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Endocrinology, doi:10.1210/en.2008-0408
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Endocrinology Vol. 149, No. 12 5972-5983
Copyright © 2008 by The Endocrine Society

EGCG Stabilizes p27kip1 in E2-Stimulated MCF-7 Cells through Down-Regulation of the Skp2 Protein

Hsiu-Chen Huang, Tzong-Der Way, Chih-Li Lin and Jen-Kun Lin

Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan 100

Address all correspondence and requests for reprints to: Jen-Kun Lin, Ph.D., Institute of Biochemistry and Molecular Biology, National Taiwan University Medicine College, Room 947, No. 1, Section 1, Jen-Ai Road, Taipei 100, Taiwan. E-mail: jklin{at}ha.mc.ntu.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Loss of p27Kip1 is associated with a poor prognosis in breast cancer. According to previous findings, a decrease in p27Kip1 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S-phase kinase protein 2 (Skp2). Epigallocatechin-3-gallate (EGCG), the main constituent of green tea, was found to stabilize p27Kip1 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression remains unclear. Here we investigated the mechanisms involved in EGCG’s growth inhibition of estrogen-responsive human breast cancer MCF-7 cells. In our results, EGCG increased p27Kip1 and decreased Skp2 in a time- and dose-dependent manner, suggesting that p27Kip1 and Skp2 may be involved in the growth inhibition by EGCG in estrogen-stimulated MCF-7 cells. Interestingly, mRNA levels of p27Kip1 and Skp2 did not significantly change in estrogen-stimulated MCF-7 cells after EGCG treatments. Moreover, overexpression of Skp2 in MCF-7 cells prevented accumulation of p27Kip1 and promoted resistance to the antiproliferative effects of EGCG. This suggests that the down-regulation of the F-box protein Skp2 is the mechanism underlying p27Kip1 accumulation. Furthermore, both tamoxifen and paclitaxel significantly and synergistically enhanced the growth inhibition of MCF-7 cells by EGCG through the down-regulation of Skp2 protein. However, the down-regulation of Skp2 was not always correlate with the up-regulation of p27, suggesting that EGCG-dependent Skp2 down-regulation can influence cell growth in several ways. The therapeutic strategies designed to reduce Skp2 may therefore play an important clinical role in treatment of breast cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GREEN TEA IS thought to exert a possible inhibitory effect against tumorigenesis and tumor growth because of the biological activities of its polyphenols. Epigallocatechin-3-gallate (EGCG) is the major polyphenol component of green tea and a potential component for anticarcinogenesis. The beneficial effect of EGCG (Fig. 1AGo) on breast cancer has been extensively studied using nude mice xenograft models (1, 2). A previous study found that treatment with EGCG reduced the growth of the implanted breast tumors MCF-7 and MDAMB-231 in nude mice (3). Additionally, epidemiological studies have suggested that increased consumption of green tea is also related to improved prognoses for human breast cancer (4, 5). Therefore, EGCG may have beneficial characteristics that could be exploited in the treatment of breast cancer. However, the mechanism of EGCG-mediated inhibition of human breast cancer cell proliferation has yet to be defined.


Figure 1
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  		FIG. 1. Changes in cell cycle phase distribution and Rb phosphorylation after estradiol and EGCG treatment. A, The structure of EGCG. B, Cells were growth arrested for 48 h with 10 nM ICI 182780 and then treated at time 0 with DMSO alone (CON), 100 nM estradiol (E2), 100 nM estradiol plus 10 µM EGCG (E2+EGCG), or 10 µM EGCG alone (EGCG) for the indicated periods. Cells were harvested and stained with propidium iodide, and the cell cycle distribution was analyzed by flow cytometry. C, Total cell lysates were prepared from growth-arrested MCF-7 cells treated with DMSO alone (CON), 100 nM estradiol (E2), or 100 nM estradiol plus 10 µM EGCG (E2+EGCG), and Western blot analysis was performed. The experiment was performed thrice with pRB antibody, with each yielding similar results.

 
Estrogens are key molecules in breast cancer development and can influence breast cancer cell growth. About 60% of breast cancers are hormone dependent, contain estrogen receptors, and require estrogen for tumor growth (6, 7). Estrogens and antiestrogens exert growth-regulatory actions in normal and malignant breast epithelial cells through regulation of events in the G1 phase of the cell cycle. p27 is a key regulator of G1 to S phase progression. p27 binds and inhibits cyclin E/ cyclin-dependent kinase (Cdk)-2 and thereby negatively regulates S phase entry. Although p27 protein is strongly expressed in normal mammary epithelial tissue, decreased levels of p27 protein in primary breast cancers are correlated with poor prognoses (8, 9, 10). Most, if not all, cases of decreased p27 protein in human cancers are a reflection of accelerated p27 proteolysis. A major mechanism of p27 proteolysis involves its recognition by S-phase kinase protein 2 (Skp2), a component of the Skp1-Cullin 1-F-box protein (SCF) ubiquitin ligase complex. Indeed, mice lacking Skp2 have shown high levels of p27 and free cyclin E, suggesting a pivotal role for Skp2 in the degradation of p27 in vivo (11). Additionally, high Skp2 expression has been associated with down-regulation of p27 protein in several malignant diseases such as breast cancer, prostate cancer, gastric carcinoma, lymphomas, non-small cell lung cancer, and colorectal carcinomas (12, 13, 14, 15, 16, 17, 18). Furthermore, recent studies show that Skp2 is oncogenic and is overexpressed in human breast cancers (19, 20, 21). This implies that Skp2 may be a specific therapeutic target in a subset of aggressive breast carcinomas.

Our previous study has demonstrated that the initial growth inhibition of MCF-7 by EGCG correlates with an increase in the levels of the cyclin-dependent kinase inhibitors p21 and p27, a profound decrease in cyclin-dependent kinase 2 and 4 activity, and accumulation of cells in the G1 phase of the cell cycle (22). These findings led us to further investigate proteins involved in regulation of the G1 phase of the cell cycle, including the SCF ubiquitin ligase complex and cyclin-dependent kinase inhibitors like p27.

