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Guggulsterone Inhibits NF-κB and IκBα Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis

Guggulsterone Inhibits NF-κB and IκBα Kinase Activation, Suppresses Expression of Anti-apoptotic... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 45, Issue of November 5, pp. 47148–47158, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Guggulsterone Inhibits NF-B and IB Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis* Received for publication, July 16, 2004, and in revised form, August 17, 2004 Published, JBC Papers in Press, August 17, 2004, DOI 10.1074/jbc.M408093200 Shishir Shishodia and Bharat B. Aggarwal‡ From the Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 were discovered by investigating a folklore claim (1, 2). Taxol is Guggulsterone, derived from Commiphora mukul and used to treat obesity, diabetes, hyperlipidemia, athero- perhaps the most recent example. Between 1981 and 2002, 48 sclerosis, and osteoarthritis, has been recently shown to of 65 drugs approved for the therapy of cancer were natural antagonize the farnesoid X receptor and decrease the products, were based on natural products, or mimicked natural expression of bile acid-activated genes. Because activa- products in one form or another (3). These phytochemicals are tion of NF-B has been closely linked with inflammatory commonly called chemotherapeutic or chemopreventive agents. diseases affected by guggulsterone, we postulated that it These phytochemicals may fight disease through suppression must modulate NF-B activation. In the present study, of the inflammatory response. Dysregulated inflammation is we tested this hypothesis by investigating the effect of the cause of a great many diseases, including cancer (4, 5). It this steroid on the activation of NF-B induced by in- stands to reason, then, that suppression of inflammation, flammatory agents and carcinogens. Guggulsterone sup- whether by phytochemicals or other means, should delay the pressed DNA binding of NF-B induced by tumor necro- onset of disease (1, 2). sis factor (TNF), phorbol ester, okadaic acid, cigarette One phytochemical that has aroused considerable interest is smoke condensate, hydrogen peroxide, and interleu- guggulsterone (4,17(20)-pregnadiene-3,16-dione), a plant sterol kin-1. NF-B activation was not cell type-specific, be- derived from the gum resin (guggulu) of the tree Commiphora cause both epithelial and leukemia cells were inhibited. mukul. The resin has been used in Ayurvedic medicine for Guggulsterone also suppressed constitutive NF-B acti- centuries to treat a variety of ailments, including obesity, bone vation expressed in most tumor cells. Through inhibi- fractures, arthritis, inflammation, cardiovascular disease, and tion of IB kinase activation, this steroid blocked IB lipid disorders (6, 7). The anti-arthritic and anti-inflammatory phosphorylation and degradation, thus suppressing p65 activity of gum guggul was demonstrated as early as 1960, by phosphorylation and nuclear translocation. NF-B-de- Gujral et al. (8). Sharma et al. showed its activity in experi- pendent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK was also blocked mental arthritis induced by mycobacterial adjuvant (9). The by guggulsterone but without affecting p65-mediated effectiveness of guggul for treating osteoarthritis of the knee gene transcription. In addition, guggulsterone de- has also been demonstrated (10). Recent studies have shown creased the expression of gene products involved in that guggulsterone is an antagonist for bile acid receptor far- anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, and nesoid X receptor (11, 12). Other studies have shown that survivin), proliferation (cyclin D1 and c-Myc), and me- guggulsterone enhances transcription of the bile salt export tastasis (MMP-9, COX-2, and VEGF); this correlated with pump (13), thus regulating cholesterol homeostasis. enhancement of apoptosis induced by TNF and chemo- An understanding of the molecular mechanisms underlying therapeutic agents. Overall, our results indicate that guggulsterone is just now emerging. In 2003, Meselhy et al. (14) guggulsterone suppresses NF-B and NF-B-regulated showed that guggulsterone can suppress inflammation by in- gene products, which may explain its anti-inflammatory hibiting inducible nitric-oxide synthetase expression induced activities. by lipopolysaccharide in macrophages. Because most inflam- matory diseases are mediated through the activation of NF- B, a nuclear transcription factor (15, 16), we hypothesize that Of the 121 prescription drugs in use today for cancer treat- it is involved in the activity of guggulsterone. Because expres- ment, 90 are derived from plant species. Almost 74% of these sion of inducible nitric-oxide synthetase requires NF-B acti- vation, Meselhy’s demonstration that guggulsterone down-reg- ulates the expression of inducible nitric-oxide synthetase * This work was supported in part by the Clayton Foundation for Research (to B. B. A.), Department of Defense U.S. Army Breast Cancer supports this hypothesis. Research Program Grant BC010610 (to B. B. A.), Grant PO1 CA91844 NF-B is present in all cells in a resting state in the cyto- from the National Institutes of Health (NIH) on lung chemoprevention (to B. B. A.), and a P50 Head and Neck Specialized Programs of Re- search Excellence grant from the NIH (to B. B. A.). The costs of publi- cation of this article were defrayed in part by the payment of page The abbreviations used are: NF-B, nuclear factor-B; IB, inhibitory charges. This article must therefore be hereby marked “advertisement” subunit of NF-B; TNF, tumor necrosis factor; SEAP, secretory alkaline in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phosphatase; IKK, IB kinase; COX-2, cyclooxygenase-2; MMP-9, ma- ‡ A Ransom Horne Jr. Distinguished Professor of Cancer Research. trix metalloproteinase-9; TNFR, TNF receptor; TRADD, TNFR-associated To whom correspondence should be addressed: Cytokine Research Lab- death domain; TRAF, TNFR-associated factor; NIK, NF-B-inducing ki- oratory, Dept. of Experimental Therapeutics, The University of Texas nase; PMA, phorbol myristate acetate; FBS, fetal bovine serum; EMSA, M. D. Anderson Cancer Center, Box 143, 1515 Holcombe Blvd., Hous- electrophoretic mobility shift assay; TPCK, L-1-tosylamido-2-phenylethyl ton, TX 77030. Tel.: 713-792-3503/6459; Fax: 713-794-1613; E-mail: chloromethyl ketone; MTT, 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyl- [email protected]. tetrazolium bromide; PARP, polyadenosine ribose polymerase. 47148 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Inhibition of TNF-induced NF-B by Guggulsterone 47149 (Jurkat) and myelogenous leukemia (KBM-5) cells were obtained from plasm; only when activated and translocated to the nucleus is the American Type Culture Collection (Manassas, VA). A549, Jurkat, the sequence of events leading to activation initiated (15, 16). and H1299 cells were cultured in RPMI 1640 medium, and KBM-5 cells Currently NF-B is considered a family of Rel-domain-contain- were cultured in Iscove’s modified Dulbecco’s modified Eagle’s medium. ing proteins, namely Rel A (also called p65), Rel B, c-Rel, p50 For Jurkat and A549 cells, RPMI 1640 was supplemented with 10% (also called NF-B1), and p52 (also called NF-B2). Similarly, a FBS, 100 units/ml penicillin, and 100 g/ml streptomycin; for KBM-5 family of anchorin-domain-containing proteins has been iden- cells, Dulbecco’s modified Eagle’s medium was supplemented with 15% FBS plus penicillin and streptomycin. tified that keeps the NF-B in its inactive state within the NF-B Activation—To determine NF-B activation by TNF, which nucleus. These include IB,IB,IB,IB, bcl-3, p105, and has a well established role in inflammation, tumor proliferation, pro- p100. Most carcinogens, inflammatory agents, and tumor pro- motion, invasion, and metastasis (24), we carried out EMSA essentially moters, including cigarette smoke, phorbol ester, okadaic acid, as previously described (25). The dried gels were visualized, and radio- H O , and TNF have been shown to activate NF-B. active bands were quantitated using a PhosphorImager (Molecular 2 2 Under resting conditions, NF-B consists of a heterotrimer of Dynamics, Sunnyvale, CA) using ImageQuaNT software. The -fold ac- tivation, over that of untreated control lane, was then calculated. p50, p65, and IB in the cytoplasm. The phosphorylation, Western Blot Analysis—To determine the effect of guggulsterone on ubiquitination, and degradation of IB and phosphorylation TNF-dependent IB degradation, p65 translocation, and p65 phospho- of p65 leads to the translocation of NF-B to the nucleus where rylation, cytoplasmic extracts were prepared as previously described it binds to specific response elements in the DNA. The phos- 6 (26) from H1299 cells (2  10 /ml) that had been pretreated with 50 M phorylation of IB is catalyzed by IKK, which consists of three guggulsterone for 4 h and then exposed to 0.1 nM TNF for various times. subunits IKK, IKK, and IKK. Gene deletion studies have For determining the phosphorylation of IB, H1299 cells (2  10 /ml) were first treated with 100 M N-acetyl-Leu-Leu-norleucinal (a protea- shown that IKK is essential for NF-B activation by most some inhibitor) for 1 h, then treated with 50 M guggulsterone for 4 h agents. The identity of the kinase that induces the phosphoryl- and then exposed to 0.1 nM TNF for various time points. Thirty micro- ation of p65 is controversial, but IKK, protein kinase C, and grams of cytoplasmic protein was resolved on 10% SDS-PAGE gel, protein kinase A have been implicated (for references see Refs. transferred to a nitrocellulose membrane, blocked with 5% nonfat milk, 17 and 18). NF-B has been shown to regulate the expression of and probed with specific antibody against IB, phosphorylated IB, p65, and phosphorylated p65. To determine the expression of cyclin D1, a number of genes whose products are involved in tumorigen- COX-2, MMP-9, cIAP-1, xIAP, TRAF1, Bcl-2, Bfl-1, cFLIP, and survivin esis (15–18). These include anti-apoptotic genes (e.g. ciap, su- in whole cell extracts of treated cells (2  10 cells in 2 ml medium), vivin, traf, bcl-2, and bcl-xl); cox2; mmp-9; genes encoding 30–50 g of protein was resolved on SDS-PAGE and probed by Western adhesion molecules, chemokines, and inflammatory cytokines; blot with specific antibodies as per the manufacturer’s recommended and cell cycle regulatory genes (e.g. cyclin d1). protocol. The blots were washed, exposed to horseradish peroxidase- Furthermore, NF-B has been implicated in obesity (19), conjugated secondary antibodies for 1 h, and finally detected by ECL reagent (Amersham Biosciences). The bands were quantitated using a hyperlipidemia (20), atherosclerosis (21), osteoarthritis (22), Personal Densitometer Scan version 1.30 using ImageQuaNT software and bone loss (23), all of which can be modulated by guggul- version 3.3 (Amersham Biosciences). sterone. In the present report we investigated the effect of Immunolocalization of NF-B p65—The effect of guggulsterone on guggulsterone on NF-B activation induced by a variety of cigarette smoke-induced nuclear translocation of p65 was examined by inflammatory agents and carcinogens. The aim of the study an immunocytochemical method using an epifluorescence microscope was to determine whether guggulsterone can suppress NF-B (Labophot-2; Nikon, Tokyo, Japan) and a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) as described previously (27). activation induced by inflammatory agents and carcinogens IKK Assay—To determine the effect of guggulsterone on TNF-in- and block NF-B-regulated gene expression that mediates in- duced IKK activation, we analyzed IKK by a method essentially as flammation and carcinogenesis. We found that guggulsterone described previously (28). inhibited activation of NF-B through suppression of IB NF-B-dependent Reporter Gene Transcription—The effect of gug- kinase, IB phosphorylation, and degradation, p65 nuclear gulsterone on TNF-induced NF-B-dependent reporter gene transcrip- translocation, and NF-B-dependent reporter gene expression. tion in H1299 cells was measured as previously described (29). COX-2 Promoter-dependent Reporter Luciferase Gene Expression— Guggulsterone also abrogated the expression of NF-B-regu- COX-2 promoter activity was examined as described elsewhere (28). To lated gene products that inhibit apoptosis and promote inflam- further determine the effect of guggulsterone on COX-2 promoter, A293 mation and tumor metastasis. cells were seeded at a concentration of 1.5  10 cells per well in six-well plates. After overnight culture, the cells in each well were transfected EXPERIMENTAL PROCEDURES with 2 g of DNA consisting of COX-2 promoter-luciferase reporter Materials—Z-Guggulsterone, obtained from Steraloids, Inc. (New- plasmid, along with 6 l of LipofectAMINE 2000 according to the port, RI), was dissolved in Me SO as a 100 mM stock solution and stored manufacturer’s protocol. The COX-2 promoter (375 to 59), which at 20 °C. Bacteria-derived human tumor necrosis factor (TNF), puri- was amplified from human genomic DNA by using the primers 5- fied to homogeneity with a specific activity of 5  10 units/mg, was GAGTCTCTTATTTATTTTT-3 (sense) and 5-GCTGCTGAGGAGTTC- kindly provided by Genentech, Inc. (South San Francisco, CA). Penicil- CTGGACGTGC-3 (antisense), was kindly provided by Dr. Xiao-Chun lin, streptomycin, RPMI 1640 medium, FBS, and LipofectAMINE 2000 Xu (M. D. Anderson Cancer Center). After a 6-h exposure to the trans- were obtained from Invitrogen. The following polyclonal antibodies fection mixture, the cells were incubated in medium containing guggul- were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): sterone for 12 h. The cells were exposed to TNF (0.1 nM) for 24 h and anti-p65, against the epitope corresponding to amino acids mapping then harvested. Luciferase activity was measured by using the Pro- within the amino-terminal domain of human NF-B p65; anti-p50, mega luciferase assay system according to the manufacturer’s protocol against a peptide 15 amino acids long mapping at the nuclear localiza- and detected by using Monolight 2010 (Analytical Luminescence Lab- tion sequence region of NF-B p50; anti-IB, against amino acids oratory, San Diego, CA). All experiments were performed in triplicate 297–317 mapping at the carboxyl terminus of IB/MAD-3; anti-c-Rel, and repeated at least twice to prove their reproducibility. anti-cyclin D1 against amino acids 1–295, which represents full-length Cytotoxicity Assay—Cytotoxicity was assayed by the modified tetra- cyclin D1 of human origin; anti-MMP-9, anti-polyadenosine ribose poly- zolium salt 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyl-tetrazolium bro- merase (PARP); anti-IAP1; anti-IAP2; anti-Bcl-2; anti-Bfl-1/A1; and mide (MTT) assay as described previously (27). anti-TRAF1. Anti-COX-2, anti-MMP9, and anti-XIAP antibodies were PARP Cleavage Assay—For detection of cleavage products of PARP, obtained from BD Biosciences, and phospho-specific anti-IB (Ser32) whole cell extracts were prepared by subjecting guggulsterone-treated antibody was from Cell Signaling (Beverly, MA). Anti-IKK and anti- cells to lysis in lysis buffer (20 mM Tris, pH 7.4; 250 mM NaCl; 2 mM IKK antibodies were kindly provided by Imgenex (San Diego, CA). EDTA, pH 8.0; 0.1% Triton-X 100; 0.01 g/ml aprotinin; 0.005 g/ml Cell Lines—The cell lines used in our studies included human non- leupeptin; 0.4 mM phenylmethylsulfonyl fluoride; and 4 mM NaVO ). small cell lung carcinoma (H1299) cells and human lung epithelial cell Lysates were spun at 14,000 rpm for 10 min to remove insoluble carcinoma (A549) cells, both kindly provided by Dr. Reuben Lotan (The material, resolved by 10% SDS-PAGE, and probed with PARP University of Texas M. D. Anderson Cancer Center). Human leukemia antibodies. 47150 Inhibition of TNF-induced NF-B by Guggulsterone FIG.1. A, structure of guggulsterone. B, guggulsterone blocks NF-B activation induced by PMA, interleukin-1, okadaic acid, cigarette smoke condensate (CSC), and H O . H1299 cells (2  10 /ml) were preincubated for4hat37 °C with 50 M guggulsterone and then treated with PMA 2 2 (100 ng/ml, 1 h), interleukin-1 (100 ng/ml, 1 h), okadaic acid (500 nM, 4 h), H O (250 M, 1 h), or cigarette smoke condensate (10 g/ml, 30 min). 2 2 Nuclear extracts were prepared and tested for NF-B activation as described under “Experimental Procedures.” The numbers at the bottom of each lane indicate -fold activation over that of untreated control. Live and Dead Assay—To measure apoptosis, we used the Live and NF-B by these agents in H1299 cells. A DNA-binding assay Dead assay (Molecular Probes), which determines intracellular esterase (EMSA) showed that guggulsterone suppressed the NF-B ac- activity and plasma membrane integrity. This assay employs calcein, a tivation induced by all these agents (Fig. 1B). These results polyanionic dye, which is retained within the live cells and provides green suggest that guggulsterone acted at a step in the NF-B acti- fluorescence (30). It also employs the ethidium monomer dye (red fluores- vation pathway that is common to all these agents. cence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of Inhibition of NF-B Activation by Guggulsterone Is Not Cell live cells. Briefly, 1  10 cells were incubated with 10 M guggulsterone Type-specific—Because some reports suggest that distinct for 24 h and then treated with 1 nM TNF for 16 h at 37 °C. Cells were signal transduction pathways mediate NF-B induction in stained with the Live and Dead reagent (5 M ethidium homodimer, 5 M epithelial and lymphoid cells (31), we determined whether gug- calcein-AM) and then incubated at 37 °C for 30 min. Cells were analyzed gulsterone inhibited NF-B activation in three cell types. Gug- under a fluorescence microscope (Labophot-2). gulsterone completely inhibited TNF-induced NF-B activation RESULTS in lung epithelial cell carcinoma (A549), T cell leukemia (Jurkat), and myeloid leukemia (KBM-5) cells (Fig. 2A), indi- The guggulsterone used in these studies, which has a ste- cating a lack of cell type specificity. roid-like structure (Fig. 1A), was dissolved in Me SO. The Guggulsterone Inhibits Constitutive NF-B Activation—We concentration of guggulsterone used and the duration of expo- next tested the effect of guggulsterone on NF-B activation in sure had minimal effect on the viability of these cells as deter- human multiple myeloma (U266) and head and neck squamous mined by trypan blue dye exclusion test (data not shown). Guggulsterone Blocks NF-B Activation Induced by TNF, cell carcinoma (MDA 1986) tumor cells, which both express constitutively active NF-B (27, 32). Guggulsterone completely Interleukin-1, PMA, H O , Cigarette Smoke Condensate, and 2 2 Okadaic Acid—Because TNF, interleukin-1, PMA, H O , cig- inhibited this constitutively active NF-B (Fig. 2B). 2 2 arette smoke condensate, and okadaic acid activate NF-B, we The Suppression of NF-B by Guggulsterone Is Dose- and examined the effect of guggulsterone on the activation of Time-dependent—Guggulsterone inhibited TNF-mediated Inhibition of TNF-induced NF-B by Guggulsterone 47151 FIG.2. Guggulsterone suppresses inducible and constitutive NF-B ac- tivation. A, suppression of inducible ac- tivation is not cell-type specific. Two mil- lion A549, Jurkat, or KBM-5 cells were pre-treated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for 30 min. The nuclear extracts were then prepared and assayed for NF-Bby EMSA as described under “Experimental Procedures.” B, two million multiple my- eloma and head and neck squamous cell carcinoma cells were exposed to 50 M guggulsterone for 4 h, and then nuclear extracts were prepared and assayed for NF-B by EMSA as described under “Ex- perimental Procedures.” The numbers at the bottom of each lane indicate -fold ac- tivation over that of untreated control. NF-B activation in a dose-dependent manner, with maximum binding ability of NF-B proteins prepared from cells by treat- inhibition occurring at 50 M (Fig. 3A). The minimum time ment with TNF (Fig. 3D). Therefore, guggulsterone must inhibit required for complete inhibition of NF-B activation was 4 h NF-B activation by a different mechanism. (Fig. 3B). Guggulsterone Inhibits TNF-dependent IB Degradation— Suppressed NF-B Consists of Both p50 and p65—When Because IB degradation is normally a condition for translo- nuclear extracts from TNF-activated cells were incubated with cation of NF-B to the nucleus (36), we determined whether the antibodies to the p50 (NF-B1) and the p65 (RelA) subunit of guggulsterone’s inhibition of TNF-induced NF-B activation NF-B, the resulting bands were shifted to higher molecular was due to inhibition of IB degradation. We found that TNF masses (Fig. 3C), suggesting that the TNF-activated complex induced IB degradation in control cells as early as 10 min, consisted of p50 and p65. Neither preimmune serum nor irrel- but in guggulsterone-pretreated cells TNF had no effect on evant antibody had any effect. Addition of excess unlabeled IB degradation (Fig. 4A, upper panel). NF-B (cold oligonucleotide; 100-fold) caused complete disap- Guggulsterone Inhibits TNF-dependent IB Phosphoryla- pearance of the band, whereas mutated oligonucleotide had no tion—We next determined whether guggulsterone affected effect on the DNA binding. TNF-induced IB phosphorylation, another condition for Guggulsterone Does Not Directly Affect Binding of NF-Btothe NF-B translocation. Western blot