Departments of 1Pathology and 2Cellular Biology and Anatomy, Louisiana State University Health Sciences Center in Shreveport, Shreveport, Louisiana 71130
Submitted 31 October 2002 ; accepted in final form 12 March 2003
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ABSTRACT |
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apoptosis; antioxidants; fibrogenesis
Although underlying mechanisms remain incompletely understood, it is widely
accepted that oxidative stress plays crucial roles in HSC activation during
liver injury (17,
31,
48). Oxidative stress is
formed by an excessive production of reactive oxygen species, which are
generated endogenously by all aerobic cells as byproducts of a number of
metabolic reactions (16).
Oxidative stress has been implicated in many human diseases such as cancer,
cardiovascular diseases, and aging (reviewed by Halliwell; Ref.
22). Studies have shown that
oxidative stress stimulates HSC entry into S phase, nuclear factor
(NF)-B activation, and gene expression
(31). The antioxidant vitamin
E inhibits the activation of HSC
(31) and represses
iron-induced rat hepatic fibrogenesis
(41). The predominant
mechanism of antioxidant protective action is to destroy free radicals. The
therapeutic efficacy of current well-known antioxidants, including superoxide
dismutase, vitamin E, and ascorbic acid, in treatment of human hepatic
fibrosis is, however, generally unimpressive
(23). Many polyphenolic
compounds in plants, including those found in vegetables, fruits, wine, and
tea, exhibit antioxidant activities and are beneficial to human health.
The polyphenol compound curcumin is the main yellow pigment of a popular
spice, turmeric, and is widely used as a food colorant. Turmeric is the major
ingredient in curry. Besides its dietary use, turmeric has been used in
Chinese herbal medicine for skin and gut diseases and wound healing. Curcumin
is a potent antioxidant (44).
It has shown its ability to inhibit lipid peroxidation
(42,
47), nitric oxide synthetase
activity (4), production of
reactive oxygen species (28),
protein kinase C activity
(32), and NF-B activity
(46). Curcumin has received
attention as a promising dietary supplement for cancer prevention
(44) and liver protection
(11). A recent study indicated
that dietary administration of curcumin improved both acute and subacute rat
liver injury caused by carbon tetrachloride
(39). The protective
mechanisms of curcumin remain poorly addressed.
The peroxisome proliferator-activated receptors (PPARs) belong to the
superfamily of nuclear receptors
(21). PPAR forms heterodimers
with the retinoid X receptor and binds to specific response elements to induce
transcription in response to a variety of endogenous and exogenous ligands,
including fatty acids, arachidonic acid metabolites, and synthetic drugs, as
reviewed by Forman et al.
(15). Of the PPAR isoforms,
PPAR- is the most widely studied
(1). Previous studies indicated
that expression of PPAR-
inhibited PDGF-induced proliferation and
migration of vascular smooth muscle cells
(18). Three recent studies
independently demonstrated that the level of PPAR-
and its
trans-activating activity were diminished during HSC activation in
vitro, whereas NF-
B and activator protein-1 (AP-1) activities were
increased (19,
33,
36). PPAR-
ligands
inhibited cell proliferation and collagen-
1(I) expression in
primary HSC (34 days)
(36). The dramatic reduction
in the abundance of PPAR-
results in a significant decline in response
to exogenous PPAR-
ligands in activated HSC
(19,
33,
36). These findings implied a
potential therapeutic value of PPAR-
ligands in treatment of liver
fibrosis if the expression of PPAR-
can be induced in activated
HSC.
The aims of this study were to evaluate effects of curcumin on
culture-activated HSC growth and to begin exploring the underlying mechanisms.
Our results indicated that curcumin significantly inhibited cell proliferation
and induced apoptosis of activated HSC in vitro. In addition, we demonstrated,
for the first time, that curcumin dramatically induced the expression of
PPAR- at levels of transcription and translation as well as revived
PPAR-
trans-activating activity in activated HSC. Furthermore,
activation of PPAR-
by curcumin resulted in inhibition of transcription
factor NF-
B trans-activating activity. Blocking PPAR-
activation by a specific PPAR-
antagonist caused a marked reduction in
inhibition of activated HSC proliferation. Together, our results have
indicated that PPAR-
activation by curcumin plays critical and
significant roles in inhibition of activated HSC growth in vitro.
