Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects. How silymarin produces these effects is not understood, but it may involve suppression of NF-κB, 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-κB activation induced by various inflammatory agents. Silymarin blocked TNF-induced activation of NF-κB in a dose- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of ΙκBα, an inhibitor of NF-κB. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA. NF-κB-dependent reporter gene transcription was also suppressed by silymarin. Silymarin also blocked NF-κB activation induced by phorbol ester, LPS, okadaic acid, and ceramide, whereas H2O2-induced NF-κB activation was not significantly affected. The effects of silymarin on NF-κB 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-κB 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.
Extensive research within the last few years has shown that certain fruits, vegetables, herbs, and plants exhibit chemopreventive effects (Ref. 1 and references therein). One of these, silymarin, is a polyphenolic flavanoid isolated from the fruits and seeds of the milk thistle (also called artichoke) (Silybum marianum) (for references, see Ref. 2). Various studies indicate that silymarin exhibits strong antioxidant activity (3, 4, 5), increases cellular glutathione content (6), and induces superoxide dismutase (SOD)3 (4). By inhibiting lipid peroxidation, silymarin protects against hepatic toxicity induced by a wide variety of agents (7, 8, 9, 10, 11). Pharmacological studies indicate that silymarin is not toxic even at high doses (12). The flavanoid is used clinically for the treatment of alcoholic liver diseases (12).
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-κB 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-κB (21), including phorbol ester, okadaic acid, and TNF. In addition, several genes that are involved in tumorigenesis, metastasis, and inflammation are regulated by NF-κB (21). Recent reports indicate that NF-κB protects cells from undergoing apoptosis (22). The activation of NF-κB is regulated by several kinases which belong to the mitogen-activated protein kinase (MAPK) family (23). Furthermore, activators of NF-κB are also known to induce apoptosis (21). The activation of NF-κB 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-κB, members of the MAPK, and caspase-mediated apoptosis. Our results demonstrate that silymarin is a potent inhibitor of NF-κB activation. It also inhibits TNF-induced c-Jun N-terminal protein kinase (JNK) and MAPK kinase (MEK) activation and caspase-induced apoptosis.
Materials and Methods
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 × 107 U/mg, was kindly provided by Genentech (South San Francisco, CA). Ab against IκBα and double-stranded oligonucleotide having the AP-1 consensus sequence were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-IκBα (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-κB 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.
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 1× antibiotic-antimycotics. Cells were free from Mycoplasma as detected by the Gen-Probe Mycoplasma Rapid Detection kit (Fisher Scientific, Pittsburgh, PA).
NF-κB activation assay
To assay NF-κB activation, EMSA were conducted essentially as described (25). Briefly, nuclear extracts prepared from TNF-treated cells (2 × 106/ml) were incubated with 32P end-labeled 45-mer double-stranded NF-κB oligonucleotide (6 μg protein with 16 fmol DNA) from the HIV-LTR, 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (bold indicates NF-κB 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-κB 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 IκBα
To assay IκBα, 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 IκBα, or IκBα 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 × 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 × 400 μl) and kinase buffer (2 × 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 [γ-32P]ATP. Reactions were stopped with the addition of 15 μl of 2× 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-κB-mediated reporter gene transcription, U-937 cells were transiently transfected by the calcium phosphate method with the plasmids 243RMICAT (contains wild-type NF-κB binding site) and -243RMICAT-κm (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 assays were performed as indicated previously (28). Briefly, U-937 cells (5 × 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 × 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 × 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 × 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 × 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).
Silymarin is a polyphenolic flavanoid with chemical structure (2-[2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-1,4-benzodioxin-6-yl]-2,3-dihydro-3,5,7-trihydroxy-4H-1-benzopyran-4-one) as described (2). The concentration and duration of treatment with silymarin used in these studies had no effect on the viability of U-937 cells (97% cell viability after exposure of cells to 50 μM silymarin for 2 h).
