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Clinical Cancer Research Vol. 12, 5910-5918, October 1, 2006
© 2006 American Association for Cancer Research

Cancer Susceptibility and Prevention

Isodeoxyelephantopin, a Novel Sesquiterpene Lactone, Potentiates Apoptosis, Inhibits Invasion, and Abolishes Osteoclastogenesis through Suppression of Nuclear Factor-{kappa}B (NF-{kappa}B) Activation and NF-{kappa}B-Regulated Gene Expression

Haruyo Ichikawa1, Mangalam S. Nair2, Yasunari Takada1, D.B. Alan Sheeja2, M.A. Suresh Kumar3, Oommen V. Oommen3 and Bharat B. Aggarwal1

Authors' Affiliations: 1 Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and 2 Organic Chemistry Division, Regional Research Laboratory; 3 Department of Zoology, University of Kerala, Thiruvananthapuram, India

Requests for reprints: Bharat B. Aggarwal, Department of Experimental Therapeutics, Unit 143, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1817; Fax: 713-794-1613; E-mail: aggarwal{at}mdanderson.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Deoxyelephantopin (ESD) and isodeoxyelephantopin (ESI) are two sesquiterpene lactones derived from the medicinal plant Elephantopus scaber Linn. (Asteraceae). Although they are used for the treatment of a wide variety of proinflammatory diseases, very little is known about their mechanism of action. Because most genes that control inflammation are regulated by activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), we postulated that ESD and ESI mediate their activities through modulation of the NF-{kappa}B activation pathway.

Experimental Design: We investigated the effect of ESI and ESD on NF-{kappa}B activation by electrophoretic mobility shift assay and NF-{kappa}B-regulated gene expression by Western blot analysis.

Results: We found that ESI suppressed NF-{kappa}B activation induced by a wide variety of inflammatory agents, including tumor necrosis factor (TNF), interleukin-1ß, phorbol 12-myristate 13-acetate, and lipopolysaccharide. The suppression was not cell type specific, and both inducible and constitutive NF-{kappa}B activation was blocked. ESI did not interfere with the binding of NF-{kappa}B to DNA but rather inhibited I{kappa}B{alpha} kinase, I{kappa}B{alpha} phosphorylation, I{kappa}B{alpha} degradation, p65 phosphorylation, and subsequent p65 nuclear translocation. ESI also suppressed the expression of TNF-induced NF-{kappa}B-regulated, proliferative, antiapoptotic, and metastatic gene products. These effects correlated with enhancement of apoptosis induced by TNF and suppression of TNF-induced invasion and receptor activator of NF-{kappa}B ligand-induced osteoclastogenesis.

Conclusion: Our results indicate that ESI inhibits NF-{kappa}B activation and NF-{kappa}B-regulated gene expression, which may explain the ability of ESI to enhance apoptosis and inhibit invasion and osteoclastogenesis.

The use of plant principles for the treatment of a wide variety of diseases is practiced in most cultures. However, the active ingredients in these plants and their mechanisms of action are poorly understood. Delineation of this knowledge could provide safer, more efficacious treatment of various human ailments. In particular, the extract of the plant Elephantopus scaber Linn. (Asteraceae; called Elephant's foot in English, Gobhi in Hindi, and Didancao in China) is used by various tribal groups in India for treatment of diarrhea, ulcers, eczema, nephritis, edema, dampness, chest pain, and stomach ache. In Chinese medicine, the extract of this plant is used as an antidiuretic, antifebrile, antiviral, and antibacterial agent as well as in the treatment of hepatitis, bronchitis, cough associated with pneumonia, and arthralgia (1–4). Isodeoxyelephantopin (ESI) and deoxyelephantopin (ESD) are two sesquiterpene lactones that have been isolated from E. scaber (5–10). ESI and ESD have been shown to possess anti-inflammatory activities, as indicated by inhibition of carrageenan- and adjuvant-induced paw edema in rats (3) and carbon tetrachloride–induced chronic liver dysfunction (1, 2). What is the mechanism of action for both ESI and ESD is poorly understood. Because various gene products regulated by the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) are known to regulate tumorigenesis and inflammation, we postulated that ESI and ESD must mediate their effects through modulation of NF-{kappa}B activation (11).