The aim of this study was to elucidate molecular mechanisms that allow for the broad antimitogenic function of EGCG. We found that EGCG attenuated Skp2 protein levels, thereby inhibiting p27 ubiquitination and promoting p27 accumulation in estrogen-stimulated MCF-7 cells. The results further demonstrate that Skp2 overexpression prevents the EGCG-mediated growth arrest of MCF-7 cells by down-regulating p27 expression. Furthermore, both tamoxifen and paclitaxel significantly and synergistically enhanced growth inhibition of MCF-7 cells by EGCG. We also found that this inhibition was mediated through down-regulation of Skp2 and up-regulation of p27 protein expression. Taken together, our results suggest that the Skp2/p27 index plays an important role in the growth inhibition of MCF-7 cells mediated by EGCG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
EGCG, paclitaxel, tamoxifen, 17β-estradiol (E2), LY294002, PD98059, cycloheximide, N-acetyl-Leu-Leu-norleu-al (LLnL), and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO). ICI 182780, c-Jun N-terminal kinase (JNK) inhibitor II, and SB203580 were purchased from Calbiochem (San Diego, CA). Antibodies for Skp2, Cullin 1, p21, p27 (C-19), cyclin A, cyclin D, cyclin E, cdk2, and cdk4 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies for p27 (clone 57) came from BD Transduction Laboratories (Lexington, KY). Antibodies for Akt, p44/42 MAPK, p38 MAPK, JNK1, RB, phospho-mammalian target of rapamycin (mTOR) (Ser2448), phospho-p70 S6 kinase (Thr389), phospho-Akt (Ser-473), phospho-p44/p42 MAPK (Thr202/Tyr204), phosphor-p38 MAPK (Thr180/Tyr182), phosphor-JNK (Thr183/Tyr185), phospho-protein kinase C (PKC), and phospho-RB were purchased from Cell Signaling Technology (Beverly, MA). β-Actin antibody was from Abcam Inc. (Cambridge, MA). Antimouse and antirabbit antibodies conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology. All other reagents and chemicals were purchased from Sigma and were of analytical grade.

Cell culture
The human breast cancer cell lines used in this study were MDA-MB-453, AU565, BT483, BT474, HBL-100, and MCF-7. MCF-7 was cultured in DMEM supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT) and 1% penicillin-streptomycin, and other cell lines were cultured in DMEM/F-12. These cells were grown at 37 C in a humidified atmosphere of 5% CO2. For assays requiring growth arrest, MCF-7 cells were plated in 10-cm dishes and grown in DMEM-10% FCS for 2–3 d, at which time the medium was changed to phenol red-free DMEM with 0.1% FCS and 10 nM ICI 182,780. After 48 h of ICI 182780 pretreatment and without a change of medium, estradiol (100 nM) or vehicle was added directly to the medium. This protocol results in the arrest of greater than 85% of the cells in the G0/G1 phase, as described previously (23, 24, 25, 26, 27). Stock solutions of E2 and ICI 182,780 were prepared in ethanol and added to growth-arrested cultures as indicated in the text. Control cultures received equal amounts of ethanol or dimethyl sulfoxide (DMSO) as vehicle controls where appropriate.

Flow cytometry
Cells (1 x 106) were cultured in 10-cm petri dishes and incubated for various times. They were then harvested, washed with PBS, resuspended in 200 µl of PBS, and fixed in 800 µl of iced 100% ethanol at –20 C. After being left to stand overnight, cell pellets were collected by centrifugation, resuspended in 1 ml of hypotonic buffer (0.5% Triton X-100 in PBS and 0.5 µg/ml ribonuclease), and incubated at 37 C for 30 min. Then 1 ml of propidium iodide solution (50 µg/ml) was added, and the mixture was allowed to stand on ice for 30 min. Fluorescence emitted from the propidium iodide-DNA complex was quantitated after excitation of the fluorescent dye by FACScan cytometry (BD Biosciences, San Jose, CA).

Immunoblotting (IB) and Western blotting
Cells were treated with various agents as indicated in figure legends. After treatment, cells were placed on ice, washed with cold PBS, and lysed in lysis buffer [1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 25 mM NaF, 0.5 mM sodium orthovanadate, 1 mM dithiothreitol, 1 µg/ml pepstanin, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.1 mg/ml phenylmethylsulfonyl fluoride]. Cell lysates were centrifuged twice at 12,000 rpm for 30 min at 4 C. Protein content was determined against a standardized control, using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Each lane was loaded with 50 µg protein separated on SDS-PAGE and electrotransferred to polyvinylidene difluoride membrane (Immobilonp, Millipore, Bedford, MA). The membrane was preincubated in PBS containing 0.01% Tween 20, 1% BSA, and 0.2% NaN3 overnight at 4 C. It was then incubated with a different primary antibody, followed by secondary antirabbit/goat/mouse IgG conjugated with horseradish peroxidase. The immunoreactive bands were visualized with enhanced chemiluminescent reagents (Amersham, Piscataway, NJ).

Immunofluorescence assay
MCF-7cells were plated on coverslips placed in six-well plates. Experiments were performed 24 h after cell attachment. Cells were fixed in PBS containing 4% paraformaldehyde for 10–15 min at room temperature. Cells were rinsed with PBS two to three times and then blocked with 1% normal goat serum for 30 min. Incubations were performed with primary antibodies diluted in blocking buffer at 4 C overnight, after which coverslips were washed and incubated for 30 min with the fluorescein isothiocyanate-conjugated secondary antibodies diluted in blocking buffer. Coverslips were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA) and viewed under a Leica TCS SP2 confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany).