Silymarin inhibits TNF-induced NF-κB 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-κB by EMSA. As shown in Fig. 1,A, silymarin inhibited TNF-mediated NF-κB 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-κB. We next tested the length of incubation required for silymarin to block TNF-induced NF-κB 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-κB activation observed, and the inhibition decreased gradually with a shorter preincubation time (Fig. 1,B). Cotreatment or posttreatment with silymarin did not inhibit NF-κB activation (Fig. 1,B). Previous studies from our laboratory have shown that a high concentration of TNF (10 nM) can activate NF-κB 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-κB 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-κB was assayed by EMSA. Although the activation of NF-κB by 10,000 pM of TNF was strong, silymarin inhibited it just as efficiently as it did the 0.1 nM TNF concentration (Fig. 1 C). These results suggest that silymarin is a very potent inhibitor of NF-κB activation.
Activated NF-κB inhibited by silymarin consists of p50 and p65 subunits
Various combinations of Rel/NF-κB proteins can constitute an active NF-κB 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-κB, we incubated nuclear extracts from TNF-activated cells with Ab to either p50 (NF-κBI) or p65 (Rel A) subunits and then conducted EMSA. Abs to either subunit of NF-κB shifted the band to a higher m.w. (Fig. 2 A), 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-κB. Excess unlabeled NF-κB (100-fold) caused complete disappearance of the band, indicating the specificity of NF-κB.
Silymarin does not interfere with the DNA binding ability of NF-κB 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-κB activation by modifying the NF-κB subunits, thus preventing its binding to DNA (31, 32). To determine whether silymarin also directly modifies NF-κB 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 IκBα subunit, thus releasing NF-κB for binding to the DNA. As seen in Fig. 2, B and C, silymarin did not modify the DNA-binding ability of NF-κB proteins prepared by treatment with either DOC or TNF. Therefore, silymarin’s inhibition of NF-κB 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-κB activation by silymarin is not cell-type specific
Besides U-937 cells, we also examined the ability of silymarin to block TNF-induced NF-κB activation in other myeloid (ML-1a), lymphoid (Jurkat), and epithelial (HeLa) cells (Fig. 3). The results of these experiments indicate that silymarin inhibited NF-κB 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.
Silymarin blocks phorbol ester-, LPS-, okadaic acid-, and ceramide-mediated activation of NF-κB
Besides TNF, NF-κB 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-κB by these various agents. The results shown in Fig. 4 indicate that silymarin completely blocked the activation of NF-κB induced by all those agents except H2O2. The activation of NF-κB 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-κB activation.
Silymarin inhibits TNF-dependent phosphorylation and degradation of IκBα
The translocation of NF-κB to the nucleus is preceded by the phosphorylation and proteolytic degradation of IκBα (23). To determine whether the inhibitory action of silymarin was due to an effect on IκBα degradation, the cytoplasmic levels of IκBα proteins were examined by Western blot analysis. IκBα 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 IκBα (Fig. 5 A).
We also investigated whether silymarin inhibits IκBα degradation by blocking its phosphorylation. The serine phosphorylation of IκBα induced by TNF was stabilized by pretreatment of cells for 1 h with ALLN, a proteosome inhibitor (33). The hyperphosphorylated form of IκBα appeared as a slow migrating band on SDS-PAGE (Fig. 5,B), which disappeared when cells were pretreated with silymarin (compare lane 4 with lane 8), indicating that silymarin blocks IκBα phosphorylation. This was further examined by the use of Abs which detect only serine phosphorylated form of IκBα. These results shown in Fig. 5 C further confirm that TNF-induces IκBα phosphorylation and silymarin inhibits it quite effectively.
Pretreatment of cells with silymarin also completely abolished the TNF-induced nuclear translocation of p65 (Fig. 5 D).
Silymarin represses NF-κB-CAT reporter gene expression
To determine the effect of silymarin on TNF-induced NF-κB-dependent reporter gene expression, the promoter of the rat mdr1b gene containing a NF-κB 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-κB sequence were pretreated with silymarin for 2 h before TNF treatment. Transfection with the MDR gene containing the mutated NF-κB binding site did not result in induction of CAT by TNF. These results demonstrate that silymarin also represses NF-κB-dependent gene expression induced by TNF.
