Am J Physiol Cell Physiol 288: C1094 –C1108, 2005.
First published January 12, 2005; doi:10.1152/ajpcell.00569.2004.
Antimitogenic effect of green tea (⫺)-epigallocatechin gallate on 3T3-L1
preadipocytes depends on the ERK and Cdk2 pathways
Pei-Fang Hung, Bo-Tsung Wu,* Hui-Chian Chen,* Yen-Hang Chen, Chia-Lin Chen,
Ming-Hua Wu, Hsien-Chun Liu, Meng-Jung Lee, and Yung-Hsi Kao
Department of Life Science, College of Science, National Central University, Chung-Li City, Taoyuan, Taiwan
Submitted 23 November 2004; accepted in final form 5 January 2005
Hung, Pei-Fang, Bo-Tsung Wu, Hui-Chian Chen, Yen-Hang
Chen, Chia-Lin Chen, Ming-Hua Wu, Hsien-Chun Liu, Meng-Jung
Lee, and Yung-Hsi Kao. Antimitogenic effect of green tea (⫺)-epigal-
locatechin gallate on 3T3-L1 preadipocytes depends on the ERK and
Cdk2 pathways. Am J Physiol Cell Physiol 288: C1094 –C1108, 2005.
First published January 12, 2005; doi:10.1152/ajpcell.00569.2004.—
Green tea catechins, especially (⫺)-epigallocatechin gallate (EGCG),
have been proposed as a chemopreventative for obesity, diabetes,
cancer, and cardiovascular diseases. However, relatively little is
known about the mechanism of the action of EGCG on fat cell
function. This study was designed to investigate the pathways of
EGCG’s modulation of the mitogenesis of 3T3-L1 preadipocytes.
Preadipocyte proliferation as indicated by an increased number of
cells and greater incorporation of bromodeoxyuridine (BrdU) was
inhibited by EGCG in dose-, time-, and growth phase-dependent
manners. Also, EGCG dose and time dependently decreased levels of
phospho-ERK1/2, Cdk2, and cyclin D1 proteins, reduced Cdk2 activ-
ity, and increased levels of G0/G1 growth arrest, p21waf/cip, and
p27kip1, but not p18ink, proteins and their associations to Cdk2.
However, neither MEK1, ERK1/2, p38 MAPK, phospho-p38, JNK,
nor phospho-JNK was changed. Increased phospho-ERK1/2 content
and Cdk2 activity, respectively, via the transfection of MEK1 and
Cdk2 cDNA into preadipocytes prevented EGCG from reducing cell
numbers. These data demonstrate the ERK- and Cdk2-dependent
antimitogenic effects of EGCG. Moreover, EGCG was more effective
than epicatechin, epicatechin gallate, and epigallocatechin in changing
the mitogenic signals. The signal of EGCG in reducing growth of
3T3-L1 preadipocytes differed from that of 3T3 fibroblasts. Results of
this study may relate to the mechanism by which EGCG modulates
body weight.
3T3-L1 preadipocyte; mitogen-activated protein kinase; cyclin-depen-
dent kinase
OBESITY is a common disease associated with risks of cancer,
diabetes, hypertension, and cardiovascular disease (21). Maybe
for this reason, estimates of the economic costs, prevalence,
morbidity, and mortality associated with more-modest degrees
of being overweight and obese are rising (2, 21, 44). The
development of obesity is characterized by increased number
of fat cells and their lipids due to the processes of so-called
mitogenesis and differentiation, which are regulated by ge-
netic, endocrine, metabolic, neurological, pharmacological, en-
vironmental, and nutritional factors (21). Accordingly, an un-
derstanding of the mechanism through which a particular
nutrient affects the mitogenesis of preadipocytes and their
differentiation to adipocytes would help prevent the initiation
* P.-F. Hung and B.-T. Wu contributed equally to this work.
Address for reprint requests and other correspondence: Y.-H. Kao, Dept. of
Life Science, College of Science, National Central Univ., Chung-Li City,
Taoyuan, Taiwan 32054 (E-mail: ykao@cc.ncu.edu.tw).
C1094 0363-6143/05 $8.00 Copyright © 2005 the American Physiological Society
and progression of obesity and its associated diseases in hu-
mans.
Green tea catechins (GTCs) are polyphenolic flavonoids
once called vitamin P (34). Since the discoveries that they have
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unique chemical structures (Fig. 1) and are major ingredients
of unfermented tea (24, 33), they have been found to possess
widespread biological functions and health benefits (1, 24, 25,
28, 45). In vivo, GTCs, especially (⫺)-epigallocatechin gallate
(EGCG; Fig. 1), lower the incidence of cancers (1, 24, 25, 28,
45), collagen-induced arthritis (16), oxidative stress-induced
neurodegenerative diseases (27), and streptozotocin-induced
diabetes (38). Also, EGCG can reduce body weight and body
fat (18). In support of this antiobese effect of EGCG, other in
vivo data have shown that EGCG or EGCG-containing green
tea extract reduces food uptake, lipid absorption, and blood
triglyceride, cholesterol, and leptin levels as well as stimulating
energy expenditure, fat oxidation, HDL levels, and fecal lipid
excretion (10, 18, 19, 24). These in vivo observations may be
explained by in vitro findings that EGCG and caffeine syner-
gistically with norepinephrine stimulate the thermogenesis of
brown adipose tissue (11), that EGCG regulates various en-
zymes related to lipid anabolism and catabolism, such as
acetyl-CoA carboxylase, fatty acid synthase, pancreatic lipase,
gastric lipase, and lipoxygenase (24, 48), that EGCG is a potent
prooxidant and antioxidant (24, 42, 43), and that EGCG re-
duced serum- or insulin-induced increases in cell numbers and
the triacylglycerol content during a 9-day period of differenti-
ation (19). These in vivo and in vitro observations suggest that
green tea EGCG appears to modulate the mitogenic, endocrine,
and metabolic functions of fat cells.
Despite the importance of EGCG, relatively little is known
about the mechanism of its action in regulating the mitogenesis
of preadipocytes and their differentiation to adipocytes. The
fact that the EGCG receptor, the so-called laminin receptor,
discovered in cancer cells (39), has not been identified in fat
cells and the fact that fat cells have different isoforms of
laminins (30) have also caused much controversy. Accord-
ingly, a thorough examination of the signal element through
which EGCG executes its modulation of preadipocyte mito-
genesis should help clarify these observations. MAPK and Cdk
are essential mitogenic signal transducers in most cells (4, 17,
31), including 3T3-L1 preadipocytes (5, 40), and they have
been proposed as being important signals of EGCG in modu-
lating the growth of cancer cells based on various studies (1,
22–28, 43, 46). Further studies are required to determine
whether any of them are responsible for EGCG signaling in
preadipocyte antimitogenesis.
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
http://www.ajpcell.org
 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
Fig. 1. Structures of four major green tea catechins. Differences among these
catechins occur in the number of hydroxyl groups, the presence of a galloyl
group, and the molecular weight.
The present study was designed to understand the mecha-
nism of how EGCG acts in reducing the number of 3T3-L1
preadipocytes as they grow. Our specific aim was to investigate
whether EGCG-regulated preadipocyte proliferation is depen-
dent on the ERK MAPK and Cdk2 pathways. The mechanistic
results of this study may have possible utility in the treatment
of obesity using this compound.
