From the Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute and Interdisciplinary Oncology Program and Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida 33612
Received for publication, May 17, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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It has been discovered that proteasome
inhibitors are able to induce tumor growth arrest or cell death and
that tea consumption is correlated with cancer prevention. Here, we
show that ester bond-containing tea polyphenols, such as
( Previous epidemiological studies have suggested that tea
consumption may have a protective effect against human cancer (1-4). Recent animal studies have also demonstrated that green tea polyphenols could suppress the formation and growth of human cancers, including skin (5, 6), lung (7), liver (8), esophagus (9), and stomach (10). The
major components of green and black tea include
epigallocatechin-3-gallate
(EGCG)1, epigallocatechin
(EGC), epicatechin-3-gallate (ECG), epicatechin (EC), and their epimers
(see Fig. 1A). EGCG among those polyphenols has been most
extensively examined because of its relative abundance and strong
cancer-preventive properties (1, 11). EGCG has been shown to inhibit
several cancer-related proteins, including urokinase (12), nitric-oxide
synthase (13), teromerase (14), and tumor necrosis factor- The 20S proteasome, a multicatalytic complex (700 kDa), constitutes the
catalytic key component of the ubiquitous proteolytic machinery 26S
proteasome (16-20). There are three major proteasomal activities:
chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide
hydrolyzing (PGPH) activities (16, 21). The ubiquitin-proteasome system
plays a critical role in the specific degradation of cellular proteins
(22), and two of the proteasome functions are to allow tumor cell cycle
progression and to protect tumor cells against apoptosis (23). The
chymotrypsin-like but not trypsin-like activity of the proteasome is
associated with tumor cell survival (24, 25). Many cell cycle and cell
death regulators have been identified as targets of the
ubiquitin-proteasome-mediated degradation pathway. These proteins
include p53 (26), pRB (27), p21 (28), p27Kip1 (29),
I Here, we report for the first time that ester bond-containing tea
polyphenols potently and selectively inhibit the proteasomal chymotrypsin-like but not trypsin-like activity in vitro and
in vivo. Among the tea polyphenols examined, EGCG showed the
strongest inhibitory activity against purified 20S proteasome, 26S
proteasome of tumor cell extracts, and 26S proteasome in intact tumor
cells. Furthermore, the inhibition of the proteasome in vivo
was able to accumulate the natural proteasome substrates
p27Kip1 and I Materials--
Highly purified tea polyphenols EGCG
(>95%), ECG (>98%), EGC (>98%), EC (>98%), GCG (>98%), GC
(>97%), CG (>98%), and C (>98%) were purchased from Sigma and
used directly without further purification. A green tea extract was a
gift from the Lipton Company (Englewood Cliffs, NJ) that contained
51.5% EGCG, 14.7% ECG, 8.3% EGC, 8.5% EC, 4.4% GCG, 2.4% GC,
1.6% C, and 1.6% caffeine. A black tea extract was also a gift from
Lipton that contained 19.7% EGCG, 14.9% ECG, 0.9% EGC, 4.8% EC,
0.0% GCG, 0.5% GC, 2.0% C, and 1.2% caffeine. Purified 20S
proteasome (Methanosarcina thermophile, Recombinant,
Escherichia coli) and purified calpain I (human
erythrocytes) were purchased from Calbiochem. Fluorogenic peptide
substrates Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal
chymotrypsin-like activity), benzyloxycarbonyl
(Z)-Leu-Leu-Glu-AMC (for the proteasomal PGPH activity),
Suc-Leu-Tyr-AMC (for the calpain I activity), and
Ac-Asp-Glu-Val-Asp-AMC (for the caspase-3 activity) were also obtained
from Calbiochem, and Z-Gly-Arg-AMC (for the proteasomal trypsin-like
activity) was from Bachem (King of Prussia, PA). The specific calpain
inhibitor calpeptin and the specific caspase-3 inhibitor Ac-DEVD-CHO
were obtained from Calbiochem. Monoclonal antibody to
p27Kip was purchased from PharMingen (San Diego, CA),
polyclonal antibodies to I Cell Culture and Cell Extract Preparation--
Human Jurkat T
and prostate cancer (LNCaP, PC-3) cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 units/ml of
penicillin, and 100 µg/ml streptomycin. Human breast cancer MCF7
cells and normal (WI-38) and SV40-transformed (VA-13) human fibroblasts
were grown in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum, penicillin, and streptomycin. All cells were
maintained in a 5% CO2 atmosphere at 37 °C. A whole cell extract was prepared as described previously (24). Cells were
harvested, washed with phosphate-buffered saline twice, and homogenized
in a lysis buffer (50 mM Tris-HCl, pH 8.0, 5 mM
EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 0.5 mM
phenylmethylsulfonyl fluoride, and 0.5 mM dithiothreitol)
for 30 min at 4 °C. Afterward, the lysates were centrifuged at
14,000 × g for 30 min, and the supernatants were
collected as whole cell extracts.
