phosphoinositol-3-kinase (PI-3-kinase)-dependent activation of Akt through phosphorylation of specific threonine (Thr 308) and serine (Ser 473) residues (Figure 1
). The downstream effects of Akt activation, including increased cellular proliferation and protection from apoptotic stimuli, have been attributed in part to enhanced phosphorylation of Bad, glycerol synthase kinase-3 (GSK-3), Caspase-9 and Forkhead proteins, and increased cyclin D protein levels (7).

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Fig. 1. IGF-I-mediated activation of Akt and subsequent inactivation of GSK-3. IRS, insulin receptor substrate; PI, phosphatidylinositol; PDK, protein dependent kinase; P, phosphorylation event (7,27,34).
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Green tea is consumed at high levels in Asian countries, and research has shown that green tea polyphenols such as epigallocatechin gallate (EGCG) are capable of reducing tumor burden in animal models, and inhibiting proliferation and inducing apoptosis in cell culture models (813). Although green tea polyphenols are generally considered to be more potent in influencing these processes, there are a number of studies that have reported that polyphenols from black tea are similarly effective (1417). As black tea is more readily consumed in western nations, it is worth investigating the effects of black tea polyphenols (BTPs) on mechanisms involved in prostate cancer development.
An analysis of the effects of tea polyphenols on IGF-I signaling has not been reported previously; therefore, we analyzed the ability of a BTP fraction to inhibit IGF-I signal transduction pathways. We hypothesized that BTPs would inhibit IGF-I-induced DNA synthesis in prostate cells, and that this inhibition would occur at least in part through the inhibition of the Akt signal transduction pathway. The BTP fraction used in these experiments was provided by the T.J. Lipton Tea Company (Englewood Cliffs, NJ). The approximate composition of this BTP fraction is: gallic acid, 3%; catechin, 2%; epicatechin (EC), 7%; epigallocatechin (EGC), 2%; epicatechin gallate (ECG), 13%; EGCG, 17%; theaflavins, 8%; unknown polyphenols, 32% (18).
To test whether or not BTPs could inhibit IGF-I-induced proliferative events in prostate cells, we pre-treated serum-starved Du145 prostate cancer cells with varying concentrations of BTP before stimulation with IGF-I (Figure 2
). Cell-cycle analysis of propidium iodide stained cells from this experiment revealed a significant G1 block (84%) resulting from serum starvation of Du145 cells (Figure 2A
). Treatment with 25 ng/ml IGF-I induced a 2-fold increase in the number of cells in S-phase, and a concomitant decrease in the number of cells in G1 (Figure 2B
). This result is in agreement with previous reports that IGF-I can stimulate DNA synthesis and proliferation of Du145 cells (5). Treatment of Du145 cells with as low as 10 µg/ml of BTP completely inhibited the IGF-I-induced progression of cells into S-phase (Figure 2C
F). Others have found that EGCG inhibits serum-induced DNA synthesis in Du145 cells (8,9), but this is the first report of an inhibition of IGF-I-stimulated DNA synthesis by BTPs. Interestingly, BTPs were as effective in our studies as similar concentrations of purified EGCG were in previous reports (8,9,19).

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Fig. 2. BTPs inhibit IGF-I-induced DNA synthesis in Du145 prostate carcinoma cells. Du145 cells were seeded at 3.5x105 cells/well and allowed to attach overnight in 6 well dishes in Dubellco's modified medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were then starved for 20 h in DMEM, following which they were treated with BTP and IGF-I for 24 h at the following concentrations: (A) control (no IGF-I, no BTP); (B) 25 ng/ml IGF-I, no BTP; (C) 25 ng/ml IGF-I, 10 µg/ml BTP; (D) 25 ng/ml IGF-I, 20 µg/ml BTP; (E) 25 ng/ml IGF-I, 40 µg/ml BTP; (F) 25 ng/ml IGF-I, 80 µg/ml BTP. Cells were then trypsinized, fixed in ice cold 70% ethanol and stained with propidium iodide in the presence of 5 µg/ml RNase. Stained cells were analyzed on a Coulter EPICS Elite flow cytometer using a 610 nm bypass filter and aggregate discrimination. Cell-cycle analysis was carried out using Multicycle DNA cell cycle analysis software from Phoenix Flow Systems. Results are an average of two independent experiments.