Silymarin did not inhibit TNF-induced AP-1 activation
Silymarin inhibits TNF-induced JNK and MEK activation
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. 8,A). MEK is known to activate JNK. We found that silymarin also inhibited TNF-induced MEK activation in a dose-dependent manner (Fig. 8 B).
Silymarin blocks TNF-induced apoptosis
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. 9,A, 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. 9 B, 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.
Silymarin blocks TNF-induced ROI generation and lipid peroxidation
Previous reports have shown that TNF activates NF-κB through generation of ROI (22). Whether or not silymarin suppresses NF-κB activation through suppression of ROI generation was examined by flow cytometry. As shown in Fig. 10,A, 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-κB activation (29), we also examined the effect of silymarin on TNF-induced lipid peroxidation. Results in Fig. 10 B 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.
Because silymarin exhibits anticarcinogenic, anti-inflammatory, and cytoprotective effects, we hypothesized that these effects of silymarin are mediated through suppression of NF-κB activation, an early mediator of the pleiotropic effects of TNF. Our results clearly demonstrate that silymarin is a potent inhibitor of NF-κB activation induced by a wide variety of inflammatory agents. The inhibition of NF-κB activation by silymarin correlated with suppression of IκBα phosphorylation and degradation, p65 nuclear translocation, and NF-κB-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-κB activation. We showed that silymarin does not interfere with the binding of NF-κB to the consensus DNA binding site. NF-κB activation requires sequential phosphorylation, ubiquitination, and degradation of IκBα. Because silymarin blocks IκBα phosphorylation and degradation, it suggests that the effects of silymarin on NF-κB is through inhibition of phosphorylation and thus the proteolysis of IκBα. The phosphorylated form of IκBα is known to appear on the gel as a band with retarded mobility (23, 31). The lack of detection of IκBα band with slow migration or by Abs against the phosphorylated form of IκBα after treatment of cells with silymarin suggests that silymarin blocked the phosphorylation of IκBα. The phosphorylation of IκBa is regulated by a large number of kinases, including IκBα kinase (IKKα), IKKβ, IKKγ, NF-κB-inducing kinase, TGF-β-activated kinase-1, and MEKK1 (23, 31, 35, 36, 37, 38). Besides MEKK1, MEKK2 and MEKK3 have been implicated in NF-κB 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 IκBα phosphorylation by inhibiting the activity of MEKK1 or other kinases.
We found that silymarin blocked NF-κB activation induced by a wide variety of agents including TNF, okadaic acid, ceramide, LPS, and PMA in U-937 cells. H2O2-induced NF-κB activation was unaffected by silymarin, suggesting a difference in the pathway leading to NF-κB activation by different activators. A recent report indicated that silymarin blocked okadaic acid-induced NF-κB activation but not that induced by TNF in HepG2 cells (40). Although the effects of silymarin on okadaic acid-induced NF-κB 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-κB 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-κB in regulation of apoptosis is controversial. Several studies indicate that NF-κB activation blocks apoptosis (for references, see Ref. 22), whereas others show that NF-κB activation has no effect on TNF-induced apoptosis (44, 45, 46, 47); then there are reports which indicate that NF-κB activation is required for apoptosis (48, 49). The inhibition of NF-κB 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-κB activation or that NF-κB and apoptosis are inhibited independent of each other.
Because silymarin inhibited TNF-induced activation of both NF-κB 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 γ-glutamylcysteine synthetase, a rate-limiting enzyme in the glutathione biosynthesis pathway (51), blocks both NF-κB 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-κB 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-κB-dependent reporter gene expression. Several genes are involved in tumor promotion that are regulated by NF-κB. 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-κB-dependent gene expression. Because NF-κB-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-κB (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-κB by preventing the degradation of IκBα (53). The effects of salicylate on NF-κB 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.
This work was supported by the Clayton Foundation for Research.
Abbreviations used in this paper: SOD, superoxide dismutase; IκB, inhibitory subunit of NF-κB; 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, IκB-α kinase; D-PBS, Dulbecco’s PBS.