analysis using antibody DNA—Although TPCK (the serine protease inhibitor), herbimy- that detects only the serine-phosphorylated form of IB indi- cin A (protein tyrosine kinase inhibitor), and caffeic acid phenyl cated that TNF induced IB phosphorylation as early as 5 ethyl ester directly modify NF-B to suppress its activation (33– min, and guggulsterone almost completely suppressed it (Fig. 35), EMSA showed that guggulsterone did not modify the DNA- 4A, middle panel). Thus guggulsterone inhibited TNF-induced 47152 Inhibition of TNF-induced NF-B by Guggulsterone FIG.3. A, guggulsterone inhibits TNF-dependent NF-B activation in a dose-dependent manner. H1299 cells (2  10 /ml) were preincubated with the indicated concentrations of guggulsterone for4hat37 °Cand 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  10 /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  10 /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  10 /ml) treated or not treated with 0.1 nM TNF for 30 min were treated with the indicated concentrations of guggulsterone for4hat 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. NF-B activation by inhibiting phosphorylation and degrada- tion, which is required for TNF-induced phosphorylation of tion of IB. IB. As shown in Fig. 4B (upper panel), guggulsterone com- Guggulsterone Inhibits TNF-induced IKK Activation—Be- pletely suppressed TNF-induced activation of IKK. TNF or cause guggulsterone inhibits the phosphorylation of IB,we guggulsterone had no direct effect on the expression of either tested the effect of guggulsterone on TNF-induced IKK activa- IKK (middle panel)orIKK (lower panel) proteins. However, Inhibition of TNF-induced NF-B by Guggulsterone 47153 FIG.4. A, guggulsterone inhibits TNF-induced phosphorylation and degradation of IB. H1299 cells (2  10 /ml) were incubated with 50 M guggulsterone for4hat37 °C, treated with 0.1 nM TNF for the indicated times at 37 °C, and then tested for IB (upper panel) and phosphorylated IB (middle panel) in cytosolic fractions by Western blot analysis. Equal protein loading was evaluated by -actin (lower panel). B, guggulsterone inhibits TNF-induced IB kinase activity. H1299 cells (2  10 /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  10 /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. we did find that guggulsterone inhibited the phosphorylation of Guggulsterone Represses TNF-induced NF-B-dependent Re- GST IB (at a 50 M concentration) by directly interfering porter Gene Expression—Although we showed by EMSA that with IKK activity (Fig. 4C). guggulsterone blocked NF-B activation, DNA binding alone Guggulsterone Inhibits TNF-induced Phosphorylation and does not always correlate with NF-B-dependent gene tran- Nuclear Translocation of p65—We also tested the effect of scription, suggesting that there are additional regulatory steps guggulsterone on TNF-induced phosphorylation of p65, be- (38). Transient transfection of H1299 cells with the NF-B- cause phosphorylation is also required for transcriptional ac- regulated SEAP reporter construct followed by stimulation tivity of p65 (37). As shown in Fig. 5A, guggulsterone sup- with TNF produced an almost 13-fold increase in SEAP activity pressed p65 phosphorylation almost completely. Likewise, over vector control activity (Fig. 6A). TNF-induced SEAP ac- Western blot analysis (Fig. 5B) and immunocytochemistry (Fig. tivity was abolished by dominant-negative IB, indicating 5C) indicated that guggulsterone abolished TNF-induced nu- specificity. When the cells were pretreated with guggulsterone, clear translocation of p65. TNF-induced NF-B-dependent SEAP expression was inhib- 47154 Inhibition of TNF-induced NF-B by Guggulsterone FIG.5. A, guggulsterone inhibits TNF-induced phosphorylation of p65. H1299 cells (2  10 /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  10 /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  10 /ml) were first treated with 50 M guggulsterone for4hat37 °Cand then exposed to 0.1 nM TNF. After cytospin, immunocytochemical analysis was performed as described under “Experimental Procedures.” ited by guggulsterone in a dose-dependent manner. These re- Guggulsterone Represses TNF-induced COX-2 Promoter Ac- sults demonstrate that guggulsterone inhibits NF-B-depend- tivity—We next determined whether guggulsterone affected ent reporter gene expression induced by TNF. COX-2 promoter activity, which is regulated by NF-B (41). As We next determined where guggulsterone acts in the se- shown in Fig. 6C, guggulsterone significantly reduced the TNF- quence of TNFR1, TRADD, TRAF2, NIK, and IKK recruitment induced COX-2 promoter activity in a dose-dependent manner. that characterizes TNF-induced NF-B activation (39, 40). In Guggulsterone Inhibits TNF-induced COX-2, MMP-9, and cells transfected with TNFR, TRADD, TRAF2, NIK, IKK, and VEGF Expression and TNF-induced Cyclin D1 and c-myc Ex- p65 plasmids, NF-B-dependent SEAP expression was in- pression—Guggulsterone abolished, in a dose-dependent fash- duced; 50 M guggulsterone significantly suppressed SEAP ion, the TNF-induced expression of COX-2, MMP-9, and VEGF expression in all cells except those transfected with p65. Be- (Fig. 7A), which are known to be NF-B-regulated gene prod- cause IKK activation can cause the phosphorylation of IB ucts (41–43). Expression of the NF-B-regulated gene products and p65, we suggest that guggulsterone inhibits NF-B activa- cyclin D1 and c-Myc (44, 45) was also abolished by guggul- tion through inhibition of IKK. sterone (Fig. 7B). Inhibition of TNF-induced NF-B by Guggulsterone 47155 FIG.6. A, guggulsterone inhibits TNF- induced NF-B-dependent reporter gene (SEAP) expression. H1299 cells were transiently transfected with an NF-B- containing plasmid linked to the SEAP gene and then treated with the indicated concentrations of guggulsterone. After 24 h in culture with 0.1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described under “Ex- perimental Procedures.” Results are ex- pressed as -fold activity over the activity of the vector control. B, guggulsterone in- hibits NF-B-dependent reporter gene ex- pression induced by TNFR, TRADD, TRAF, NIK, and IKK. H1299 cells were transiently transfected with the indicated plasmids along with an NF-B-containing plasmid linked to the SEAP gene and then left either untreated or treated with 50 M guggulsterone for 4 h. Cell super- natants were assayed for secreted alka- line phosphatase activity as described un- der “Experimental Procedures.” Results are expressed as -fold activity over the activity of the vector control. Bars indi- cate standard deviation. C, guggulsterone inhibits TNF-induced COX-2 promoter activity. H1299 cells were transiently transfected with a COX-2 promoter plas- mid linked to the luciferase gene and then treated with the indicated concentrations of guggulsterone. After 24 h in culture with 0.1 nM TNF, cell supernatants were collected and assayed for luciferase activ- ity as described under “Experimental Pro- cedures.” Results are expressed as -fold activity over the activity of the vector control. Guggulsterone Inhibits TNF-induced Activation of Anti-apo- tory stimuli (hydrogen peroxide, TNF, and interleukin-1) ptotic Gene Products—NF-B up-regulates the expression of a through inhibition of IKK, IB phosphorylation, and IB number of genes implicated in facilitating tumor cell survival, degradation, which led to abrogation of p65 phosphorylation including cIAP1, xIAP, Bfl-1, BCl-2, TRAF1, cFLIP, and sur- and nuclear translocation. NF-B-dependent reporter gene vivin (46–54). We found that guggulsterone inhibited the TNF- transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, induced expression of all of these proteins (Fig. 8). and IKK, was also blocked. The expression of gene products Guggulsterone Potentiates the Cytotoxic Effects of TNF and involved in anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, Chemotherapeutic Drugs—Because NF-B-regulated products and survivin), proliferative genes (cyclin d1 and c-Myc), and suppress TNF- and chemotherapy-induced apoptosis (55, 56), metastatic genes (MMP-9, COX-2, and VEGF) was down-regu- we examined the effects of guggulsterone on the apoptotic lated by guggulsterone, and this down-regulation correlated effects of TNF and the chemotherapeutic drugs paclitaxel and with enhancement of apoptosis induced by TNF and chemo- doxorubicin. Guggulsterone enhanced the cytotoxic effects of therapeutic agents. TNF, Taxol, and doxorubicin (Fig. 9A) and the caspase-induced The present study was undertaken to investigate the potential cleavage of PARP activated by TNF (Fig. 9B). The Live and mechanism for the anti-inflammatory effects of guggulsterone, Dead assay also showed that TNF-induced apoptosis was sig- which has been used to treat obesity, diabetes, hyperlipidemia, nificantly enhanced by guggulsterone (Fig. 9C). atherosclerosis, and osteoarthritis. Although guggulsterone can DISCUSSION antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes (11), these effects do not explain its In this study, we have demonstrated that guggulsterone anti-inflammatory attributes. Because activation of NF-B has suppressed NF-B activated by carcinogens (phorbol ester, okadaic acid, and cigarette smoke condensate) and inflamma- been found in most inflammatory diseases, we hypothesized that 47156 Inhibition of TNF-induced NF-B by Guggulsterone FIG.7. A, guggulsterone inhibits COX-2, MMP-9, and VEGF expression in- duced by TNF. H1299 cells (2  10 /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, guggul- sterone inhibits cyclin D1 and c-Myc ex- pression induced by TNF. H1299 cells (2  10 /ml) were left untreated or incu- bated 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 ly- sate was analyzed by Western blotting using antibodies against cyclin D1 and c-Myc. all these agents. We identified IKK as a target site: cells that were exposed to guggulsterone failed to activate IKK in re- sponse to TNF. Surprisingly, incubation of IKK with guggul- sterone 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. Au- phan et al. (57) showed that glucocorticoids inhibit NF-B activation and that this inhibition is mediated through induc- tion of the IB, which traps activated NF-B in inactive cy- toplasmic complexes. Interestingly, estradiol was found to ac- tivate 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, FIG.8. Guggulsterone inhibits the expression of anti-apo- ptotic gene products IAP1, XIAP, Bfl-1/A1, Bcl-2, TRAF1, cFLIP, be completely ruled out by the results of our study. and survivin. H1299 cells (2  10 /ml) were left untreated or incu- Various tumor cell types express constitutively active NF- bated with 50 M guggulsterone for 4 h and then treated with 0.1 nM B, and it is critical for their proliferation (18, 27, 32). The