Inhibition of Purified 20S Proteasome Activity by Tea
Polyphenols--
The chymotrypsin-like activity of purified 20S
proteasome was measured as follows. 0.5 µg of purified 20S proteasome
was incubated with 20 µM fluorogenic peptide substrate,
Suc-Leu-Leu-Val-Tyr-AMC (for the proteasomal chymotrypsin-like
activity), for 30 min at 37 °C in 100 µl of assay buffer (20 mM Tris-HCl, pH 8.0) with or without a tea polyphenol or
tea extract. After incubation, the reaction mixture was diluted to 200 µl with the assay buffer followed by a measurement of the hydrolyzed
7-amido-4-methyl-coumarin (AMC) groups using a VersaFluorTM
Fluorometer with an excitation filter of 380 nm and an emission filter
of 460 nm (Bio-Rad).
Inhibition of the Proteasome Activity in Whole Cell Extracts by
Tea Polyphenols--
A whole cell extract (3.5 µg) of Jurkat T cells
was incubated for 90 min at 37 °C with 20 µM
fluorogenic peptide substrates for various activities of the
proteasomes, Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Glu-AMC, and
Z-Gly-Gly-Arg-AMC, in 100 µl of the assay buffer with or without EGCG
or EGC. The hydrolyzed AMCs were quantified as described earlier.
Inhibition of the Proteasome Activity in Intact Tumor Cells by
Tea Polyphenols--
To measure the inhibition of proteasome activity
in living tumor cells, Jurkat T (1 × 105
cells/ml/well), MCF-7, or PC-3 cells (1 × 104
cells/ml/well) were cultured in 24-well plates. These cells were first
incubated for 12 h with various concentrations of EGCG, EGC,
Assays for Calpain I and Caspase-3 Activities--
To measure
the activity of calpain I, 3 µg of purified calpain I was incubated
with 40 µM fluorogenic peptide calpain substrate, Suc-Leu-Tyr-AMC, for 30 min at 37 °C in 100 µl of assay buffer (50 mM Tris-HCl, pH 7.5, containing 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM
Atomic Orbital Energy Analysis--
The electron density surface
colored by nucleophilic susceptibility was created using the Cache
Worksystem version 3.2 (Oxford Molecular Ltd.). After geometrical
optimization (using augmented MM3), the electron distribution between
the highest occupied molecular orbital and the lowest unoccupied
molecular orbital was evaluated, and a three-dimensional isosurface of
susceptibility to nucleophilic attack (generated by an extended Huckel
wave function) was calculated and superimposed over the molecule. A
colored "bullseye" with a white center is characteristic of atoms
that are highly susceptible to nucleophilic attack.
Western Blot Analysis--
Jurkat T, LNCaP, WI-38, or VA-13
cells were treated with various concentrations of EGCG or EGC for
indicated hours (see figure legends). To measure the changes in protein
stability, Jurkat T cells were pretreated with 10 µg/ml cycloheximide
for 2 h (to inhibit translation) followed by coincubation with
EGCG or the proteasome inhibitor LLnV (for a positive control). This
was followed by the preparation of whole cell extracts. The enhanced
chemiluminescence Western blot analysis was then performed using
specific antibodies to p27Kip, I Flow Cytometry--
Cell cycle analysis based on DNA content was
performed as follows. At each time point, cells were harvested,
counted, and washed twice with phosphate-buffered saline. Cells (5 × 106) were suspended in 0.5 ml of phosphate-buffered
saline, fixed in 5 ml of 70% ethanol for at least 2 h at
High Performance Liquid Chromatography (HPLC) Analysis--
EGCG
or EGC (1 mM) was incubated with either the purified 20S
proteasome (45 µg) or its buffer for the indicated hours at 37 °C
in 100 µl of a reaction buffer (20 mM Tris-HCl, 1 mM dithiothreitol, pH 7.2). After each reaction, the sample
was filtered with a 0.45 µm of nylon syringe filter (Nalge Co.,
Rochester, NY), and 20 µl of filtered sample was injected to HPLC
equipped with a C-18 reverse phase column (0.46 × 25 cm,
Separation Group, Hesperia, CA). The solvent system was 12%
acetonitrile, 2% ethyl acetate, and 0.05% phosphoric acid, the flow
rate was 1 ml/min, and the proteasome cleavage products were monitored
at 280 nm. The standard controls also included gallic acid without
incubation and the purified proteasome alone.
Inhibition of Chymotrypsin-like Activity of Purified 20S Proteasome
by Ester Bond-containing Tea Polyphenols--
It has been reported
that lactacystin, when converted to its active form
clasto-lactacystin
Three other ester bond-containing tea polyphenols, ECG, GCG, and CG
(Fig. 1A), were also found to be strong inhibitors of the
chymotrypsin-like activity of the purified 20S proteasome (IC50 values were 194, 187, and 124 nM,
respectively). In contrast, all the corresponding polyphenols that do
not contain ester bonds, EC, GC, and C (Fig. 1A), could not
inhibit the proteasomal chymotrypsin-like activity. These results
indicate that the ester bonds contained in tea polyphenols are
essential for potent inhibition of the proteasomal chymotrypsin-like
activity. Furthermore, a green or black tea extract, which contains
significant portions of EGCG (51.5 and 19.7%, respectively) and ECG
(14.7 and 14.9%, respectively, see under "Experimental
Procedures"), also strongly inhibited the chymotrypsin-like activity
of the 20S proteasome (IC50 values were 0.1 and 0.3 µg/ml, respectively).
The electrophilic ester bond carbon of Inhibition of the Proteasomal Chymotrypsin-like Activity in Tumor
Cell Extracts and Intact Tumor Cells by EGCG--
We then tested if
EGCG or EGC could inhibit the 26S proteasome activity in a tumor cell
extract. We found that 10 µM EGCG inhibited ~70% of
the proteasomal chymotrypsin-like activity in a Jurkat T cell extract,
whereas EGC at the same concentration had little effect (Fig.