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MATERIALS AND METHODS |
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Lactate dehydrogenase release assays. Lactate dehydrogenase (LDH) assays were performed as recently described (9). In brief, preconfluent HSC were treated with curcumin at the indicated concentrations for 24 h. LDH in conditioned media was determined as medium LDH. LDH in cell lysates was analyzed as cellular LDH. LDH in DMEM with 10% FBS was defined as contamination arising from FBS and subtracted from medium and cellular LDH. LDH activities were determined by an LDH assay kit (Sigma). Results were shown as percentage of total LDH, i.e., medium LDH%/(medium LDH + cellular LDH).
Determination of cell growth. Semiconfluent HSC (5.5 x 104) grown in DMEM containing 10% FBS were treated with curcumin at the indicated concentrations for the indicated times. After cells were washed, cell growth was determined by attached cell numbers counted by a computer-equipped cell counter (Coulter, Miami, FL). Each treatment was given in triplicate. The experiment was repeated at least three times.
[3H]thymidine incorporation assays. The assay was performed as recently described (9). Briefly, semiconfluent HSC (5.5 x 104) grown in DMEM containing 10% FBS were treated with curcumin at the indicated concentrations for 24 h and subsequently pulsed for 4 h with methyl-[3H]thymidine (1 µCi/ml) (Amersham Life Science, Arlington Heights, IL). Whole lysates were mixed with the scintillation fluid Soluscint O (National Diagnostics, Highland Park, NJ) and were counted by a liquid scintillation analyzer. Results were expressed as counts per minute from triplicate experiments.
Bromodeoxyuridine staining. Preconfluent HSC in slide flasks were incubated in DMEM with 10% FBS with or without curcumin at 30 µM for the indicated times. Two hours before cells were harvested, bromodeoxyuridine (BrdU) was added at a final concentration of 25 µg/ml. Cells were fixed and stained by using a BrdU-staining kit from Zymed, following the protocol provided by the manufacturer.
Detection of apoptotic HSC by TUNEL. Preconfluent HSC cultured in DMEM with 10% FBS in slide flasks were treated with or without curcumin (30 µM) for the indicated times. Cells were washed three times with cold PBS before fixation. Apoptotic HSC were detected by the DeadEnd Colorimetric TUNEL System (Promega), following the protocol provided by the manufacturer.
Caspase-3 activity assays. Caspase-3 activities were measured by using a kit purchased from Promega and by following the protocol provided by the manufacturer. Briefly, semiconfluent HSC were treated with curcumin (30 µM) for the indicated times. Cell lysates were incubated with the substrate DEVD-p-nitroanilide (pNA) at 37°C for 6090 min. Results of the reaction were read by a spectrophotometer at 405 nm. The level of caspase enzymatic activity in the cell lysates is directly proportional to the color reaction. A standard calibration curve of pNA was established by a series dilution of pNA solution provided with the kit. Each treatment was performed at least three times.
Western blotting analysis. Whole cell extracts were prepared from preconfluent HSC. SDS-PAGE with 10% resolving gel was used to separate proteins (25 µg/well). Separated proteins were detected by using primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized by utilizing chemiluminescence reagent (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
Plasmids and transient transfection. The NF-B reporter
plasmid pNF-
B-Luc was purchased from Clontech Laboratories (Palo Alto,
CA). The PPAR-
reporter plasmid pPPRE-TK-Luc contains three copies of
the PPAR-
-response elements from acyl-CoA oxidase gene linked to the
herpes virus thymidine kinase promoter (105/+51) and luciferase vector,
which was a gift from Dr. Kevin J. McCarthy (Louisiana State University Health
Sciences Center in Shreveport). Semiconfluent HSC in six-well plastic plates
were transiently transfected using the LipofectAMINE reagent (Life
Technologies, Grand Island, NY). Each sample (3 µg/well) treatment had
three repeats in each experiment. Luciferase assays were performed as
previously described (8).
Transfection efficiency was determined by cotransfection of a
-galactosidase reporter, pSV-
-gal (0.5 µg/well; Promega).
-Galactosidase activities were measured by a chemiluminescence assay kit
(Tropix, Bedford, MA) according to the manufacturer's instructions. Results
were combined from three independent experiments.