TNF for different times. Whole cell extracts were prepared, and 50 g potential mechanism of constitutive activation of NF-Bisnot of the whole cell lysate was analyzed by Western blotting using anti- bodies against IAP1, XIAP, Bfl-1/A1, bcl-2, TRAF1, cFLIP, and survivin fully understood. Overexpression of IB without inhibition of as indicated. NF-B activity and mutations in the ib gene in Reed-Stern- berg cells (59) and enhanced IB degradation in mature mu- rine B-cell lines (60) have been demonstrated as potential guggulsterone modulates NF-B activation. The results of the study validate this hypothesis. mechanisms. Our laboratory showed that constitutive expres- sion of TNF in T-cell lymphoma (32) and interleukin-1 in acute Very little is known about the mechanism of action of gug- gulsterone. Our results clearly indicate that guggulsterone can myelogenous leukemia (61) are potential mechanisms. We found that human multiple myeloma cells and head and neck suppress NF-B activation induced by a wide variety of agents, suggesting that the site of action of guggulsterone is common to squamous cell carcinoma cells also express constitutive NF-B, Inhibition of TNF-induced NF-B by Guggulsterone 47157 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  S.D. from triplicate cultures. B, KBM-5 cells (2  10 /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  10 /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.” and guggulsterone suppressed the activation. It is very likely expression of numerous anti-apoptotic gene products, all that this inhibition also occurs through inhibition of IKK. known to be regulated by NF-B activation. The overexpres- That activation of NF-B regulates genes that control prolif- sion of IAP1, xIAP, Bfl-1/A1, bcl-2, cFLIP, and survivin has eration and metastasis of cancer has been well established (17, been found in numerous tumors and has been linked to sur- 18, 43). Our results demonstrate that guggulsterone can sup- vival, chemoresistance, and radioresistance. Because most of press the expression of COX-2, MMP-9, VEGF, cyclin D1, and these gene products are down-regulated by guggulsterone, we c-Myc, all regulated by NF-B. We found that COX-2 promoter believe that guggulsterone has potential as an agent for over- activity was also significantly down-regulated by this steroid. coming radioresistance and chemoresistance. Indeed, guggul- The lack of complete inhibition of COX-2 promoter activity by sterone potentiated the apoptotic effects of TNF and Taxol and guggulsterone suggests that this promoter is also regulated by doxorubicin. Thus guggulsterone, which is a pharmacologically transcription factors other than NF-B. These results imply safe agent (6), could be used as an anticancer agent on its own, that guggulsterone has a potential to suppress proliferation of a preventive agent, or an enhancer of chemotherapy/radiother- tumor cells and their metastasis. apy activity. Our demonstration, that guggulsterone sup- Our results indicate that guggulsterone also suppresses the presses NF-B and NF-B-regulated gene expression, may 47158 Inhibition of TNF-induced NF-B by Guggulsterone 23. Abu-Amer, Y., and Tondravi, M. M. (1997) Nat. Med. 3, 1189–1190 explain its effects against atherosclerosis, diabetes, osteoar- 24. Aggarwal, B. B. (2003) Nat. Rev. Immunol. 3, 745–756 thritis, and other inflammatory diseases. 25. Chaturvedi, M. M., Mukhopadhyay, A., and Aggarwal, B. B. (2000) Methods Enzymol. 319, 585–602 Cancer is a hyperproliferative disorder characterized by the 26. Majumdar, S., and Aggarwal, B. B. (2001) J. Immunol. 167, 2911–2920 up-regulation of genes responsible for transformation, prolifer- 27. Bharti, A. C., Donato, N., Singh, S., and Aggarwal, B. B. (2003) Blood 101, ation, invasion, angiogenesis, and metastasis. Most of these 1053–1062 28. Shishodia, S., Majumdar, S., Banerjee, S., and Aggarwal, B. B. (2003) Cancer activities are influenced by the aberrant activity of NF-B; Res. 63, 4375–4383 therefore, the main objective of this study was to evaluate the 29. Manna, S. K., Mukhopadhyay, A., and Aggarwal, B. B. (2000) J. Immunol. 165, 4927–4934 efficacy of guggulsterone as an inhibitor of NF-B. Our study 30. Takada, Y., Khuri, F. R., and Aggarwal, B. B. (2004) J. Biol. Chem. 279, for the first time demonstrates that guggulsterone can sup- 26287–26299 press NF-B activation induced by various carcinogens, inflam- 31. Bonizzi, G., Piette, J., Merville, M. P., and Bours, V. (1997) J. Immunol. 159, 5264–5272 matory agents, and tumor promoters. This effect was not cell 32. Giri, D. K., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 14008–14014 type-specific and equally pronounced in cells where NF-Bis 33. Finco, T. S., Beg, A. A., and Baldwin, A. S., Jr. (1994) Proc. Natl. Acad. Sci. constitutively active. TNF-induced activation of IKK and phos- U. S. A. 91, 11884–11888 34. Mahon, T. M., and O’Neill, L. A. (1995) J. Biol. Chem. 270, 28557–28564 phorylation and degradation IB were also inhibited by gug- 35. Natarajan, K., Singh, S., Burke, T. R., Jr., Grunberger, D., and Aggarwal, B. B. gulsterone. Guggulsterone was quite effective in blocking the (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9090–9095 36. Miyamoto, S., Maki, M., Schmitt, M. J., Hatanaka, M., and Verma, I. M. (1994) phosphorylation and translocation of TNF-induced p65, which Proc. Natl. Acad. Sci. U. S. A. 91, 12740–12744 led to the abrogation of NF-B-dependent reporter gene expres- 37. Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661–671 sion. TNF-induced NF-B reporter activity induced by TNFR, 38. Nasuhara, Y., Adcock, I. M., Catley, M., Barnes, P. J., and Newton, R. (1999) J. Biol. Chem. 274, 19965–19972 TRADD, TRAF, NIK, and IKK, but not that activated by p65, 39. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299–308 was also blocked. 40. Simeonidis, S., Stauber, D., Chen, G., Hendrickson, W. A., and Thanos, D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 49–54 Acknowledgment—We thank Walter Pagel for a careful review of 41. Yamamoto, K., Arakawa, T., Ueda, N., and Yamamoto, S. (1995) J. Biol. Chem. the manuscript. 270, 31315–31320 42. Esteve, P. O., Chicoine, E., Robledo, O., Aoudjit, F., Descoteaux, A., Pot- REFERENCES worowski, E. F., and St-Pierre, Y. (2002) J. Biol. Chem. 277, 35150–35155 43. Huang, S., Robinson, J. B., Deguzman, A., Bucana, C. D., and Fidler, I. J. 1. Craig, W. J. (1997) J. Am. Diet. Assoc. 97, S199–S204 (2000) Cancer Res. 60, 5334–5339 2. Craig, W. J. (1999) Am. J. Clin. Nutr. 70, 491S–499S 44. Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and Baldwin, 3. Newman, D. J., Cragg, G. M., and Snader, K. M. (2003) J. Nat. Prod. 66, A. S., Jr. (1999) Mol. Cell. Biol. 19, 5785–5799 1022–1037 45. Wittekindt, N. E., Hortnagel, K., Geltinger, C., and Polack, A. (2000) Nucleic 4. Coussens, L. M., and Werb, Z. (2002) Nature 420, 860–867 Acids Res. 28, 800–808 5. Balkwill, F., and Mantovani, A. (2001) Lancet 357, 539–545 46. Zhu, L., Fukuda, S., Cordis, G., Das, D. K., and Maulik, N. (2001) FEBS Lett. 6. Urizar, N. L., and Moore, D. D. (2003) Annu. Rev. Nutr. 23, 303–313 508, 369–374 7. Sinal, C. J., and Gonzalez, F. J. (2002) Trends Endocrinol. Metab. 13, 275–276 47. Schwenzer, R., Siemienski, K., Liptay, S., Schubert, G., Peters, N., Scheurich, 8. Gujral, M. L., Sareen, K., Tangri, K. K., Amma, M. K., and Roy, A. K. (1960) P., Schmid, R. M., and Wajant, H. (1999) J. Biol. Chem. 274, 19368–19374 Indian J. Physiol. Pharmacol. 4, 267–273 48. Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J., Malim, M. H., and Ballard, 9. Sharma, J. N. (1977) Arzneimittelforschung 27, 1455–1457 D. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10057–10062 10. Singh, B. B., Mishra, L. C., Vinjamury, S. P., Aquilina, N., Singh, V. J., and 49. You, M., Ku, P. T., Hrdlickova, R., and Bose, H. R., Jr. (1997) Mol. Cell. Biol. Shepard, N. (2003) Altern. Ther. Health Med. 9, 74–79 17, 7328–7341 11. Urizar, N. L., Liverman, A. B., Dodds, D. T., Silva, F. V., Ordentlich, P., Yan, 50. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J. A., Binder, B. R., and Y., Gonzalez, F. J., Heyman, R. A., Mangelsdorf, D. J., and Moore, D. D. Lipp, J. (1998) J. Exp. Med. 188, 211–216 (2002) Science 296, 1703–1706 51. Catz, S. D., and Johnson, J. L. (2001) Oncogene 20, 7342–7351 12. Wu, J., Xia, C., Meier, J., Li, S., Hu, X., and Lala, D. S. (2002) Mol. Endocrinol. 52. Grumont, R. J., Rourke, I. J., and Gerondakis, S. (1999) Genes Dev. 13, 16, 1590–1597 400–411 13. Cui, J., Huang, L., Zhao, A., Lew, J. L., Yu, J., Sahoo, S., Meinke, P. T., Royo, 53. Zong, W. X., Edelstein, L. C., Chen, C., Bash, J., and Gelinas, C. (1999) Genes I., Pelaez, F., and Wright, S. D. (2003) J. Biol. Chem. 278, 10214–10220 Dev. 13, 382–387 14. Meselhy, M. R. (2003) Phytochemistry 62, 213–218 54. Kreuz, S., Siegmund, D., Scheurich, P., and Wajant, H. (2001) Mol. Cell. Biol. 15. Yamamoto, Y., and Gaynor, R. B. (2001) Curr. Mol. Med. 1, 287–296 21, 3964–3973 16. Aggarwal, B. B., Takada, Y., Shishodia, S., Gutierrez, A. M., Oommen, O. V., 55. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. Ichikawa, H., Baba, Y., and Kumar, A. (2004) Indian J. Exp. Biol. 42, (1996) Science 274, 787–789 341–353 56. Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr. (1996) Science 274, 784–787 17. Karin, M., Cao, Y., Greten, F. R., and Li, Z. W. (2002) Nat. Rev. Cancer 2, 57. Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A., and Karin, M. (1995) 301–310 Science 270, 286–290 18. Garg, A., and Aggarwal, B. B. (2002) Leukemia 16, 1053–1068 58. Shyamala, G., and Guiot, M. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 19. Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z. W., Karin, M., and Shoelson, S. E. (2001) Science 293, 1673–1677 10628–10632 59. Emmerich, F., Meiser, M., Hummel, M., Demel, G., Foss, H. D., Jundt, F., 20. Thurberg, B. L., and Collins, T. (1998) Curr. Opin. Lipidol. 9, 387–396 21. Brand, K., Page, S., Rogler, G., Bartsch, A., Brandl, R., Knuechel, R., Page, M., Mathas, S., Krappmann, D., Scheidereit, C., Stein, H., and Dorken, B. (1999) Blood 94, 3129–3134 Kaltschmidt, C., Baeuerle, P. A., and Neumeier, D. (1996) J. Clin. Invest. 97, 1715–1722 60. Miyamoto, S., Chiao, P. J., and Verma, I. M. (1994) Mol. Cell. Biol. 14, 3276–3282 22. Miagkov, A. V., Kovalenko, D. V., Brown, C. E., Didsbury, J. R., Cogswell, J. P., Stimpson, S. A., Baldwin, A. S., and Makarov, S. S. (1998) Proc. Natl. Acad. 61. Estrov, Z., Manna, S. K., Harris, D., Van, Q., Estey, E. H., Kantarjian, H. M., Sci. U. S. A. 95, 13859–13864 Talpaz, M., and Aggarwal, B. B. (1999) Blood 94, 2844–2853 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Guggulsterone Inhibits NF-κB and IκBα Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis

Shishodia, Shishir; Aggarwal, Bharat B.