2A). The addition of EGCG to
the Jurkat cell extract also potently inhibited another proteasomal
activity, the PGPH activity, but did not affect the proteasomal
trypsin-like activity (Fig. 2A). To investigate whether EGCG
specifically inhibits the proteasome activity, its effects on other
protease activities were examined. The activity of purified calpain I
enzyme was inhibited by the specific calpain inhibitor calpeptin (35)
but not EGCG (Fig. 2B). Similarly, a caspase-3-like activity
in Jurkat T cell extract was blocked by the specific caspase-3
inhibitor Ac-DEVD-CMK (36) but not EGCG (Fig. 2C). It
appears that EGCG selectively inhibits the proteasomal chymotrypsin
(and PGPH) activity over other protease activities.
To determine whether EGCG could also inhibit the living cell
proteasomal activity, Jurkat T cells were first incubated with various
concentrations of EGCG or EGC followed by an additional incubation with
a fluorogenic proteasome peptide substrate. Afterward, the cell medium
was collected for the measurement of hydrolyzed products (free AMCs).
By performing this assay, we found that EGCG significantly inhibited
the proteasomal chymotrypsin-like activity in intact Jurkat cells in a
concentration-dependent manner (IC50 = 18 µM), whereas EGC had a much less effect (Fig.
3A).
We noticed that the concentrations of EGCG needed to inhibit the
proteasome activity in Jurkat cell extracts (Fig. 2A), and intact Jurkat cells (Fig. 3A) were much higher than were
needed for the inhibition of purified 20S proteasome activity (Fig.
1A). We suspected that higher concentrations of other
proteasome inhibitors might be needed to reach their in vivo
cellular target, the proteasome. If true, a specific authentic
proteasome inhibitor should display differential potencies between
purified proteasome and living cell proteasome activity. To test this
idea, the effects of
We also found that EGCG inhibited the proteasomal chymotrypsin-like
activity in intact breast (MCF-7) and prostate (PC-3 and LNCaP) cancer
cells (Fig. 4D and data not
shown). However, EGCG did not inhibit the proteasomal trypsin-like
activity in living Jurkat T cells (Fig. 3C). Taken together,
our data suggest that EGCG but not EGC can selectively inhibit the
chymotrypsin-like activity of purified 20S proteasome, 26S proteasome
of tumor cell extracts, and 26S proteasome of living tumor cells.
To determine the molecular target(s) responsible for the
cancer-preventative effects of green tea, one must adhere to the concentrations of the molecules, which are found physiologically in
green tea drinkers. Previous studies indicate that EGCG or other
catechins are present in low micromolar ranges in the plasma and saliva
of human volunteers (3, 38) and in mice that had been fed with tea
(38). Here we found that EGCG in low micromolar ranges acts as a potent
proteasome inhibitor in vitro and in vivo (Figs.
1-3), indicating that EGCG at physiological levels could inhibit the
proteasomal chymotrypsin-like activity in intact cancer cells and bring
about the resultant tumor growth arrest (see below).
Accumulation of the Proteasome Target Proteins p27Kip1
and I
Because most of the proteasome-mediated protein degradation pathways
require ubiquitination (22), we expected that the inhibition of
proteasome activity by EGCG should increase the levels of
polyubiquitinated proteins. Indeed, when lysates of EGCG-treated Jurkat
T cells were immunoblotted with an antiserum to ubiquitin, increased
levels of several ubiquitinated proteins were detected (Fig.
4C).
To determine whether other cancer cell lines are also responsive to
EGCG treatment, human prostate cancer LNCaP cells were treated with
EGCG at 1 or 10 µM for 12 h. Again, EGCG at 1 µM increased the levels of p27 and I
To rule out possible stimulatory effects of EGCG on the syntheses of
p27 and I
Coincubation of the cycloheximide-pretreated cells with 10 µM EGCG also greatly increased the levels of I
The following arguments support that inhibition of the proteasome
activity by EGCG is responsible for the accumulation of p27 and
I EGCG Induces Tumor Cell Growth Arrest in G1 Phase of
the Cell Cycle--
It has been well documented that overexpression of
either p27 (39, 40) or I
Exposure of LNCaP prostate cancer cells to 10 µM EGCG for
12 h initiated G1 arrest by a 7% increase (Fig.
6B), which was correlated with p27 and I Normal Human WI-38 Fibroblasts Are More Resistant to EGCG-induced
p27 Accumulation and G1 Arrest Than Their SV40-transformed
Counterpart--
Previously, we reported that proteasome inhibitors
selectively accumulated p27 protein and induced apoptosis in tumor and transformed abnormal human cells (24). To investigate whether EGCG has any differential effects on transformed and normal cells, the
normal human fibroblast cell line WI-38 and its SV40-transformed derivative (VA-13) were treated with 10 µM EGCG followed
by the measurement of p27 and I
Similar to Jurkat T and LNCaP tumor cells (Fig. 4), the treatment of
the transformed VA-13 cells with 10 µM EGCG significantly increased p27 levels (Fig.
7A). A 12-h treatment with
EGCG increased p27 expression by 2.8-fold; after 36 or 48 h, p27
levels were further increased by 7.6- and 9.2-fold, respectively (Fig.