Immunoprecipitation
A 500-µg sample of MCF-7 total cellular protein was first precleared by being incubated with protein A agarose 20 µl (Amersham Pharmacia Biotech, Piscataway, NJ) for 30 min. The clarified supernatants were collected by microfugation at 12,000 rpm for 5 min and then incubated with primary antibody for 2 h at 4 C. The reaction mixtures were supplemented with 20 µl of protein A agarose to absorb the immunocomplexes at 4 C overnight. Beads were then washed thrice with lysis buffer. Proteins were eluted with 20 µl electrophoresis buffer and fractionated using SDS-PAGE. Western blots were also performed.

Transient transfections
Vectors (pMyc3 CMV14) expressing the human Skp2 (pMyc3 CMV14 hSkp2) and His-tagged ubiquitin (Ub) were kindly provided by Hungwen Chen (Academia Sinica, Nankang, Taipei, Taiwan). One day before transfection, cells were seeded in a six-well plate without antibiotics and with a density of 30–40%. For plasmid transfections, 2 µg of plasmid DNA were premixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in Opti-MEM (Invitrogen) and added to the wells for 6 h. Cells were incubated for an additional 48 h before being analyzed by Western blot as previously described.

RNA interference suppression of Skp2
The Skp2 p45 small interfering RNA (siRNA) gene silencer (human) double-stranded RNA was obtained from Santa Cruz Biotechnology (sc-36499). MCF-7 cells were transfected with small interfering RNAs using siRNA transfection reagent (Santa Cruz) and incubated for 6 h. Afterward the cells were analyzed by immunoblot for Skp2 expression. For suppression of cellular p27 expression, short hairpin interfering RNA (shRNAi) was designed to target specific sequences of human p27 (Academia Sinica, Taipei, Taiwan, Clone ID: TRCN0000009856; NM ID: NM_004064; target sequence: 5'-AGCAATGCGCAGGAATAAGG-3'). MCF-7cells were transfected with 100 nM p27 shRNAi or mismatched shRNAi using Lipofectamine 2000 (Invitrogen) and cellular expression analyzed 24 h later. Afterward the cells were analyzed by MTT assay for cell growth determination.

MTT assay
Cells were seeded at 2 x 104 cells/well in a 24-well plate for 24 h; treated with varying concentrations of EGCG, tamoxifen, or paclitaxel; and incubated for an additional 24 h. The effect of EGCG, tamoxifen, or paclitaxel on cell growth was examined by the MTT assay. Briefly, 20 µl of MTT solution (5 mg/ml; Sigma) was added to each well and incubated for 4 h at 37 C. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 200 µl of DMSO. Finally, the absorbance was monitored by a microplate reader at a wavelength of 550 nm.

RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) as recommended by the manufacturer’s instructions. Total RNA (5 µg) was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase and oligo (deoxythymidine) 18 primer by incubating the reaction mixture (25 µl) at 37 C for 90 min. Amplification of cDNA was performed by PCR in a final volume of 50 µl containing 2 µl of reverse transcription product, deoxynucleotide triphosphates (each at 200 µM), 1x reaction buffer, a 1 µM concentration of each primer (Skp2, forward, 5'-ACAGTGAGAACATCCCCCAG-3', reverse, 5'-GGTCCATAAATGATCGTGGG-3'; p27, forward, 5'-ATGTCAAACGTGCGAG TGT-CTAA, reverse, 5'-TTACGTTTGACGTCTTCTGAGG-3'; glyceraldehyde-3-phosphate dehydrogenase, forward, 5'-TGA AGGTCGGTGTGAACGGATTTGGC-3', reverse, 5'-CATGTAGGCCATGAGGTCCACCAC-3'), and 50 U/ml ProTaq DNA polymerase. After an initial denaturation for 5 min at 95 C, 30 cycles of amplification (95 C for 30 sec, 58 C for 1 min, and 72 C for 2 min) were performed, followed by 72 C for 10 min. A 5-µl sample of each PCR product was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

Statistical analysis
The results obtained were expressed as means ± SE. Each value is the mean of at least three separate experiments in each group. The significance of the difference (P value) was statistically analyzed by ANOVA followed by Dunnett’s multiple comparison test to assess the statistical significance (*, P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
EGCG induces cell cycle arrest in the G1 phase
To establish a sensitive experimental system for the study of specific estradiol-regulated events associated with cell cycle progression, cells were growth arrested with the pure estrogen antagonist ICI 182780 and then rescued by the addition of estradiol. A representative time course for changes in cell cycle phase distribution after this treatment strategy is shown in Fig. 1BGo. With ICI 182780 pretreatment for 48 h, cells accumulated in the G1 phase such that the phase distribution changed to 86.3, 5.5, and 6.8%, respectively. After estradiol treatment, the proportion of G1 phase cells decreased from 86.3 to a minimum of 45.7% at 24 h. This decrease was mirrored by a 6-fold increase in the proportion of cells in the S phase from 5.5 to 34.2%, indicating a synchronized population of cells progressing from the G1 to the S phase. No significant increase in S phase fraction was evident in cultures cotreated with estradiol and EGCG. These data suggest that EGCG-treated MCF-7 cells were arrested in the G1 phase of the cell cycle, indicating 80.1% of the cells were accumulated in the G1 phase at 24 h of the treatment. Cells that possess wild-type retinoblastoma protein (RB) require its inactivation by phosphorylation for progress through the G1 phase. We therefore resolved to determine whether cotreated estradiol and EGCG altered pRB phosphorylation in growth-arrested MCF-7 cells. Treatment with estradiol did demonstrably increase RB phosphorylation by 24 h after release from growth arrest. Furthermore, the hyperphosphorylation of Rb induced by estradiol was inhibited by coadministration of EGCG (Fig. 1CGo).