Electrophoretic mobility shift assay. Electrophoretic mobility
shift assay (EMSA) was performed as previously described
(8). The integrity of nuclear
extracts was tested by EMSA with a 32P-labeled specificity
protein-1 (SP-1) consensus probe, resulting in distinct SP-1 shifts from all
extracts (data not shown). The NF-B probe containing consensus
NF-
B binding sites was purchased from Santa Cruz Biotechnology.
RNA isolation and real-time PCR. Total RNA was isolated by
TRI-Reagent (Sigma), following the protocol provided by the manufacturer.
Real-time PCR was carried out as recently described
(9). mRNA fold changes in
target genes relative to the endogenous GAPDH control were calculated as
suggested by Schmittgen et al.
(45). Primers used in
real-time PCR were: collagen-1(I) forward 5'-CCT CAA
GGG CTC CAA CGA G-3', reverse 5'-TCA ATC ACT GTC TTG CCC
CA-3';
-SMA forward 5'-CCG ACC GAA TGC AGA AGG A-3',
reverse 5'-ACA GAG TAT TTG CGC TCC GGA-3'; fibronectin forward
5'-TGT CAC CCA CCA CCT TGA-3', reverse 5'-CTG ATT GTT CTT
CAG TGC GA-3'; PPAR-
forward 5'-ATT CTG GCC CAC CAA CTT
CGG-3', reverse 5'-TGG AAG CCT GAT GCT TTA TCC CCA-3'; bcl-2
forward 5'-ATG GGG TGA ACT GGG GGA TTG-3', reverse 5'-TTC
CGA ATT TGT TTG GGG CAG GTC-3'; GAPDH forward 5'-GGC AAA TTC AAC
GGC ACA GT-3', reverse 5'-AGA TGG TGA TGG GCT TCC C-3'.
Statistical analysis. Differences between means were evaluated using an unpaired two-sided Student's t-test (P < 0.05 was considered significant). Where appropriate, comparisons of multiple treatment conditions with controls were analyzed by ANOVA with the Dunnett's test for post hoc analysis.
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RESULTS |
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Curcumin inhibits the proliferation of passaged HSC. To begin elucidating mechanisms underlying the inhibition of cultured HSC growth by curcumin, we hypothesized that this antioxidant might reduce HSC proliferation and/or induce HSC apoptosis. To test this hypothesis, cell proliferation was assessed by analyzing the incorporation of methyl-[3H]thymidine or BrdU into chromosomal DNA. Passaged HSC were treated with or without curcumin for 24 h at the indicated concentrations and pulsed with methyl-[3H]thymidine or BrdU for 4 or 2 h, respectively. As shown in Fig. 2A, compared with control (0 µM), curcumin at 30, 50, and 100 µM significantly reduced [3H]thymidine incorporation by 45.7, 50.6, and 52.9%, respectively, suggesting that curcumin, in a dose-dependent manner, inhibited DNA synthesis and cell proliferation. These results were confirmed by BrdU staining of cultured HSC (Fig. 2B). Western blotting analyses were carried out to further evaluate the effect of curcumin on the expression of proteins related to the cell cycle. As shown in Fig. 3, curcumin, in a time-dependent manner, markedly reduced the abundance of cell cycle-stimulating proteins, including cyclin D1, D2, and E. In addition, this polyphenol enhanced the levels of cell cycle inhibitory proteins, including p21WAF1/Cip1 and p27Kip1. Together, these results indicated that curcumin inhibited cell proliferation of activated HSC.
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Curcumin induces apoptosis in activated HSC. Additional experiments were performed to evaluate the effect of curcumin on HSC survival. Passaged HSC, treated with curcumin, were stained by TUNEL. As shown in Fig. 4A, curcumin increased the number of TUNEL-stained cells in a time-dependent manner, suggesting an increase in apoptotic cells. Caspase-3 is an executive enzyme for cell apoptosis. Caspase-3 activity assays demonstrated that curcumin, compared with control, significantly increased the enzyme activity by 6.2-fold after 24 h of treatment (Fig. 4B). Real-time PCR further showed that curcumin reduced the mRNA level of Bcl-2, an antiapoptotic protein, by 82% after 24 h of treatment (Fig. 4C). Together, these results demonstrated that curcumin induced time-dependent apoptosis in activated HSC.