7A). In contrast, the treatment of normal WI-38 cells with
10 µM EGCG for up to 48 h did not increase p27
levels (Fig. 7A).
EGCG treatment of VA-13 cells also increased levels of I
Correlated with the selective p27 accumulation in the transformed cells
over normal WI-38 cells by EGCG (Fig. 7A), VA-13 cells were
found to be more sensitive to EGCG-induced G1 arrest than WI-38 cells. After a 12-h treatment with EGCG, the G1
population of VA-13 cells was increased by 22% (Fig.
8, A and C). In
contrast, no apparent G1 arrest was observed in WI-38 cells
under this condition (Fig. 8, B and C). At
24 h, the G1 population of the transformed cells was
further increased (by ~25%); the WI-38 G1 population began to increase (by <5%) (Fig. 8C). After a 36-h
treatment, the VA-13 G1 population continued to increase
(by 33%, Fig. 8, A and C); only at this time, a
16% increase in the G1 population of WI-38 cells was also
detected (Fig. 8, B and C). The results from
several independent experiments confirmed that the transformed VA-13
cells were more sensitive to EGCG-induced G1 arrest than the normal WI-38 cells (Fig. 8C). It appears that the
delayed EGCG-induced G1 arrest in WI-38 cells is associated
with the lack of p27 accumulation and the partial induction of
I HPLC Analysis of EGCG After Reaction with Purified
Proteasome--
The ester bond carbon in
To test this hypothesis, a highly purified EGCG (for review see Fig.
9D) was incubated with
purified 20S proteasome for various hours followed by HPLC analysis.
After a 2-h incubation, a gallic acid-like peak (retention time 4.78)
associated with a 40% decrease in the level of EGCG was detected in
the HPLC chromatogram whose level was corresponded to a
concentration of <5% EGCG (Fig. 9, A and C and
Fig. 10). The gallic acid-like peak was
not produced from EGCG in the absence of the proteasome (Fig.
9D). The incubation of EGCG with purified proteasome for
4 h resulted in the disappearance of an ~80% EGCG, associated
with an increase in the level of the gallic acid-like peak that is
equivalent to a concentration of 13% EGCG (Fig. 9, B versus
A and Fig. 10). After a 6-10 h incubation with the
proteasome, >95% EGCG disappeared, whereas the level of the gallic
acid-like product linearly increased to a level equivalent to 20-25%
EGCG (Fig. 10).
The incubation of EGCG with the 20S proteasome also resulted in the
appearance of an EGC-like product (retention time 6.62 min), although
its level was very low (Fig. 9, A and B
versus G), suggesting that the produced EGC could
be further degraded by the proteasome. Indeed, purified EGC was
degraded almost completely by purified 20S proteasome (Fig. 9,
F versus G). Several unknown products
of EGCG, including one with a retention time of 5.75 min between gallic
acid and EGC peaks, were observed (Fig. 9, A and
B). Another unknown peak at a retention time of ~8 min
resulted from the mixture of the buffer and purified proteasome
(compare Fig. 9 A and B with E). Taken
together, the disappearance of most of the free EGCG prior to the
appearance of low levels of gallic acid-like product suggests that EGCG
might form a tight complex with the proteasome. This hypothesis is
consistent with the observed potency of EGCG as a proteasome
inhibitor in vitro (Figs. 1 and 2) and in vivo
(Figs. 3-5 and 7). The HPLC data also suggest that the
proteasome-bound EGCG could be slowly cleaved at one or more places
including the ester bond, which leads to the production of gallic acid,
EGC, and other products. Finally, a complete cleavage of EGC by the
purified proteasome (Fig. 9F) is consistent with failure of
EGC to inhibit the purified proteasome activity (Fig. 1A)
and the proteasome activity in Jurkat cell extracts (Fig. 2A) and intact Jurkat T cells (Figs. 3A and
4A).
Based on our current study, we propose the following molecular
mechanisms by which EGCG inhibits the proteasome. The two nucleophilic electrons located on the N-terminal threonine hydroxyl group of the
proteasome subunit X (32-34) could attack the ester bond carbon of
EGCG after binding to the proteasome active site. A tight
EGCG-proteasome complex could be generated, thereby inactivating the
proteasome. This complex would slowly disassociate to free EGC and
gallic acid. Further studies are needed to understand the nature of
ester bond-containing polyphenols as a potent proteasome inhibitor.
In summary, for many years it has been shown through epidemiological
studies that green tea is a cancer-preventative agent (1-4). It has
also been shown that the proteasome plays an important role in the
development and progression of cancer (22, 23). Our study has
demonstrated for the first time that the compounds found in tea and in
the bodies of green tea drinkers can inhibit the proteasome at or near
physiological concentrations. Our results also indicate that the
inhibition of the proteasome activity by EGCG can selectively control
the growth of tumor and transformed cells. We suggest that the
cancer-preventative properties of green tea could be attributed, at
least in part, to its ability to inhibit the proteasome activity. Our
finding along with the low toxicity of EGCG also implicates the role of
tea in a potential clinical therapy in combination with current
anticancer drugs.