MATERIALS AND METHODS
Chemical reagents. Green tea EGCG and other catechins (⬎98%
pure) were isolated from green tea (Camellia sinensis) in our labora-
tory as described previously (18). Catechins were dissolved in 0.1%
DMSO and sterile medium for cell treatment. Other materials (i.e.,
PD98059) were purchased from Sigma (St. Louis, MO) unless other-
wise mentioned. Penicillin-streptomycin, DMEM, FBS, trypsin, aga-
rose, and a 1-kb plus DNA ladder marker were purchased from
GIBCO-BRL Life Technologies (New York, NY). The 3⬘-RACE
system, TRIzol, Taq polymerase, and a BenchMark prestained protein
ladder were purchased from Invitrogen Life Science Technologies
(Carlsbad, CA). Except for the phospho-histone H1 and cyclin D1
antibodies, which were obtained from Calbiochem and Merck (Darm-
stadt, Germany), all other antibodies (i.e., ERK-1, ERK-2, phospho-
ERKs, p21waf1/cip, p27Kip1, p18INK, donkey anti-rabbit IgG-horse-
radish peroxidase) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
Cell culture. According to a published method (19), 3T3 and
3T3-L1 cells (American Type Culture Collection; Manassas, VA)
were grown in DMEM (pH 7.4) containing 10% FBS, 100 U/ml of
penicillin, and 100 ␮g/ml streptomycin (GIBCO-BRL) in a humidi-
fied atmosphere of 95% air-5% CO2 at 37°C. Medium (10 ml) was
replaced every 2 days. Because serum components contain the factors
for facilitating 3T3-L1 differentiation from preadipocytes to adipo-
cytes when they are confluent, these cells were subcultured before
reaching confluency. Confluent 3T3 were subcultured.
Growth inhibition experiments. 3T3 and 3T3-L1 cells
(15,000⬃20,000 cells/cm2) were plated in triplicate wells of a 12-well
plate. To determine whether a dose- or growth phase-dependent effect
of GTCs on the growth of 3T3-L1 preadipocytes exists, we treated
different growth phases (days 1⬃6 with day 1 being the day of cell
inoculum) of cells with epicatechin (EC), epigallocatechin (EGC),
AJP-Cell Physiol • VOL 288 • MAY 2005 •
C1095
epicatechin gallate (ECG), or EGCG at various concentrations
(0⬃400 ␮M) for indicated time periods. After a particular time course
of incubation, cells were trypsinized and counted with a hemocytom-
eter using the 0.4% trypan blue exclusion method. To measure cellular
proliferation, we modified the method reported by Vollenweider et al.
(41) and used a commercially available bromodeoxyuridine (BrdU)
ELISA kit (Roche Applied Science; Mannheim, Germany) as follows.
3T3-L1 cells (2,000 cells/well) were plated into a 96-well microplate
(tissue culture grade) with 100 ␮l DMEM supplemented with 10%
FBS. After 24 h were allowed for attachment, cells were starved with
serum-free DMEM for 36 h, which was then replaced with fresh
DMEM containing 10% FBS, the thymidine analog BrdU (10 ␮M),
and each GTC (at the indicated concentrations) for 4 h at 37°C. This
allowed BrdU to be incorporated into newly synthesized DNA of
dividing cells during their S phase. After incubation, residual cells
were washed with 10 mM PBS and then collected by centrifugation at
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1,500 rpm for 5 min. Cell pellets were dried at 60°C for 1 h, fixed with
200 ␮l FixDenat solution/well for 30 min at 15⬃25°C, probed with
mouse anti-BrdU-POD for 1 h, and visualized with the addition of 100
␮l 3,3,5,5-tetramethylbenzidine substrate to each well for 5 min for
color development. An aliquot of 100 ␮l of 1 N H2SO4 was added to
stop the reaction of each well for the 1-min incubation on a 300-rpm
shaker. The absorbance was read at 450 nm using a MRX microtiter
plate reader (Dynatech Laboratories; Chantilly, VA). Culture medium
alone and cells incubated with anti-BrdU-POD in the absence of BrdU
were used as blank controls for nonspecific binding.
MAPK inhibitor treatment. 3T3-L1 preadipocytes, cultured in
DMEM containing 10% FBS for 2 days and synchronized with
serum-free DMEM for 1 day, were pretreated for 2 h with EGCG
(20⬃50 ␮M), PD98059 (50 ␮M) (12), and/or U0126 (10 ␮M) (13).
These compounds were dissolved in 100% DMSO (at a final concen-
tration of 0.1%) and then added to fresh DMEM containing 10% FBS
during the experiment. After 4 h of incubation, protein amounts of
ERK1/2 and phospho-ERK1/2 were measured by Western blot anal-
ysis while the number of cells was examined by the trypan blue
exclusion method after 48 h of incubation.
Cdk2 plasmid constructs. cDNA encoding wild-type murine Cdk2
(Cdk2⫹/⫹) and the dominant negative form of murine Cdk2 (dnCdk2;
with a mutation of Asp145 in Cdk2 to Asn145), as described by Heuvel
and Harlow (17), was amplified by RT-PCR. Total RNA was isolated
from 3T3-L1 preadipocytes with the TRIzol kit, and cDNA was then
synthesized from equal amounts of RNA using Moloney murine
leukemia virus reverse transcriptase (Invitrogen; Carlsbad, CA). The
forward and reverse primers for obtaining Cdk2⫹/⫹ cDNA were
5⬘-ATGGAGAACTTTCAAAAGGTG-3⬘ and 5⬘-TCAGAGCCGA-
AGGTGGGGGC-3⬘, respectively. To obtain dnCdk2 cDNA, four
primers were needed to introduce a site-specific mutation by overlap
exclusion (42), and they were 5⬘-ATGGAGAACTTTCAAAAGGTG-
3⬘, 5⬘-TCAGAGCCGAAGGTGGGGGC-3⬘, 5⬘-GTCCAAAGTTT-
GCCAGCTTG-3⬘, and 5⬘-CAAGCTGGCAAACTTTGGAC-3⬘. PCR
was performed under the following conditions: an initial denaturing
cycle at 94°C for 3 min, followed by 30 cycles of amplification
consisting of denaturation at 94°C for 45 s, annealing at 53°C for 30 s,
and extension at 72°C for 90 s. A final extension at 72°C for 10 min
was added after the last cycle. The PCR product was run on a 1.5%
(wt/vol) agarose gel using 40 mM Tris-acetate buffer (pH 8.0)
containing 1 mM EDTA and was visualized using 0.5 ␮g/ml ethidium
bromide. The Cdk2⫹/⫹ and dnCdk2 products (predicted to be about
900 bp) were cloned to the pTargetT vector (Promega) as described by
Sambrook and Russell (36). Before their transfection into the preadi-
pocytes, they were verified with nucleotide sequencing performed at
the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The
molecular weights of overexpressed Cdk2⫹/⫹ and dnCdk2 proteins
were verified to be about 34 kDa by Western blot analysis, and their
activities were also confirmed to catalyze the phosphorylation of the
histone H1 substrate (20).
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C1096 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
Cell transfection and overexpression experiments. We modified the
methods reported by Yang et al. (46) to perform our transfection
experiments. In some experiments, 3T3-L1 preadipocytes were tran-
siently (24 h) transfected with 15 ␮g of either the pcDNA3.1 plasmid,
pBabe puro vector, pcDNA3.1-MKK6EE (the constitutively active
MKK6 mutant designated MKK6EE, a gift from Dr. S. L. Chen),
pCMV-MEKK1 (a constitutively active MEKK1-activating JNK ki-
nase, a gift from Dr. S. L. Chen), pBabe puro-MEK1, or pBabe
puro-MEK1S217E/S221E (a constitutively active MEK1 mutant des-
ignated MEK1EE; a gift from Dr. J. J. Yang). The overexpressed
preadipocytes were cotransfected with pSV-␤-galactosidase cDNA
whose transfection efficiency was determined by analyzing the ␤-ga-
lactosidase activity. There were insignificant changes in ␤-galactosi-
dase production from all transfected cells (data not shown). Activities
of MEK1, MEKK1, and MKK6 proteins were assessed as measured
by Western blot analysis of changes in the amounts of phospho-
ERK1/2, phospho-JNK, and phospho-p38, respectively. In other ex-
periments, 3T3-L1 preadipocytes in a 10-cm plate were stably trans-
fected with 15 ␮g of the pTargetT vector constructed with Cdk2⫹/⫹
and dnCdk2 cDNAs. Stable clones were selected with 1 mg/ml of the
antibiotic G-418 (BD Biosci Clontech Lab; Palo Alto, CA). Amounts
and activities of Cdk2⫹/⫹ and dnCdk2 proteins expressed in the stable
clones were later determined by immunoblotting and immunoprecipi-
tation, respectively. A 45-␮l volume of the TransFast transfection
reagent was used during the stable and transient transfections and was
comprised of the synthetic cationic lipid (⫹)-N,N-bis(2-hydroxy-
ethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium io-
dide and the neutral lipid L-dioleoyl-phosphatidylethanolamine.