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Curcumin reduces mRNA levels of
collagen-1(I), fibronectin, and
-SMA in passaged HSC. As mentioned earlier, HSC activation is
characterized by expression of
-SMA and overproduction of ECM
components, including collagen-
1(I) and fibronectin. Further
experiments were to evaluate the effect of curcumin on the expression of ECM
and
-SMA. Passaged HSC were treated with curcumin (30 µM) for the
indicated times. Total RNA was prepared for real-time PCR assays. Endogenous
GAPDH was used as an internal control. As shown in
Fig. 5, curcumin, in a
time-dependent manner, reduced the steady-state levels of mRNA of
collagen-
1(I), fibronectin, and
-SMA in passaged
HSC.
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Curcumin induces the expression of PPAR- and activates
its trans-activating activity in HSC. Additional studies were focused on
elucidating the mechanisms by which the antioxidant curcumin inhibited cell
proliferation of activated HSC. Inhibition of cell growth and proliferation by
activation of PPAR-
has been reported in several cell types
(20,
30). Recent studies have shown
that PPAR-
is highly expressed in quiescent HSC in normal livers
(19,
33,
36). However, its abundance
and its trans-activating activity are diminished during HSC
activation (19,
33,
36). PPAR-
ligands
inhibit HSC proliferation and collagen-
1(I) expression in
vitro (36). The antioxidants
troglitazone and
-tocopherol induce the expression of PPAR-
in
nonadipose tissues and cell lines
(12,
14,
40). These results prompted us
to hypothesize that the antioxidant curcumin might induce the expression of
PPAR-
in activated HSC in vitro and that activation of PPAR-
might contribute to the growth-inhibitory effect of curcumin on activated HSC.
To test the hypotheses, initial experiments were designed to determine the
capability of curcumin in inducing the expression of PPAR-
and in
activating its trans-activating activity in activated HSC in vitro.
Passaged HSC were treated with curcumin at 30 µM for the indicated times.
Total RNA or protein extracts were prepared from the cells. As demonstrated by
real-time PCR in Fig.
6A, compared with no treatment (0 h), curcumin
significantly enhanced the abundance of PPAR-
mRNA by 145, 210, and
390% in passaged HSC after the treatment for 8, 16, and 24 h, respectively.
Western blotting analyses confirmed the role of curcumin in increasing the
protein level of PPAR-
in passaged HSC
(Fig. 6B). These
results collectively demonstrated that curcumin induced the expression of
PPAR-
in activated HSC.
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Further experiments were to verify the effect of curcumin on stimulating
the trans-activating activity of PPAR-. Passaged HSC were
transfected with the PPAR-
reporter plasmid pPPRE-TK-Luc, containing
three copies of PPAR-
response elements inserted into a luciferase
reporter vector. After recovery, cells were treated with various
concentrations of curcumin or PGJ2, a natural PPAR-
ligand.
As shown in Fig. 6C,
compared with control (0 µM), curcumin at 20, 30, and 50 µM caused
dose-dependent, significant increases in luciferase activity by 210, 450, and
510%, respectively. PGJ2 alone at 5 and 10 µM, however, led to
relatively smaller increases in luciferase activity by 150 and 190%,
respectively, suggesting that PGJ2 might only activate endogenous
PPAR-
expressed at a relatively low level in activated HSC.
PGJ2 does not induce PPAR-
gene expression in activated HSC
(data not shown). Further transfection analyses demonstrated that pretreatment
of the transfected cells with PD-68235 (20 µM), a specific PPAR-
antagonist (5), apparently
blocked the increase in luciferase activity induced by curcumin (30 µM),
whereas PD-68235 itself had no significant effect on luciferase activity
(Fig. 6D). In
addition, compared with cells treated with curcumin or PGJ2 alone,
cells treated with both curcumin and PGJ2 showed a further
significant increase in luciferase activity, which was completely abolished by
PD-68235 pretreatment (Fig.
6D). Together, these results demonstrated that curcumin
induced the expression of PPAR-
and activated its
trans-activating activity in activated HSC. It is presumed that
PPAR-
ligands exist in the media with 10% FBS
(34).
