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B, c-Jun N-Terminal Kinase, and Apoptosis1
Departments of
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Molecular Oncology and
Bone Marrow Transplantation, Cytokine Research Laboratory, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, a nuclear transcription factor, which regulates the expression
of various genes involved in inflammation, cytoprotection, and
carcinogenesis. In this report, we investigated the effect of silymarin
on NF-B activation induced by various inflammatory agents. Silymarin
blocked TNF-induced activation of NF-B in a dose- and time-dependent
manner. This effect was mediated through inhibition of phosphorylation
and degradation of IB
, an inhibitor of NF-B. Silymarin
blocked the translocation of p65 to the nucleus without affecting its
ability to bind to the DNA. NF-B-dependent reporter gene
transcription was also suppressed by silymarin. Silymarin also blocked
NF-B activation induced by phorbol ester, LPS, okadaic acid, and
ceramide, whereas H2O2-induced NF-B
activation was not significantly affected. The effects of silymarin on
NF-B activation were specific, as AP-1 activation was unaffected.
Silymarin also inhibited the TNF-induced activation of
mitogen-activated protein kinase kinase and c-Jun N-terminal kinase and
abrogated TNF-induced cytotoxicity and caspase activation. Silymarin
suppressed the TNF-induced production of reactive oxygen intermediates
and lipid peroxidation. Overall, the inhibition of activation of
NF-B and the kinases may provide in part the molecular basis for the
anticarcinogenic and anti-inflammatory effects of silymarin, and
its effects on caspases may explain its role in
cytoprotection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Besides its hepatoprotective principle, silymarin is a potent chemopreventive agent (13, 14, 15). It provides substantial protection against different stages of UVB-induced carcinogenesis (14) and blocks phorbol ester-induced tumor promotion (15). How silymarin mediates its anticarcinogenic effects is not fully understood, but it has been shown that some of the effects are mediated through inhibition of receptor tyrosine kinases (16), cyclin-dependent kinases (17, 18), TNF mRNA expression (19), and ornithine decarboxylase activity (13). Besides anticarcinogenic effects, silymarin also exerts anti-inflammatory action in vivo (20).
Because silymarin exhibits anticarcinogenic, anti-inflammatory, and
growth-modulatory effects, much as TNF does, we hypothesized that these
effects of silymarin are mediated through suppression of NF-B
activation, the mediator of many of the TNF effects. Numerous lines of
evidence suggest this possibility. For example, various agents that
promote tumorigenesis are known to activate NF-B (21),
including phorbol ester, okadaic acid, and TNF. In addition, several
genes that are involved in tumorigenesis, metastasis, and inflammation
are regulated by NF-B (21). Recent reports indicate
that NF-B protects cells from undergoing apoptosis
(22). The activation of NF-B is regulated by several
kinases which belong to the mitogen-activated protein kinase (MAPK)
family (23). Furthermore, activators of NF-B are also
known to induce apoptosis (21). The activation of NF-B
and kinases in most cases is dependent on the production of reactive
oxygen species (21, 22, 23).
Because silymarin has been described to be an antioxidant with
anti-inflammatory, cytoprotective, and anticarcinogenic effects, we
tested the hypothesis that these effects are mediated through its
modulation of activation of NF-B, members of the MAPK, and
caspase-mediated apoptosis. Our results demonstrate that silymarin is a
potent inhibitor of NF-B activation. It also inhibits TNF-induced
c-Jun N-terminal protein kinase (JNK) and MAPK kinase (MEK) activation
and caspase-induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Silymarin was obtained from Aldrich Chemical (Milwaukee, WI) and
was dissolved in 100% DMSO at 48 mg/ml (100 mM; m.w. = 482.4). All
subsequent dilutions were made in the media. Antibiotics-antimycotics
(contains penicillin, streptomycin, and amphotericin B), RPMI 1640
medium, and FBS were obtained from Life Technologies (Grand Island,
NY). Glycine, PMA, LPS, ceramide, NaCl, thiobarbituric acid, calpain
inhibitor I (ALLN, N-acetylleucylleucylnorleucinal), and BSA
were obtained from Sigma (St. Louis, MO). Bacteria-derived recombinant
human TNF, purified to homogeneity with a specific activity of 5
x 107 U/mg, was kindly provided by Genentech
(South San Francisco, CA). Ab against IB and double-stranded
oligonucleotide having the AP-1 consensus sequence were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-IB
(Ser32) Ab was purchased from New England Biolabs
(Beverly, MA). Poly(ADP) ribose polymerase (PARP) Ab was purchased from
PharMingen (San Diego, CA). The rat MDR1bCAT plasmid, 243RMICAT,
containing the chloramphenicol acetyltransferase (CAT) gene with either
wild-type or mutated NF-B binding site was kindly supplied by Dr. M.
