Guggulsterone Inhibits NF- (cid:1) B and I (cid:1) B (cid:2) Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis*

Guggulsterone, derived from Commiphora mukul and used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, has been recently shown to antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes. Because activation of NF- (cid:1) B has been closely linked with inflammatory diseases affected by guggulsterone, we postulated that it must modulate NF- (cid:1) B activation. In the present study, we tested this hypothesis by investigating the effect of this steroid on the activation of NF- (cid:1) B induced by inflammatory agents and carcinogens. Guggulsterone suppressed DNA binding of NF- (cid:1) B induced by tumor necrosis factor (TNF), phorbol ester, okadaic acid, cigarette smoke condensate, hydrogen peroxide, and interleu-kin-1. NF- (cid:1) B activation was not cell type-specific, because both epithelial and leukemia cells were inhibited. Guggulsterone also suppressed constitutive NF- (cid:1) B activation expressed in most tumor cells. Through inhibition of I (cid:1) B kinase activation, this steroid blocked I (cid:1) B (cid:2) phosphorylation and degradation, thus suppressing p65 phosphorylation (cid:6) guggulsterone for 4 0.1 TNF various time Thirty micro-grams cytoplasmic resolved on 10% SDS-PAGE gel, nitrocellulose blocked 5% nonfat milk, and probed with specific antibody against I (cid:1) B (cid:2) phosphorylated I (cid:1) B (cid:2) , p65, phosphorylated p65. To determine the expression of cyclin D1, COX-2, MMP-9, cIAP-1, xIAP, TRAF1, Bcl-2, Bfl-1, cFLIP, and survivin in whole cell extracts of treated cells (2 (cid:2) 10 6 cells in 2 ml 30–50 (cid:6) g on SDS-PAGE and probed by Western blot with specific antibodies as per the manufacturer’s recommended protocol. The blots washed, to horseradish peroxidase- 1 h, and finally ECL reagent (Amersham The were quantitated using Scan 1.30 ImageQuaNT

plasm; only when activated and translocated to the nucleus is the sequence of events leading to activation initiated (15,16). Currently NF-B is considered a family of Rel-domain-containing proteins, namely Rel A (also called p65), Rel B, c-Rel, p50 (also called NF-B1), and p52 (also called NF-B2). Similarly, a family of anchorin-domain-containing proteins has been identified that keeps the NF-B in its inactive state within the nucleus. These include IB␣, IB␤, IB␥, IB⑀, bcl-3, p105, and p100. Most carcinogens, inflammatory agents, and tumor promoters, including cigarette smoke, phorbol ester, okadaic acid, H 2 O 2 , and TNF have been shown to activate NF-B.
Under resting conditions, NF-B consists of a heterotrimer of p50, p65, and IB␣ in the cytoplasm. The phosphorylation, ubiquitination, and degradation of IB␣ and phosphorylation of p65 leads to the translocation of NF-B to the nucleus where it binds to specific response elements in the DNA. The phosphorylation of IB␣ is catalyzed by IKK, which consists of three subunits IKK␣, IKK␤, and IKK␥. Gene deletion studies have shown that IKK␤ is essential for NF-B activation by most agents. The identity of the kinase that induces the phosphorylation of p65 is controversial, but IKK␤, protein kinase C, and protein kinase A have been implicated (for references see Refs. 17 and 18). NF-B has been shown to regulate the expression of a number of genes whose products are involved in tumorigenesis (15)(16)(17)(18). These include anti-apoptotic genes (e.g. ciap, suvivin, traf, bcl-2, and bcl-xl); cox2; mmp-9; genes encoding adhesion molecules, chemokines, and inflammatory cytokines; and cell cycle regulatory genes (e.g. cyclin d1).