Effects of EGCG on expression levels of cell cycle regulatory proteins in estrogen-stimulated MCF-7 cells
To sort out the specific cell cycle regulatory proteins responsible for the induced cell cycle arrest after EGCG treatment, Western analysis using antibodies specific to cyclin A, cyclin E, cyclin D, Cdk2, Cdk4, p21, p27, Skp2, and Cullin1 was performed (Fig. 2Go). Cyclin D3 levels increased 6 h after estradiol rescue to reach maximum levels at 24–36 h. Cyclin E, cyclin A, and Cdk4 remained at control levels until 12 h after estradiol rescue and were further increased at 24–36 h. The increased expression of cyclin A and Cdk4 protein after estrogen treatment of MCF-7 cells has been reported previously (25). Several studies have shown that the increasing Skp2 expression thus correlates with the decline in p27 levels (23, 24, 25). This is evident in estrogen-treated MCF-7 cells and remains so throughout the S phase. In this study, we found that the level of Skp2 protein was shown to increase to its highest level at 24 h and decreased gradually up to 36 h after treatment with estradiol. Conversely, the protein level of p27 was found to continue to decrease in growth-arrested MCF-7 cells 24–36 h after treatment with estradiol. Our results suggest that the down-regulation of Skp2 was not always correlated with the up-regulation of p27 (Fig. 2BGo). Next, we resolved to determine whether cotreated estradiol and EGCG altered cell cycle-regulatory proteins in growth-arrested MCF-7 cells. As shown in Fig. 2CGo, Skp2 and cyclin D3 levels decreased in culture cotreated with estradiol and EGCG. p21, cyclin A, cyclin E, Cdk2, and Cdk4 protein levels remained relatively unchanged in culture cotreated with estradiol and EGCG. In contrast, the protein p27 was found to decrease in growth-arrested MCF-7 cells 24–36 h after treatment with estradiol but remain constant in cells cotreated with estradiol and EGCG. The EGCG effect on p27 level was clearly dose and time dependent. In addition, up-regulation of p27 was accompanied by a reduction in Skp2 after EGCG exposure, whereas no significant change in Cullin 1 protein level was detected. These data indicated that EGCG modulated the expression of cell cycle-regulatory proteins inducing G1 growth arrest.


Figure 2
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  		FIG. 2. Effects of EGCG on expression levels of cell cycle regulatory proteins in estrogen-stimulated MCF-7 cells. Growth-arrested MCF-7 cells were treated with Me2SO alone (CON), 100 nM estradiol (E2), 100 nM estradiol plus varying concentrations of EGCG for 24 h (A), or 100 nM estradiol plus 10 µM EGCG (E2+EGCG) for the indicated times (B and C). Total cell lysates were prepared, and Western blot analysis was performed. The experiment was performed thrice with each antibody, with each yielding similar results. β-Actin was used as an internal control for equivalent protein loading. The values below the figures represent the change in protein expression of the bands normalized to actin. D, Knockdown of p27 expression by p27 shRNAi attenuates EGCG-induced cell growth inhibition. MCF-7 cells were plated on a 24-well plate and transfected with p27 shRNAi or with the same volume of medium as a control. After transfection, cells were incubated in the medium in the presence of 10 µM EGCG or the same volume of medium containing DMSO as a control at 37 C for the indicated times. At the time point, cells were harvested, and cell viability was determined by MTT assay, and the number of viable cells after treatment is expressed as a percentage of the estrogen-stimulated MCF-7 cells. Data are the mean ± SE of three independent experiments (*, P < 0.05 by Student’s t test).

 
Previous studies have shown that cyclin D1 levels rise 2- to 4-fold above control 4–8 h after estradiol treatment of growth-arrested MCF-7 cells and fall to control levels by 24 h after treatment (24, 25, 26). In supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org, we found that cyclin D1 levels did not increase in cultures treated with estradiol alone at 24 h. In contrast, cyclin D1 levels decreased in cultures cotreated with estradiol and EGCG at 24 h. Therefore, the loss of Skp2 after EGCG cotreatment at 24 h would have been expected to also lead to a modest stabilization of cyclin D1. In contrast, EGCG cotreatment decreased the expression level of cyclin D1, suggesting that the EGCG-mediated degradation of Skp2 and decreasing cyclin D1 levels defines a distinct pathway. Furthermore, experiments would be required to determine this possibility.

Next, we used a p27 shRNAi approach for inhibition of EGCG-mediated p27 induction to determine the effect of p27 inhibition on EGCG-induced cell growth inhibition in estrogen-stimulated MCF-7 cells. As shown in Fig. 2DGo, cell growth assessment demonstrated that down-regulation of p27 expression by p27 shRNAi resulted in a significant reduction of EGCG-induced cell growth inhibition in estrogen-stimulated MCF-7 cells. The data suggest that the increase in p27 expression by EGCG contributes to cell growth inhibition in estrogen-stimulated MCF-7 cells.

Effects of EGCG on the mRNA level of p27 and Skp2 in estrogen-stimulated MCF-7 cells
To address the mechanism by which EGCG increased the protein level of p27 and decreased the protein level of Skp2, we first examined the mRNA level of p27 and Skp2. As shown in Fig. 3AGo, no significant change in Skp2 or p27 mRNA levels were observed in culture cotreated with estradiol and EGCG. These results indicated that EGCG-induced increase in p27 protein levels and decrease in Skp2 protein levels involves a posttranscriptional mechanism.