)
epigallocatechin-3-gallate (EGCG), potently and specifically
inhibit the chymotrypsin-like activity of the proteasome in
vitro (IC50 = 86-194 nM) and in vivo (1-10 µM) at the concentrations found in the
serum of green tea drinkers. Atomic orbital energy analyses and high
performance liquid chromatography suggest that the carbon of the
polyphenol ester bond is essential for targeting, thereby inhibiting
the proteasome in cancer cells. This inhibition of the proteasome by
EGCG in several tumor and transformed cell lines results in the
accumulation of two natural proteasome substrates, p27Kip1
and I
B-
, an inhibitor of transcription factor NF-
B, followed by growth arrest in the G1 phase of the cell cycle.
Furthermore, compared with their simian virus-transformed counterpart,
the parental normal human fibroblasts were much more resistant to EGCG-induced p27Kip1 protein accumulation and
G1 arrest. Our study suggests that the proteasome is a
cancer-related molecular target of tea polyphenols and that inhibition
of the proteasome activity by ester bond-containing polyphenols may
contribute to the cancer-preventative effect of tea.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(15). However, nonphysiological concentrations of EGCG
(i.e., concentrations higher than those found in human serum
after tea consumption) were used in some earlier studies. Whether one
or more of these proteins are the real molecular targets of EGCG and
other tea polyphenols under physiological conditions needs further investigations.
B-
(30), and Bax (31).
B-
as well as induce the arrest of tumor
cells in the G1 phase. Finally, normal human WI-38
fibroblasts were more resistant to the EGCG treatment than their
SV40-transformed counterpart.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B-
and actin were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA), and polyclonal antibodies to
ubiquitin were from Sigma.
-lactone, or LLnL followed by an additional 2-h incubation with the
fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC or
Z-Gly-Gly-Arg-AMC. Afterward, the cell medium (200 µl/sample) was
collected and used for measurement of free AMCs.
-mercaptoethanol, 5 mM CaCl2, and 0.1%
CHAPS) with or without EGCG or the specific calpain inhibitor
calpeptin. For caspase-3 activity assay, a Jurkat T cell extract (3.5 µg) was incubated for 90 min at 37 °C with 20 µM
fluorogenic peptide substrate, Ac-Asp-Glu-Val-Asp-AMC, with or without
EGCG or the specific caspase-3 inhibitor Ac-DEVD-CHO. After incubation,
the reaction mixture was diluted to 200 µl with the assay buffer, and
the hydrolyzed AMCs were quantified as described above.
B-
, ubiquitin, or
actin as described previously (24).
20 °C, centrifuged, resuspended again in 1 ml of propidium iodide
staining solution (50 µg of propidium iodide, 100 units of RNase A,
and 1 mg of glucose/ml of phosphate-buffered saline), and incubated at
room temperature for 30 min. The cells were then analyzed with FACScan (Becton Dickinson Immunocytometry, San Jose, CA) and ModFit LT cell cycle analysis software (Verity Software, Topsham, ME). The cell
cycle distribution is presented as the percentage of cells containing
G1, S, G, S, and G2/M DNA content as
judged by propidium iodide staining.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-lactone (
-lactone), is a highly specific and irreversible inhibitor of the proteasome (32-34). This
-lactone contains an ester bond (Fig.
1A) that is responsible for
interacting with and inhibiting the proteasome (32-34). We noticed a
similar ester bond present in several tea polyphenols including EGCG,
ECG, GCG, and CG (Fig. 1A). We hypothesized that tea
polyphenols containing ester bonds would inhibit the proteasome activity, whereas tea polyphenols without ester bonds would not. We
tested this hypothesis by performing a cell-free proteasome activity
assay in the presence of tea polyphenols. The chymotrypsin-like activity of purified 20S proteasome (the catalytic core of 26S proteasome) (22) was significantly inhibited by EGCG (Fig.
1B) whose IC50 value was calculated to be 86 nM (Fig. 1A). In contrast, EGC (IC50 = 1.2 mM) and gallic acid (IC50 = 7.1 mM), the two moieties of EGCG linked by an ester bond, were
14,000- and 83,000-fold less potent than EGCG, respectively (Fig. 1,
A and B). As a positive control,
-lactone also
potently inhibited the proteasomal chymotrypsin activity
(IC50 = 600 nM in Fig. 1, A and
B) (34). The shape of the inhibition curve of EGCG was
similar to that of
-lactone (Fig. 1B).
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Fig. 1.
Structure-activity relationships of tea
polyphenols. A, the structure and potency of
polyphenols. IC50 of each tea polyphenol toward the
chymotrypsin-like activity of the purified 20S proteasome was measured
as described under "Experimental Procedures." N/A
indicates that the inhibitory activity of the corresponding polyphenol
at 50 µM was <10%. B,
concentration-dependent inhibition of the chymotrypsin-like
activity of the purified 20S proteasome by EGCG, EGC, and -lactone.
C, the susceptibility of EGCG, EGC, and
-lactone to a
nucleophilic attack was calculated as described under "Experimental
Procedures."