Cells transfected with the vehicle or the recombinant plasmid
containing MEK1, MEK1EE, or MKK6EE cDNAs were incubated
with or without 50 ␮M EGCG for 48 h. After incubation, the cell
number of preadipocytes was examined by trypan blue dye exclusion.
Unless otherwise noted, transfected cells with the vehicle, Cdk2⫹/⫹
cDNA, and dnCdk2 cDNA were incubated with and without 20⬃100
␮M EGCG for the indicated time periods.
Flow cytometric analysis. Changes in the kinetics of the cell cycle
were analyzed by flow cytometry as described by Kokontis et al. (20).
3T3-L1 cells (15,000⬃20,000 cells/cm2) transfected with and without
the vehicle, Cdk2⫹/⫹, and dnCdk2 plasmids were plated in a 10-cm
dish containing 10 ml DMEM supplemented with 10% FBS. One day
after inoculation, the medium was replaced with serum-free DMEM to
obtain the homogenous cell population. After a 1-day starvation, cells
were treated with fresh medium in the presence and absence of EC,
ECG, EGC, or EGCG at various concentrations and time periods. The
harvested cell pellets were fixed in 70% ethanol (dissolved in 10 mM
PBS, pH 7.4) and stored at ⫺20°C until later analysis. Cell pellets
were washed with 10 mM cold PBS (pH 7.4), incubated at 37°C for
30 min with 200 ␮g/ml RNase A (Sigma), and then stained with 4
␮g/ml propidium iodide (Sigma) in PBS containing 1% Triton X-100
(Sigma). Cell cycle profiles and distributions were determined by flow
cytometric analysis of 104 cells using the CELLQuest program on a
FACS Calibur flow cytometer (Becton-Dickinson; San Jose, CA).
Clumped cells were excluded from the cell cycle distribution analysis
by gating.
Immunoprecipitation. Cdk2 protein was immunoprecipitated ac-
cording to the method described by Kokontis et al. (20). After
experimental treatments, preadipocytes were washed twice in 10 mM
PBS and then lysed in 1 ml buffer A [20 mM Tris 䡠 HCl (pH 7.6), 1 mM
EDTA, 1 mM Na3VO4, 0.2% Triton X-100, and 1 mM PMSF]. The
lysate was agitated for 15 min at 4°C and then centrifuged at 14,000
rpm for 10 min to collect the supernatant. The protein content of the
supernatant was determined in duplicate by the dye-binding method
(6) using a Bio-Rad (Richmond, CA) microplate reader and BSA
(Sigma) as a standard. An aliquot of the supernatant (1 mg protein)
was preincubated for 1 h at 4°C with Cdk2 antibody or preimmunized
normal rabbit serum (NRS; as the control) for 1 h at room temperature
or overnight at 4°C. The mixture was incubated with 20 ␮l protein
AJP-Cell Physiol • VOL
A-agarose (Santa Cruz Biotechnology) overnight at 4°C. The total
amounts of Cdk2, p18, p21, and p27 in the immunoprecipitates were
measured by Western blot analysis with each antibody. After normal-
ization to the Cdk2 protein, the amounts of each cyclin-dependent
kinase inhibitor (CKI) protein were expressed as a percentage of the
control, and changes in their bindings to Cdk2 were indicated. Data
obtained from NRS were not shown because of the insignificant
changes.
Western blot analysis. Western immunoblot analysis was per-
formed on supernatant fractions of preadipocytes as described by
Kokontis et al. (20). An aliquot of 50 ␮g of supernatant protein was
separated by 12% SDS-PAGE with 2⫻ gel-loading buffer [100 mM
Tris 䡠 HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue,
and 10% ␤-mercaptoethanol] and then blotted onto Immobilon-NC
transfer membranes (Millipore; Bedford, MA). The immunoblots
were blocked for 1 h at room temperature with 10 mM PBS containing
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0.1% Tween 20 (PBST) and 5% defatted milk. After the immunoblots
were washed with PBST, immunoblot analyses were performed. All
primary antibodies (ERK-1, ERK-2, phospho-ERKs, MEK1, p38,
phospho-p38, JNK, phospho-JNK, ␤-actin, Cdc2, Cdk2, p18, p21,
p27, cyclin D1, and phospho-histone H1 antisera) were used at a
dilution of 1:1,000 (⬃0.2 ␮g/ml). Donkey anti-rabbit IgG, donkey
anti-mouse IgG, donkey anti-goat IgG, or goat anti-guinea pig IgG
conjugated with horseradish peroxidase was used as the secondary
antibodies at a dilution of 1:2,000 (⬃0.2 ␮g/ml). The immunoblots
were visualized using the Western Lightning chemiluminescence
reagent plus kit (Perkin-Elmer Life Science; Boston, MA) for 3 min
followed by exposure to Fuji film for 2⬃3 min. Blots were quantified
using the FX Pro Plus Molecular Imager (Bio-Rad Laboratories).
After normalization to ␤-actin protein, levels of these intracellular
proteins were expressed as a percentage of the control unless other-
wise noted.
Cdk2 activity assay. After immunoprecipitation, Cdk2 activity was
determined as modified from the method of Kokontis et al. (20).
Assays were performed at 37°C for 30 min in a final volume of 25 ␮l.
The final substrate mixture per tube contained 20 mM ATP, 10 ␮g
histone H1, 20 mM Tris 䡠 HCl buffer (pH 7.5), 4 mM MgCl2, 0.8 mM
EGTA, and Cdk2 immunoprecipitates. The reaction was terminated
by the addition of 50 ␮l SDS-PAGE sample buffer as described
above. We removed a 20-␮l aliquot of the solution to load onto
SDS-PAGE and used anti-phospho-histone H1 as the primary anti-
body to immunoblot the samples as described above. Changes in the
amounts of phospho-histone H1, after normalization to the immuno-
precipitated Cdk2 protein, indicated alterations in Cdk2 activity.
Statistical analysis. Data are expressed as means ⫾ SE unless
otherwise noted. An unpaired Student’s t-test was used to examine
differences between the control and EGCG-treated groups. One-way
ANOVA followed by the Student-Newman-Keuls multiple-range test
was used to examine differences among multiple groups. Differences
were considered significant at P ⬍ 0.05. All statistical analyses were
performed using SigmaStat (Jandel Scientific; Palo Alto, CA).
RESULTS
Mitogenesis of 3T3-L1 preadipocytes was inhibited by
GTCs. There were significant variations in the measured cell
number when the distinct phases of 3T3-L1 preadipocytes were
incubated with different concentrations of EC, ECG, EGC, or
EGCG (Fig. 2). In general, EGCG was more effective in
reducing the cell number than EC, ECG, or EGC. Latent (days
1–2), log-phase (days 3– 4), and confluent (days 5– 6) preadi-
pocytes had different sensitivities to individual GTCs depend-
ing on the dosage and duration of treatment (Fig. 2, A–I). For
example, the IC50 values of EC, EGC, and ECG in day 3
preadipocytes were all ⬎50 ␮M during the 72 h of treatment
except for 10 –20 ␮M EGCG at 24⬃72 h (Fig. 2, D–F). In day
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Fig. 2. Reductive effect of green tea catechins on the cell number of 3T3-L1 preadipocytes depending on the dosage (0⬃400 ␮M) and duration of treatment
and on the growth phases of cells. The different growth phases of cells included day 1 (A–C; the day when cells were plated), day 3 (D–F), and day 5 (G–I)
cells. The cell number was counted using the trypan blue exclusion method. F, EC;
, EGC; E, ECG; ƒ, EGCG. Data are expressed as means ⫾ SE from triplicate
determinations. For clarity, SE bars and statistical significances are not shown.