Figure 3
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  		FIG. 3. A, Effects of EGCG on Skp2 and p27 mRNA level in estrogen-stimulated MCF-7 cells. Growth-arrested MCF-7 cells were treated with DMSO alone (CON), 100 nM estradiol (E2), or 100 nM estradiol plus varying concentrations of EGCG for 24 h. Total RNA was isolated, and the mRNA expression was analyzed by RT-PCR as described in Materials and Methods. Skp2 and p27 mRNA levels were analyzed by RT-PCR and quantitated with Bio-Rad PDQuest Image software. The values below the figures represent change in mRNA expression of the bands normalized to glycerol-3-phosphate dehydrogenase (G3PDH). B, Measurement of Skp2 protein degradation after inhibition of protein synthesis by CHX. Growth arrest MCF-7 cells were pretreated with 10 µg/ml CHX for 2 h and subsequently coincubated with estradiol and EGCG as indicated. The expression of Skp2 was determined by Western blotting analysis. β-Actin was used as an internal control for equivalent protein loading. C, In vivo ubiquitination of hSkp2. Skp2 was immunoprecipitated (IP) from MCF-7 cells transfected with the His-tagged Ub expression plasmid pHis-Ub and/or pMyc3 CMV14 hSkp2 in the absence or presence of EGCG. Ubiquitylated Skp2 was visualized by IB with the anti-His antibody (top panel), Skp2 levels were monitored by Western blotting (bottom panel). D, In vivo effects of proteasome inhibitors on p27 protein stability. Estrogen-stimulated MCF-7 cells were incubated with 10 µM EGCG for 24 h in the presence or absence of the proteasome inhibitor LLnL. Total cell lysates corresponding to 50 µg of proteins were subjected to IB using p27 antibody. E, Effects of EGCG on p27 ubiquitination in vivo. Ubiquitinated forms of p27 were analyzed by immunoprecipitation of p27 from 1 mg of total cell lysate with the P27 (C-19) antibody and IB with the p27 (clone 57) antibody. Steady-state levels of p27 in the samples were monitored by IB. EGCG-treated cells showed impaired p27 ubiquitination. F, MCF-7 cells grown on coverslips were treated with medium alone (CON), 100 nM estradiol (E2), or 100 nM estradiol plus 10 µM EGCG (E2+EGCG) for 24 h. Cells were fixed with 4% paraformaldehyde and stained with p27 antibody, followed by fluorescein isothiocyanate-conjugated secondary antibody (red). Analysis of subcellular distribution was performed by confocal microscopy. The bar on the images represents 20 µm in length.

 
To determine whether Skp2 degradation is accelerated by EGCG, we treated growth-arrested MCF-7 cells with the translation inhibitor cycloheximide (CHX) and then measured the relative Skp2 level in these cells. As shown in Fig. 3BGo, this level decreased faster in cells treated with CHX-chased estradiol and EGCG than in cells treated with CHX plus estradiol only. This result suggests that a posttranslational mechanism contributes to EGCG-induced Skp2 depletion in the estrogen-stimulated MCF-7 cells. We therefore tested whether EGCG regulates Skp2 ubiquitylation in vivo. MCF-7 cells were transfected with plasmids expressing His-tagged ubiquitin and Skp2, and the presence of ubiquitylated forms of Skp2 was analyzed by Western blotting. As shown in Fig. 3CGo, the polyubiquitylated Skp2 intermediates accumulated heavily in EGCG-treated cells. This raised the possibility that Skp2 may also be a target of EGCG-mediated ubiquitylation.

To determine the mechanism underlying EGCG-induced p27 up-regulation, MCF-7 cells were treated with the proteasome inhibitor LLnL for up to 24 h. As shown in Fig. 3DGo, proteasome inhibitors induced an increase of p27 in the estrogen-stimulated MCF-7 cells. No relevant additive effect was observed in cells cotreated with EGCG and LLnL, suggesting that these compounds probably increase p27 protein levels by acting on a common target: the ubiquitin/proteasome degradation pathway. We therefore asked whether the EGCG-induced p27 up-regulation was related to the protein’s loss of ubiquitination. p27 was then immunoprecipitated from cells treated with DMSO, estradiol, and estradiol plus EGCG, and all were cultured with LLnL to increase the half-life of their ubiquitinated intermediates and analyzed by IB. Whereas proteasome inhibition caused a marked increase in slowly migrating p27 derivatives in treated estrogen-stimulated MCF-7 cells, EGCG-cotreated cells showed no detectable levels of ubiquitinated derivatives (Fig. 3EGo). The data suggest that EGCG induces the defect in p27 ubiquitination in the estrogen-stimulated MCF-7 cells. Recent reports suggested that p27-mediated G1 phase arrest and suppression of cell growth were associated with subcellular localization of p27 from the cytoplasm to nucleus (28). To determine whether EGCG-induced cell G1/S arrest could be involved in the alteration of p27 subcellular localization, we performed immunofluorescence staining experiments to observe the localization of p27 in MCF-7 cells. As shown in Fig. 3FGo, estradiol decreased the nuclear localization of p27. However, this mislocalization was counteracted by coadministration of EGCG, suggesting that EGCG-mediated growth inhibition involves increased p27 nuclear localization.

Overexpression of Skp2 inhibits the antiproliferative effect of EGCG
To substantiate the role of EGCG-dependent Skp2 down-regulation in mediating the antiproliferative effect of these compounds, we sought to verify whether the enforced expression of Skp2 was able to counteract EGCG-induced growth inhibition. As shown in Fig. 4AGo, MCF-7 cells infected with the pMyc3 CMV14 hsSkp2 vector showed a strong up-regulation of Skp2 protein levels, and this correlated with decreased p27 expression. We also examined the level of the p27 protein in MCF-7 transfected with Skp2-siRNA. Skp2-siRNA transfection induced the down-regulation of Skp2 protein (Fig. 4BGo). As expected, p27 protein had accumulated in Skp2-siRNA transfectant cells. Moreover, this protein was more stable than in control siRNA transfectant cells. We then treated MCF-7 overexpressing Skp2 with EGCG and determined the effect of this overexpression on p27 levels. Immunoblot analysis showed that p27 accumulation had been counteracted in this Skp2 overexpressed clone after EGCG treatment (Fig. 4CGo). As expected from the results of immunoblot analysis, flow cytometry identified an impaired G1 arrest in these Skp2 overexpressed cells (Fig. 4DGo). This finding demonstrated the Skp2 level to be an important regulator of the p27 up-regulation induced by EGCG in MCF-7 cells.