Furthermore, NF-B has been implicated in obesity (19), hyperlipidemia (20), atherosclerosis (21), osteoarthritis (22), and bone loss (23), all of which can be modulated by guggulsterone. In the present report we investigated the effect of guggulsterone on NF-B activation induced by a variety of inflammatory agents and carcinogens. The aim of the study was to determine whether guggulsterone can suppress NF-B activation induced by inflammatory agents and carcinogens and block NF-B-regulated gene expression that mediates inflammation and carcinogenesis. We found that guggulsterone inhibited activation of NF-B through suppression of IB␣ kinase, IB␣ phosphorylation, and degradation, p65 nuclear translocation, and NF-B-dependent reporter gene expression. Guggulsterone also abrogated the expression of NF-B-regulated gene products that inhibit apoptosis and promote inflammation and tumor metastasis.
Cell Lines-The cell lines used in our studies included human nonsmall cell lung carcinoma (H1299) cells and human lung epithelial cell carcinoma (A549) cells, both kindly provided by Dr. Reuben Lotan (The University of Texas M. D. Anderson Cancer Center). Human leukemia (Jurkat) and myelogenous leukemia (KBM-5) cells were obtained from the American Type Culture Collection (Manassas, VA). A549, Jurkat, and H1299 cells were cultured in RPMI 1640 medium, and KBM-5 cells were cultured in Iscove's modified Dulbecco's modified Eagle's medium. For Jurkat and A549 cells, RPMI 1640 was supplemented with 10% FBS, 100 units/ml penicillin, and 100 g/ml streptomycin; for KBM-5 cells, Dulbecco's modified Eagle's medium was supplemented with 15% FBS plus penicillin and streptomycin.
NF-B Activation-To determine NF-B activation by TNF, which has a well established role in inflammation, tumor proliferation, promotion, invasion, and metastasis (24), we carried out EMSA essentially as previously described (25). The dried gels were visualized, and radioactive bands were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuaNT software. The -fold activation, over that of untreated control lane, was then calculated.
Western Blot Analysis-To determine the effect of guggulsterone on TNF-dependent IB␣ degradation, p65 translocation, and p65 phosphorylation, cytoplasmic extracts were prepared as previously described (26) from H1299 cells (2 ϫ 10 6 /ml) that had been pretreated with 50 M guggulsterone for 4 h and then exposed to 0.1 nM TNF for various times. For determining the phosphorylation of IB␣, H1299 cells (2 ϫ 10 6 /ml) were first treated with 100 M N-acetyl-Leu-Leu-norleucinal (a proteasome inhibitor) for 1 h, then treated with 50 M guggulsterone for 4 h and then exposed to 0.1 nM TNF for various time points. Thirty micrograms of cytoplasmic protein was resolved on 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with specific antibody against IB␣, phosphorylated IB␣, p65, and phosphorylated p65. To determine the expression of cyclin D1, COX-2, MMP-9, cIAP-1, xIAP, TRAF1, Bcl-2, Bfl-1, cFLIP, and survivin in whole cell extracts of treated cells (2 ϫ 10 6 cells in 2 ml medium), 30 -50 g of protein was resolved on SDS-PAGE and probed by Western blot with specific antibodies as per the manufacturer's recommended protocol. The blots were washed, exposed to horseradish peroxidaseconjugated secondary antibodies for 1 h, and finally detected by ECL reagent (Amersham Biosciences). The bands were quantitated using a Personal Densitometer Scan version 1.30 using ImageQuaNT software version 3.3 (Amersham Biosciences).
Immunolocalization of NF-B p65-The effect of guggulsterone on cigarette smoke-induced nuclear translocation of p65 was examined by an immunocytochemical method using an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan) and a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) as described previously (27).
IKK Assay-To determine the effect of guggulsterone on TNF-induced IKK activation, we analyzed IKK by a method essentially as described previously (28).
NF-B-dependent Reporter Gene Transcription-The effect of guggulsterone on TNF-induced NF-B-dependent reporter gene transcription in H1299 cells was measured as previously described (29).