Tien Kuo (University of Texas M. D. Anderson Cancer Center,
Houston, TX). The characterization of these plasmids has been described
previously in detail (24). The fluorescent reactive oxygen
intermediate (ROI) probe dihydrorhodamine 123 purchased from Molecular
Probes (Eugene, OR) was supplied by Dr. M. Tien Kuo.
Cell lines
Most of the studies were performed with human histiocytic lymphoma U-937 cells because various cellular response on these cells are well characterized in our laboratory (25, 26, 27, 28). U-937 (human histiocytic lymphoma), Jurkat (T cells), and HeLa (epithelial cells) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). ML-1a (myeloid cells) was a gift from Dr. Ken Takeda (Showa University, Showa, Japan). Cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1x antibiotic-antimycotics. Cells were free from Mycoplasma as detected by the Gen-Probe Mycoplasma Rapid Detection kit (Fisher Scientific, Pittsburgh, PA).
NF-B activation assay
To assay NF-B activation, EMSA were conducted essentially as
described (25). Briefly, nuclear extracts prepared from
TNF-treated cells (2 x 106/ml) were
incubated with 32P end-labeled 45-mer
double-stranded NF-B oligonucleotide (6 µg protein with 16 fmol
DNA) from the HIV-LTR,
5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3'
(bold indicates NF-B binding sites) for 15 min at 37°C, and the
DNA-protein complex formed was resolved from free oligonucleotide on
6.6% native polyacrylamide gels. A double-stranded mutated
oligonucleotide,
5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGG
CGTGG-3', was used to examine the specificity of binding of
NF-B to the DNA. The specificity of binding was also examined by
competition with the unlabeled oligonucleotide. The dried gels were
visualized, and radioactive bands were quantitated by a PhosphorImager
using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
AP-1 activation assay
To assay AP-1 activation, 6 µg nuclear extract prepared as indicated above was incubated with 16 fmol of the 32P-end-labeled AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (bold indicates AP-1 binding site) for 15 min at 37°C and was analyzed using 6% native polyacrylamide gel. The specificity of binding was examined by competition with unlabeled oligonucleotide. Visualization and quantitation of radioactive bands were done as indicated above.
Western blot for IB
To assay IB, postnuclear (cytoplasmic) extracts were
prepared (26) from TNF-treated cells either in the
presence or absence of ALLN, a calpain inhibitor, and then resolved on
10% SDS-polyacrylamide gels. After electrophoresis, the proteins were
electrotransferred to nitrocellulose filters, probed with rabbit
polyclonal Abs against either IB, or IB phosphorylated at
serine 32, and detected by enhanced chemiluminescence (Amersham
Pharmacia Biotech, Piscataway, NJ).
c-Jun kinase assay
The c-Jun kinase assay was performed by a modified method as
described earlier (27). Briefly, after treatment of cells
(3 x 106/ml) with TNF for 10 min, cell
extracts were prepared by lysing cells in buffer containing 20 mM HEPES
(pH 7.4), 2 mM EDTA, 250 mM NaCl, 1% Nonidet P-40, 2 µg/ml
leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and
1 mM DTT. Cell extracts (150–250 µg/sample) were immunoprecipitated
with 0.03 µg anti-JNK Ab for 60 min at 4°C. Immune complexes
were collected by incubation with protein A-G Sepharose beads for 45
min at 4°C. The beads were washed with lysis buffer (4 x 400
µl) and kinase buffer (2 x 400 µl: 20 mM HEPES (pH 7.4), 1 mM
DTT, and 25 mM NaCl). Kinase assays were performed for 15 min at 30°C
with GST-Jun1–79(1–79) as a substrate (2 µg/sample) in 20 mM HEPES
(pH 7.4), 10 mM MgCl2, 1 mM DTT, and 10 µCi
[
-32P]ATP. Reactions were stopped with the
addition of 15 µl of 2x SDS sample buffer, boiled for 5 min and
subjected to SDS-PAGE (9%). GST-Jun1–79(1–79) was visualized by staining
with Coomassie blue, and the dried gel was analyzed by a PhosphorImager
(Molecular Dynamics).