Activation of PPAR- plays a critical role in inhibition
of NF-
B activity by curcumin. Transcription factor
NF-
B has been described as a primary regulator and mediator of
oxidative stress. Although the causal relationship remains unknown, previous
studies demonstrated that activation and survival of HSC were closely
associated with the activation of NF-
B
(24,
31,
43). It was, therefore, of
interest to elucidate the effect of the antioxidant curcumin on NF-
B
activity in HSC. Passaged HSC were treated with curcumin at 30 µM for the
indicated times. Nuclear extracts were prepared for EMSA using a
32P-labeled probe containing consensus NF-
B binding sites.
As shown in Fig. 7A,
compared with no treatment (lane 3), curcumin caused an apparent
reduction in the density of the protein-DNA complex in a time-dependent
pattern (lanes 1 and 2). Fiftyfold excess of the unlabeled
probe competitively caused a marked, if not complete, reduction in the binding
band (lane 4), suggesting the specificity of the protein binding to
the probe. Anti-p50 antibodies, but not normal rabbit IgG (data not shown),
resulted in a significant supershift and abolished the NF-
B binding
band (lane 5). This result illustrated that curcumin reduced
NF-
B DNA binding activity in passaged HSC. Further experiments were
carried out to evaluate effects of curcumin on NF-
B
trans-activating activity. HSC were transfected with the NF-
B
reporter plasmid pNF-
B-Luc and were treated with or without curcumin at
indicated concentrations (Fig.
7B). Luciferase assays demonstrated that curcumin, in a
dose-dependent manner, significantly reduced luciferase activities, indicating
that curcumin inhibited NF-
B trans-activating activity in
cultured HSC. Previous studies have demonstrated an association between
activation of PPAR-
and inhibition of NF-
B
(10,
26,
50). It is plausible to
evaluate roles of PPAR-
activated by curcumin in inhibition of
NF-
B in activated HSC. As demonstrated in
Fig. 7C, pretreatment
of cells with the specific PPAR-
antagonist PD-68235 abolished, in a
dose-dependent manner, the effect of curcumin (30 µM) on inhibition of
NF-
B trans-activating activity. PD-68235 itself increased,
although not significantly, luciferase activities at both 5 and 20 µM,
which might result from blockade of endogenous PPAR-
activation by the
antagonist. Together, these results demonstrated that the antioxidant curcumin
inhibited NF-
B activities mediated by activation of PPAR-
in
activated HSC.
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Blocking the activation of PPAR- abrogates the
growth-inhibitory effect of curcumin on HSC. Additional experiments were
carried out to examine our hypothesis that inhibition of activated HSC growth
by curcumin might be mediated by activation of PPAR-
. Semiconfluent HSC
were pretreated with or without the specific PPAR-
antagonist PD-68235
at the indicated concentrations for 30 min before the addition of curcumin (30
µM) for an additional 24 h. Cells or cell extracts were prepared for
determination of cell numbers or Western blotting analyses, respectively. As
demonstrated in Fig.
8A, curcumin significantly reduced cell numbers, as
expected. PD-68235 itself at 10 or 20 µM had no detectable effect on cell
numbers. Pretreatment of cells with PD-68235 apparently abrogated the
inhibitory effect of curcumin on cell numbers, indicating that blocking the
activation of PPAR-
by PD-68235 abolished the growth-inhibitory effect
of curcumin on activated HSC proliferation. We have demonstrated that curcumin
altered the expression of proteins related to the cell cycle
(Fig. 3). Further experiments
were aimed at the role of PPAR-
activated by curcumin in the expression
of cell cycle-related proteins (Fig.
8B). Compared with control (lane 1), Western
blotting analyses confirmed that curcumin reduced the protein levels of cyclin
D1 and cyclin E (lane 2). PD-68235 alone at either 10 or
20 µM had no evident effect on the protein levels (lanes 3 and
4). However, PD-68235 pretreatment dramatically abolished the role of
curcumin in reducing the protein levels of cyclin D1 and cyclin E
(lanes 5 and 6). Similarly, PD-68235 abrogated the role of
curcumin in increasing the protein level of p21WAF1/Cip1 (lanes
5 and 6), a cell cycle-inhibitory protein. These results implied
that PPAR-
activation might be involved in the inhibitory effect of
curcumin on cell growth by alteration of levels of proteins involved in cell
cycles.