COX-2 Promoter-dependent Reporter Luciferase Gene Expression-COX-2 promoter activity was examined as described elsewhere (28). To further determine the effect of guggulsterone on COX-2 promoter, A293 cells were seeded at a concentration of 1.5 ϫ 10 5 cells per well in six-well plates. After overnight culture, the cells in each well were transfected with 2 g of DNA consisting of COX-2 promoter-luciferase reporter plasmid, along with 6 l of LipofectAMINE 2000 according to the manufacturer's protocol. The COX-2 promoter (Ϫ375 to ϩ59), which was amplified from human genomic DNA by using the primers 5Ј-GAGTCTCTTATTTATTTTT-3Ј (sense) and 5Ј-GCTGCTGAGGAGTTC-CTGGACGTGC-3Ј (antisense), was kindly provided by Dr. Xiao-Chun Xu (M. D. Anderson Cancer Center). After a 6-h exposure to the transfection mixture, the cells were incubated in medium containing guggulsterone for 12 h. The cells were exposed to TNF (0.1 nM) for 24 h and then harvested. Luciferase activity was measured by using the Promega luciferase assay system according to the manufacturer's protocol and detected by using Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA). All experiments were performed in triplicate and repeated at least twice to prove their reproducibility.
Live and Dead Assay-To measure apoptosis, we used the Live and Dead assay (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity. This assay employs calcein, a polyanionic dye, which is retained within the live cells and provides green fluorescence (30). It also employs the ethidium monomer dye (red fluorescence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of live cells. Briefly, 1 ϫ 10 5 cells were incubated with 10 M guggulsterone for 24 h and then treated with 1 nM TNF for 16 h at 37°C. Cells were stained with the Live and Dead reagent (5 M ethidium homodimer, 5 M calcein-AM) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2).

RESULTS
The guggulsterone used in these studies, which has a steroid-like structure (Fig. 1A), was dissolved in Me 2 SO. The concentration of guggulsterone used and the duration of exposure had minimal effect on the viability of these cells as determined by trypan blue dye exclusion test (data not shown).
Guggulsterone Blocks NF-B Activation Induced by TNF, Interleukin-1␤, PMA, H 2 O 2 , Cigarette Smoke Condensate, and Okadaic Acid-Because TNF, interleukin-1␤, PMA, H 2 O 2 , cigarette smoke condensate, and okadaic acid activate NF-B, we examined the effect of guggulsterone on the activation of NF-B by these agents in H1299 cells. A DNA-binding assay (EMSA) showed that guggulsterone suppressed the NF-B activation induced by all these agents (Fig. 1B). These results suggest that guggulsterone acted at a step in the NF-B activation pathway that is common to all these agents.
Inhibition of NF-B Activation by Guggulsterone Is Not Cell Type-specific-Because some reports suggest that distinct signal transduction pathways mediate NF-B induction in epithelial and lymphoid cells (31), we determined whether guggulsterone inhibited NF-B activation in three cell types. Guggulsterone completely inhibited TNF-induced NF-B activation in lung epithelial cell carcinoma (A549), T cell leukemia (Jurkat), and myeloid leukemia (KBM-5) cells ( Fig. 2A), indicating a lack of cell type specificity.
Guggulsterone Inhibits Constitutive NF-B Activation-We next tested the effect of guggulsterone on NF-B activation in human multiple myeloma (U266) and head and neck squamous cell carcinoma (MDA 1986) tumor cells, which both express constitutively active NF-B (27,32). Guggulsterone completely inhibited this constitutively active NF-B (Fig. 2B).
The Suppression of NF-B by Guggulsterone Is Dose-and Time-dependent-Guggulsterone inhibited TNF-mediated NF-B activation in a dose-dependent manner, with maximum inhibition occurring at 50 M (Fig. 3A). The minimum time required for complete inhibition of NF-B activation was 4 h (Fig. 3B).