Under normal conditions, NF-{kappa}B is present in the cytoplasm as an inactive heterotrimer consisting of three subunits: p50, p65, and I{kappa}B{alpha}. On activation, I{kappa}B{alpha} undergoes phosphorylation- and ubiquitination-dependent degradation by the 26S proteasome, thus exposing nuclear localization signals on the p50-p65 heterodimer, leading to nuclear translocation and binding to a specific consensus sequence in the DNA, 5'-GGGACTTTC-3' (12). This binding activates NF-{kappa}B gene expression, which results in transcription of NF-{kappa}B-regulated genes (11). The phosphorylation of I{kappa}B{alpha} is mediated through activation of the I{kappa}B{alpha} kinase (IKK) complex, which consists of IKK-{alpha}, IKK-ß, IKK-{gamma} (also called NF-{kappa}B essential modulator), IKK-associated protein 1, FIP-3 (type 2 adenovirus E3 14.7-kDa interacting protein), heat shock protein 90, and glutamine, leucine, lysine, and serine-rich protein (13).

Because of the critical role of NF-{kappa}B in inflammatory diseases, we investigated the effect of ESI and ESD on the NF-{kappa}B activation pathway induced by various inflammatory agents. Our results strongly suggest that ESI is a potent suppressor of NF-{kappa}B activation induced by various agents and that this suppression is mediated through inhibition of IKK. Consequently, the expression of gene products that regulate apoptosis, proliferation, angiogenesis, invasion, and osteoclastogenesis is suppressed.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Reagents. ESD and ESI were isolated from the chloroform extract of E. scaber Linn. (collected at the Regional Research Laboratory, Thiruvananthapuram, India) using successive chromatography with a solvent system of increasing polarity containing hexane/ethylacetate mixtures. Both compounds were obtained as pure white crystals. The structures of both compounds were confirmed by comparing the ionizing radiation, 1H nuclear magnetic resonance, and 13C nuclear magnetic resonance spectral data as well as the melting points found in the literature (6–8). Ten millimolar solutions of ESI and ESD were prepared in 100% dimethyl sulfoxide, stored as small aliquots at –20°C, and then diluted as needed in a cell culture medium. The final concentration of DMSO used in most experiments was 0.1%, which had no effect on NF-{kappa}B activation.

Bacteria-derived human recombinant tumor necrosis factor (TNF), purified to homogeneity with a specific activity of 5 x 107 units/mg, was provided by Genentech (South San Francisco, CA). Penicillin, streptomycin, RPMI 1640, DMEM, Iscove's modified Dulbecco's medium, DMEM/F-12, and fetal bovine serum (FBS) were obtained from Invitrogen (Grand Island, NY). Phorbol 12-myristate 13-acetate (PMA) and an anti-ß-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO). Anti-p65, anti-p50, anti-I{kappa}B{alpha}, anti-cyclin D1, anti-matrix metalloproteinase (MMP)-9, anti-c-Myc, anti-poly (ADP-ribose) polymerase (PARP), anti-intercellular adhesion molecule-1 (ICAM-1), anti-TNF receptor-associated factor 1 (TRAF1), anti-inhibitor of apoptosis protein (IAP) 1, anti-IAP2, anti-Bcl-2, anti-Bcl-xL, and anti-Bfl-1/A1 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-survivin antibody was obtained from R&D Systems (Minneapolis, MN). Anti-cyclooxygenase (COX)-2 antibodies and goat anti-mouse horseradish peroxidase were obtained from BD Biosciences (San Diego, CA). Phosphorylated specific anti-I{kappa}B{alpha} (Ser32/36) and anti-p65 (Ser536) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-IKK-{alpha}, anti-IKK-ß, and anti-FADD-like interleukin-1ß-converting enzyme (FLIP)/caspase-8-inhibitory protein antibodies were provided by Imgenex (San Diego, CA). Goat anti-rabbit horseradish peroxidase conjugate was purchased from Bio-Rad (Hercules, CA). Protein A/G-Sepharose beads were obtained from Pierce Biotechnology (Rockford, IL). [{gamma}-32P]ATP was purchased from ICN Pharmaceuticals (Costa Mesa, CA).

Cell lines. KBM-5 human chronic myeloid leukemia, H1299 lung adenocarcinoma, HL60 human promyelocytic leukemia, A293 human embryonic kidney carcinoma, MCF-7 human breast adenocarcinoma, MM.1S human multiple myeloma, and U266 human multiple myeloma cell lines and RAW 264.7 mouse macrophages were obtained from the American Type Culture Collection (Manassas, VA). LICR-LON-HN5 and SCC4 human squamous cell carcinoma cell lines were provided by Dr. M. J. O'Hare (Haddow Laboratories, Institute of Cancer Research, Sutton, Surrey, United Kingdom). KBM-5 cells were cultured in Iscove's modified Dulbecco's medium with 15% FBS. HL60, H1299, MM.1S, and U266 cells were cultured in RPMI 1640, and A293 cells were cultured in DMEM supplemented with 10% FBS. LICR-LON-HN5 and SCC4 cells were cultured in DMEM containing 10% FBS, 100 µmol/L nonessential amino acids, 1 mmol/L pyruvate, 6 mmol/L L-glutamine, and 1x vitamins. Culture media were also supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin. RAW 264.7 cells were cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics.