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To begin to determine the mechanism by which BTPs inhibit IGF-I-induced DNA synthesis, we analyzed the effect of BTP treatment on two major signaling pathways activated by IGF-I binding to the IGFR-1 receptor, the Erk and Akt pathways. IGF-I has been reported to cause phosphorylation and activation of Erk1/Erk2 in Du145 cells (20), but there have been no reports on IGF-I-mediated phosphorylation of Akt in these cells. As seen in Figure 3A
, treatment of serum-starved Du145 cells with 25 ng/ml IGF-I causes a rapid phosphorylation of Akt on serine residue 473 within 10 min, and this phosphorylation was maintained for at least 60 min. Phosphorylation of threonine residue 308 was analyzed in the same manner and showed a similar result (data not shown). Phosphorylation of these two amino acids has been demonstrated conclusively to result in the activation of Akt kinase activity (7). In our hands, levels of Erk1/Erk2 phosphorylation were high in serum-starved Du145 cells and IGF-I did not further stimulate their phosphorylation (data not shown). Treatment of Du145 cells with up to 80 µg/ml BTPs before IGF-I treatment did not alter Erk1/Erk2 phosphorylation (data not shown). However, IGF-I-induced Akt phosphorylation was inhibited by BTPs beginning at between 10 and 20 mg/ml and achieving near total inhibition with 40 mg/ml BTP (Figure 3B
). The data suggest that inhibition of the Akt pathway may be an important mechanism by which BTPs inhibit IGF-I stimulation in Du145 cells. We also tested the effect of BTPs on IGF-I-mediated Akt phosphorylation in normal human prostate epithelial cells (PrEC). Nearly identical results were found in PrEC cells as were observed with Du145 cells with regard to BTP inhibition of Akt phosphorylation (Figure 3C
). These results indicate that Du145 cells behave similarly to PrEC cells with regard to inhibition of IGF-I-induced Akt phosphorylation by BTPs, and that this inhibition is likely to occur by a similar mechanism in the two cell types.

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Fig. 3. BTPs inhibit IGF-I-mediated Akt phosphorylation in prostate cells. (A) Du145 cells were seeded at 3.5x105 cells/well in 6 well dishes in DMEM with 10% FBS and allowed to attach overnight. Following overnight serum starvation, cells were treated with 25 ng/ml IGF-I and incubated for the indicated times. (B) Du145 cells were seeded at 3.5x105 cells/well in 6 well dishes in DMEM with 10% FBS and allowed to attach overnight. Following overnight serum starvation, cells were treated with BTPs for 15 min, followed by a 20 min treatment with IGF-I as indicated. (C) Normal human prostate PrEC cells (Clonetics, Walkersville, MD) were seeded at 3.5x105 cells/well in 6 well dishes in PrEGM (Clonetics) and allowed to attach overnight. Following overnight growth factor starvation in PrEBM, cells were treated with BTPs for 15 min, followed by a 20 min treatment with IGF-I as indicated. Following treatments, cell lysates were collected directly in sample buffer [62.5 mM Tris (pH 6.8), 2% sodium dodecylsulfate (SDS), 10% glycerol, 50 mM dithiothreitol], sonicated, separated on an 8% polyacrylamide (PAGE) gel, transferred to a PVDF membrane and probed with a primary antibody specific for phospho-Akt (Ser 473, Cell Signaling Technology). Blots were then stripped and re-probed with a primary antibody for Akt (Cell Signaling Technology). Bound primary antibody was detected with horseradish peroxidase conjugated secondary antibody and chemiluminescent substrate. A representative blot from at least three independent experiments is shown.