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Troglitazone further enhances the curcumin effect on inhibition of
activated HSC. We have observed that, compared with either curcumin or
PGJ2 alone, the combination of curcumin and PGJ2
resulted in a further increase in luciferase activity in HSC transfected with
pPPRE-TK-Luc (Fig.
6D). We assumed that the PPAR- ligand might take
advantage of the increase in the abundance of PPAR-
induced by curcumin
and further stimulate the receptor trans-activating activity. To
study this assumption, passaged HSC were pretreated with curcumin at the
indicated concentrations for 24 h to increase the abundance of PPAR-
.
Cells were subsequently treated with or without troglitazone (10 µM), a
synthetic PPAR-
ligand, for an additional 24 h. As shown in
Fig. 9, without curcumin
pretreatment, troglitazone itself did not significantly reduce cell numbers.
In contrast, with curcumin pretreatment at 10 or 30 µM, in addition to the
inhibition by curcumin, troglitazone caused a further significant reduction in
cell numbers by 27.5 or 36.4%, respectively. MTS assay (Promega), a
colorimetric method for determining the number of viable cells, obtained a
similar result. These results demonstrated that troglitazone further enhanced
the curcumin effect on inhibition of activated HSC and implied that
troglitazone might take advantage of the increase in the level of PPAR-
induced by curcumin to further inhibit the proliferation of activated HSC.
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DISCUSSION |
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Previous studies have suggested that D-type cyclins might play critical
roles in cell cycle progression, especially at the early
G0/G1 phase
(25,
38). Inhibition of cyclin
D1 expression by microinjection of anti-cyclin D1
antibodies or antisense cyclin D1 cDNA prevented cells from
entering S phase (2). In this
study, we observed that curcumin significantly altered the expression of
proteins related to the cell cycle in activated HSC. This antioxidant markedly
reduced the abundance of cell cycle-stimulating proteins, including cyclin
D1,D2, and E. In addition, this polyphenol increased the
protein levels of cell cycle-inhibitory proteins, including
p21WAF1/Cip1 and p27Kip1, in passaged HSC. A recent
study demonstrated that cyclin D1, D2, and E played a
key role in the transition of the HSC cell cycle from G1 to S phase
(29). Interestingly, blockade
of PPAR- activation by the antagonist PD-68235 dramatically, if not
completely, abrogated the ability of curcumin to alter the expression of cell
cycle-related proteins, suggesting that alteration of the expression of cell
cycle-related proteins by curcumin might be mediated by PPAR-
. This
result was consistent with a previous observation that PPAR-
activated
by either natural (PGJ2 and PGD2) or synthetic ligands
(BRL-49653 and troglitazone) selectively inhibited the expression of cyclin
D1 gene mediated by AP-1
(49).
We had previously demonstrated that curcumin blocked JNK activation and inhibited AP-1 activity in passaged HSC (7). We recently observed that another antioxidant ()-epigallocatechin-3-gallate (EGCG), a major and active component in green tea extracts, also inhibited passaged HSC proliferation and altered the expression of proteins related to the cell cycle (9). Further experiments are necessary to elucidate the mechanisms by which antioxidants regulate the expression of cell cycle-related proteins.
The reduction of levels of PPAR- is coupled with the activation of
HSC (19,
33,
36), implying a role of
PPAR-
in inhibiting the activation of HSC. In this study, we
demonstrated, for the first time, that the antioxidant curcumin induced the
expression of PPAR-
and revived the activation of its
trans-activating activity in activated HSC in vitro. Induction of
PPAR-
expression is not unique to curcumin. Recent studies showed a
unique capability of troglitazone among thiazolidinediones of inducing the
expression of PPAR-
in nonadipose tissues and cell lines
(12,
40). The unique antioxidant
-tocopherol moiety in the chemical structure of troglitazone was
assumed to be responsible for it
(13,
27). Further studies
demonstrated that
-tocopherol was also able to induce PPAR-
expression (14). We recently
observed that the antioxidant EGCG in green tea extracts also induced the
expression of PPAR-
in activated HSC (Chen and Zhang, unpublished
observations). The mechanisms by which antioxidants induce PPAR-
gene
expression in activated HSC remain poorly understood.