Suppressed NF-B Consists of Both p50 and p65-When nuclear extracts from TNF-activated cells were incubated with antibodies to the p50 (NF-B1) and the p65 (RelA) subunit of NF-B, the resulting bands were shifted to higher molecular masses (Fig. 3C), suggesting that the TNF-activated complex consisted of p50 and p65. Neither preimmune serum nor irrelevant antibody had any effect. Addition of excess unlabeled NF-B (cold oligonucleotide; 100-fold) caused complete disappearance of the band, whereas mutated oligonucleotide had no effect on the DNA binding.
Guggulsterone Does Not Directly Affect Binding of NF-B to the DNA-Although TPCK (the serine protease inhibitor), herbimycin A (protein tyrosine kinase inhibitor), and caffeic acid phenyl ethyl ester directly modify NF-B to suppress its activation (33)(34)(35), EMSA showed that guggulsterone did not modify the DNA-binding ability of NF-B proteins prepared from cells by treatment with TNF (Fig. 3D). Therefore, guggulsterone must inhibit NF-B activation by a different mechanism.
Guggulsterone Inhibits TNF-dependent IB␣ Degradation-Because IB␣ degradation is normally a condition for translocation of NF-B to the nucleus (36), we determined whether the guggulsterone's inhibition of TNF-induced NF-B activation was due to inhibition of IB␣ degradation. We found that TNF induced IB␣ degradation in control cells as early as 10 min, but in guggulsterone-pretreated cells TNF had no effect on IB␣ degradation (Fig. 4A, upper panel).
Guggulsterone Inhibits TNF-dependent IB␣ Phosphorylation-We next determined whether guggulsterone affected TNF-induced IB␣ phosphorylation, another condition for NF-B translocation. Western blot analysis using antibody that detects only the serine-phosphorylated form of IB␣ indicated that TNF induced IB␣ phosphorylation as early as 5 min, and guggulsterone almost completely suppressed it (Fig.  4A, middle panel). Thus guggulsterone inhibited TNF-induced Guggulsterone Inhibits TNF-induced IKK Activation-Because guggulsterone inhibits the phosphorylation of IB␣, we tested the effect of guggulsterone on TNF-induced IKK activa-tion, which is required for TNF-induced phosphorylation of IB␣. As shown in Fig. 4B (upper panel), guggulsterone completely suppressed TNF-induced activation of IKK. TNF or guggulsterone had no direct effect on the expression of either IKK␣ (middle panel) or IKK␤ (lower panel) proteins. However, FIG. 3. A, guggulsterone inhibits TNF-dependent NF-B activation in a dose-dependent manner. H1299 cells (2 ϫ 10 6 /ml) were preincubated with the indicated concentrations of guggulsterone for 4 h at 37°C and then treated with 0.1 nM TNF for 30 min. Nuclear extracts were prepared and tested for NF-B activation, as described under "Experimental Procedures." B, guggulsterone inhibits TNF-dependent NF-B activation in a time-dependent manner. H1299 cells (2 ϫ 10 6 /ml) were preincubated with 50 M guggulsterone for the indicated times at 37°C and then treated with 0.1 nM TNF for 30 min at 37°C. Nuclear extracts were prepared and then tested for NF-B activation. C, TNF-induced NF-B consists of p50 and p65 subunits. Nuclear extracts from H1299 cells (2 ϫ 10 6 /ml) treated or not treated with 0.1 nM TNF for 30 min were incubated with the antibodies indicated for 30 min at room temperature, and the complex was analyzed by supershift assay. D, guggulsterone does not modulate the ability of NF-B to bind to the DNA. Nuclear extracts from H1299 cells (2 ϫ 10 6 /ml) treated or not treated with 0.1 nM TNF for 30 min were treated with the indicated concentrations of guggulsterone for 4 h at room temperature and then assayed for DNA binding by EMSA. The numbers at the bottom of each lane indicate -fold activation over that of untreated control.
we did find that guggulsterone inhibited the phosphorylation of GST IB␣ (at a 50 M concentration) by directly interfering with IKK activity (Fig. 4C).