2, day 4, and day 6 preadipocytes, the IC50 values of EC, EGC,
and ECG at 48 h were all ⬎200 ␮M except for 50 –300 ␮M
EGCG (data not shown).
The determination of whether the reduction in cell number
induced by GTCs was due to their influences on the cell
viability of 3T3-L1 preadipocytes was examined by the trypan
blue exclusion method. High doses of 100⬃400 ␮M EGCG
decreased the cell viability of day 3 preadipocytes by 15⬃30%.
However, at concentrations of green tea EC, ECG, EGC, and
EGCG below 50 ␮M, the cell viability of these log-phase cells
still remained at 90⬃100% (Fig. 3A). Similar effects of these
catechins on the latent and confluent preadipocytes were ob-
served (data not shown). A further BrdU incorporation test on
AJP-Cell Physiol • VOL
log-phase preadipocytes showed that EGCG inhibited cell
proliferation in a dose-dependent manner (Fig. 3B). The IC50
of EGCG was about 50 ␮M. At this concentration, EGCG was
more effective than EC, ECG, and EGC at inhibiting preadi-
pocyte proliferation (Fig. 3B). Moreover, flow cytometric anal-
ysis indicated that EGCG, but not EC, ECG, or EGC, changed
the percentages in the four different phases of the cell cycle of
preadipocytes (Fig. 3C). EGCG arrested preadipocytes in the
G0/G1 phase of the cell cycle.
Antimitogenic effect of EGCG depends on ERK pathways.
To elucidate the signaling through which EGCG acts on the
mitogenesis of 3T3-L1 preadipocytes, we examined whether
the effect of EGCG was dependent on the ERK and MEK1
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Fig. 3. Effects of green tea catechins on cell viability (A), cell
proliferation (B), and different phases of the cell cycle (C) in
the log-phase (day 3) of 3T3-L1 preadipocytes. A: dose-depen-
dent effect of green tea catechins on cell viability, as examined
by the trypan blue exclusion method, was observed 48 h after
treatment. B: effect of green tea catechins on cell proliferation,
as measured by bromodeoxyuridine (BrdU) incorporation, was
observed after 48 h of treatment. *Significant difference (P ⬍
0.05) from the control. a– cGroups with different letters are
significantly different (P ⬍ 0.05) from each other. C: effect of
green tea catechins on four different phases of the cell cycle, as
measured by flow cytometry, was observed. Data are expressed
as means ⫾ SE of triplicate determinations. For clarity, SE bars
and statistical significances are not shown in A and C.

pathways. In log-phase preadipocytes, total protein levels of
neither ERK1/2 nor MEK1 were significantly changed by 50
␮M EGCG over the 24-h course (Fig. 4A). However, the
amounts of phospho-ERK-1 and phospho-ERK-2 were signif-
icantly reduced by EGCG. The effect of EGCG was time
dependent (Fig. 4A) and dose dependent (Fig. 4B); for a given
4-h treatment, the IC50 of EGCG was about 50 ␮M (Fig. 4B).
In contrast, EGCG increased the levels of phospho-ERKs in
latent preadipocytes (day 1), whereas it had no effect on their
amounts in confluent (day 5) preadipocytes (Fig. 4C).
To determine whether EGCG’s effect in reducing MEK1
activity is selective, changes in the activities of MEK1,
MKK3/6, and MKK4/7 were assessed by changing the
amounts of the phosphorylated form of their own protein
substrates, ERK-1 and ERK-2, p38 MAPK, and JNK MAPK
(Fig. 5A), respectively, after EGCG treatment. EGCG at 50
␮M for 4 h did not significantly alter the amounts of ERK1/2,
p38, phospho-p38, JNK, or phospho-JNK but did significantly
decrease the amounts of phospho-ERKs by about 60%.
AJP-Cell Physiol • VOL
We further enhanced MEK1 activity by overexpressing
MEK1 or its constitutively active mutant, MEK1EE, in prea-
dipocytes to demonstrate whether overexpression of such ki-
nases could prevent EGCG-induced decreases in cell numbers
and MEK1 activity (Fig. 5). We observed that preadipocytes
overexpressed with either MEK1 or MEK1EE revealed no
significant changes in their cell number (Fig. 5B) between the
presence and absence of 50 ␮M EGCG for 48 h (Fig. 5B). In
contrast, the numbers of preadipocytes that were transfected
with the empty vector, MKK6EE (Fig. 5B), or MEKK1 (data
not shown) were still significantly reduced by EGCG. In-
creased expression and activity of MEK1 protein in MEK1- or
MEK1EE-transfected preadipocytes are confirmed in Fig. 5C.
Differences of EGCG with other specific MEK1 inhibitors.
On the basis of IC50 values of EGCG (20⬃50 ␮M), PD98059
(50 ␮M), and U0126 (10 ␮M) for inhibiting preadipocyte
proliferation or reducing MEK1 activity, we tested whether the
inhibitory effects of EGCG on preadipocyte growth and MEK1
activity differed from those of other specific inhibitors of the
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Fig. 4. Effects of EGCG on protein amounts of ERK-1, ERK-2, phospho-ERK-1, phospho-ERK-2, and MEK1 in 3T3-L1 preadipocytes (day 3). A:
time-dependent effect of EGCG at 50 ␮M was observed. B: dose-dependent effect of EGCG was observed after 4 h of treatment. C: EGCG at 50 ␮M altered
MEK1 activity, as indicated by decreased phospho-ERKs, which was dependent on the growth phases of cells after 4 h of treatment. Day 1, the day when cells
were plated; latent, day 3; log-phase, day 5 (confluent); ERK1/2, ERK-1 ⫹ ERK-2; pERK1/2 or pERKs, phospho-ERK-1 ⫹ phospho-ERK-2. These kinases were
measured by Western blot analysis and then expressed after normalization to actin. Data are expressed as means ⫾ SE from triplicate determinations. In some
data, SE bars are too small to be seen. *P ⬍ 0.05 vs. the control.

MEK1 protein (Fig. 6). Proliferation of preadipocytes was
inhibited by either EGCG, PD98059, or U0126 alone. In
addition, their growth was inhibited by a combination of
EGCG with either PD98059 or U0126 (Fig. 6A). Total amounts
of neither of the proteins, ERK-1 or ERK-2, changed after
treatment with each inhibitor (Fig. 6B). Interestingly, EGCG
tended to be additive with either PD98059 or U0126 in de-
creasing MEK1 activity, as indicated by the reduced amounts
of phospho-ERK1/2 proteins.
Effect of EGCG on the insulin-like growth factor-induced
increases in the activity of MEK1. Insulin-like growth factors
(IGFs) have been implicated in the proliferation of preadipo-
cytes through an increased activity of MEK1. The possibility
that the EGCG-induced reduction in MEK1 activity is related
to its decreasing the IGF-induced stimulation in MEK1 activity
was also examined (Fig. 7). EGCG dose dependently reduced
MEK1 activity, as indicated by decreased phospho-ERK1/2
proteins, stimulated by either IGF-I (1 nM; Fig. 7A) or IGF-II
(10 nM; Fig. 7B).
Differences in MEK1 activity by EGCG compared with other
GTCs. When 50 ␮M of the four GTCs of EC, EGC, ECG, and
EGCG were individually added to preadipocytes for 4 h,
AJP-Cell Physiol • VOL
EGCG was the most effective in reducing the amounts of
phospho-ERK1/2 proteins (Fig. 8). The total amounts of
ERK-1, ERK-2, and MEK1 proteins did not change after
treatment with each catechin.