Transient transfection and CAT assay
To measure TNF-induced NF-B-mediated reporter gene
transcription, U-937 cells were transiently transfected by the calcium
phosphate method with the plasmids 243RMICAT (contains wild-type
NF-B binding site) and -243RMICAT-m (mutated binding site)
according to the instructions supplied by the manufacturer (Life
Technologies). After 12 h of transfection, the cells were
stimulated with different concentrations of TNF for 2 h, washed,
and examined for CAT activity as described (24).
Cytotoxicity assay
Cytotoxicity assays were performed as indicated previously (28). Briefly, U-937 cells (5 x 103/0.1 ml) were pretreated with 50 µM silymarin for 2 h and then exposed to different concentrations of TNF for 72 h at 37°C in a CO2 incubator. Cell viability was then determined by the MTT dye uptake assay by incubating the cells with the MTT dye (25 µl of 5 mg/ml) for 2 h at 37°C. We lysed the granules in lysis buffer (20% SDS in 50% dimethylformamide) by overnight incubation at 37°C and detected the absorbance at 590 nm using a 96-well multiscanner autoreader (Dynatech MR 5000; Dynatech Laboratories, Chantilly, VA).
Immunoblot analysis of PARP degradation
TNF-induced apoptosis was examined by proteolytic cleavage of PARP (28). Briefly, U-937 cells (2 x 106/ml) were pretreated with different concentrations of silymarin for 2 h and then activated with 1 nM TNF in the presence of cycloheximide (2 µg/ml). After 2 h at 37°C, cell extracts were prepared by incubating the cells for 30 min on ice in 0.05 ml buffer containing 20 mM HEPES (pH 7.4), 2 mM EDTA, 250 mM NaCl, 0.1% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM PMSF, 0.5 µg/ml benzamidine, and 1 mM DTT for 30 min. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 µg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with mouse anti-PARP Ab, and then detected by enhanced chemiluminescence (Amersham). Apoptosis was represented by the cleavage of 116-kDa PARP into a 85-kDa peptide product.
Determination of lipid peroxidation
TNF-induced lipid peroxidation was determined by detection of thiobarbituric acid-reactive malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters, as described (29). U-937 cells (3 x 106/ml) pretreated with either media or silymarin (50 µM) for 2 h were stimulated with different concentrations of TNF for 1 h. Then cells were washed with PBS before undergoing three cycles of freeze thawing in 200 µl water. After protein determination, 300 µg protein (in 0.1 ml) was added to 800 µl assay mix containing 0.4% (w/v) thiobarbituric acid, 0.5% (w/v) SDS, and 9.4% (v/v) acetic acid (pH 3.5). After incubation for 1 h at 95°C, samples were cooled to room temperature, centrifuged at 14,000 x g for 10 min, and the absorbance of the supernatants was read at 532 nm. Results were normalized with the amount of MDA equivalents/mg protein and expressed as a percentage of thiobarbituric acid-reactive substances above control values. Untreated cells showed 0.571 ± 0.126 nmol of MDA equivalents/mg protein.