Guggulsterone Inhibits TNF-induced Phosphorylation and Nuclear Translocation of p65-We also tested the effect of guggulsterone on TNF-induced phosphorylation of p65, because phosphorylation is also required for transcriptional activity of p65 (37). As shown in Fig. 5A, guggulsterone suppressed p65 phosphorylation almost completely. Likewise, Western blot analysis (Fig. 5B) and immunocytochemistry (Fig.  5C) indicated that guggulsterone abolished TNF-induced nuclear translocation of p65.

Guggulsterone Represses TNF-induced NF-B-dependent Reporter Gene
Expression-Although we showed by EMSA that guggulsterone blocked NF-B activation, DNA binding alone does not always correlate with NF-B-dependent gene transcription, suggesting that there are additional regulatory steps (38). Transient transfection of H1299 cells with the NF-Bregulated SEAP reporter construct followed by stimulation with TNF produced an almost 13-fold increase in SEAP activity over vector control activity (Fig. 6A). TNF-induced SEAP activity was abolished by dominant-negative IB␣, indicating specificity. When the cells were pretreated with guggulsterone, TNF-induced NF-B-dependent SEAP expression was inhib-  (lower panel). B, guggulsterone inhibits TNF-induced IB␣ kinase activity. H1299 cells (2 ϫ 10 6 /ml) were treated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for the indicated time intervals. Whole cell extracts were prepared, and 200 g of extract was immunoprecipitated with antibodies against IKK␣ and IKK␤. Thereafter immune complex kinase assay was performed as described under "Experimental Procedures." To examine the effect of guggulsterone on the level of expression of IKK proteins, 30 g of whole cell extract was run on 10% SDS-PAGE, electrotransferred, and immunoblotted with indicated antibodies as described under "Experimental Procedures." C, guggulsterone directly inhibits IKK activity. Whole cell extracts were prepared from untreated and TNF (0.1 nM)-treated H1299 cells (2 ϫ 10 6 /ml); 200 g of protein/sample whole cell extract was immunoprecipitated with antibodies against IKK␣ and IKK␤. The immune complex was treated with the indicated concentrations of guggulsterone for 30 min at 30°C, and then a kinase assay was performed as described under "Experimental Procedures." Equal protein loading was evaluated by IKK␤.
ited by guggulsterone in a dose-dependent manner. These results demonstrate that guggulsterone inhibits NF-B-dependent reporter gene expression induced by TNF.
We next determined where guggulsterone acts in the sequence of TNFR1, TRADD, TRAF2, NIK, and IKK recruitment that characterizes TNF-induced NF-B activation (39,40). In cells transfected with TNFR, TRADD, TRAF2, NIK, IKK␤, and p65 plasmids, NF-B-dependent SEAP expression was induced; 50 M guggulsterone significantly suppressed SEAP expression in all cells except those transfected with p65. Because IKK activation can cause the phosphorylation of IB␣ and p65, we suggest that guggulsterone inhibits NF-B activation through inhibition of IKK.
Guggulsterone Represses TNF-induced COX-2 Promoter Activity-We next determined whether guggulsterone affected COX-2 promoter activity, which is regulated by NF-B (41). As shown in Fig. 6C, guggulsterone significantly reduced the TNFinduced COX-2 promoter activity in a dose-dependent manner.