Electrophoretic mobility shift assays. To determine NF-{kappa}B activation, electrophoretic mobility shift assay (EMSA) was done as described previously (14). Briefly, nuclear extracts prepared from TNF-treated cells were incubated with a 32P-end-labeled 45-mer double-stranded NF-{kappa}B oligonucleotide (15 µg protein with 16 fmol DNA) from the HIV long-terminal repeat 5'-TTGTTESIAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (boldface indicates NF-{kappa}B binding sites) for 30 minutes at 37°C. The DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTESIACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-{kappa}B to the DNA. The binding specificity was also examined using competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with antibodies against either p50 or p65 of NF-{kappa}B for 15 minutes at 37°C before the complex was analyzed using EMSA. Antibodies against cyclin D1 and preimmune serum were included as negative controls. The dried gels were visualized, and the radioactive bands were quantitated using a Storm 820 PhosphorImager with the ImageQuant software program (Amersham, Piscataway, NJ).

Western blot analysis. To determine the levels of protein expression in the cytoplasm or nucleus in KBM-5 cells, we prepared extracts (15) and fractionated them using SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each antibody, and detected using enhanced chemiluminescence reagent (Amersham).

IKK assay. To determine the effect of ESI on TNF-induced IKK activation, IKK assay was done as described previously (16). Briefly, the IKK complex from whole-cell extracts (400 µg protein) was precipitated with an antibody against IKK-{alpha} and then treated with protein A/G-Sepharose beads. After 2 hours, the beads were washed with lysis buffer and then resuspended in a kinase assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 µCi [{gamma}-32P]ATP, 10 µmol/L unlabeled ATP, and 2 µg substrate glutathione S-transferase-I{kappa}B{alpha} (amino acids 1-54). After incubation at 30°C for 30 minutes, the reaction was terminated by boiling with an SDS sample buffer for 5 minutes. Finally, the protein was resolved on a 10% SDS-PAGE gel, the gel was dried, and the radioactive bands were visualized using a Storm 820 PhosphorImager. To determine the total amounts of IKK-{alpha} and IKK-ß in each sample, 50 µg whole-cell proteins was resolved on a 7.5% SDS-PAGE gel, electrotransferred to a nitrocellulose membrane, and then blotted with either an anti-IKK-{alpha} or anti-IKK-ß antibody.

Immunocytochemistry for NF-{kappa}B p65 localization. The effect of ESI on the nuclear translocation of p65 was examined using immunocytochemistry as described previously (15). Briefly, treated cells were plated on poly-l-lysine–coated glass slides by centrifugation using a Cytospin 4 cytocentrifuge (Thermo Shandon, Pittsburgh, PA), air dried, and fixed with 4% paraformaldehyde after permeabilization with 0.2% Triton X-100. After being washed in PBS, the slides were blocked with 5% normal goat serum for 1 hour and then incubated with a rabbit polyclonal anti-human p65 antibody at a dilution of 1:200. After an overnight incubation at 4°C, the slides were washed, incubated with goat anti-rabbit IgG-Alexa 594 (Molecular Probes, Eugene, OR) at a dilution of 1:200 for 1 hour, and counterstained for nuclei with Hoechst 33342 (50 ng/mL) for 5 minutes. Stained slides were mounted using a mounting medium purchased from Sigma-Aldrich and analyzed under a fluorescence microscope (Labophot 2, Nikon, Tokyo, Japan). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and the MetaMorph software program (version 4.6.5; Universal Imaging, Downingtown, PA).

LIVE/DEAD assay. To measure apoptosis, we also used the LIVE/DEAD assay (Molecular Probes), which determines the intracellular esterase activity and plasma membrane integrity. This assay was done as described previously (17). This assay uses the green fluorescent polyanionic dye calcein, which is retained within live cells. It also uses the red fluorescent monomer dye ethidium, which can enter cells only through damaged membranes to bind to nucleic acids but is excluded by the intact plasma membrane of live cells. Briefly, 5 x 105 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours at 37°C. Cells were stained with the LIVE/DEAD assay reagent (5 µmol/L ethidium homodimer and 5 µmol/L calcein-AM) and then incubated at 37°C for 30 minutes. Cells were then analyzed under a fluorescence microscope (Labophot 2).