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It is of interest that Du145 and PrEC cells have low basal levels of phosphorylated Akt, whereas two other prostate cancer cell lines, PC-3 and LNCaP, have constitutively high basal levels of phospho-Akt. This observation has been attributed to the loss of PTEN function in PC-3 and LNCaP cells (21,22). PTEN is a lipid phosphatase that functions by dephosphorylating phosphatidylinositol-3,4,5-triphosphate, the product of PI-3-kinase, thereby inactivating the signal for Akt phosphorylation (Figure 1
). In experiments with PC-3 cells, we found that IGF-I treatment results in only a minor increase in phospho-Akt over high basal levels, and a 30 min treatment with BTPs inhibits only the induced phospho-Akt without affecting the high basal levels (data not shown). These results indicate that BTP treatment inhibited the generation of new signaling events critical for IGF-I-induced phosphorylation of Akt, but did not affect mechanisms involved in maintaining Akt in its active phosphorylated state.
It has been reported that purified EGCG and theaflavin-3,3'-digallate (TF-3) are potent inhibitors of epidermal growth factor receptor and platelet-derived growth factor receptor autophosphorylation in response to their respective ligands (17,23,24). These tea polyphenols were demonstrated to inhibit binding of the cognate ligand to its receptor. In order to determine if reduced receptor autophosphorylation might be responsible for the observed inhibition of Akt phosphorylation by BTP in IGF-I-treated Du145 cells, we tested the effect of these treatments on insulin-like growth receptor-1 (IGFR-1) autophosphorylation. Treatment of serum-starved Du145 cells with IGF-I resulted in a strong increase in IGFR-1 phosphorylation as determined by IGFR-1 immunoprecipitation and western blotting for phosphorylated tyrosine residues (Figure 4
). A small decrease in IGFR-1 receptor autophosphorylation was observed in cells treated with as low as 10 µg/ml BTP; however, complete inhibition was observed only at a dose of 80 µg/ml BTP (Figure 4
). It appears from these results that the inhibition of IGFR-1 autophosphorylation probably accounts for part of the decrease in Akt phosphorylation seen in BTP-treated cells, but as higher doses were required to achieve complete inhibition of receptor phosphorylation, it is unlikely that this is the only mechanism involved. Other potential mechanisms may include inhibition of kinase activities downstream of the IGFR-1 receptor (17).

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Fig. 4. Du145 cells were seeded at 3.5x105 cells/well in 6 well dishes in DMEM with 10% FBS and allowed to attach overnight. Following overnight serum starvation in DMEM, cells were pre-treated with BTPs at the indicated concentrations for 15 min followed by a 20 min treatment with 25 ng/ml IGF-I. Immunoprecipitation for IGFR-1 was carried out with a rabbit polyclonal antibody against IGFR-1 (Oxford Biomedical, Oxford, MI) according to the manufacturers immunoprecipitation protocol. Briefly, the cells were rinsed with phosphate-buffered saline (PBS) and lyzed on ice in PBSTSD buffer (PBS, Tween-20, SDS, deoxycholate) with protease and phosphatase inhibitors. The immunoprecipitated protein was eluted from the protein Gagarose beads by boiling in sample buffer, separated on an 8% PAGE gel and transferred to a PVDF membrane. The blot was then probed with a phospho-tyrosine specific primary antibody (P-Tyr-100, Cell Signaling Technology) to detect phosphorylated IGFR-1. Bound primary antibody was detected with horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. A representative blot from at least three independent experiments is shown.