Guggulsterone Inhibits TNF-induced COX-2, MMP-9, and VEGF Expression and TNF-induced Cyclin D1 and c-myc Expression-Guggulsterone abolished, in a dose-dependent fashion, the TNF-induced expression of COX-2, MMP-9, and VEGF (Fig. 7A), which are known to be NF-B-regulated gene products (41)(42)(43). Expression of the NF-B-regulated gene products cyclin D1 and c-Myc (44,45) was also abolished by guggulsterone (Fig. 7B). 5. A, guggulsterone inhibits TNF-induced phosphorylation of p65. H1299 cells (2 ϫ 10 6 /ml) were incubated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for the indicated times. The cytoplasmic extracts were analyzed by Western blotting using antibodies against the phosphorylated form of p65. B, guggulsterone inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 ϫ 10 6 /ml) were either untreated or pretreated with 50 M guggulsterone for 4 h at 37°C and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic and nuclear extracts were prepared and analyzed by Western blotting using antibodies against p65. C, guggulsterone inhibits TNF-induced nuclear translocation of p65. H1299 cells (1 ϫ 10 6 /ml) were first treated with 50 M guggulsterone for 4 h at 37°C and then exposed to 0.1 nM TNF. After cytospin, immunocytochemical analysis was performed as described under "Experimental Procedures." Guggulsterone Inhibits TNF-induced Activation of Anti-apoptotic Gene Products-NF-B up-regulates the expression of a number of genes implicated in facilitating tumor cell survival, including cIAP1, xIAP, Bfl-1, BCl-2, TRAF1, cFLIP, and survivin (46 -54). We found that guggulsterone inhibited the TNFinduced expression of all of these proteins (Fig. 8).
Guggulsterone Potentiates the Cytotoxic Effects of TNF and Chemotherapeutic Drugs-Because NF-B-regulated products suppress TNF-and chemotherapy-induced apoptosis (55,56), we examined the effects of guggulsterone on the apoptotic effects of TNF and the chemotherapeutic drugs paclitaxel and doxorubicin. Guggulsterone enhanced the cytotoxic effects of TNF, Taxol, and doxorubicin (Fig. 9A) and the caspase-induced cleavage of PARP activated by TNF (Fig. 9B). The Live and Dead assay also showed that TNF-induced apoptosis was significantly enhanced by guggulsterone (Fig. 9C).
The present study was undertaken to investigate the potential mechanism for the anti-inflammatory effects of guggulsterone, which has been used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis. Although guggulsterone can antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes (11), these effects do not explain its anti-inflammatory attributes. Because activation of NF-B has been found in most inflammatory diseases, we hypothesized that guggulsterone modulates NF-B activation. The results of the study validate this hypothesis.
Very little is known about the mechanism of action of guggulsterone. Our results clearly indicate that guggulsterone can suppress NF-B activation induced by a wide variety of agents, suggesting that the site of action of guggulsterone is common to all these agents. We identified IKK as a target site: cells that were exposed to guggulsterone failed to activate IKK in response to TNF. Surprisingly, incubation of IKK with guggulsterone was sufficient to suppress its activity, suggesting that guggulsterone is a direct inhibitor of IKK. Ours is the first report to suggest that a steroid can suppress NF-B activation through inhibition of IKK activity.
Although steroids exhibit anti-inflammatory activity, whether it is through suppression of NF-B is less clear. Auphan et al. (57) showed that glucocorticoids inhibit NF-B activation and that this inhibition is mediated through induction of the IB␣, which traps activated NF-B in inactive cytoplasmic complexes. Interestingly, estradiol was found to activate NF-B (58).
By NF-B gene reporter assay, we found that guggulsterone suppressed NF-B activation induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK but not that activated by p65. Although this pathway is restricted to TNF-induced NF-B activation, these results again confirm that IKK is a potential target of guggulsterone. Whether guggulsterone suppresses NF-B activation through other mechanisms cannot, however, be completely ruled out by the results of our study.
Various tumor cell types express constitutively active NF-B, and it is critical for their proliferation (18,27,32). The potential mechanism of constitutive activation of NF-B is not fully understood. Overexpression of IB␣ without inhibition of NF-B activity and mutations in the ib␣ gene in Reed-Sternberg cells (59) and enhanced IB␣ degradation in mature murine B-cell lines (60) have been demonstrated as potential mechanisms. Our laboratory showed that constitutive expression of TNF in T-cell lymphoma (32) and interleukin-1 in acute myelogenous leukemia (61) are potential mechanisms. We found that human multiple myeloma cells and head and neck squamous cell carcinoma cells also express constitutive NF-B,  7. A, guggulsterone inhibits COX-2, MMP-9, and VEGF expression induced by TNF. H1299 cells (2 ϫ 10 6 /ml) were left untreated or incubated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for different times. Whole cell extracts were prepared, and 80 g of the whole cell lysate was analyzed by Western blotting using antibodies against COX-2 and MMP-9. B, guggulsterone inhibits cyclin D1 and c-Myc expression induced by TNF. H1299 cells (2 ϫ 10 6 /ml) were left untreated or incubated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for different times. Whole cell extracts were prepared, and 80 g of the whole cell lysate was analyzed by Western blotting using antibodies against cyclin D1 and c-Myc. and guggulsterone suppressed the activation. It is very likely that this inhibition also occurs through inhibition of IKK.