Annexin V assay. An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of Annexin V. To identify apoptosis, cells were stained with an Annexin V antibody conjugated with the fluorescent dye FITC. Briefly, 5 x 105 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours at 37°C and then stained with FITC. Cells were washed in PBS, resuspended in 100 µL binding buffer containing a FITC-conjugated anti-Annexin V antibody, and then analyzed using a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. The cytotoxicity was asssayed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, which examines DNA strand breaks during apoptosis, with an In situ Cell Death Detection reagent (Roche Molecular Biochemicals, Mannheim, Germany). Briefly, 5 x 105 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours at 37°C. Thereafter, cells were incubated with a reaction mixture for 60 minutes at 37°C. Stained cells were analyzed using a flow cytometer (FACSCalibur).

Invasion assay. Invasion through the extracellular matrix is a crucial step in tumor metastasis. A Matrigel basement membrane matrix extracted from an Englebreth-Holm-Swarm mouse tumor was used as a reconstituted basement membrane for in vitro invasion assays. The BD BioCoat tumor invasion system used for these assays has a chamber with a light-tight polyethylene terephthalate membrane with 8-µm pores coated with a reconstituted basement membrane gel (BD Biosciences). H1299 cells (2.5 x 104 per well) were resuspended in serum-free medium, and the suspension was seeded into the upper wells of chamber. After overnight incubation, cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for an additional 24 hours in the presence of 1% FBS. The cells that passed through the Matrigel were labeled with 4 µg/mL calcein-AM (Molecular Probes) in PBS for 30 minutes at 37°C and subjected to scanning fluorescence with a VICTOR3 luminometer (Perkin-Elmer Life And Analytical Sciences, Wellesley, MA).

Osteoclast differentiation assay. RAW 264.7 cells were cultured in 24-well dishes at a density of 1 x 104 per well and allowed to adhere overnight. The medium was then replaced, and the cells were treated with 5 nmol/L (100 ng/mL) receptor activator of NF-{kappa}B ligand (RANKL) and ESI. On day 5, cultures were stained for tartrate-resistant acid phosphatase (TRAP) expression as described previously (18) using an acid phosphatase kit, and the total TRAP-positive multinucleated (three or more nuclei) osteoclasts per well were counted.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The aim of the present study was to investigate the effect of ESI and ESD on the NF-{kappa}B activation pathway induced by various carcinogens and inflammatory stimuli and on NF-{kappa}B-regulated gene expression. Because the TNF-induced NF-{kappa}B activation pathway has been well characterized, we investigated the effects of ESI and ESD on TNF-induced NF-{kappa}B activation in detail. The structures of ESI and ESD are shown in Fig. 1A .


Figure 1
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  		Fig. 1. ESI suppressed activator-induced NF-{kappa}B in a dose-dependent manner. A, structures of ESI and ESD. B, KBM-5 cells were coincubated with ESI at the indicated concentrations and 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and analyzed for NF-{kappa}B activation as described in Materials and Methods. C, KBM-5 cells were coincubated with ESD at the indicated concentrations and 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and analyzed for NF-{kappa}B activation as described in Materials and Methods. D, NF-{kappa}B induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with the indicated antibodies, an unlabeled NF-{kappa}B oligo probe, or a mutant oligo probe and then assayed for NF-{kappa}B activation using EMSA. E, ESI blocks NF-{kappa}B activation induced by TNF, lipopolysaccharide (LPS), interleukin-1ß (IL-1ß), and PMA. KBM-5 cells were coincubated with 10 µmol/L ESI for 30 minutes, 0.1 nmol/L TNF for 30 minutes, 10 µg/mL lipopolysaccharide for 30 minutes, 100 ng/mL interleukin-1ß for 2 hours, and 15 ng/mL PMA for 1 hour and then analyzed for NF-{kappa}B activation as described in Materials and Methods.