Figure 4
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  		FIG. 4. Adoptive expression of p45Skp2 in MCF-7 cells. A, MCF-7 cells were infected with the pMyc3 CMV14 or pMyc3 CMV14 hSkp2 vectors. After infection, proteins were extracted and the expression of p45Skp2 and p27 was analyzed by IB. B, MCF-7 cells were untransfected or transfected with Skp2-siRNA as described in Materials and Methods. Western blot analysis of Skp2 and p27 protein expression was performed 48 h after transfection. C, MCF-7-pMyc3 CMV14 and MCF-7-pMyc3 CMV14 hsSkp2 cells were incubated with EGCG (10–20 µM) or DMSO for 24 h. Total lysates were analyzed by IB for p45Skp2 and p27 expression. D, Cell-cycle profile of Skp2-overexpressed MCF-7 cells after EGCG treatment. Flow cytometric analysis displayed an impairment in the EGCG-induced G1 arrest through the abolished accumulation of p27 in the Skp2-overexpressed cells.

 
EGCG down-regulates phospho-Akt, phospho-mTOR, phospho-S6K, and phospho-JNK levels in estrogen-stimulated MCF-7 cells
Akt, ERK, JNK, p38, and PKC have been shown to be involved in Skp2 regulation in various cell types (23, 29). To examine whether the activities of Akt, MAPKs, and PKC are affected by EGCG, we analyzed the phosphorylation of Akt, MAPKs, and PKC in estrogen-stimulated MCF-7 cells after treatment with EGCG (5–20 µM) for 24 h. Immunoblot analysis with antiphospho-specific antibody was then performed. As shown in Fig. 5AGo, EGCG inhibited the phosphorylation of Akt, mTOR, and JNK. EGCG did not affect S6K, ERK, p38, or PKC phosphorylation. In further studies, we examined whether the down-regulation of Skp2 and up-regulation of p27 protein expression in estrogen-stimulated MCF-7 cells by EGCG was mediated through the modulation of Akt and JNK phosphorylation. As shown in Fig. 5BGo, treating the cells with the inhibitors of phosphatidylinositol 3-kinase (PI3K) (LY294002) significantly decreased Skp2 and increased p27 protein expression. Taken together, it suggests that EGCG decreased Skp2 and increased p27 protein expression by repressing the phosphorylation of Akt in estrogen-stimulated MCF-7 cells.


Figure 5
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  		FIG. 5. Effects of EGCG on the Akt, S6K, mTOR, PKC, and MAPKs phosphorylation in estrogen-stimulated MCF-7 cells. A, Estrogen-stimulated MCF-7 cells were treated with the JNK inhibitor SP600125 (SP; 20 µM) or p38 MAPK inhibitor SB203580 (SB; 20 µM); PI3K inhibitors LY294002 (LY; 10 µM) or MEK1 inhibitor PD98059 (PD; 20 µM); PKC inhibitor GF109203X (GF; 1 µM); or EGCG (5, 10, and 20 µM) at 37 C for 24 h. Levels of phosphorylated Akt, S6K, mTOR, ERK1/2, p38 MAPK, JNK1/2, and PKC were analyzed by IB with phopho-Akt (Ser-473), S6K (pThr389), phopho-mTOR (Ser-2448), phopho-p44/42 MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), and phopho-JNK (Thr183/Tyr185) antibodies. B, The protein levels of Skp2, p27, and β-actin were analyzed by IB with specific antibody.

 
Enhancing effects of EGCG with paclitaxel or tamoxifen
Tamoxifen and paclitaxel are both very effective agents used in the treatment of breast cancer (30, 31, 32). To extend the study of synergistic effects, we examined whether tamoxifen and paclitaxel agents could enhance anticancer activity by EGCG. As expected, both tamoxifen and paclitaxel significantly and synergistically enhanced growth inhibition of MCF-7 cells by EGCG. After 24 h of incubation with 5 µM EGCG alone, the number of viable cells was 98.3% of control, whereas the number with 200 nM tamoxifen alone was 90.8%. Cotreatment with 5 µM EGCG and 200 nM tamoxifen reduced the number to 16.8% (Fig. 6AGo). These findings indicate that EGCG and tamoxifen have a synergistic effect on the inhibition of MCF-7 cell growth. We also found that cotreatment with 5 µM EGCG and 50 nM paclitaxel enhanced this inhibition more strongly than either paclitaxel or EGCG alone. These mechanisms seem related to the down-regulation of Skp2 protein level (Fig. 6BGo). However, cotreatment with tamoxifen and EGCG or paclitaxel and EGCG produced no change in p27 protein expression, even though Skp2 expression and cell proliferation fell dramatically (Fig. 6Go B). Similar results were also observed in AU565 cells. AU565 cells that overexpress HER2 showed higher susceptibility to EGCG and paclitaxel than did BT483, BT474, MDAMB453, HBL-100, or MCF-7 cells, resulting in lower cell viability (supplemental Fig. S2A). Moreover, high-dose tamoxifen (100–400 nM) and paclitaxel (50–200 nM) significantly and synergistically enhanced growth inhibition of AU565 cells by EGCG, but low-dose tamoxifen and paclitaxel cotreatment did not (supplemental Fig. S2B). Likewise, the growth inhibition induced by the down-regulation of Skp2 was not due to increased p27 levels in AU565 cancer cells, suggesting that EGCG-dependent Skp2 down-regulation can influence on cell growth in several ways (supplemental Fig. S2C).