That activation of NF-B regulates genes that control proliferation and metastasis of cancer has been well established (17,18,43). Our results demonstrate that guggulsterone can suppress the expression of COX-2, MMP-9, VEGF, cyclin D1, and c-Myc, all regulated by NF-B. We found that COX-2 promoter activity was also significantly down-regulated by this steroid. The lack of complete inhibition of COX-2 promoter activity by guggulsterone suggests that this promoter is also regulated by transcription factors other than NF-B. These results imply that guggulsterone has a potential to suppress proliferation of tumor cells and their metastasis.
Our results indicate that guggulsterone also suppresses the expression of numerous anti-apoptotic gene products, all known to be regulated by NF-B activation. The overexpression of IAP1, xIAP, Bfl-1/A1, bcl-2, cFLIP, and survivin has been found in numerous tumors and has been linked to survival, chemoresistance, and radioresistance. Because most of these gene products are down-regulated by guggulsterone, we believe that guggulsterone has potential as an agent for overcoming radioresistance and chemoresistance. Indeed, guggulsterone potentiated the apoptotic effects of TNF and Taxol and doxorubicin. Thus guggulsterone, which is a pharmacologically safe agent (6), could be used as an anticancer agent on its own, a preventive agent, or an enhancer of chemotherapy/radiotherapy activity. Our demonstration, that guggulsterone suppresses NF-B and NF-B-regulated gene expression, may FIG. 9. Guggulsterone enhances apoptosis induced by TNF and chemotherapeutic agents. A, KBM-5 cells (5000 cells/0.1 ml) were incubated at 37°C with TNF, Taxol, or doxorubicin in the presence and absence of 50 M guggulsterone, as indicated for 72-h duration, and the viable cells were assayed using MTT reagent. The results are shown as the mean Ϯ S.D. from triplicate cultures. B, KBM-5 cells (2 ϫ 10 6 /ml) were serum-starved for 24 h and then incubated with TNF alone or in combination with guggulsterone for the indicated times, and PARP cleavage was determined by Western blot analysis as described under "Experimental Procedures." C, KBM-5 cells (2 ϫ 10 6 /ml) were serum-starved for 24 h and then incubated with TNF alone or in combination with guggulsterone as indicated for 24 h. Cell death was determined by calcein-AM-based Live and Dead assay as described under "Experimental Procedures." explain its effects against atherosclerosis, diabetes, osteoarthritis, and other inflammatory diseases.
Cancer is a hyperproliferative disorder characterized by the up-regulation of genes responsible for transformation, proliferation, invasion, angiogenesis, and metastasis. Most of these activities are influenced by the aberrant activity of NF-B; therefore, the main objective of this study was to evaluate the efficacy of guggulsterone as an inhibitor of NF-B. Our study for the first time demonstrates that guggulsterone can suppress NF-B activation induced by various carcinogens, inflammatory agents, and tumor promoters. This effect was not cell type-specific and equally pronounced in cells where NF-B is constitutively active. TNF-induced activation of IKK and phosphorylation and degradation IB␣ were also inhibited by guggulsterone. Guggulsterone was quite effective in blocking the phosphorylation and translocation of TNF-induced p65, which led to the abrogation of NF-B-dependent reporter gene expression. TNF-induced NF-B reporter activity induced by TNFR, TRADD, TRAF, NIK, and IKK␤, but not that activated by p65, was also blocked.