Effects of EGCG on preadipocyte mitogenesis and MEK1
activity varied with cell types. At the doses of 20 and 100 ␮M,
EGCG significantly reduced the numbers of 3T3-L1 and 3T3
cells (Fig. 9A) to a greater extent than it did human KB oral
cancer cells (data not shown). A change in the protein expres-
sions of ERK-1 and ERK-2 by EGCG was not observed in any
of the three types of cells (Fig. 9B). However, protein amounts
of phospho-ERK1/2 in both 3T3 and 3T3-L1 cells were dose-
dependently reduced by EGCG.
Effect of EGCG on Cdk of preadipocytes differed from those
of other GTCs and from other cell types. Cdk2 is well known
as a downstream protein regulating cell mitogenesis via con-
trolling the procession of the four different phases of the cell
cycle, thereby resulting in changes in cell numbers. Accord-
ingly, we investigated whether EGCG alters the protein ex-
pression and activity of Cdk2 (Fig. 10). Treatment with EGCG
for 48 h reduced the level (Fig. 10A) and activity (Fig. 10B) of
Cdk2 protein in a dose-dependent manner. Although EGCG
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C1100 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH

Fig. 5. Antimitogenic effect of EGCG on prea-

dipocytes was dependent on MEK1 pathways.
A: EGCG at 50 ␮M for 4 h reduced protein
amounts of phospho-ERK-1/2 but did not alter
protein amounts of p38, phospho-p38 (pp38),
JNK, or phospho-JNK (pJNK) in 3T3-L1 prea-
dipocytes (day 3). B: transient overexpression of
MEK1 and its constitutively active MEK1S217E/
S221E (MEK1EE) mutant, but neither MKK6EE
nor the vehicles (pBabe and pcDNA3.1), pre-
vented the reduced numbers of 3T3-L1 preadi-
pocytes by 50 ␮M EGCG for 48 h. C: trans-
fected preadipocytes with the vehicle, MEK1,

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and MEK1EE in the presence and absence of
EGCG (50 ␮M) were confirmed by changes in
the protein expression and activity of MEK1, as
assessed by changes in the amounts of pERK
proteins. These kinases were measured by West-
ern blot analysis and then expressed after nor-
malization to actin. Data are expressed as
means ⫾ SE from triplicate determinations. In
some data, SE bars are too small to be seen.
*P ⬍ 0.05 vs. the control.

did not alter Cdk2 protein expression at 4 and 24 h of incuba-
tion, it was found to dose dependently decrease the activity of
Cdk2. In contrast to EGCG, neither EC, EGC, nor ECG (data
not shown) significantly altered Cdk2 protein levels after 48 h
of treatment. This indicates the existence of catechin-specific
effects of green tea.
In a further examination of whether the long-term (48 h)
effects of EGCG on Cdks vary with cell type, we assessed
changes in the protein expression of Cdk2 and Cdc2 (also
called Cdk1) by 3T3-L1 and 3T3 cells (Fig. 10C). At a low
dose of 20 ␮M, EGCG induced significant decreases in the
protein expression of Cdk2, but not Cdc2, by 3T3-L1 preadi-
pocytes. In contrast, EGCG tended to increase Cdk2 protein
expression in 3T3 cells (Fig. 10C) but decreased Cdc2 protein
expression in 3T3 cells. At a high dose of 100 ␮M, EGCG
reduced protein levels of Cdk2 and Cdc2 in 3T3-L1 cells,
whereas in 3T3 cells, it enhanced Cdk2 protein expression and
reduced Cdc2 protein expression.
Overexpression of Cdk2⫹/⫹ with EGCG-inhibited growth of
preadipocytes. In a further demonstration of whether Cdk2
protein is required for the effect of EGCG on 3T3-L1 preadi-
pocytes, we stably cloned preadipocytes that overexpressed
Cdk2⫹/⫹ and examined whether overexpression of Cdk2⫹/⫹
could prevent EGCG-induced growth inhibition of preadipo-
cytes after treatment with 20⬃100 ␮M EGCG for 5 days (Fig.
11). As indicated by the increased cell number, Cdk2⫹/⫹-
transfected preadipocytes grew more rapidly than vehicle-
transfected cells during the 5-day incubation. The growth of
the former cells, but not the latter cells, was not changed by
EGCG (Fig. 11A). However, overexpression of dnCdk2 with a
mutation of Asp145 in Cdk2 to Asn145 slowed down growth of
preadipocytes after a 5-day incubation no matter the presence
and absence of EGCG (data not shown). When examined at 24
AJP-Cell Physiol • VOL
and 48 h of EGCG treatment, EGCG’s alteration of the four
different phases of the cell cycle (Fig. 11B), EGCG’s reduction
of levels (Fig. 11C) and activity (Fig. 11D) of Cdk2 protein and
the EGCG-altered binding of Cdk2 to p18, p21, and p27 (Fig.
11E) were still observed in vehicle-transfected preadipocytes
but not in Cdk2⫹/⫹-transfected preadipocytes.
Effects of EGCG on CKIs. Because Cdk2 activity is nega-
tively regulated by CKIs, we investigated whether EGCG
changes the protein expressions of CKIs, such as p21waf/cip,
p27Kip1, and p18INK, in 3T3-L1 preadipocytes as well as the
binding of Cdk2 to these proteins (Fig. 12). Following 4 h of
EGCG incubation, the protein levels of p21 or p27 were not
significantly altered, even though up to 100 ␮M EGCG was
used (Fig. 12A). However, this catechin significantly induced
increases in the protein levels of p21 and p27, but not p18, after
24 and 48 h of treatment. Although EGCG did not alter the
protein expression of p21 and p27 at 4 h, it dose dependently
increased their bindings to Cdk2 (Fig. 12B). EGCG also
increased the binding of p21 and p27, but not p18, to Cdk2 at
24 and 48 h after treatment. With 24- and 48-h incubations,
neither EC, ECG, nor EGC significantly changed the protein
expressions of p18, p21, or p27 (data not shown). There was a
trend of EGCG increasing protein expression of p27 in 3T3
fibroblast and 3T3-L1 preadipocytes after 48 h of treatment
(Fig. 12C).
Effect of GTCs on cyclin D1 protein expression. It is well
known that cyclin D protein is one of the G1 cyclins that
controls the G1/S transition of the cell cycle. Accordingly,
whether EGCG changes the protein expression of cyclin D1
was assessed after cells were treated with EGCG for 4⬃48 h
(Fig. 13). We observed that EGCG reduced the total amount of
cyclin D1 protein in a dose-dependent manner after 24 (Fig.
13B) and 48 h (Fig. 13C) but not after 4 h (Fig. 13A) of
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 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
Fig. 6. Differences in EGCG from specific inhibitors of MEK1, PD98059 (PD;
50 ␮M) and U0126 (10 ␮M), in reducing the cell number (A; 20⬃50 ␮M) and
protein amounts (B; 50 ␮M EGCG) of phospho-ERK-1/2, but not ERK1/2, in
3T3-L1 preadipocytes (day 3). After 48 h of treatment, cells were examined by
the trypan blue exclusion method. After 4 h of treatment, the amounts of
kinases were measured by Western blot analysis and then expressed after
normalization to actin. Data are expressed as means ⫾ SE from triplicate
determinations. In some data, SE bars are too small to be seen. a– dGroups with
different letters are significantly different (P ⬍ 0.05) from each other.
treatment. Neither EC, ECG, nor EGC at 20⬃100 ␮M signif-
icantly reduced cyclin D1 protein expression after 24 h of
treatment. However, at a high dose of 100 ␮M, ECG signifi-
cantly reduced cyclin D1 protein levels after 48 h of treatment.
DISCUSSION
Green tea EGCG has been proposed as an obesity chemo-
preventative and a fat cell modulator based on various labora-
tory studies (24). Unlike a preliminary report (19), this study
not only details the reductive effect of EGCG on the number of
preadipocytes with dose-, time-, and growth phase-dependent
manners but also provides certain in-depth understanding of
the mechanism of EGCG’s action in the regulation of mito-
genesis of preadipocytes.