Activation of PPAR- by curcumin makes a significant contribution to
the inhibitory effect of the antioxidant on activated HSC growth. Blocking
PPAR-
activation by PD-68235 significantly, if not completely,
abolished the inhibitory effects of curcumin on cell growth. PPAR-
has
shown its ability to inhibit cell growth and to regulate gene expression in
several cell types, including HSC
(19,
20,
30,
33,
36). The PPAR-
ligands
troglitazone and PGJ2 significantly decreased PDGF-induced
proliferation in activated human HSC and inhibited
-SMA expression
during HSC activation (19).
Our preliminary results suggested that activation of PPAR-
might be
involved in inhibition of the expression of the
-SMA gene, but not the
collagen-
1(I) and fibronectin genes, by curcumin in
activated HSC (Xu and Chen, unpublished data). It is presumed that
PPAR-
ligands existing in media with 10% FBS initiate the activation of
PPAR-
induced by curcumin in activated HSC. This assumption is
supported by a recent observation that platelet-derived lysophosphatidic acid
in serum is a transcellular PPAR-
agonist
(34). The effect of curcumin
on production of endogenous PPAR-
ligand(s) is completely unknown.
NF-B has been described as a primary regulator and mediator of
oxidative stress. It has been implicated in cell proliferation, cell cycle
regulation, and apoptosis
(35). Although the causal
relationship remains unknown, previous studies demonstrated that activation
and survival of HSC were closely associated with activation of NF-
B
(24,
31,
43). It was suggested that
inhibition of NF-
B activation might be a potential strategy for
prevention and/or treatment of hepatic fibrogenesis
(24). In the present studies,
we demonstrated that the antioxidant curcumin reduced NF-
B activity in
cultured HSC. Previous studies have shown that activation of PPAR-
resulted in inhibition of NF-
B activity
(10,
26,
50). Experiments in this study
demonstrated that the PPAR-
antagonist PD-68235 abrogated the
inhibitory effect of curcumin on NF-
B trans-activating
activity. These results suggested, for the first time, that the inhibitory
effect of curcumin on NF-
B activity in activated HSC might be mediated
by PPAR-
activation, which provided a novel insight into the mechanism
of the inhibitory effects of curcumin on NF-
B activity. Further studies
are necessary to evaluate whether it is unique in activated HSC. It remains
incompletely understood how PPAR-
activation could result in inhibition
of NF-
B. It was previously reported that PPAR-
agonists,
including Wy-14643, clofibrate, carbaprostacyclin, and ciglitazone, inhibited
NF-
B activity by prevention of I
B from phosphorylation and
subsequent degradation in cytokine-stimulated mesangial cells
(6). A recent study showed that
overexpression of the coactivator p300 restored NF-
B
trans-activating activity suppressed by a PPAR-
ligand
(37), implying that
PPAR-
might interfere with NF-
B trans-activating
activity via coactivator competition. Additional studies are necessary to
elucidate the causal relationship among NF-
B activities, PPAR-
activation, and the expression of genes regulated by curcumin.
In summary, our results demonstrated that the antioxidant curcumin
inhibited the growth of passaged HSC by reducing cell proliferation and
inducing apoptosis. In addition, the polyphenol suppressed the activation of
passaged HSC demonstrated by repressing the activity of NF-B and
reducing mRNA levels of collagen-
1(I) and fibronectin, as
well as
-SMA. Furthermore, we reported, for the first time, that
curcumin dramatically induced the expression of PPAR-
and revived its
trans-activating activity in activated HSC in vitro. Activation of
PPAR-
contributed to the inhibitory effect of curcumin on activated HSC
proliferation. It should be emphasized that the results in this study were
generated from cultured HSC and that they might not necessarily and
comprehensively reflect facts in quiescent HSC in vivo. Further experiments,
beyond the scope of this study, are required to elucidate the underlying
mechanisms of PPAR-
in inhibition of HSC proliferation. Our results in
this study provided a novel insight into mechanisms of inhibition of activated
HSC proliferation by antioxidants, including curcumin. The characteristics of
curcumin, including antioxidant potential, inhibition of activated HSC
proliferation, induction of apoptosis, activation of PPAR-
, as well as
the long history of dietary consumption of curry without adverse health
effects, make it a potential antifibrotic candidate for treatment and
prevention of hepatic fibrogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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