Measurement of ROI
The production of ROI on treatment of cells with TNF was determined by flow cytometry as described (30). Briefly U-937 cells (5 x 105) were incubated either with RPMI 1640 medium supplemented with 10% FBS or with media containing 50 µM silymarin for 2 h at 37°C. Cells were then stimulated with 1 nM TNF for different times, washed with D-PBS, and resuspended in 1 ml D-PBS. To detect ROI production, cells were exposed to dihydrorhodamine 123 (5 mM stock in DMSO) at a final concentration of 1 µM for 1 h at 37°C with moderate shaking (100 rpm) and then washed with D-PBS three times and resuspended in 1 ml D-PBS. Rhodamine 123 fluorescence intensity resulting from dihydrorhodamine 123 oxidation was measured by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) with excitation at 488 nm and was detected between 515 and 550 mm. Data analysis was performed using LYSYSII software (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Silymarin inhibits TNF-induced NF-B activation
U-937 cells were pretreated with indicated concentrations of
silymarin for 2 h and then stimulated with 100 pM TNF for 30 min,
and nuclear extracts were prepared and assayed for NF-B by EMSA. As
shown in Fig. 1
A, silymarin
inhibited TNF-mediated NF-B activation in a dose-dependent manner
with maximum inhibition at 50 µM. Silymarin or the DMSO solvent
(0.4% v/v) by themselves did not activate NF-B. We next tested the
length of incubation required for silymarin to block TNF-induced
NF-B activation. The cells were incubated with silymarin for 120
min, 60 min, 30 min, and 15 min before the addition of TNF, at the same
time as the addition of TNF, or 5, 15, and 30 min after the addition of
TNF. The cells were treated with TNF for 30 min. Only when the cells
were pretreated for 120 min with silymarin was maximum inhibition of
NF-B activation observed, and the inhibition decreased gradually
with a shorter preincubation time (Fig. 1B). Cotreatment or
posttreatment with silymarin did not inhibit NF-B activation (Fig. 1B). Previous studies from our laboratory have shown that a
high concentration of TNF (10 nM) can activate NF-B within 5 min,
and this induction is more intense than that obtained with cells using
a 100-fold lower concentration of TNF for a longer time
(25). To determine the effect of silymarin on NF-B
activation at even higher concentration, both untreated and
silymarin-pretreated cells were incubated with various concentrations
of TNF (0–10,000 pM) for 30 min, and then the NF-B was assayed by
EMSA. Although the activation of NF-B by 10,000 pM of TNF was
strong, silymarin inhibited it just as efficiently as it did the 0.1 nM
TNF concentration (Fig. 1C). These results suggest that
silymarin is a very potent inhibitor of NF-B activation.
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B inhibited by silymarin consists of p50 and p65
subunits
Various combinations of Rel/NF-B proteins can constitute an
active NF-B heterodimer that binds to specific sequences in DNA
(21). To show that the retarded band visualized by EMSA in
TNF-treated cells was indeed NF-B, we incubated nuclear extracts
from TNF-activated cells with Ab to either p50 (NF-BI) or p65 (Rel
A) subunits and then conducted EMSA. Abs to either subunit of NF-B
shifted the band to a higher m.w. (Fig. 2A), thus suggesting that the
TNF-activated complex consists of p60 and p65 subunits. Neither
preimmune serum nor such irrelevant Abs as anti-cRel or
anti-cyclin DI had any effect on the mobility of NF-B. Excess
unlabeled NF-B (100-fold) caused complete disappearance of the band,
indicating the specificity of NF-B.
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B proteins
It has been shown that both
N-tosyl-L-phenylalanyl chloromethyl ketone, a
serine protease inhibitor, and herbimycin A, a protein tyrosine kinase
inhibitor, down-regulate NF-B activation by modifying the NF-B
subunits, thus preventing its binding to DNA (31, 32). To
determine whether silymarin also directly modifies NF-B proteins, we
incubated cytoplasmic extracts from untreated cells, those treated with
deoxycholate (DOC) (0.8%) for 15 min at room temperature, or nuclear
extracts from TNF-triggered cells with various concentrations of
silymarin and then measured DNA-binding activity using EMSA. The DOC
treatment has been shown to dissociate the IB subunit, thus
releasing NF-B for binding to the DNA. As seen in Fig. 2
, B and C, silymarin did not modify the DNA-binding
ability of NF-B proteins prepared by treatment with either DOC or
TNF. Therefore, silymarin’s inhibition of NF-B activation is not
due to interference with its DNA binding, thus distinguishing its
activity from that of N-tosyl-L-phenylalanyl
chloromethyl ketone or herbimycin A.
Inhibition of NF-B activation by silymarin is not cell-type
specific
Besides U-937 cells, we also examined the ability of silymarin to
block TNF-induced NF-B activation in other myeloid (ML-1a), lymphoid
(Jurkat), and epithelial (HeLa) cells (Fig. 3). The results of these experiments
indicate that silymarin inhibited NF-B
activation in all cell types, thus suggesting that this effect of
silymarin is not cell-type specific. Almost complete inhibition,
however, required 10- to 100-fold M excess of silymarin.