The observed decrease in the number of preadipocytes by
EGCG could be attributable to its inhibition of cell mitogene-
sis. This is supported by decreased BrdU incorporation, a
measure of DNA replication, and by increased G0/G1 growth
arrest of the cell cycle when preadipocytes were incubated with
EGCG. However, the observed decreases in the cell number by
high doses (100⬃400 ␮M) of EGCG could also be explained
by its induction of cell apoptosis. This is evident by the fact
that such high doses of EGCG reduced the cell viability of
preadipocytes by 15⬃30%, induced the appearance of DNA
AJP-Cell Physiol • VOL
C1101
fragmentation (data not shown), and increased the activity of
the caspase-3 protein (data not shown), an apoptotic enzyme.
Taken together, green tea EGCG may act at different concen-
trations in regulating mitogenesis and apoptosis of 3T3-L1
preadipocytes. This contention is similar to the reported dose-
dependent effect of EGCG on neuroblastoma cells (22, 26 –27,
42). However, the possibility still remains that EGCG acts at
10⬃400 ␮M to induce antimitogenesis and apoptosis of
3T3-L1 preadipocytes via oxidative stress as reported for
hepatoma (42) and neuroblastoma cells (22, 26, 43).
Antimitogenic effect of EGCG on preadipocytes depends on
ERK pathway. To our knowledge, the MAPK family is an
essential part of the signal transduction machinery in signal
transmissions from cell surface receptors and environmental
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stimulation, and it contains three major MAPK subfamilies:
ERK, p38, and JNK (4, 31). They have been proposed to serve
as signal elements in several types of cells through which
EGCG may regulate cell growth (1, 3, 8, 24 –25, 35, 45) and
found to modulate the mitogenic and adipogenic signalings of
IGF-I in 3T3-L1 preadipocytes (5). We observed herein that
acute (4 h) exposure to EGCG induced a decrease in phosphor-
ylated ERK1/2 in 3T3-L1 preadipocytes but did not alter the
total levels of MEK1, ERK-1, ERK-2, p38, phospho-p38, JNK,
or phospho-JNK. This suggests that EGCG acts on a specific
type of MAPK, especially in the ERK MAPK family. This
contention is also partially supported by the fact that chronic
(24 or 48 h) exposure to EGCG induced a decrease in the
phosphorylated ERK1/2 of preadipocytes, although it did not
alter total levels of MEK1 or ERK1/2 proteins. In addition,
transient amplification of phospho-ERK1/2 content by trans-
fecting MEK1 cDNA or its active mutant cDNA to 3T3-L1
preadipocytes prevented EGCG-induced decreases in their cell
number. The total levels of MEK1 protein in vehicle-, MEK1-,
or MEK1EE-transfected preadipocytes were not affected by
any of the EGCG treatments. In contrast, overexpression of
either MKK6EE, a constitutively active mutant of MKK6 to
activate p38 MAPK kinase, or MEKK1 (data not shown), a
MEKK1 construct favoring the activation of JNK MAPK
kinase, did not prevent EGCG-induced decreases in the num-
ber of preadipocytes. Taken together, these findings demon-
strate that a suppressive effect of EGCG on preadipocyte
proliferation is likely mediated via ERK MAPK-dependent and
p38 MAPK- and JNK MAPK-independent pathways and con-
firms that the ERK MAPK subfamily is important in preadi-
pocyte proliferation, as reported by Boney et al. (5).
The ERK-dependent effect of EGCG observed in 3T3-L1
preadipocytes is also strengthened by our findings that two
specific inhibitors of Erk MAPK, PD98059 (12) and U0126
(13), alone inhibit cell growth and MEK1 activity, as shown
with reduced phospho-ERK1/2 (5, 40), and that either of them
used to treat preadipocytes within the IC50 range speeds up
EGCG-induced reduction in the amounts of phosphorylated
ERK1/2 and, to a lesser extent, in the number of cells. It
appears that EGCG works differently from PD98059 and
U0126 in reducing levels of phosphorylated ERK1/2 proteins.
Whereas PD98059 prevents MEK1 activation by Raf (12),
U0126 directly protects ERK from being phosphorylated by
MEK1 (13). In cell-free systems, the inhibition of EGCG on
MAPK activity is competitive with the myelin basic protein
substrate and is noncompetitive with ATP (47). In contrast, the
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C1102 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH

Fig. 7. Effect of EGCG on insulin-like growth factor (IGF)-I-

and IGF-II-induced stimulation of the amounts of phospho-
ERK1/2 proteins. Twelve-hour-starved day 3 preadipocytes
were pretreated with EGCG (0 –100 mM) for 2 h and then
stimulated with either IGF-I (1 nM; A) or IGF-II (10 nM; B).
After 1 h of treatment, the amounts of phospho-ERK1/2,
ERK1/2, and actin proteins were analyzed by Western blot
analysis and then expressed after normalization to actin. Data
are expressed as means ⫾ SE from triplicate determinations.
For some data, SE bars are too small to be seen. *P ⬍ 0.05 vs.
the control (the group treated with IGF in the absence of
EGCG).

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activities of certain protein phosphatases are stimulated by phosphorylated ERK1/2 from preadipocytes via reducing the
15% by 10⬃50 ␮M EGCG (47). In cultured cells, phosphor- phosphorylation of MEK1 and the association of Raf with
ylation of ERK1/2 can be regulated by a variety of factors, MEK1 as reported for H-Ras-transformed cells (9). However,
including growth factors, G protein-coupled receptors, tyrosine confirming this requires more thorough studies.
kinase receptors, and Raf and MEK1 kinases (4, 31). We
measured the amounts of phospho-ERK1/2 protein after
3T3-L1 preadipocytes were treated with either 1 nM IGF-I or
10 nM IGF-II in the presence and absence of 50 ␮M EGCG.
EGCG did significantly prevent the increase in phosphorylated
ERK1/2 by either IGF-I or IGF-II (Fig. 7) and concomitantly
reduced IGF-I receptor activity, as indicated by a decrease in
the phosphotyrosine-IGF-I receptor and an association of the
IGF-II receptor with Gi␣-2 protein (unpublished observations).
Alternatively, it is possible that EGCG induces a decrease in

Fig. 8. Catechin-specific effects of green tea on protein amounts of ERK-1/2,
phospho-ERK1/2, and MEK1 in 3T3-L1 preadipocytes. Day 3 preadipocytes
were treated with 50 ␮M EC, EGC, ECG, and EGCG for 4 h. These kinases
were measured by Western blot analysis (A) and then expressed after normal-
ization to actin (B). Data are expressed as means ⫾ SE from triplicate
determinations. In some data, SE bars are too small to be seen. a– cGroups with
different letters are significantly different (P ⬍ 0.05) from each other.
Fig. 9. Differential effects of EGCG on altering the cell number (A) and
MEK1 activity (B) of 3T3-L1 preadipocytes and 3T3 fibroblasts (day 3)
observed after 48 h of treatment. The number of cells was determined using the
trypan blue exclusion method. The amounts of ERK1/2 and phospho-ERK1/2
proteins were measured by Western blot analysis and then expressed after
normalization to actin. Data are expressed as means ⫾ SE from triplicate
determinations. In some data, SE bars are too small to be seen. *P ⬍ 0.05 vs.
the control for a given type of cell line.