-lactone is responsible for
its biological inhibition of the proteasome (32-34), supported by
previous studies using x-ray crystallography (16). When the atomic
orbital energy was analyzed, the ester bond carbon of
-lactone showed a high susceptibility toward a nucleophilic attack with an
arbitrary value of 1.1 (Fig. 1C). We then determined if the levels of nucleophilic susceptibility found in tea polyphenols correlate with their proteasome inhibitory activities. The ester bond
carbon of EGCG was found to have the highest susceptibility toward a
nucleophilic attack among all the other atoms with a value of 0.7, whereas the carbon with the highest nucleophilic susceptibility on EGC
was found to have a low value of 0.2 (Fig. 1C). Similarly, a
high nucleophilic susceptibility was found in other ester
bond-containing polyphenols, ECG, GCG, and CG (all with values of
0.7), whereas low nucleophilic susceptibility was found in
nonester bond-containing polyphenols, EC, GC, and C (with values of
0.3, 0.2, and 0.3, respectively). Thus, the nucleophilic susceptibility
of tea polyphenols correlated with their ability to inhibit the
proteasome chymotrypsin-like activity. These data support the essential
role of polyphenol ester bonds in the inhibition of the proteasome activity.
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Fig. 2.
Specific inhibition of the proteasome
activity by EGCG in Jurkat cell extracts. A Jurkat cell extract
(3.5 µg/reaction) was incubated for 90 min with various fluorogenic
peptide substrates for the proteasomal chymotrypsin-like, PGPH,
trypsin-like activity (A), or caspase-3 activity
(C), or a purified calpain I (3 µg) was incubated for 90 min with a fluorogenic calpain substrate (B) in the absence
or presence of EGCG (10 µM), EGC (10 µM),
calpeptin (1 µM), or Ac-DEVD-CHO (10 µM) as
indicated followed by the measurement of free AMC groups as described
under "Experimental Procedures" (p < 0.05).
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Fig. 3.
Inhibition of the proteasome activity by EGCG
but not EGC in intact Jurkat cells. Human Jurkat T cells were
preincubated for 12 h with either the solvent (indicated by 0 or
cont. for control) or EGCG, EGC, -lactone, or LLnL
at the indicated concentrations followed by an additional 2-h
incubation with the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (for
the chymotrypsin-like activity of the proteasome) or Z-Gly-Gly-Arg-AMC
(for the trypsin-like activity of the proteasome) (A and
B). C, the medium was collected, and
the free AMC groups were measured as described under "Experimental
Procedures." Most of the data from A were derived from
three independent experiments, except for EGCG and EGC at 10 µM derived from six independent experiments to most
accurately determine the validity of this important
concentration.
-lactone, LLnL, and EGCG were measured on
inhibition of the proteasomal chymotrypsin-like activity in intact
Jurkat T cells. Fig. 3B demonstrates that
-lactone, LLnL,
and EGCG at 10 µM inhibited 20, 40, and 24% of the
proteasomal chymotrypsin-like activity in living Jurkat cells,
respectively, with the assay system used. The IC50 value of
-lactone to inhibit the chymotrypsin-like activity of a purified 20S
proteasome was 0.6 µM under our conditions (Fig.
1A) and 0.1-0.2 µM under other conditions
(34), and the IC50 value of LLnL to inhibit the 20S
proteasome chymotrypsin-like activity was 0.14 µM (37).
Therefore, it appears that even for a specific proteasome inhibitor,
higher concentrations are necessary for the inhibition of the living
cell proteasome activity. Because both
-lactone and EGCG showed
greater potencies to purified 20S proteasome (IC50 values
were 600 and 86 nM, respectively, Fig. 1A) than
to intact cellular proteasome activity (20 and 24% inhibition at 10 µM, respectively, Fig. 3B), EGCG seemed to be
able to target, thereby inhibiting the proteasome in Jurkat T cells.
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Fig. 4.
Accumulation of p27,
I B-
, and
ubiquitinated proteins by EGCG. A-C, lane 1, Jurkat T
cells were treated with solvent, 1 or 10 µM EGCG or EGC
for 12 h (A), or 25 µM EGCG for the
indicated hours (B and C), or prostate cancer
LNCaP cells were treated with solvent (lane 1 in
A-D), 1 or 10 µM EGCG, or
EGC for 12 h (D), followed by a Western blot assay
using specific antibodies to p27, I
B-
, actin, or ubiquitin.
Molecular masses of I
B-
and actin are 40 and 43 kDa,
respectively. The band of 56 kDa, indicated by an arrow in
B might be an I
B-
-related protein. The bands indicated
in C are ubiquitin-containing proteins. Relative density
(RD) values are normalized ratios of the intensities of p27
or I
B-
band to the corresponding actin band.
B-
in Tumor Cells Treated with EGCG--
To further confirm
that EGCG inhibits the proteasome activity in vivo, Jurkat T
cells were treated with various concentrations of EGCG or EGC for
different hours followed by measuring levels of the
cyclin-dependent kinase inhibitor p27Kip1 and
I
B-
, two well known target proteins of the proteasome (29, 30). A
12-h treatment of Jurkat cells with 1 µM EGCG increased p27 levels by ~3-fold (Fig. 4A, lane 2 versus lane 1), and the same treatment with 10 µM EGCG increased p27 expression by ~4-fold (lane
3 versus lane 1). In contrast, EGC at the
same concentrations had no such effect (Fig. 4A). EGCG
treatment also increased I
B-
levels by 2.7-fold after a 2-h
treatment and by ~ 4-fold after 4-8 h of treatment (Fig.
4B). A band of 56 kDa, detectable by the anti-I
B-
antibody used, was increased significantly during EGCG treatment
(indicated by an arrow, Fig. 4B), suggesting that it might be an I
B-
-related protein.