Journal of Biological Chemistry – Nov 1, 2004
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 45, Issue of November 5, pp. 47148–47158, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Guggulsterone Inhibits NF-B and IB Kinase Activation, Suppresses Expression of Anti-apoptotic Gene Products, and Enhances Apoptosis* Received for publication, July 16, 2004, and in revised form, August 17, 2004 Published, JBC Papers in Press, August 17, 2004, DOI 10.1074/jbc.M408093200 Shishir Shishodia and Bharat B. Aggarwal‡ From the Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 were discovered by investigating a folklore claim (1, 2). Taxol is Guggulsterone, derived from Commiphora mukul and used to treat obesity, diabetes, hyperlipidemia, athero- perhaps the most recent example. Between 1981 and 2002, 48 sclerosis, and osteoarthritis, has been recently shown to of 65 drugs approved for the therapy of cancer were natural antagonize the farnesoid X receptor and decrease the products, were based on natural products, or mimicked natural expression of bile acid-activated genes. Because activa- products in one form or another (3). These phytochemicals are tion of NF-B has been closely linked with inflammatory commonly called chemotherapeutic or chemopreventive agents. diseases affected by guggulsterone, we postulated that it These phytochemicals may fight disease through suppression must modulate NF-B activation. In the present study, of the inflammatory response. Dysregulated inflammation is we tested this hypothesis by investigating the effect of the cause of a great many diseases, including cancer (4, 5). It this steroid on the activation of NF-B induced by in- stands to reason, then, that suppression of inflammation, flammatory agents and carcinogens. Guggulsterone sup- whether by phytochemicals or other means, should delay the pressed DNA binding of NF-B induced by tumor necro- onset of disease (1, 2). sis factor (TNF), phorbol ester, okadaic acid, cigarette One phytochemical that has aroused considerable interest is smoke condensate, hydrogen peroxide, and interleu- guggulsterone (4,17(20)-pregnadiene-3,16-dione), a plant sterol kin-1. NF-B activation was not cell type-specific, be- derived from the gum resin (guggulu) of the tree Commiphora cause both epithelial and leukemia cells were inhibited. mukul. The resin has been used in Ayurvedic medicine for Guggulsterone also suppressed constitutive NF-B acti- centuries to treat a variety of ailments, including obesity, bone vation expressed in most tumor cells. Through inhibi- fractures, arthritis, inflammation, cardiovascular disease, and tion of IB kinase activation, this steroid blocked IB lipid disorders (6, 7). The anti-arthritic and anti-inflammatory phosphorylation and degradation, thus suppressing p65 activity of gum guggul was demonstrated as early as 1960, by phosphorylation and nuclear translocation. NF-B-de- Gujral et al. (8). Sharma et al. showed its activity in experi- pendent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, and IKK was also blocked mental arthritis induced by mycobacterial adjuvant (9). The by guggulsterone but without affecting p65-mediated effectiveness of guggul for treating osteoarthritis of the knee gene transcription. In addition, guggulsterone de- has also been demonstrated (10). Recent studies have shown creased the expression of gene products involved in that guggulsterone is an antagonist for bile acid receptor far- anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, and nesoid X receptor (11, 12). Other studies have shown that survivin), proliferation (cyclin D1 and c-Myc), and me- guggulsterone enhances transcription of the bile salt export tastasis (MMP-9, COX-2, and VEGF); this correlated with pump (13), thus regulating cholesterol homeostasis. enhancement of apoptosis induced by TNF and chemo- An understanding of the molecular mechanisms underlying therapeutic agents. Overall, our results indicate that guggulsterone is just now emerging. In 2003, Meselhy et al. (14) guggulsterone suppresses NF-B and NF-B-regulated showed that guggulsterone can suppress inflammation by in- gene products, which may explain its anti-inflammatory hibiting inducible nitric-oxide synthetase expression induced activities. by lipopolysaccharide in macrophages. Because most inflam- matory diseases are mediated through the activation of NF- B, a nuclear transcription factor (15, 16), we hypothesize that Of the 121 prescription drugs in use today for cancer treat- it is involved in the activity of guggulsterone. Because expres- ment, 90 are derived from plant species. Almost 74% of these sion of inducible nitric-oxide synthetase requires NF-B acti- vation, Meselhy’s demonstration that guggulsterone down-reg- ulates the expression of inducible nitric-oxide synthetase * This work was supported in part by the Clayton Foundation for Research (to B. B. A.), Department of Defense U.S. Army Breast Cancer supports this hypothesis. Research Program Grant BC010610 (to B. B. A.), Grant PO1 CA91844 NF-B is present in all cells in a resting state in the cyto- from the National Institutes of Health (NIH) on lung chemoprevention (to B. B. A.), and a P50 Head and Neck Specialized Programs of Re- search Excellence grant from the NIH (to B. B. A.). The costs of publi- cation of this article were defrayed in part by the payment of page The abbreviations used are: NF-B, nuclear factor-B; IB, inhibitory charges. This article must therefore be hereby marked “advertisement” subunit of NF-B; TNF, tumor necrosis factor; SEAP, secretory alkaline in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phosphatase; IKK, IB kinase; COX-2, cyclooxygenase-2; MMP-9, ma- ‡ A Ransom Horne Jr. Distinguished Professor of Cancer Research. trix metalloproteinase-9; TNFR, TNF receptor; TRADD, TNFR-associated To whom correspondence should be addressed: Cytokine Research Lab- death domain; TRAF, TNFR-associated factor; NIK, NF-B-inducing ki- oratory, Dept. of Experimental Therapeutics, The University of Texas nase; PMA, phorbol myristate acetate; FBS, fetal bovine serum; EMSA, M. D. Anderson Cancer Center, Box 143, 1515 Holcombe Blvd., Hous- electrophoretic mobility shift assay; TPCK, L-1-tosylamido-2-phenylethyl ton, TX 77030. Tel.: 713-792-3503/6459; Fax: 713-794-1613; E-mail: chloromethyl ketone; MTT, 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyl- [email protected]. tetrazolium bromide; PARP, polyadenosine ribose polymerase. 47148 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Inhibition of TNF-induced NF-B by Guggulsterone 47149 (Jurkat) and myelogenous leukemia (KBM-5) cells were obtained from plasm; only when activated and translocated to the nucleus is the American Type Culture Collection (Manassas, VA). A549, Jurkat, the sequence of events leading to activation initiated (15, 16). and H1299 cells were cultured in RPMI 1640 medium, and KBM-5 cells Currently NF-B is considered a family of Rel-domain-contain- were cultured in Iscove’s modified Dulbecco’s modified Eagle’s medium. ing proteins, namely Rel A (also called p65), Rel B, c-Rel, p50 For Jurkat and A549 cells, RPMI 1640 was supplemented with 10% (also called NF-B1), and p52 (also called NF-B2). Similarly, a FBS, 100 units/ml penicillin, and 100 g/ml streptomycin; for KBM-5 family of anchorin-domain-containing proteins has been iden- cells, Dulbecco’s modified Eagle’s medium was supplemented with 15% FBS plus penicillin and streptomycin. tified that keeps the NF-B in its inactive state within the NF-B Activation—To determine NF-B activation by TNF, which nucleus. These include IB,IB,IB,IB, bcl-3, p105, and has a well established role in inflammation, tumor proliferation, pro- p100. Most carcinogens, inflammatory agents, and tumor pro- motion, invasion, and metastasis (24), we carried out EMSA essentially moters, including cigarette smoke, phorbol ester, okadaic acid, as previously described (25). The dried gels were visualized, and radio- H O , and TNF have been shown to activate NF-B. active bands were quantitated using a PhosphorImager (Molecular 2 2 Under resting conditions, NF-B consists of a heterotrimer of Dynamics, Sunnyvale, CA) using ImageQuaNT software. The -fold ac- tivation, over that of untreated control lane, was then calculated. p50, p65, and IB in the cytoplasm. The phosphorylation, Western Blot Analysis—To determine the effect of guggulsterone on ubiquitination, and degradation of IB and phosphorylation TNF-dependent IB degradation, p65 translocation, and p65 phospho- of p65 leads to the translocation of NF-B to the nucleus where rylation, cytoplasmic extracts were prepared as previously described it binds to specific response elements in the DNA. The phos- 6 (26) from H1299 cells (2  10 /ml) that had been pretreated with 50 M phorylation of IB is catalyzed by IKK, which consists of three guggulsterone for 4 h and then exposed to 0.1 nM TNF for various times. subunits IKK, IKK, and IKK. Gene deletion studies have For determining the phosphorylation of IB, H1299 cells (2  10 /ml) were first treated with 100 M N-acetyl-Leu-Leu-norleucinal (a protea- shown that IKK is essential for NF-B activation by most some inhibitor) for 1 h, then treated with 50 M guggulsterone for 4 h agents. The identity of the kinase that induces the phosphoryl- and then exposed to 0.1 nM TNF for various time points. Thirty micro- ation of p65 is controversial, but IKK, protein kinase C, and grams of cytoplasmic protein was resolved on 10% SDS-PAGE gel, protein kinase A have been implicated (for references see Refs. transferred to a nitrocellulose membrane, blocked with 5% nonfat milk, 17 and 18). NF-B has been shown to regulate the expression of and probed with specific antibody against IB, phosphorylated IB, p65, and phosphorylated p65. To determine the expression of cyclin D1, a number of genes whose products are involved in tumorigen- COX-2, MMP-9, cIAP-1, xIAP, TRAF1, Bcl-2, Bfl-1, cFLIP, and survivin esis (15–18). These include anti-apoptotic genes (e.g. ciap, su- in whole cell extracts of treated cells (2  10 cells in 2 ml medium), vivin, traf, bcl-2, and bcl-xl); cox2; mmp-9; genes encoding 30–50 g of protein was resolved on SDS-PAGE and probed by Western adhesion molecules, chemokines, and inflammatory cytokines; blot with specific antibodies as per the manufacturer’s recommended and cell cycle regulatory genes (e.g. cyclin d1). protocol. The blots were washed, exposed to horseradish peroxidase- Furthermore, NF-B has been implicated in obesity (19), conjugated secondary antibodies for 1 h, and finally detected by ECL reagent (Amersham Biosciences). The bands were quantitated using a hyperlipidemia (20), atherosclerosis (21), osteoarthritis (22), Personal Densitometer Scan version 1.30 