AJP-Cell Physiol • VOL 288 • MAY 2005 • www.ajpcell.org

 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
Antimitogenic effect of EGCG on preadipocytes depends on
Cdk2 pathway. Cdks are key regulators of the cell cycle in
vertebrate cells. They are related to the effects of EGCG in
modulating cell mitogenesis and growth arrest of most cancer
cells (1, 23–25) and can serve as the main controller of
mitogenesis and mitotic clonal expansion of preadipocytes
(40). We observed herein that doses of 20⬃100 ␮M EGCG
decreased Cdk2 activity at 4, 24, and 48 h and reduced its
protein levels at 48 h but not at 4 and 24 h. Also, EGCG dose
dependently induced G1 growth arrest at 24 and 48 h after
treatment. In addition, increased Cdk2 activity via the trans-
fection of Cdk2⫹/⫹ cDNA to preadipocytes prevented EGCG-
induced decreases in their Cdk2 activity and cell number and
EGCG-induced increases of G1 arrest, whereas decreased
Cdk2 activity via the transfection of dnCdk2 cDNA to preadi-
pocytes slowed down the 5-day growth of preadipocytes and
increased their G1 growth arrest (data not shown). These
observations suggest that the effect of EGCG of inducing
preadipocyte antimitogenesis and growth arrest is dependent
on a Cdk2 pathway and requires inactivation of the Cdk2
AJP-Cell Physiol • VOL
C1103
Fig. 10. Effects of green tea EGCG on the protein
expression of Cdk2 and Cdk1 (also called Cdc2) and
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on the activity of Cdk2. A: EGCG dose and time
dependently reduced the protein expression of Cdk2
in 3T3-L1 preadipocytes (day 3). B: EGCG dose and
time dependently reduced Cdk2 activity, as measured
by decreased phosphorylation of histone H1. Cdk2
activity was expressed after normalization to the im-
munoprecipitated (IP) Cdk2 protein. C: effects of
EGCG on protein levels of Cdk2 and Cdc2 were
dependent on cell types after 48 h of treatment. The
amounts of Cdk2 and Cdc2 proteins were measured
by Western blot analysis [or immunoblotting (IB)]
and then expressed after normalization to actin. Data
are expressed as means ⫾ SE from triplicate determi-
nations. In some data, SE bars are too small to be seen.
*P ⬍ 0.05 vs. the control for a given type of cell line.
protein. Because cyclin D1 is a G1 cyclin associated with Cdk4
and Cdk6 proteins, which favor cell cycle arrest at the G1
checkpoint (4), decreased cyclin D1 protein expression by
EGCG for 24⬃48 h suggests the possibility of Cdk4- and
Cdk6-related effects of EGCG on preadipocyte growth arrest.
However, we observed herein that 20 ␮M EGCG for 48 h did
not affect the total levels of Cdc2 protein and that 100 ␮M
EGCG for 48 h reduced the levels of Cdc2 protein less than
that of Cdk2. Accordingly, EGCG appears to act on a specific
type of Cdks in preadipocytes, but further studies are required
to illustrate this contention. Because Cdc2 takes over as the
predominant Cdk activity in the early G2/M transition of the
cell cycle (4), the observed decrease in Cdc2 protein expres-
sion by 100 ␮M EGCG suggests the possibility of the action of
EGCG on the G2/M phase of preadipocytes.
Regulation of Cdk2 activity of in vivo cultured cells occurs
at multiple levels, involving the synthesis of subunits and the
association of inhibitory proteins such as p21 and p27 (29, 37).
On these bases, a decrease in the Cdk2 activity of 3T3-L1
preadipocytes induced by 4 h of EGCG treatment may have
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Fig. 11. Stable overexpression of Cdk2 prevented
EGCG-induced alterations in the cell number (A), the
phases of the cell cycle (B), Cdk2 protein levels (C),
Cdk2 activity (D), and the binding of Cdk2 to p18, p21,
and p27 (E) in 3T3-L1 preadipocytes. The number of
cells and percentages of the four different phases of the
cell cycle were examined by the 0.4% trypan blue
exclusion method and by flow cytometry, respectively.
Protein expression and activity of Cdk2 were confirmed
by Western blot analysis (or IB) and by the IP method
and were then expressed after normalization to actin and
to Cdk2, respectively. The binding of Cdk2 to p18, p21,
and p27 (E) was determined by measuring changes in
the amounts of p18, p21, and p27 after Cdk2 immuno-
precipitation and were then expressed after normaliza-
tion to the immunoprecipitated amount of Cdk2 protein.
Data are expressed as means ⫾ SE in A from triplicate
determinations and means ⫾ SD from duplicate deter-
minations in B–E, respectively. In some data, SD bars
are too small to be seen. For clarity, SE bars and
statistical significances are not shown. CKI, cyclin-
dependent kinase inhibitor.

resulted from the observed increase in the association of Cdk2
with p21 and p27 by EGCG. However, the short-term effect of
EGCG in reducing Cdk2 activity should be unrelated to the
availabilities of p21, p27, and Cdk2 because EGCG did not
alter their protein levels in this period. However, increased
levels of p21 and p27, but not of p18 or Cdk2, observed with
the 24-h EGCG treatment may be responsible for the increased
association of Cdk2 with p21 and p27, but not p18, by EGCG,
thereby leading to low Cdk2 activity and a subsequent rise in
the percentage of G0/G1 arrest. In addition, the decreased levels
of Cdk2 protein and increased levels of p21 and p27, but not
AJP-Cell Physiol • VOL
p18, observed with the 48-h EGCG treatment may explain the
EGCG-induced increase in the association of Cdk2 with p21
and p27, but not p18, thereby resulting in decreases in Cdk2
activity and increases in the percentage of G0/G1 arrest. These
results suggest that EGCG may act on a particular type of
preadipocyte in the CKI family to reduce Cdk2 activity. It
would be of interest if other types of CKIs, such as p53 (4),
were also found to be involved in the action of EGCG on
preadipocyte growth arrest as reported in cervical cells (35). As
the sequestration of p21 and p27 is mediated via the induction
of cyclin D1 and cyclin D2 protein synthesis rates (32), the 24-
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 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
and 48-h decreases in cyclin D1 protein expression of preadi-
pocytes by EGCG may also explain the EGCG-induced in-
crease in the association of Cdk2 with p21 and p27. Further
studies to determine whether EGCG affects the association of
cyclin D1 with these CKIs would help clarify this notion.
The Cdk2 activity of in vivo cultured cells is also regulated
at phosphorylation-dephosphorylation levels (29, 37). In rat
aortic smooth muscle cells, increased Cdk2 activity by endo-
thelin is mediated via either the activation of Erk and Cdc25A
(a phosphatase) or the inactivation of WEE1 (an inhibitory
kinase) but is prevented by the inactivation of ERK and the
activation of WEE1 (7). Whereas Cdk2 activity is inactivated
through phosphorylation at Tyr15 by WEE1, it is restored
through dephosphorylation at Tyr15 by Cdc25A (7). Activities
of WEE1 and Cdc25A are, respectively, inactivated and acti-
vated through phosphorylation by the activation of ERK, and
vice versa (7). Accordingly, the observed EGCG reduction in
Cdk2 activity in 3T3-L1 preadipocytes may be mediated by the
inactivation of ERK. This explanation is supported by obser-
vations that decreased amounts of phospho-ERK1/2 proteins
by EGCG occur in parallel to the reduced activity, but not
protein levels, of Cdk2 by EGCG over a 48-h period. Because
Cdk2 activity in in vivo cells is also regulated by the associ-
ation of stimulatory proteins, such as cyclin E (4, 29), more
AJP-Cell Physiol • VOL
C1105
Fig. 12. Effects of green tea EGCG on increasing the protein
expression (A) of p21 and p27, but not p18, and the binding (B)
of Cdk2 to p21 and p27, but not p18, in 3T3-L1 preadipocytes
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(day 3). The amounts of these CKIs were measured by Western
blot analysis (or IB). In A, each CKI was directly measured
from cell lysates and then expressed after normalization to
actin. In B, Cdk2 and each CKI were measured after the IP of
Cdk2 protein and were then expressed after normalization to the
immunoprecipitated Cdk2 protein. C: effect of EGCG on the
protein expressions of p21 and p27, but not p18, was dependent
on the cell type after 48 h of treatment. Data are expressed as
means ⫾ SE from triplicate determinations. In some data, SE
bars are too small to be seen. *P ⬍ 0.05 vs. the control for a
given cell line.
investigations are needed to clarify whether the production of
cyclin E protein and its association with Cdk2 are altered by
EGCG and thereafter cause decreases in Cdk2 activity. How-
ever, an interesting observation not shown here is that the
cyclin D1 protein is able to associate with Cdk2, and such an
association can be altered with EGCG treatment (unpublished
observations). This suggests that EGCG may result in altered
cyclin-Cdk2 protein complexes in 3T3-L1 preadipocytes as
reported in mouse liver cells (14).