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B
Besides TNF, NF-B is also activated by a wide variety of other
agents including phorbol ester, LPS, okadaic acid, and ceramide
(21). However, the signal transduction pathway induced by
these agents differ. We therefore examined the effect of silymarin on
the activation of NF-B by these various agents. The results shown in
Fig. 4 indicate that silymarin completely blocked the activation of
NF-B induced by all those agents except
H2O2. The activation of
NF-B by H2O2 was
partially affected. These results suggest that silymarin may act at a
step where all these agents (except
H2O2) converge in the
signal transduction pathway leading to NF-B activation.
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B
The translocation of NF-B to the nucleus is preceded by the
phosphorylation and proteolytic degradation of IB
(23). To determine whether the inhibitory action of
silymarin was due to an effect on IB degradation, the cytoplasmic
levels of IB proteins were examined by Western blot analysis.
IB began to degrade 5 min after TNF treatment of U-937 cells and
disappeared completely within 15 min. The band reappeared by 30 min.
The pretreatment of cells with silymarin completely abolished the
TNF-induced degradation of IB (Fig. 5A).
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B degradation by
blocking its phosphorylation. The serine phosphorylation of IB
induced by TNF was stabilized by pretreatment of cells for 1 h
with ALLN, a proteosome inhibitor (33). The
hyperphosphorylated form of IB appeared as a slow migrating band
on SDS-PAGE (Fig. 5B), which disappeared when cells were
pretreated with silymarin (compare lane 4 with lane
8), indicating that silymarin blocks IB phosphorylation.
This was further examined by the use of Abs which detect only serine
phosphorylated form of IB. These results shown in Fig. 5C further confirm that TNF-induces IB phosphorylation
and silymarin inhibits it quite effectively.
Pretreatment of cells with silymarin also completely abolished the
TNF-induced nuclear translocation of p65 (Fig. 5D).
Silymarin represses NF-B-CAT reporter gene expression
To determine the effect of silymarin on TNF-induced
NF-B-dependent reporter gene expression, the promoter of the rat
mdr1b gene containing a NF-B binding site linked to the
CAT reporter gene was used. U-937 cells were transiently
transfected with the CAT reporter construct and then stimulated with
TNF either in the presence or absence of silymarin. An almost 5-fold
increase in CAT activity was noted upon stimulation with TNF (Fig. 6). However, TNF-induced CAT activity was
reduced significantly when the cells transfected with the wild-type
NF-B sequence were pretreated with silymarin for 2 h before TNF
treatment. Transfection with the MDR gene containing the
mutated NF-B binding site did not result in induction of CAT by TNF.
These results demonstrate that silymarin also represses
NF-B-dependent gene expression induced by TNF.
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TNF is also a potent activator of AP-1 (34). TNF
induced AP-1 expression by 3-fold in U-937 cells at 1 nM concentration.
The activation of AP-1 was not inhibited by silymarin up to 50 µM
(Fig. 7).
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TNF is a potent activator of JNK and MEK. Whether or not these
kinases are modulated by silymarin was also examined.The U-937 cells
were pretreated with different concentrations of silymarin for 2 h
and stimulated with TNF (1 nM) for 10 min; activation of JNK was then
measured. TNF activated JNK by about 7-fold, an activation that
gradually decreased with increasing concentrations of silymarin.
Silymarin (50 µM) inhibited most of the JNK induced by TNF (Fig. 8A). MEK is known to activate
JNK. We found that silymarin also inhibited TNF-induced MEK activation
in a dose-dependent manner (Fig. 8B).
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Among the cytokines, TNF is one of the most potent inducers of
apoptosis. We first investigated the effects of silymarin on
TNF-induced cytotoxicity against U-937 cells. Cells were incubated with
various concentrations of TNF for 72 h either in the presence or
absence of silymarin and then examined for cell viability by the MTT
method. As shown in Fig. 9A,
TNF induced cytotoxicity in U-937 in a dose-dependent manner, and this
effect was completely abolished by the presence of silymarin. These
results indicate that silymarin is cytoprotective. TNF induces
cytotoxic effects through activation of caspases, which can cleave
various cellular proteins including PARP. Whether or not silymarin
affects TNF-induced PARP cleavage was also examined. As shown in Fig. 9B, TNF induced cleavage of PARP, and this cleavage was
abolished by pretreatment of cells with silymarin in a dose-dependent
manner. Thus, these results suggest that silymarin is a potent
inhibitor of TNF-induced apoptosis.