using ImageQuaNT software and bone loss (23), all of which can be modulated by guggul- version 3.3 (Amersham Biosciences). sterone. In the present report we investigated the effect of Immunolocalization of NF-B p65—The effect of guggulsterone on guggulsterone on NF-B activation induced by a variety of cigarette smoke-induced nuclear translocation of p65 was examined by inflammatory agents and carcinogens. The aim of the study an immunocytochemical method using an epifluorescence microscope was to determine whether guggulsterone can suppress NF-B (Labophot-2; Nikon, Tokyo, Japan) and a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) as described previously (27). activation induced by inflammatory agents and carcinogens IKK Assay—To determine the effect of guggulsterone on TNF-in- and block NF-B-regulated gene expression that mediates in- duced IKK activation, we analyzed IKK by a method essentially as flammation and carcinogenesis. We found that guggulsterone described previously (28). inhibited activation of NF-B through suppression of IB NF-B-dependent Reporter Gene Transcription—The effect of gug- kinase, IB phosphorylation, and degradation, p65 nuclear gulsterone on TNF-induced NF-B-dependent reporter gene transcrip- translocation, and NF-B-dependent reporter gene expression. tion in H1299 cells was measured as previously described (29). COX-2 Promoter-dependent Reporter Luciferase Gene Expression— Guggulsterone also abrogated the expression of NF-B-regu- COX-2 promoter activity was examined as described elsewhere (28). To lated gene products that inhibit apoptosis and promote inflam- further determine the effect of guggulsterone on COX-2 promoter, A293 mation and tumor metastasis. cells were seeded at a concentration of 1.5  10 cells per well in six-well plates. After overnight culture, the cells in each well were transfected EXPERIMENTAL PROCEDURES with 2 g of DNA consisting of COX-2 promoter-luciferase reporter Materials—Z-Guggulsterone, obtained from Steraloids, Inc. (New- plasmid, along with 6 l of LipofectAMINE 2000 according to the port, RI), was dissolved in Me SO as a 100 mM stock solution and stored manufacturer’s protocol. The COX-2 promoter (375 to 59), which at 20 °C. Bacteria-derived human tumor necrosis factor (TNF), puri- was amplified from human genomic DNA by using the primers 5- fied to homogeneity with a specific activity of 5  10 units/mg, was GAGTCTCTTATTTATTTTT-3 (sense) and 5-GCTGCTGAGGAGTTC- kindly provided by Genentech, Inc. (South San Francisco, CA). Penicil- CTGGACGTGC-3 (antisense), was kindly provided by Dr. Xiao-Chun lin, streptomycin, RPMI 1640 medium, FBS, and LipofectAMINE 2000 Xu (M. D. Anderson Cancer Center). After a 6-h exposure to the trans- were obtained from Invitrogen. The following polyclonal antibodies fection mixture, the cells were incubated in medium containing guggul- were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): sterone for 12 h. The cells were exposed to TNF (0.1 nM) for 24 h and anti-p65, against the epitope corresponding to amino acids mapping then harvested. Luciferase activity was measured by using the Pro- within the amino-terminal domain of human NF-B p65; anti-p50, mega luciferase assay system according to the manufacturer’s protocol against a peptide 15 amino acids long mapping at the nuclear localiza- and detected by using Monolight 2010 (Analytical Luminescence Lab- tion sequence region of NF-B p50; anti-IB, against amino acids oratory, San Diego, CA). All experiments were performed in triplicate 297–317 mapping at the carboxyl terminus of IB/MAD-3; anti-c-Rel, and repeated at least twice to prove their reproducibility. anti-cyclin D1 against amino acids 1–295, which represents full-length Cytotoxicity Assay—Cytotoxicity was assayed by the modified tetra- cyclin D1 of human origin; anti-MMP-9, anti-polyadenosine ribose poly- zolium salt 3-(4,5-dimethylthiozol-2-yl)2,5-diphenyl-tetrazolium bro- merase (PARP); anti-IAP1; anti-IAP2; anti-Bcl-2; anti-Bfl-1/A1; and mide (MTT) assay as described previously (27). anti-TRAF1. Anti-COX-2, anti-MMP9, and anti-XIAP antibodies were PARP Cleavage Assay—For detection of cleavage products of PARP, obtained from BD Biosciences, and phospho-specific anti-IB (Ser32) whole cell extracts were prepared by subjecting guggulsterone-treated antibody was from Cell Signaling (Beverly, MA). Anti-IKK and anti- cells to lysis in lysis buffer (20 mM Tris, pH 7.4; 250 mM NaCl; 2 mM IKK antibodies were kindly provided by Imgenex (San Diego, CA). EDTA, pH 8.0; 0.1% Triton-X 100; 0.01 g/ml aprotinin; 0.005 g/ml Cell Lines—The cell lines used in our studies included human non- leupeptin; 0.4 mM phenylmethylsulfonyl fluoride; and 4 mM NaVO ). small cell lung carcinoma (H1299) cells and human lung epithelial cell Lysates were spun at 14,000 rpm for 10 min to remove insoluble carcinoma (A549) cells, both kindly provided by Dr. Reuben Lotan (The material, resolved by 10% SDS-PAGE, and probed with PARP University of Texas M. D. Anderson Cancer Center). Human leukemia antibodies. 47150 Inhibition of TNF-induced NF-B by Guggulsterone FIG.1. A, structure of guggulsterone. B, guggulsterone blocks NF-B activation induced by PMA, interleukin-1, okadaic acid, cigarette smoke condensate (CSC), and H O . H1299 cells (2  10 /ml) were preincubated for4hat37 °C with 50 M guggulsterone and then treated with PMA 2 2 (100 ng/ml, 1 h), interleukin-1 (100 ng/ml, 1 h), okadaic acid (500 nM, 4 h), H O (250 M, 1 h), or cigarette smoke condensate (10 g/ml, 30 min). 2 2 Nuclear extracts were prepared and tested for NF-B activation as described under “Experimental Procedures.” The numbers at the bottom of each lane indicate -fold activation over that of untreated control. Live and Dead Assay—To measure apoptosis, we used the Live and NF-B by these agents in H1299 cells. A DNA-binding assay Dead assay (Molecular Probes), which determines intracellular esterase (EMSA) showed that guggulsterone suppressed the NF-B ac- activity and plasma membrane integrity. This assay employs calcein, a tivation induced by all these agents (Fig. 1B). These results polyanionic dye, which is retained within the live cells and provides green suggest that guggulsterone acted at a step in the NF-B acti- fluorescence (30). It also employs the ethidium monomer dye (red fluores- vation pathway that is common to all these agents. cence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of Inhibition of NF-B Activation by Guggulsterone Is Not Cell live cells. Briefly, 1  10 cells were incubated with 10 M guggulsterone Type-specific—Because some reports suggest that distinct for 24 h and then treated with 1 nM TNF for 16 h at 37 °C. Cells were signal transduction pathways mediate NF-B induction in stained with the Live and Dead reagent (5 M ethidium homodimer, 5 M epithelial and lymphoid cells (31), we determined whether gug- calcein-AM) and then incubated at 37 °C for 30 min. Cells were analyzed gulsterone inhibited NF-B activation in three cell types. Gug- under a fluorescence microscope (Labophot-2). gulsterone completely inhibited TNF-induced NF-B activation RESULTS in lung epithelial cell carcinoma (A549), T cell leukemia (Jurkat), and myeloid leukemia (KBM-5) cells (Fig. 2A), indi- The guggulsterone used in these studies, which has a ste- cating a lack of cell type specificity. roid-like structure (Fig. 1A), was dissolved in Me SO. The Guggulsterone Inhibits Constitutive NF-B Activation—We concentration of guggulsterone used and the duration of expo- next tested the effect of guggulsterone on NF-B activation in sure had minimal effect on the viability of these cells as deter- human multiple myeloma (U266) and head and neck squamous mined by trypan blue dye exclusion test (data not shown). Guggulsterone Blocks NF-B Activation Induced by TNF, cell carcinoma (MDA 1986) tumor cells, which both express constitutively active NF-B (27, 32). Guggulsterone completely Interleukin-1, PMA, H O , Cigarette Smoke Condensate, and 2 2 Okadaic Acid—Because TNF, interleukin-1, PMA, H O , cig- inhibited this constitutively active NF-B (Fig. 2B). 2 2 arette smoke condensate, and okadaic acid activate NF-B, we The Suppression of NF-B by Guggulsterone Is Dose- and examined the effect of guggulsterone on the activation of Time-dependent—Guggulsterone inhibited TNF-mediated Inhibition of TNF-induced NF-B by Guggulsterone 47151 FIG.2. Guggulsterone suppresses inducible and constitutive NF-B ac- tivation. A, suppression of inducible ac- tivation is not cell-type specific. Two mil- lion A549, Jurkat, or KBM-5 cells were pre-treated with 50 M guggulsterone for 4 h and then treated with 0.1 nM TNF for 30 min. The nuclear extracts were then prepared and assayed for NF-Bby EMSA as described under “Experimental Procedures.” B, two million multiple my- eloma and head and neck squamous cell carcinoma cells were exposed to 50 M guggulsterone for 4 h, and then nuclear extracts were prepared and assayed for NF-B by EMSA as described under “Ex- perimental Procedures.” The numbers at the bottom of each lane indicate -fold ac- tivation over that of untreated control. NF-B activation in a dose-dependent manner, with maximum binding ability of NF-B proteins prepared from cells by treat- inhibition occurring at 50 M (Fig. 3A). The minimum time ment with TNF (Fig. 3D). Therefore, guggulsterone must inhibit required for complete inhibition of NF-B activation was 4 h NF-B activation by a different mechanism. (Fig. 3B). Guggulsterone Inhibits TNF-dependent IB Degradation— Suppressed NF-B Consists of Both p50 and p65—When Because IB degradation is normally a condition for translo- nuclear extracts from TNF-activated cells were incubated with cation of NF-B to the nucleus (36), we determined whether the antibodies to the p50 (NF-B1) and the p65 (RelA) subunit of guggulsterone’s inhibition of TNF-induced NF-B activation NF-B, the resulting bands were shifted to higher molecular was due to inhibition of IB degradation. We found that TNF masses (Fig. 3C), suggesting that the TNF-activated complex induced IB degradation in control cells as early as 10 min, consisted of p50 and p65. Neither preimmune serum nor irrel- but in guggulsterone-pretreated cells TNF had no effect on evant antibody had any effect. Addition of excess unlabeled IB degradation (Fig. 4A, upper panel). NF-B (cold oligonucleotide; 100-fold) caused complete disap- Guggulsterone Inhibits TNF-dependent IB Phosphoryla- pearance of the band, whereas mutated oligonucleotide had no tion—We next determined whether guggulsterone affected effect on the DNA binding. TNF-induced IB phosphorylation, another condition for Guggulsterone Does Not Directly Affect Binding of NF-Btothe NF-B translocation. Western blot analysis using antibody DNA—Although