Figure 6
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  		FIG. 6. Effects of EGCG, tamoxifen, and paclitaxel on cell proliferation and Skp2 expression in MCF-7 cells. A, MCF-7 cells were cultured in DMEM supplemented with 10% FCS for 24 h. After 24 h culture, cells were synchronized by serum-free medium (without antiestrogen ICI 182,780) for 24 h, followed by addition of 2% FCS and different concentrations of EGCG, tamoxifen, or paclitaxel, as indicated. Cell growth inhibition was determined by MTT assays. The number of viable cells after treatment is expressed as a percentage of the vehicle-only control. Data are means of three independent experiments. Bars represent the SE. The combination of EGCG and tamoxifen or EGCG and paclitaxel was more effective than either agent alone. *, P < 0.05. B, MCF-7 cells were incubated with DMSO (Con), EGCG, tamoxifen, paclitaxel, EGCG plus tamoxifen, or EGCG plus paclitaxel at 37 C for 24 h. Immunoblotting was used to measure protein levels of Skp2, p27, Cullin 1, and β-actin. C, Growth-arrested MCF-7 cells were treated with Me2SO alone (CON), 100 nM estradiol (E2), 100 nM estradiol plus 5 µM EGCG, 100 nM estradiol plus 2.5 µM EGCG and 12.5–100 nM tamoxifen, or 100 nM estradiol plus 2.5 µM EGCG and 10–40 nM paclitaxel for 24 h. Total cell lysates were prepared, and Western blot analysis was performed. The experiment was performed thrice with each antibody, with each yielding similar results. β-Actin was used as an internal control for equivalent protein loading. D, Knockdown of p27 expression by p27 shRNAi attenuates cell growth inhibition induced by cotreatment with EGCG and either tamoxifen or paclitaxel. MCF-7 cells were plated on a 24-well plate and transfected with p27 shRNAi or the same volume of medium as a control. After transfection, cells were incubated in the medium in the presence of 100 nM estradiol, 100 nM estradiol plus 10 µM EGCG, 100 nM estradiol plus 100 nM tamoxifen, or 100 nM estradiol plus 200 nM paclitaxel plus 100 nM estradiol plus 10 µM EGCG and 100 nM tamoxifen, or 100 nM estradiol plus 10 µM EGCG and 200 nM paclitaxel or the same volume of medium containing DMSO as a control at 37 C for the indicated times. At the time point, cells were harvested, and cell viability was determined by MTT assay; the number of viable cells after treatment is expressed as a percentage of the estrogen-stimulated MCF-7 cells. Data are the mean ± SE of three independent experiments (*, P < 0.05 by Student’s t test).

 
Furthermore, we wondered whether regulation of both skp2 and p27 by EGCG was specific to estrogen-stimulated cell. In an effort to answer this, proliferating estrogen-stimulated MCF-7 cells were cotreated with tamoxifen and EGCG or paclitaxel and EGCG, and the protein levels of skp2 and p27 were examined by Western blot analysis. The results showed that cotreatment with tamoxifen and EGCG or paclitaxel and EGCG decreased Skp2 protein and induced the accumulation of p27 protein in estrogen-stimulated MCF-7 cells (Fig. 6CGo). These results indicated that EGCG had the capacity to overpower the negative effect of Skp2 and p27 expression in estrogen-stimulated MCF-7 cells.

Next, we used shRNAi knockdown of p27 to determine the effect of p27 inhibition on cell growth inhibition induced by cotreatment with EGCG and either tamoxifen or paclitaxel. As shown in Fig. 6Go D, cell growth assessment demonstrated that down-regulation of p27 expression by p27 shRNAi resulted in a significant reduction of cell growth inhibition induced by cotreatment with EGCG and either tamoxifen or paclitaxel. The data suggest that the increase in p27 expression by cotreatment with EGCG and either tamoxifen or paclitaxel contributes to cell growth inhibition in estrogen-stimulated MCF-7 cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Epidemiological studies have shown that the intake of certain vegetables, fruits, and tea in the daily diet provides effective cancer prevention (33, 34). These effects have been attributed to the flavonoids and related flavanols in these plants. Several previous studies have shown that green tea polyphenol-EGCG exerts anticancer effects on cancer cells (35, 36). However, the molecular mechanism is largely unknown. We found that treatment of EGCG increased protein levels of p27 to inhibit proliferation of human breast cancer cells. The results of the present study indicate that, in MCF-7, the ubiquitin-proteasome pathway is involved in regulating the turnover of p27 and that EGCG increases p27 stability by posttranslational mechanisms, resulting in decreased proteasome-dependent degradation of the protein. Thus, it is hypothesized that EGCG may affect the F-box protein Skp2, which controls the degradation of the Cdk inhibitor p27. EGCG may enhance the protein’s stability and increase its intracellular level. Indeed, in the present study, a clear increase in p27 expression was found in the EGCG-treated MCF-7 cells, in concert with a reciprocal decrease in Skp2 expression. We demonstrated here for the first time that Skp2 is required for EGCG-induced cell cycle arrest in estrogen-stimulated MCF-7 cells (Fig. 7Go).


Figure 7
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  		FIG. 7. A diagram illustrating EGCG action in regulating skp2, p27, and G1/S transition in MCF-7 breast cancer cells in response to estrogen-dependent signaling. Estrogen stimulate S phase progression in MCF-7 cells, accompanied by the up-regulation of Skp2, with concomitant decreases in the p27 protein. The ubiquitination of p27 during S-phase progression is mediated by SCF Skp2 E3 ubiquitin ligase that captures Thr187-phosphorylated (p) p27 in estrogen-stimulated MCF-7 cells. Estrogen can also up-regulate cyclin D1, enhance p27 sequestration by cyclin D/Cdk complexes, and an associated increase in cyclin E-cdk2 kinase activity. Cotreatment with estradiol (E2) and EGCG induces a novel pathway of ubiquitylation and degradation of the F-box protein Skp2, resulting in the accumulation of p27 and cell-cycle exit.

 
Our findings are consistent with previous research showing that Skp2-deficient mice exhibit cellular accumulation of p27 (11, 37). Another finding in our study strongly supported the concept that Skp2-overexpressed MCF-7 cell clones displayed resistance to EGCG, suppressing intracellular p27 accumulation (Fig. 4Go). It is noteworthy that our results illustrate that EGCG down-regulates Skp2 expression by posttranscriptional mechanisms involving an increased ubiquitination and degradation of the protein (Fig. 3Go).

The mechanism of the EGCG-mediated degradation of Skp2 in estrogen-stimulated MCF-7 cells was not fully elucidated in this study. Recent evidence has raised the possibility that degradation of Skp2 is mediated by anaphase-promoting complex/cyclosome and its activator cdh1 (38, 39). The issue of whether or not anaphase-promoting complex/cyclosome and its activator cdh1 is a direct target for EGCG needs further study.