B-
proteins by
2.2- and 3.9-fold, respectively, and EGCG at 10 µM
increased p27 and I
B-
expression by 5.6- and 5.0-fold,
respectively, in these prostate cancer cells (Fig. 4D).
Therefore, an accumulation of p27 and I
B-
proteins by EGCG
treatment is time-dependent and
concentration-dependent.
B-
proteins, Jurkat T cells were preincubated with the
protein synthesis inhibitor cycloheximide for 2 h followed by
additional incubation with or without EGCG (in the presence of
cycloheximide) to determine whether the stability of p27 and I
B-
proteins is increased by EGCG treatment. Incubation with cycloheximide
alone significantly decreased the levels of both p27 and I
B-
proteins (Fig. 5, A and
B, lanes 2 versus lanes 1).
This decrease should be the result of degradation of these proteins in
the absence of new protein synthesis. When the cycloheximide-pretreated cells were coincubated with 10 µM EGCG, the levels of p27
protein were increased by 3-fold with respect to cycloheximide
treatment alone (Fig. 5A, lane 4 versus
lane 2). This increase should be the result of the
inhibition of p27 degradation by EGCG but not due to increased p27
synthesis because of the presence of cycloheximide. In addition, the
appearance of a band of ~70 kDa (p70) was significantly increased by
this treatment (Fig. 5A, lane 4 versus
lane 2). The p70 may contain ubiquitinated p27, because
a similar p70 containing ubiquitinated p27 was found in proteasome
inhibitor-treated human osteosarcoma MG-63 cells (29) and breast cancer
MDA-MB-231 cells (24). In fact, the sum of the levels of both p27 and
p70 was increased by 7-fold in cells cotreated with cycloheximide and EGCG (Fig. 5A, lane 4 versus lane
2).
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Fig. 5.
Accumulation of p27 and
I B-
proteins by EGCG
in cycloheximide-pretreated cells. Jurkat T cells were pretreated
with 10 µg/ml cycloheximide for 2 h followed by coincubation
with 1 or 10 µM EGCG or 10 µM LLnV for 8 or
12 h as indicated. This was followed by a Western blot assay using
specific antibodies to p27, I
B-
, and actin. The p70 is a putative
ubiquitinated p27-containing complex (29, 24). Relative density
(RD) values are normalized ratios of intensities of p27 (or
p27 plus p70) or I
B-
band to the corresponding actin band.
B-
protein 4-fold higher than that of the cells treated with cycloheximide
alone (Fig. 5B, lane 4 versus lane
2). The increase in I
B-
expression by EGCG was even
greater than that by the proteasome inhibitor LLnV at the same
concentration (4- versus 2-fold, Fig. 5B,
lane 4 versus lane 3). Therefore, the
inhibition of the proteasomal chymotrypsin-like activity in intact
tumor cells (Fig. 3) correlates well with the accumulation of p27,
I
B-
, and some ubiquitinated proteins (Figs. 4 and 5).
B-
proteins in tumor (Figs. 4 and 5) and transformed (for review
see Fig. 7) cells. First, as shown in Figs. 1-3, EGCG is a relatively
potent specific proteasome inhibitor in vitro and in
vivo. In addition, the accumulation of both p27 and I
B-
proteins was observed in an EGCG concentration-dependent
(Figs. 4A and D and 5A) and
time-dependent manner (Figs. 4B and 7).
Furthermore, after EGCG treatment, the anti-p27 antibody detected a p70
band (Fig. 5A), which may contain ubiquitinated p27 (24,
29). Finally, the coincubation of cycloheximide-pretreated cells with
EGCG demonstrated an almost complete inhibition of p27 and I
B-
protein degradation by EGCG (Fig. 5).
B-
(41, 42) suppresses the
G1-to-S phase transition. If EGCG-accumulated p27 and
I
B-
proteins (Fig. 4) were functional, the treated tumor cells
should exhibit some growth arrest at G1. To test this
possibility, Jurkat T or LNCaP cells were treated with EGCG under the
similar conditions described in Fig. 4 and harvested for analysis of
cell cycle distribution. A 12-h treatment of Jurkat T cells with 10 µM EGCG increased G1 population by 12% (Fig.
6A), consistent with the
accumulation of p27 and I
B-
proteins under the same conditions
(Fig. 4A). A 24-h treatment with EGCG remained an ~10%
increase in G1 population (Fig. 6A). The
EGCG-induced Jurkat cell G1 arrest was detected in several
independent experiments that showed statistical significance (Fig.
6C, left).
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Fig. 6.
EGCG induces G1
arrest in Jurkat T and LNCaP cancer cells. Asynchronous (0 h)
Jurkat (A) or LNCaP tumor cells (B) were treated
with 10 µM EGCG for indicated hours. At each time point,
cells were harvested and analyzed by flow cytometry. Growth arrest is
determined by the increase in the percentage of the G1
population. C, statistical analysis. The results were
derived from 3-5 independent experiments, and p values were
calculated as indicated (*, p < 0.01 as compared with
respective 0 h; **, p < 0.05 as compared with
respective 0 h).
B-
accumulation at this time (Fig. 4D). EGCG treatment of LNCaP
cells for 24 and 36 h increased the G1 population by 12 and 24%, respectively (Fig. 6B, and C,
right). Again, the EGCG-mediated G1 arrest of LNCaP
prostate cancer cells was observed in multiple independent experiments
(p < 0.01, Fig. 6C, right).