TPCK (the serine protease inhibitor), herbimy- that detects only the serine-phosphorylated form of IB indi- cin A (protein tyrosine kinase inhibitor), and caffeic acid phenyl cated that TNF induced IB phosphorylation as early as 5 ethyl ester directly modify NF-B to suppress its activation (33– min, and guggulsterone almost completely suppressed it (Fig. 35), EMSA showed that guggulsterone did not modify the DNA- 4A, middle panel). Thus guggulsterone inhibited TNF-induced 47152 Inhibition of TNF-induced NF-B by Guggulsterone FIG.3. A, guggulsterone inhibits TNF-dependent NF-B activation in a dose-dependent manner. H1299 cells (2  10 /ml) were preincubated with the indicated concentrations of guggulsterone for4hat37 °Cand 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  10 /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  10 /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  10 /ml) treated or not treated with 0.1 nM TNF for 30 min were treated with the indicated concentrations of guggulsterone for4hat 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. NF-B activation by inhibiting phosphorylation and degrada- tion, which is required for TNF-induced phosphorylation of tion of IB. IB. As shown in Fig. 4B (upper panel), guggulsterone com- Guggulsterone Inhibits TNF-induced IKK Activation—Be- pletely suppressed TNF-induced activation of IKK. TNF or cause guggulsterone inhibits the phosphorylation of IB,we guggulsterone had no direct effect on the expression of either tested the effect of guggulsterone on TNF-induced IKK activa- IKK (middle panel)orIKK (lower panel) proteins. However, Inhibition of TNF-induced NF-B by Guggulsterone 47153 FIG.4. A, guggulsterone inhibits TNF-induced phosphorylation and degradation of IB. H1299 cells (2  10 /ml) were incubated with 50 M guggulsterone for4hat37 °C, treated with 0.1 nM TNF for the indicated times at 37 °C, and then tested for IB (upper panel) and phosphorylated IB (middle panel) in cytosolic fractions by Western blot analysis. Equal protein loading was evaluated by -actin (lower panel). B, guggulsterone inhibits TNF-induced IB kinase activity. H1299 cells (2  10 /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  10 /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. we did find that guggulsterone inhibited the phosphorylation of Guggulsterone Represses TNF-induced NF-B-dependent Re- GST IB (at a 50 M concentration) by directly interfering porter Gene Expression—Although we showed by EMSA that with IKK activity (Fig. 4C). guggulsterone blocked NF-B activation, DNA binding alone Guggulsterone Inhibits TNF-induced Phosphorylation and does not always correlate with NF-B-dependent gene tran- Nuclear Translocation of p65—We also tested the effect of scription, suggesting that there are additional regulatory steps guggulsterone on TNF-induced phosphorylation of p65, be- (38). Transient transfection of H1299 cells with the NF-B- cause phosphorylation is also required for transcriptional ac- regulated SEAP reporter construct followed by stimulation tivity of p65 (37). As shown in Fig. 5A, guggulsterone sup- with TNF produced an almost 13-fold increase in SEAP activity pressed p65 phosphorylation almost completely. Likewise, over vector control activity (Fig. 6A). TNF-induced SEAP ac- Western blot analysis (Fig. 5B) and immunocytochemistry (Fig. tivity was abolished by dominant-negative IB, indicating 5C) indicated that guggulsterone abolished TNF-induced nu- specificity. When the cells were pretreated with guggulsterone, clear translocation of p65. TNF-induced NF-B-dependent SEAP expression was inhib- 47154 Inhibition of TNF-induced NF-B by Guggulsterone FIG.5. A, guggulsterone inhibits TNF-induced phosphorylation of p65. H1299 cells (2  10 /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  10 /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  10 /ml) were first treated with 50 M guggulsterone for4hat37 °Cand then exposed to 0.1 nM TNF. After cytospin, immunocytochemical analysis was performed as described under “Experimental Procedures.” ited by guggulsterone in a dose-dependent manner. These re- Guggulsterone Represses TNF-induced COX-2 Promoter Ac- sults demonstrate that guggulsterone inhibits NF-B-depend- tivity—We next determined whether guggulsterone affected ent reporter gene expression induced by TNF. COX-2 promoter activity, which is regulated by NF-B (41). As We next determined where guggulsterone acts in the se- shown in Fig. 6C, guggulsterone significantly reduced the TNF- quence of TNFR1, TRADD, TRAF2, NIK, and IKK recruitment induced COX-2 promoter activity in a dose-dependent manner. that characterizes TNF-induced NF-B activation (39, 40). In Guggulsterone Inhibits TNF-induced COX-2, MMP-9, and cells transfected with TNFR, TRADD, TRAF2, NIK, IKK, and VEGF Expression and TNF-induced Cyclin D1 and c-myc Ex- p65 plasmids, NF-B-dependent SEAP expression was in- pression—Guggulsterone abolished, in a dose-dependent fash- duced; 50 M guggulsterone significantly suppressed SEAP ion, the TNF-induced expression of COX-2, MMP-9, and VEGF expression in all cells except those transfected with p65. Be- (Fig. 7A), which are known to be NF-B-regulated gene prod- cause IKK activation can cause the phosphorylation of IB ucts (41–43). Expression of the NF-B-regulated gene products and p65, we suggest that guggulsterone inhibits NF-B activa- cyclin D1 and c-Myc (44, 45) was also abolished by guggul- tion through inhibition of IKK. sterone (Fig. 7B). Inhibition of TNF-induced NF-B by Guggulsterone 47155 FIG.6. A, guggulsterone inhibits TNF- induced NF-B-dependent reporter gene (SEAP) expression. H1299 cells were transiently transfected with an NF-B- containing plasmid linked to the SEAP gene and then treated with the indicated concentrations of guggulsterone. After 24 h in culture with 0.1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described under “Ex- perimental Procedures.” Results are ex- pressed as -fold activity over the activity of the vector control. B, guggulsterone in- hibits NF-B-dependent reporter gene ex- pression induced by TNFR, TRADD, TRAF, NIK, and IKK. H1299 cells were transiently transfected with the indicated plasmids along with an NF-B-containing plasmid linked to the SEAP gene and then left either untreated or treated with 50 M guggulsterone for 4 h. Cell super- natants were assayed for secreted alka- line phosphatase activity as described un- der “Experimental Procedures.” Results are expressed as -fold activity over the activity of the vector control. Bars indi- cate standard deviation. C, guggulsterone inhibits TNF-induced COX-2 promoter activity. H1299 cells were transiently transfected with a COX-2 promoter plas- mid linked to the luciferase gene and then treated with the indicated concentrations of guggulsterone. After 24 h in culture with 0.1 nM TNF, cell supernatants were collected and assayed for luciferase activ- ity as described under “Experimental Pro- cedures.” Results are expressed as -fold activity over the activity of the vector control. Guggulsterone Inhibits TNF-induced Activation of Anti-apo- tory stimuli (hydrogen peroxide, TNF, and interleukin-1) ptotic Gene Products—NF-B up-regulates the expression of a through inhibition of IKK, IB phosphorylation, and IB number of genes implicated in facilitating tumor cell survival, degradation, which led to abrogation of p65 phosphorylation including cIAP1, xIAP, Bfl-1, BCl-2, TRAF1, cFLIP, and sur- and nuclear translocation. NF-B-dependent reporter gene vivin (46–54). We found that guggulsterone inhibited the TNF- transcription induced by TNF, TNFR1, TRADD, TRAF2, NIK, induced expression of all of these proteins (Fig. 8). and IKK, was also blocked. The expression of gene products Guggulsterone Potentiates the Cytotoxic Effects of TNF and involved in anti-apoptosis (IAP1, xIAP, Bfl-1/A1, Bcl-2, cFLIP, Chemotherapeutic Drugs—Because NF-B-regulated products and survivin), proliferative genes (cyclin d1 and c-Myc), and suppress TNF- and chemotherapy-induced apoptosis (55, 56), metastatic genes (MMP-9, COX-2, and VEGF) was down-regu- we examined the effects of guggulsterone on the apoptotic lated by guggulsterone, and this down-regulation correlated effects of TNF and the chemotherapeutic drugs paclitaxel and with enhancement of apoptosis induced by TNF and chemo- doxorubicin. Guggulsterone enhanced the cytotoxic effects of therapeutic agents. TNF, Taxol, and doxorubicin (Fig. 9A) and the caspase-induced The present study was undertaken to investigate the potential cleavage of PARP activated by TNF (Fig. 9B). The Live and mechanism for the anti-inflammatory effects of guggulsterone, Dead assay also showed that TNF-induced apoptosis was sig- which has been used to treat obesity, diabetes, hyperlipidemia, nificantly enhanced by guggulsterone (Fig. 9C). atherosclerosis, and osteoarthritis. Although guggulsterone can DISCUSSION antagonize the farnesoid X receptor and decrease the expression of bile acid-activated genes (11), these effects do not explain its In this study, we have demonstrated that guggulsterone anti-inflammatory attributes. Because activation of NF-B has suppressed NF-B activated by carcinogens (phorbol ester, okadaic acid, and cigarette smoke condensate) and inflamma- been found in most inflammatory diseases, we hypothesized that 47156 Inhibition of TNF-induced NF-B by Guggulsterone FIG.7. A, guggulsterone inhibits COX-2, MMP-9, and VEGF expression in- duced by TNF. H1299 cells (2  10 /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, guggul- sterone inhibits cyclin D1 and c-Myc ex- pression induced by TNF. H1299 cells (2  10 /ml) were left untreated or incu- bated 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 ly- sate was analyzed by Western blotting using antibodies against cyclin D1 and c-Myc. all these agents. We identified IKK as a target site: cells that were exposed to guggulsterone failed to activate IKK in re- sponse to TNF. Surprisingly, incubation of IKK with guggul- sterone 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. Au- phan et al. (57) showed that glucocorticoids inhibit NF-B activation and that this inhibition is mediated through induc- tion of the IB, which traps activated NF-B in inactive cy- toplasmic complexes. Interestingly, estradiol was found to ac- tivate 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, FIG.8. Guggulsterone inhibits the expression of anti-apo- ptotic gene products IAP1, XIAP, Bfl-1/A1, Bcl-2, TRAF1, cFLIP, be completely ruled out by the results of our study. and survivin. H1299 cells (2  10 /ml) were left untreated or incu- Various tumor cell types express constitutively active NF- bated with 50 M guggulsterone for 4 h and then treated with 0.1 nM B, and it is