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Previous reports have shown that TNF activates NF-B through
generation of ROI (22). Whether or not silymarin
suppresses NF-B activation through suppression of ROI generation was
examined by flow cytometry. As shown in Fig. 10A, TNF induced ROI
generation in a time-dependent manner, and this was suppressed upon
pretreatment of cells with silymarin. Because the role of lipid
peroxidation has also been implicated in TNF-induced NF-B activation
(29), we also examined the effect of silymarin on
TNF-induced lipid peroxidation. Results in Fig. 10B show
that TNF induced lipid peroxidation in U-937 cells and this was
completely suppressed by silymarin. Thus, it is quite likely that
silymarin may block TNF signaling through suppression of ROI generation
and lipid peroxidation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
activation, an early mediator of the pleiotropic effects of TNF. Our
results clearly demonstrate that silymarin is a potent inhibitor of
NF-B activation induced by a wide variety of inflammatory agents.
The inhibition of NF-B activation by silymarin correlated with
suppression of IB phosphorylation and degradation, p65 nuclear
translocation, and NF-B-dependent reporter gene transcription.
Silymarin also inhibited the activation of MEK and JNK and the
apoptosis induced by TNF.
There are several possibilities for how silymarin might inhibit
TNF-induced NF-B activation. We showed that silymarin does not
interfere with the binding of NF-B to the consensus DNA binding
site. NF-B activation requires sequential phosphorylation,
ubiquitination, and degradation of IB. Because silymarin blocks
IB phosphorylation and degradation, it suggests that the effects
of silymarin on NF-B is through inhibition of phosphorylation and
thus the proteolysis of IB. The phosphorylated form of IB
is known to appear on the gel as a band with retarded mobility
(23, 31). The lack of detection of IB band with slow
migration or by Abs against the phosphorylated form of IB after
treatment of cells with silymarin suggests that silymarin blocked the
phosphorylation of IB. The phosphorylation of IBa is regulated
by a large number of kinases, including IB kinase (IKK),
IKKß, IKK, NF-B-inducing kinase, TGF-ß-activated kinase-1,
and MEKK1 (23, 31, 35, 36, 37, 38). Besides MEKK1, MEKK2 and MEKK3
have been implicated in NF-B activation, whereas MEKK4 activates JNK
(39). MEKK is known to induce the phosphorylation of MEK.
We found that silymarin inhibited the activation of MEK. Thus, it is
possible that silymarin inhibited IB phosphorylation by
inhibiting the activity of MEKK1 or other kinases.
We found that silymarin blocked NF-B activation induced by a wide
variety of agents including TNF, okadaic acid, ceramide, LPS, and PMA
in U-937 cells.
H2O2-induced NF-B
activation was unaffected by silymarin, suggesting a difference in the
pathway leading to NF-B activation by different activators. A recent
report indicated that silymarin blocked okadaic acid-induced NF-B
activation but not that induced by TNF in HepG2 cells
(40). Although the effects of silymarin on okadaic
acid-induced NF-B activation are in agreement with ours, they differ
for suppression of TNF-induced activation. This difference was not due
to cell type used. Our studies show that silymarin also inhibited
TNF-induced NF-B activation in other myeloid (ML-1a), T cells
(Jurkat), and HeLa (epithelial) cells, but higher concentrations of
silymarin were required.