 
Both ESI and ESD inhibit TNF-induced NF-{kappa}B activation. We examined the effect of ESI and ESD at different concentrations on TNF-induced NF-{kappa}B activation in KBM-5 cells. Both agents suppressed this activation, with maximum suppression occurring at 10 to 50 µmol/L (Fig. 1B and C). This concentration of ESI and ESD used for 30 minutes had >98% cell viability. Because ESI and ESD suppressed NF-{kappa}B activation to a similar extent, we investigated ESI only in all of the ensuing studies. Various combinations of Rel/NF-{kappa}B protein constitute active NF-{kappa}B heterodimers, p50 and p65, which bind to a specific DNA sequence (12). When we incubated nuclear extracts from TNF-stimulated cells with antibodies against either the p50 (NF-{kappa}B1) or p65 (RelA) subunit of NF-{kappa}B, it shifted the band to a higher molecular mass (Fig. 1D). Thus, the TNF-activated complex consisted of p50 and p65 subunits. Excess unlabeled NF-{kappa}B caused complete disappearance of the band, but a mutant oligonucleotide of NF-{kappa}B did not affect NF-{kappa}B binding activity.

ESI inhibits NF-{kappa}B activation induced by different inflammatory agents. In addition to TNF, NF-{kappa}B is activated by other inflammatory agents, such as PMA, lipopolysaccharide (LPS), and interleukin-1ß (IL-1ß) (19–22). We examined whether ESI modulates the activation of NF-{kappa}B by these agents. Our results showed that all of these agents activated NF-{kappa}B, and coincubation of cells with 10 µmol/L ESI suppressed the activation of NF-{kappa}B induced by these agents (Fig. 1E). These results suggest that ESI acts at a step in the NF-{kappa}B activation pathway that is common to all four of these agents.

Inhibition of NF-{kappa}B activation by ESI is not cell type specific. Until now, the effect of ESI on NF-{kappa}B activation was examined in human myeloid leukemia KBM-5 cells. Because the signal transduction pathway leading to NF-{kappa}B activation may be distinct in different cell types (23, 24), whether ESI could block TNF-induced NF-{kappa}B activation in MCF-7, H1299, and HL60 cells was examined (Fig. 2A ). We exposed these cells to TNF either in the presence or absence of ESI for 30 minutes and then examined them for NF-{kappa}B activation using EMSA. ESI suppressed TNF-induced NF-{kappa}B activation in all of these cells, indicating that the effect of ESI was not cell type specific.


Figure 2
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  		Fig. 2. ESI suppresses TNF-induced NF-{kappa}B activation in different cell lines. A, MCF-7, H1299, and HL60 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for 30 minutes. B, MM.1S, U266, SCC4, and LICR-LON-HN5 (HN5) cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-{kappa}B activation using EMSA. –, the time ESI was present before the addition of TNF; 0, coincubation with TNF; +, the time ESI was added after the addition of TNF. C, KBM-5 cells were preincubated with 10 µmol/L ESI for the indicated times and then tested for NF-{kappa}B activation at 37°C for 30 minutes with 0.1 nmol/L TNF using EMSA. After treatment, nuclear extracts were prepared and assayed for NF-{kappa}B activation using EMSA. D, direct effect of ESI on the NF-{kappa}B complex. Nuclear extracts were prepared from KBM-5 cells that were untreated or treated with 0.1 nmol/L TNF, incubated for 30 minutes with ESI at the indicated concentrations, and then assayed for NF-{kappa}B activation using EMSA.

 
ESI suppresses constitutive NF-{kappa}B activation. Most tumor cells express constitutively active NF-{kappa}B (21, 22), although the mechanism is not well understood. We showed that ESI suppresses constitutive activation of NF-{kappa}B in human multiple myeloma (MM.1S and U266) and head and neck squamous cell carcinoma (SCC4 and LICR-LON-HN5) cells, which are known to express constitutive active NF-{kappa}B (Fig. 2B; refs. 25, 26).

ESI is a fast-acting inhibitor of NF-{kappa}B activation. The suppression of NF-{kappa}B by most agents requires application of them before that of the NF-{kappa}B-activating agent (15, 17). However, we found that treatment with ESI 5 minutes before treatment with TNF, at the same time as treatment with TNF, and 5 or 10 minutes after treatment with TNF suppressed TNF-induced NF-{kappa}B activation (Fig. 2C), suggesting that ESI is a fast-acting inhibitor of NF-{kappa}B activation.

ESI does not directly interfere with the binding of NF-{kappa}B to DNA. Studies have shown that several NF-{kappa}B inhibitors suppress NF-{kappa}B activation by directly blocking the binding of NF-{kappa}B to DNA (27–29). When we incubated nuclear extracts from TNF-treated cells with ESI, EMSA showed that ESI had no direct effect on NF-{kappa}B binding to the DNA (Fig. 2D). Thus, ESI must inhibit NF-{kappa}B activation through an indirect mechanism.