The previous study has shown that inhibition of the mTOR pathway by rapamycin may significantly down-regulate Skp2 levels in breast cancer cells (40). In addition, constitutive activation of the PI3K/Akt pathway frequently occurs in breast cancer, and some of its oncogenic effects are mediated through the mTOR pathway. In this study, the phosphorylation of Akt and mTOR were suppressed by EGCG in estrogen-stimulated MCF-7 cells (Fig. 5AGo). Furthermore, the expression of Skp2 was also inhibited by the PI3K inhibitor LY294002 (Fig. 5BGo). Estrogen has been shown to regulate Skp2 expression through an Akt-dependent mechanism (23). Given that Akt can be either activated by PI3K or negatively regulated by phosphatase and tensin homolog deleted from chromosome 10 (13), investigations into the possible involvement of PI3K or phosphatase and tensin homolog deleted from chromosome 10 in the EGCG-induced down-regulation of Akt are currently in progress.

In the clinic, endocrine therapy is an important intervention for women with breast cancers that express estrogen receptor, and treatment with tamoxifen has enhanced patient survival (41). However, 5-yr tamoxifen use has become associated with a number of serious side effects. Therefore, alternative interventions such as herbal substances are needed to replace or to supplement current regimens. There are grounds for the belief that multiple drugs in breast cancer may be more effective than single agents. In accordance with this concept, the ideal regimen would contain at least two potent drugs (42). For instance, preclinical studies provide evidence that gefitinib in combination with tamoxifen results in an additive or synergistic effect (43). Furthermore, a recent report showed that the combination of green tea polyphenols and tamoxifen is more potent than either agent alone in suppressing breast cancer growth (44, 45, 46, 47). EGCG has also been observed to enhance the growth-inhibitory effects of paclitaxel in breast and prostate cancer cells (48, 49). Although cotreatment with EGCG and tamoxifen or EGCG and paclitaxel have produced interesting results, mechanisms of action have not been well identified.

In the present study, we found that tamoxifen and EGCG or paclitaxel and EGCG significantly and synergistically enhanced growth inhibition of MCF-7 and AU565 cells. However, MCF-7 and AU565 cells showed no elevated p27, although their expression of Skp2 plummeted and reduced cell proliferation, suggesting that other factors contribute to deregulation of p27 expression in these tumors (50, 51). Moreover, many studies have demonstrated that the down-regulation of Skp2 by siRNA caused growth arrest, growth inhibition, and apoptosis in glioblastoma and lung cancer cells (52, 53). Growth arrest induced by Skp2 down-regulation was largely due to the up-regulation of p27. However, unlike growth arrest, the apoptosis and growth inhibition induced by the down-regulation of Skp2 was not due to increased p27 levels in glioblastoma cancer cells. Skp2 has been reported as the specific ubiquitin ligase for not only p27 but also cyclin E, p21, p57, p130, E2F1, Forkhead box O (FOXO), Forkhead box O 3 (FOXO3), human Orc1 (hOrc1p), Cdk9, Cdt1, c-Myc, and B-Myb (54). Therefore, it is still important to understand that accumulation of other known Skp2 substrates may also contribute to the apoptosis and growth inhibition induction by down-regulation of Skp2. Another study showed that the down-regulation of Skp2 using siRNA increased p53-dependent apoptosis and growth inhibition pathways (55). The actual role of Skp2 in apoptosis and growth inhibition remains unclear, and further research is necessary to explore the possibilities. Here all our results point to EGCG as a suitable candidate for use in combination with cancer-preventive agents such as tamoxifen and paclitaxel to reduce their adverse effects.

It is now clear that several avenues for antitumor therapy converge at the G1/S transition. Recent work indicated that Skp2 is one of the important G1/S regulatory points because it is necessary for ubiquitin-dependent degradation of the Cdk inhibitor p27 and of the forkhead transcription factor FOXO-1 (56, 57, 58). Skp2, a newly found oncogene, can cooperate with H-RasG12V to malignantly transform primary fibroblasts in vivo (20). Similarly, Skp2 can cooperate with activated N-Ras in tumorigenesis in a mouse transgenic model, leading to significantly reduced survival times (59). In fact, Skp2 has even been suggested as a potential drug target, an idea that is significantly strengthened by the recent observation that Skp2 levels are associated with reduced survival in breast cancer (60, 61). Consequently, targeting Skp2 and realizing p27 accumulation may theoretically be a new cell cycle-based strategy for the treatment of advanced MCF-7 (62, 63). EGCG provided deep insights into this concept, opening the door to the creation of potent agents that pharmacologically target Skp2. Identifying and understanding the mechanism and molecular players that make this connection is an important direction for future investigation.


   Acknowledgments
 
The authors thank Professor Hungwen Chen (Academia Sinica, Nankang, Taipei, Taiwan) for generously providing expression plasmid CMV14 hSkp2 and His-tagged ubiquitin. We also thank Dr. Cheng Huang for their valuable discussions.


   Footnotes
 
This work was supported by the National Science Council Grants NSC 96-2311-B-002-020 and NSC96-2321-B-002-026.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 21, 2008

Abbreviations: Cdk, Cyclin-dependent kinase; CHX, cycloheximide; DMSO, dimethyl sulfoxide; EGCG, epigallocatechin-3-gallate; FCS, fetal calf serum; IB, immunoblotting; JNK, c-Jun N-terminal kinase; LLnL, N-acetyl-Leu-Leu-norleu-al; mTOR, mammalian target of rapamycin; MTT, methylthiazolyldiphenyl-tetrazolium bromide; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RB, retinoblastoma protein; S6K, phospho-p70 S6 kinase; SCF, Skp1-Cullin 1-F-box protein; shRNAi, short hairpin interfering RNA; siRNA, small interfering RNA; Skp2, S-phase kinase protein 2; Ub, ubiquitin.

Received March 24, 2008.

Accepted for publication August 8, 2008.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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