Our results also indicate that silymarin blocked TNF-induced
cytotoxicity, in agreement with previously described cytoprotective
effects (8, 9, 10). They also indicate that the inhibition of
apoptosis was mediated through suppression of caspase activation. How
silymarin suppresses caspase activation is not clear. Because redox
regulation of caspase activation has been demonstrated (41, 42), silymarin may suppress caspases through its antioxidant
activity. Recently, the role of JNK activation in TNF-induced apoptosis
was reported (43, 44, 45). Thus, it is possible that silymarin
inhibits apoptosis through inhibition of JNK. Our studies clearly
demonstrate that TNF-induced JNK activation is completely blocked by
silymarin. Because JNK activation is sensitive to the redox status of
the cell (27), the inhibition of JNK by silymarin may also
be due to its antioxidant properties. The role of NF-B in regulation
of apoptosis is controversial. Several studies indicate that NF-B
activation blocks apoptosis (for references, see Ref. 22),
whereas others show that NF-B activation has no effect on
TNF-induced apoptosis (44, 45, 46, 47); then there are reports
which indicate that NF-B activation is required for apoptosis
(48, 49). The inhibition of NF-B by silymarin did not
potentiate the apoptotic effects of TNF but rather suppressed it,
suggesting that either inhibition of apoptosis by silymarin is
dependent on inhibition of NF-B activation or that NF-B and
apoptosis are inhibited independent of each other.
Because silymarin inhibited TNF-induced activation of both NF-B and
apoptosis simultaneously, silymarin may inhibit a common step upstream
in the TNF signaling pathway. Recent studies from our laboratory showed
that overexpression of cells with either SOD (50) or with
-glutamylcysteine synthetase, a rate-limiting enzyme in the
glutathione biosynthesis pathway (51), blocks both NF-B
activation and apoptosis induced by TNF. Silymarin is known to induce
SOD and glutathione biosynthesis (4, 6). Thus, it is
possible that the effects of silymarin are mediated through quenching
of ROI, which is consistent with its known antioxidant effects
(3, 5). Our results clearly demonstrate that TNF-induced
ROI are suppressed by silymarin. The suppressive effects of silymarin
on JNK and MEK activation could also be mediated through its
antioxidant activity, as pro-oxidants are known to activate these
kinases (27, 34). Because silymarin is known to block
lipid peroxidation (3, 7, 10), and the latter has been
implicated in TNF-induced NF-B activation and apoptosis (29, 52), it is also possible that the effects of silymarin are
mediated through its antioxidant effects on lipids. Indeed, our results
confirm the inhibitory effect of silymarin on TNF-induced lipid
peroxidation.
We found that silymarin blocks NF-B-dependent reporter gene
expression. Several genes are involved in tumor promotion that are
regulated by NF-B. This includes growth factors, cyclooxygenase-2,
metalloproteases, and cell surface adhesion molecules
(21). It is possible that the anticarcinogenic effects of
silymarin are mediated through the suppression of NF-B-dependent
gene expression. Because NF-B-regulated genes also play a critical
role in inflammation, silymarin may also exhibit anti-inflammatory
effects. TNF is one of the genes involved in tumor promotion
and inflammation, whose expression is inhibited by silymarin
(19). Since replication of certain viruses such as HIV-1
is also dependent on NF-B (21), silymarin may also
abolish viral replication.
Like silymarin, the anti-inflammatory drugs sodium salicylate and
aspirin are also known to block the activation of NF-B by preventing
the degradation of IB (53). The effects of
salicylate on NF-B activation were observed, however, at a
suprapharmacological concentration (>5 mM). In contrast, silymarin in
our studies is effective at a100-fold lower concentration, suggesting
that it is a potent inhibitor. Silymarin has an established use as the
treatment for alcoholic liver diseases (11, 12). Our
results suggest that it may also have applications for various other
diseases including cancer, inflammation, and AIDS. Its lack of toxicity
even when used in large doses (54) broadens silymarin’s
potential for therapeutic use. These possibilities require further
investigation in detail.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Department of Molecular Oncology, Cytokine Research Laboratory, Box 143, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address:
3 Abbreviations used in this paper: SOD, superoxide dismutase; IB, inhibitory subunit of NF-B; MAPK, mitogen-activated protein kinase; DOC, deoxycholate; JNK, c-Jun N-terminal protein kinase; MEK, MAP/extracellular signal-related kinase kinase; PARP, poly(ADP) ribose polymerase; CAT, chloramphenicol acetyltransferase; ROI, reactive oxygen intermediates; ALLN, N-acetylleucylleucylnorleucinal; MDA, malondialdehyde; IKK, IB- kinase; D-PBS, Dulbecco’s PBS.
Received for publication May 18, 1999. Accepted for publication September 22, 1999.
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