critical for their proliferation (18, 27, 32). The TNF for different times. Whole cell extracts were prepared, and 50 g potential mechanism of constitutive activation of NF-Bisnot of the whole cell lysate was analyzed by Western blotting using anti- bodies against IAP1, XIAP, Bfl-1/A1, bcl-2, TRAF1, cFLIP, and survivin fully understood. Overexpression of IB without inhibition of as indicated. NF-B activity and mutations in the ib gene in Reed-Stern- berg cells (59) and enhanced IB degradation in mature mu- rine B-cell lines (60) have been demonstrated as potential guggulsterone modulates NF-B activation. The results of the study validate this hypothesis. mechanisms. Our laboratory showed that constitutive expres- sion of TNF in T-cell lymphoma (32) and interleukin-1 in acute Very little is known about the mechanism of action of gug- gulsterone. Our results clearly indicate that guggulsterone can myelogenous leukemia (61) are potential mechanisms. We found that human multiple myeloma cells and head and neck suppress NF-B activation induced by a wide variety of agents, suggesting that the site of action of guggulsterone is common to squamous cell carcinoma cells also express constitutive NF-B, Inhibition of TNF-induced NF-B by Guggulsterone 47157 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  S.D. from triplicate cultures. B, KBM-5 cells (2  10 /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  10 /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.” and guggulsterone suppressed the activation. It is very likely expression of numerous anti-apoptotic gene products, all that this inhibition also occurs through inhibition of IKK. known to be regulated by NF-B activation. The overexpres- That activation of NF-B regulates genes that control prolif- sion of IAP1, xIAP, Bfl-1/A1, bcl-2, cFLIP, and survivin has eration and metastasis of cancer has been well established (17, been found in numerous tumors and has been linked to sur- 18, 43). Our results demonstrate that guggulsterone can sup- vival, chemoresistance, and radioresistance. Because most of press the expression of COX-2, MMP-9, VEGF, cyclin D1, and these gene products are down-regulated by guggulsterone, we c-Myc, all regulated by NF-B. We found that COX-2 promoter believe that guggulsterone has potential as an agent for over- activity was also significantly down-regulated by this steroid. coming radioresistance and chemoresistance. Indeed, guggul- The lack of complete inhibition of COX-2 promoter activity by sterone potentiated the apoptotic effects of TNF and Taxol and guggulsterone suggests that this promoter is also regulated by doxorubicin. Thus guggulsterone, which is a pharmacologically transcription factors other than NF-B. These results imply safe agent (6), could be used as an anticancer agent on its own, that guggulsterone has a potential to suppress proliferation of a preventive agent, or an enhancer of chemotherapy/radiother- tumor cells and their metastasis. apy activity. Our demonstration, that guggulsterone sup- Our results indicate that guggulsterone also suppresses the presses NF-B and NF-B-regulated gene expression, may 47158 Inhibition of TNF-induced NF-B by Guggulsterone 23. Abu-Amer, Y., and Tondravi, M. M. (1997) Nat. Med. 3, 1189–1190 explain its effects against atherosclerosis, diabetes, osteoar- 24. Aggarwal, B. B. (2003) Nat. Rev. Immunol. 3, 745–756 thritis, and other inflammatory diseases. 25. Chaturvedi, M. M., Mukhopadhyay, A., and Aggarwal, B. B. (2000) Methods Enzymol. 319, 585–602 Cancer is a hyperproliferative disorder characterized by the 26. Majumdar, S., and Aggarwal, B. B. (2001) J. Immunol. 167, 2911–2920 up-regulation of genes responsible for transformation, prolifer- 27. Bharti, A. C., Donato, N., Singh, S., and Aggarwal, B. B. (2003) Blood 101, ation, invasion, angiogenesis, and metastasis. Most of these 1053–1062 28. Shishodia, S., Majumdar, S., Banerjee, S., and Aggarwal, B. B. (2003) Cancer activities are influenced by the aberrant activity of NF-B; Res. 63, 4375–4383 therefore, the main objective of this study was to evaluate the 29. Manna, S. K., Mukhopadhyay, A., and Aggarwal, B. B. (2000) J. Immunol. 165, 4927–4934 efficacy of guggulsterone as an inhibitor of NF-B. Our study 30. Takada, Y., Khuri, F. R., and Aggarwal, B. B. (2004) J. Biol. Chem. 279, for the first time demonstrates that guggulsterone can sup- 26287–26299 press NF-B activation induced by various carcinogens, inflam- 31. Bonizzi, G., Piette, J., Merville, M. P., and Bours, V. (1997) J. Immunol. 159, 5264–5272 matory agents, and tumor promoters. This effect was not cell 32. Giri, D. K., and Aggarwal, B. B. (1998) J. Biol. Chem. 273, 14008–14014 type-specific and equally pronounced in cells where NF-Bis 33. Finco, T. S., Beg, A. A., and Baldwin, A. S., Jr. (1994) Proc. Natl. Acad. Sci. constitutively active. TNF-induced activation of IKK and phos- U. S. A. 91, 11884–11888 34. Mahon, T. M., and O’Neill, L. A. (1995) J. Biol. Chem. 270, 28557–28564 phorylation and degradation IB were also inhibited by gug- 35. Natarajan, K., Singh, S., Burke, T. R., Jr., Grunberger, D., and Aggarwal, B. B. gulsterone. Guggulsterone was quite effective in blocking the (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9090–9095 36. Miyamoto, S., Maki, M., Schmitt, M. J., Hatanaka, M., and Verma, I. M. (1994) phosphorylation and translocation of TNF-induced p65, which Proc. Natl. Acad. Sci. U. S. A. 91, 12740–12744 led to the abrogation of NF-B-dependent reporter gene expres- 37. Zhong, H., Voll, R. E., and Ghosh, S. (1998) Mol. Cell 1, 661–671 sion. TNF-induced NF-B reporter activity induced by TNFR, 38. Nasuhara, Y., Adcock, I. M., Catley, M., Barnes, P. J., and Newton, R. (1999) J. Biol. Chem. 274, 19965–19972 TRADD, TRAF, NIK, and IKK, but not that activated by p65, 39. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299–308 was also blocked. 40. Simeonidis, S., Stauber, D., Chen, G., Hendrickson, W. A., and Thanos, D. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 49–54 Acknowledgment—We thank Walter Pagel for a careful review of 41. Yamamoto, K., Arakawa, T., Ueda, N., and Yamamoto, S. (1995) J. Biol. Chem. the manuscript. 270, 31315–31320 42. Esteve, P. O., Chicoine, E., Robledo, O., Aoudjit, F., Descoteaux, A., Pot- REFERENCES worowski, E. F., and St-Pierre, Y. (2002) J. Biol. Chem. 277, 35150–35155 43. Huang, S., Robinson, J. B., Deguzman, A., Bucana, C. D., and Fidler, I. J. 1. Craig, W. J. (1997) J. Am. Diet. Assoc. 97, S199–S204 (2000) Cancer Res. 60, 5334–5339 2. Craig, W. J. (1999) Am. J. Clin. Nutr. 70, 491S–499S 44. Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G., and Baldwin, 3. Newman, D. J., Cragg, G. M., and Snader, K. M. (2003) J. Nat. Prod. 66, A. S., Jr. (1999) Mol. Cell. Biol. 19, 5785–5799 1022–1037 45. Wittekindt, N. E., Hortnagel, K., Geltinger, C., and Polack, A. (2000) Nucleic 4. Coussens, L. M., and Werb, Z. (2002) Nature 420, 860–867 Acids Res. 28, 800–808 5. Balkwill, F., and Mantovani, A. (2001) Lancet 357, 539–545 46. Zhu, L., Fukuda, S., Cordis, G., Das, D. K., and Maulik, N. (2001) FEBS Lett. 6. Urizar, N. L., and Moore, D. D. (2003) Annu. Rev. Nutr. 23, 303–313 508, 369–374 7. Sinal, C. J., and Gonzalez, F. J. (2002) Trends Endocrinol. Metab. 13, 275–276 47. Schwenzer, R., Siemienski, K., Liptay, S., Schubert, G., Peters, N., Scheurich, 8. Gujral, M. L., Sareen, K., Tangri, K. K., Amma, M. K., and Roy, A. K. (1960) P., Schmid, R. M., and Wajant, H. (1999) J. Biol. Chem. 274, 19368–19374 Indian J. Physiol. Pharmacol. 4, 267–273 48. Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J., Malim, M. H., and Ballard, 9. Sharma, J. N. (1977) Arzneimittelforschung 27, 1455–1457 D. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10057–10062 10. Singh, B. B., Mishra, L. C., Vinjamury, S. P., Aquilina, N., Singh, V. J., and 49. You, M., Ku, P. T., Hrdlickova, R., and Bose, H. R., Jr. (1997) Mol. Cell. Biol. Shepard, N. (2003) Altern. Ther. Health Med. 9, 74–79 17, 7328–7341 11. Urizar, N. L., Liverman, A. B., Dodds, D. T., Silva, F. V., Ordentlich, P., Yan, 50. Stehlik, C., de Martin, R., Kumabashiri, I., Schmid, J. A., Binder, B. R., and Y., Gonzalez, F. J., Heyman, R. A., Mangelsdorf, D. J., and Moore, D. D. Lipp, J. (1998) J. Exp. Med. 188, 211–216 (2002) Science 296, 1703–1706 51. Catz, S. D., and Johnson, J. L. (2001) Oncogene 20, 7342–7351 12. Wu, J., Xia, C., Meier, J., Li, S., Hu, X., and Lala, D. S. (2002) Mol. Endocrinol. 52. Grumont, R. J., Rourke, I. J., and Gerondakis, S. (1999) Genes Dev. 13, 16, 1590–1597 400–411 13. Cui, J., Huang, L., Zhao, A., Lew, J. L., Yu, J., Sahoo, S., Meinke, P. T., Royo, 53. Zong, W. X., Edelstein, L. C., Chen, C., Bash, J., and Gelinas, C. (1999) Genes I., Pelaez, F., and Wright, S. D. (2003) J. Biol. Chem. 278, 10214–10220 Dev. 13, 382–387 14. Meselhy, M. R. (2003) Phytochemistry 62, 213–218 54. Kreuz, S., Siegmund, D., Scheurich, P., and Wajant, H. (2001) Mol. Cell. Biol. 15. Yamamoto, Y., and Gaynor, R. B. (2001) Curr. Mol. Med. 1, 287–296 21, 3964–3973 16. Aggarwal, B. B., Takada, Y., Shishodia, S., Gutierrez, A. M., Oommen, O. V., 55. Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R., and Verma, I. M. Ichikawa, H., Baba, Y., and Kumar, A. (2004) Indian J. Exp. Biol. 42, (1996) Science 274, 787–789 341–353 56. Wang, C. Y., Mayo, M. W., and Baldwin, A. S., Jr. (1996) Science 274, 784–787 17. Karin, M., Cao, Y., Greten, F. R., and Li, Z. W. (2002) Nat. Rev. Cancer 2, 57. Auphan, N., DiDonato, J. A., Rosette, C., Helmberg, A., and Karin, M. (1995) 301–310 Science 270, 286–290 18. Garg, A., and Aggarwal, B. B. (2002) Leukemia 16, 1053–1068 58. Shyamala, G., and Guiot, M. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 19. Yuan, M., Konstantopoulos, N., Lee, J., Hansen, L., Li, Z. W., Karin, M., and Shoelson, S. E. (2001) Science 293, 1673–1677 10628–10632 59. Emmerich, F., Meiser, M., Hummel, M., Demel, G., Foss, H. D., Jundt, F., 20. Thurberg, B. L., and Collins, T. (1998) Curr. Opin. Lipidol. 9, 387–396 21. Brand, K., Page, S., Rogler, G., Bartsch, A., Brandl, R., Knuechel, R., Page, M., Mathas, S., Krappmann, D., Scheidereit, C., Stein, H., and Dorken, B. (1999) Blood 94, 3129–3134 Kaltschmidt, C., Baeuerle, P. A., and Neumeier, D. (1996) J. Clin. Invest. 97, 1715–1722 60. Miyamoto, S., Chiao, P. J., and Verma, I. M. (1994) Mol. Cell. Biol. 14, 3276–3282 22. Miagkov, A. V., Kovalenko, D. V., Brown, C. E., Didsbury, J. R., Cogswell, J. P., Stimpson, S. A., Baldwin, A. S., and Makarov, S. S. (1998) Proc. Natl. Acad. 61. Estrov, Z., Manna, S. K., Harris, D., Van, Q., Estey, E. H., Kantarjian, H. M., Sci. U. S. A. 95, 13859–13864 Talpaz, M., and Aggarwal, B. B. (1999) Blood 94, 2844–2853

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