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A number of factors regulated by Akt have been shown to be involved in regulating proliferation and apoptosis. Cyclin proteins are involved in regulating entry into the different phases of the cell cycle, and cyclin D1 is necessary for progression through G1. Cyclin D1 is regulated at a number of different levels, but one primary mechanism of regulation is through protein degradation. Phosphorylation of cyclin D1 by GSK-3ß has been demonstrated to cause exportation from the nucleus to the cytoplasm and target it for degradation via ubiquination (2527). Akt is known to phosphorylate GSK-3ß, which results in its inactivation, which in turn results in elevated cyclin D1 protein levels (Figure 1
) (27,28). This mechanism has been proposed to play a role in proliferation induced through the Akt signaling pathway. We therefore determined if this pathway was altered by BTPs in IGF-I-stimulated Du145 cells. IGF-I treatment caused substantial phosphorylation of GSK-3 in Du145 cells as compared with untreated serum-starved cells (Figure 5A
). Treatment with as little as 10 µg/ml of BTPs resulted in a dramatic inhibition of GSK-3 phosphorylation, and 20 µg/ml completely inhibited IGF-I-mediated phosphorylation of GSK-3 in Du145 cells. Cyclin D1 levels were also increased by IGF-I treatment within 6 h (Figure 5B
). Pre-treatment of cells with BTPs inhibited the IGF-I-mediated increase in cyclin D1 protein levels in a dose-dependent manner. A dose of 10 µg/ml BTP reduced cyclin Dl levels below that of serum-starved cells that were not treated with IGF-I, whereas cyclin D1 was barely detectable in lysates from cells treated with 20 µg/ml BTP. Thus, similar doses of BTP, that are capable of inhibiting DNA synthesis in Du145 cells, also result in a substantial reduction of cyclin D1 protein levels.

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Fig. 5. Du145 cells were seeded at 3.5x105 cells/well in 6 well dishes in DMEM with 10% FBS and allowed to attach overnight. Following overnight serum starvation in DMEM, cells were pre-treated with BTPs at the indicated concentrations for 15 min followed by a 20 min treatment with 25 ng/ml IGF-I. Following treatments, cell lysates were collected directly in sample buffer, sonicated and separated on an 8% PAGE gel, and transferred to a PVDF membrane. (A) Treatment with IGF-I for 30 min and analysis western for phosphorylated GSK-3 with a phosho-GSK-3 specific primary antibody (Upstate Biotechnology, Lake Placid, NY). (B) Treatment with IGF-I for 6 h and western analysis with a Cyclin D1 specific primary antibody (Santa Cruz, Santa Cruz, CA). Bound primary antibody was detected with horseradish peroxidase conjugated secondary antibody and chemiluminescent substrate. A representative blot from at least three independent experiments is shown.
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Another mechanism that has been proposed for the involvement of Akt in cell-cycle regulation is the phosphorylation and inactivation of forkhead transcription factors that can regulate the cell-cycle inhibitor p27 (2932). An analysis of p27 protein expression in Du145 cells treated with IGF-I and BTPs under the exact same conditions as described for Figure 5B
showed no effect of these treatments on p27 protein levels by western analysis (data not shown). In addition, we could not demonstrate an increase in phosphorylation of the forkhead transcription factor FKHR in Du145 cells treated with IGF-I using phospho-specific antibodies (Cell Signaling Technology, Beverly, MD) for this protein (data not shown). There may be other forkhead receptors involved in Akt signaling in Du145 cells, but the fact that p27 levels are not altered make it unlikely that this pathway is involved in BTP inhibition of proliferation in these cells.
IGF-I has been strongly implicated in the etiology of human prostate cancer. Signaling through IGF receptors has been demonstrated to induce proliferation and prevent apoptosis in prostate cancer cell lines (5,33). The identification of agents that inhibit the IGF-I signaling pathway could lead to the development of highly successful prevention strategies for prostate cancer. The present studies clearly demonstrate that polyphenols isolated from black tea, commonly consumed in western countries, are capable of disrupting IGF-I signaling in prostate cells. Additionally, these studies indicate that inhibition of the Akt pathway may be a major mechanism by which BTPs inhibit IGF-I-induced signal transduction and proliferation.
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Notes
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1 To whom correspondence should be addressed Email: rklein{at}sprd1.mdacc.tmc.edu. 
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Acknowledgments
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Flow cytometry analysis was performed by Kent Claypool in the Cell and Tissue Analysis facility core at Science Park-Research Division, University of Texas, M.D. Anderson Cancer Center. This work was supported by the National Institutes of Health (grant number R25 97330).
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Received July 11, 2001;
revised October 5, 2001;
accepted October 9, 2001.