These results support the functional significance of inhibition of the
proteasome activity in vivo (Fig. 3) and the accumulation of
p27 and I
B-
proteins by EGCG (Figs. 4 and 5). Our study is also
consistent with previous reports that overexpression of p27 or
I
B-
causes cell arrest in G1 (39-42).
B-
protein levels and cell cycle distribution.
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Fig. 7.
Preferable accumulation of p27 protein by
EGCG in the transformed fibroblasts over the normal human
fibroblasts. Normal (WI-38) and SV40-transformed
(VA-13) human fibroblasts were treated with 10 µM EGCG for the indicated hours followed by a Western
blot assay using specific antibodies to p27 (A), I B-
(B), and actin (C) as described in the legend of
Fig. 4. The band of 56 kDa, indicated by an arrow in
B, might be an I
B-
-related protein.
B-
protein: 2.7-fold at 12 h, 8.9-fold at 36 h, and 4.2-fold at 48 h (Fig. 7B). Between 36 and 48 h, the levels of
a p56 band associated with a decrease in I
B-
expression were
increased in these transformed cells (Fig. 7B, indicated by
an arrow), again suggesting that p56 is related to I
B-
(also see Fig. 4B). Although levels of I
B-
protein were low in the untreated normal WI-38 cells (0 h), a similar
p56 protein was highly expressed (Fig. 7B). A 12-h
treatment with EGCG increased the levels of I
B-
by 4.2-fold
without affecting the p56 levels (Fig. 7B). EGCG treatment of WI-38 cells for 36-48 h did not further increase the levels of
I
B-
, although under these conditions, the levels of p56 were decreased (Fig. 7B). As a control, actin levels were
relatively unchanged during EGCG treatment in both VA-13 and WI-38
cells (Fig. 7C).
B-
expression in these normal human fibroblasts (Fig. 7,
A and B). Previously, other researchers reported
that EGCG has a pronounced growth inhibitory effect on cancerous but
not on their normal counterparts (43). Our study has extended their
observation by providing a molecular mechanism for such a selectivity
of EGCG.
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Fig. 8.
Differential sensitivity of the transformed
fibroblasts and the normal human fibroblasts to EGCG-induced
G1 arrest. Normal (WI-380) (B)
and SV40-transformed (VA-13) (A) human
fibroblasts were treated with 10 µM EGCG for the
indicated hours followed by flow cytometry analysis. Growth arrest is
determined by the increase in the percentage of G1
population. C, statistical analysis. The results were
derived from five independent experiments, and p values were
calculated as indicated (*, p < 0.01 as compared with
respective 0 h; **, p < 0.05 as compared with
respective 0 h).
-lactone can be attacked
by the strong nucleophilic hydroxyl group of N-terminal threonine
residue of the proteasome, forming a covalent complex (32, 33). We hypothesized that the ester bond of EGCG would be attacked by the
N-terminal threonine residue of the proteasome, forming a covalent (or
tight) EGCG-proteasome complex, thus inactivating the proteasome. If
so, EGCG should be quickly lost, associated with production of no (or
little) cleavage products of EGCG such as gallic acid and EGC (for
review see Fig. 1A).
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Fig. 9.
HPLC chromatograms of EGCG after incubation
with purified 20S proteasome. EGCG or EGC was incubated with
either a purified 20S proteasome (Pr.) or the buffer
(buff.) for the indicated hours followed by HPLC analysis.
The retention times of reaction products were confirmed by using tea
polyphenol standards (Std.). For details, see under
"Experimental Procedures."
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Fig. 10.
The disappearance of EGCG occurs prior to
the appearance of gallic acid-like product after incubation with
purified 20S proteasome. EGCG was incubated with purified 20S
proteasome for indicated hours followed by HPLC analysis. The levels of
EGCG and gallic acid-like product were measured and plotted.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. A. B. Pardee and R. H. Goldfarb for critical reading of this manuscript, Drs. D. C. Eichler and L. P. Solomonson for permission to use the HPLC and for valuable discussion about HPLC data, Dr. R. Lush III for initial HPLC analysis, and the Lipton Co. for providing the tea extracts.
![]() |
FOOTNOTES |
---|
* This work is supported in part by a startup fund from H. Lee Moffitt Cancer Center & Research Institute and a pilot fund of Advanced Cancer Detection Center grant from the United States Army Medical Research and Materiel Command (to Q. P. D.).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.
To whom correspondence should be addressed: Drug Discovery
Program, H. Lee Moffitt Cancer Center & Research Inst., MRC
1259C, 12902 Magnolia Dr., Tampa, FL 33612-9497. Tel.:
813-632-1437; Fax: 813-979-6748; E-mail: douqp@moffitt.usf.edu.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M004209200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EGCG, ()
epigallocatechin-3-gallate;
EGC, (
)
epigallocatechin;
ECG, (
)
epicatechin-3-gallate;
EC, (
)
epicatechin;
GCG, (
)
gallocatechin-3-gallate;
GC, (
)
gallocatechin;
CG, (
)
catechin-3-gallate;
C, (
)
catechin;
AMC, 7-amido-4-methyl-coumarin;
PGPH, peptidyl-glutamyl peptide-hydrolyzing;
HPLC, high performance liquid chromatography;
I
B-
, inhibitor of
transciption factor NF-
B;
p70, 70 kDa;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
Z, benzyloxycarbonyl.
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