Catechin-specific effect. GTCs have numerous biological
activities that can possibly provide various health benefits (1,
24 –25, 28, 45). In most cases, but not all, gallated catechins,
especially EGCG, are more active than other catechins. This
contention is supported by our findings in 3T3-L1 preadipo-
cytes that at the same dose and duration of treatment, EGCG
was generally more effective than EC, ECG, and EGC in
changing the number of cells, the amount of incorporated
BrdU, percentages of the four phases of the cell cycle, activi-
ties of MEK1 and Cdk2, and levels of Cdk2, cyclin D1, and
CKIs. The observed catechin-specific effects of green tea
suggest that EGCG may act differently from EC, EGC, and
ECG in regulating preadipocyte growth. According to the
nature of the unique structures of these catechins tested (Fig. 1)
(24, 33), EGCG contains the largest number of hydroxyl
288 • MAY 2005 • www.ajpcell.org
C1106 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
Fig. 13. Dose- and time-dependent effects of green tea catechins on the protein
expression (A) of cyclin D1 and the binding of Cdk2 to cyclin D1 (B) in 3T3-L1
preadipocytes (day 3). The amounts of cyclin D1 proteins were measured by
Western blot analysis after 4 (A), 24 (B), and 48 h (C) of treatment. Cyclin D1
was directly measured from cell lysates and then expressed after normalization
to actin. Data are expressed as means ⫾ SE of triplicate determinations. In
some data, SE bars are too small to be seen. a– cFor each time point, groups with
different letters are significantly different (P ⬍ 0.05) from each other.
groups on its three aromatic rings among the tea catechins, and
these hydroxyl groups may be important for hydrogen bonding.
Also, EGCG has both gallyl and galloyl groups, which have
some conformational flexibility, that may also be important for
interactions with other molecules. Further exploration of the
chemical basis of the antimitogenic activity on preadipocytes
by EGCG is needed to understand differences in the mecha-
nism of EGCG’s action compared with those of EC, EGC, and
ECG on these processes.
Growth phase-dependent effect. The effects of green tea
EGCG in reducing the number of 3T3-L1 preadipocytes are
dependent on the latent, proliferative, and plateau phases of
preadipocyte growth. This is evidenced by our observations
that the IC50 values of EGCG for reducing the number of day
1-6 cells differed during the 72-h treatment. This suggests that
latent, log-phase, and confluent preadipocytes have different
sensitivities to this tea catechin. This is similar to the studied
growth phase-dependent effect of IGF-I on the activation of
MAPK protein phosphorylation in 3T3-L1 preadipocytes, par-
ticularly proliferating cells (5). A possible explanation is that
various endogenous properties of the three phases of cells
occur, thereby leading to EGCG’s signal proceeding in differ-
AJP-Cell Physiol • VOL 288 • MAY 2005 •
ent phases of cells. The findings that EGCG decreased the
amounts of phospho-ERKs in day 3 cells, but not in day 1 or
5 cells, and that the basal amounts of phospho-ERKs in day 3
cells were the largest among these stages of cells (Fig. 4C)
actually support this contention. Determining whether the
number and affinity of the EGCG receptor (39) and its asso-
ciated signal proteins exhibit different presences in the latent,
proliferative, and confluent phases of preadipocytes should
help elucidate their distinct sensitivities to EGCG.
Differential effects of EGCG on 3T3 and 3T3-L1 cells.
Mechanistic studies of green tea EGCG have reported its cell
type-dependent manner (1, 24, 25, 45). This may be explained
by the fact that the sensitivity of different normal, transformed,
and cancer cell lines to green tea EGCG varies (24), although
such differences may be due to the cell culture techniques and
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.4 on October 20, 2016
assay methods employed. In this study, we used the same
experimental culture conditions and assay methods to look at
whether a cell type-specific effect of EGCG occurs. We ob-
served that the IC50 value of EGCG for reducing the cell
number was lower in 3T3-L1 cells than in 3T3 fibroblast and
human KB oral cancer cells (data not shown), supporting the
existence of different sensitivities of these cell lines to EGCG,
as similarly reviewed by Liao et al. (24). Such differences
between 3T3 fibroblasts and 3T3-L1 preadipocytes induced by
EGCG may be explained by observations that the decrease in
phosphorylated ERK1/2 of the former cells in response to 48-h
EGCG treatment was much less than that in the latter cells
(Fig. 9) and that levels of Cdc2 and Cdk2 proteins were,
respectively, decreased and increased by EGCG in the former
cells, whereas Cdc2 levels in the latter cells were decreased
much less by EGCG than were Cdk2 levels (Fig. 10). These
observations suggest that EGCG may work differently in the
two cell lines in modulating mitogenesis via altering different
phases of their cell cycles and/or the extent of apoptosis and
that the Cdk2 transducer may play different roles with EGCG
Fig. 14. Proposed mechanism of the action of green tea EGCG on mitogenesis
in 3T3-L1 preadipocytes. The signaling of EGCG to inhibit preadipocyte
proliferation was dependent on the ERK MAPK kinase and Cdk2 pathways.
Antimitogenic signals of preadipocytes induced by EGCG may be related to
the mechanism by which it exerts its antiobese effect and adipocyte regulatory
activity.
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 GREEN TEA MODULATION OF PREADIPOCYTE GROWTH
in the two cell lines. It should be noted that in certain cells,
Cdks are activated during apoptosis (49), although it is un-
known whether the increase in Cdk2 protein levels in 3T3
fibroblasts by EGCG is related to the mechanism through
which EGCG induces a decrease in the cell number. Other
different characteristics between 3T3 fibroblasts and 3T3-L1
preadipocytes have been reported, although the latter cells are
subcloned from the former cells (15). It would be worthwhile
to explore whether any of them are responsible for the differ-
ential effects of EGCG on 3T3 and 3T3-L1 cells.
We conclude that the antimitogenic effect of EGCG on
3T3-L1 preadipocytes is dependent on the Erk MAPK and
Cdk2 pathways and is likely mediated through decreases in
their activities (Fig. 14). While shown to be mediated by ERK
MAPK, signaling is demonstrated to be largely independent of
the p38 MAPK and JNK MAPK pathways. Decreases in Cdk2
activity by EGCG may be due to its effect on this particular
member of the CKI family. In general, EGCG is more effective
than other structurally related GTCs in changing mitogenic
signals. The signaling of EGCG in 3T3-L1 preadipocytes
differs from that in 3T3 fibroblasts. Changes in the endogenous
signals of preadipocytes induced by EGCG may help increase
our understanding of the modulatory mechanism of green tea
EGCG on body weight and fat cells (18 –19, 24). Future studies
on discovering the EGCG receptor in fat cells and on charac-
terizing its oxidative stress are needed to elucidate the mech-
anisms of how EGCG signals reduce the activities of MEK1
and Cdk2 proteins.
ACKNOWLEDGMENTS
We are grateful to Dr. Shutsung Liao and associates at the Ben May
Institute for Cancer Research of the University of Chicago for the gift of green
tea ECG. Also, we thank Drs. Shen-Liang Chen and Jaw-Jing Yang for the
gifts of MEK1, MEKK1, MEK1EE, and MKK6EE cDNA constructs. Finally,
we thank Drs. Lee-Young Chau and Shun-Chern Tsaur for technical assis-
tance.
GRANTS
This study was supported by Grants NSC90-2311-B-008-002 and NSC91-
2311-B-008-004 from the National Science Council, Taiwan, and the Brain
Research Center, Taiwan (to Y.-H. Kao).
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