ESI inhibits TNF-dependent I{kappa}B{alpha} degradation. We examined ESI- and TNF-treated cells for NF-{kappa}B using EMSA and for I{kappa}B{alpha} using Western blot analysis. ESI completely suppressed TNF-induced NF-{kappa}B activation (Fig. 3A ). ESI also suppressed TNF-induced I{kappa}B{alpha} degradation, although not completely (Fig. 3B). These results indicate that ESI inhibits TNF-induced NF-{kappa}B activation by preventing I{kappa}B{alpha} degradation.


Figure 3
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  		Fig. 3. Effect of ESI on I{kappa}B{alpha} degradation and phosphorylation induced by TNF. A, ESI inhibited TNF-induced activation of NF-{kappa}B in a time-dependent manner. KBM-5 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for the indicated times and then analyzed for NF-{kappa}B activation using EMSA. B, effect of ESI on TNF-induced degradation of I{kappa}B{alpha}. KBM-5 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE gels, and electrotransferred to a nitrocellulose membrane. Western blot analysis was done with an anti-I{kappa}B{alpha} antibody. C, KBM-5 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for 15 minutes after the addition of N-Ac-leu-leu-norleucinal (ALLN). Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE gels, and electrotransferred to a nitrocellulose membrane. Western blot analysis was done with anti-phosphorylated specific I{kappa}B{alpha} and anti-I{kappa}B{alpha} antibodies. D, effect of ESI on TNF-induced translocalization of p65. KBM-5 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared, fractionated on 7.5% SDS-PAGE gels, and electrotransferred to a nitrocellulose membrane. Western blot analysis was done with anti-p65 antibodies. E, immunocytochemical analysis of p65 localization. KBM-5 cells were coincubated with 10 µmol/L ESI and 1 nmol/L TNF for 15 minutes and then subjected to immunocytochemistry as described in Materials and Methods. F, effect of ESI on TNF-induced phosphorylation of p65. KBM-5 cells were coincubated with 10 µmol/L ESI and 0.1 nmol/L TNF for the indicated times. Nuclear extracts were prepared, fractionated on 7.5% SDS-PAGE gels, and electrotransferred to a nitrocellulose membrane. Western blot analysis was done with an anti-phosphorylated specific p65 antibody. G, effect of ESI on the TNF-induced activation of IKK. KBM-5 cells were pretreated with 50 µg/mL ALLN for 30 minutes and then coincubated with 10 µmol/L ESI and 1 nmol/L TNF for the indicated times. Whole-cell extracts were immunoprecipitated with an antibody against IKK-{alpha} and analyzed using immune complex kinase assay as described in Materials and Methods. To examine the effect of ESI on the level of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE gels and examined using Western blot analysis with anti-IKK-{alpha} and anti-IKK-ß antibodies. H, direct effect of ESI on the activation of IKK induced by TNF. Whole-cell extracts were prepared from cells treated with 1 nmol/L TNF and immunoprecipitated with an anti-IKK-{alpha} antibody. Immune complex kinase assay was then done in the presence or absence of ESI at the indicated concentrations.

 
ESI inhibits TNF-dependent I{kappa}B{alpha} phosphorylation. Because I{kappa}B{alpha} phosphorylation is required for I{kappa}B{alpha} degradation, we sought to determine whether ESI modulates I{kappa}B{alpha} phosphorylation. Because TNF-induced phosphorylation of I{kappa}B{alpha} leads to its rapid degradation, we blocked I{kappa}B{alpha} phosphorylation and degradation using the proteasome inhibitor N-Ac-leu-leu-norleucinal. Western blot analysis using an antibody specific for the serine-phosphorylated form of I{kappa}B{alpha} showed that ESI suppressed TNF-induced phosphorylation of I{kappa}B{alpha} (Fig. 3C), although the inhibition was incomplete.

ESI inhibits TNF-induced nuclear translocation of p65. As shown in Fig. 3D, Western blot analysis indicated that ESI significantly inhibited TNF-induced nuclear translocation of p65. Immunocytochemistry also confirmed the suppression of TNF-induced nuclear translocation of p65 by ESI (Fig. 3E).

ESI inhibits TNF-induced phosphorylation of p65. TNF also induces the phosphorylation of p65, which is required for the transcriptional activity (12). As shown in Fig. 3F, coincubation of cells with ESI significantly inhibited TNF-induced phosphorylation of p65.

ESI inhibits TNF-induced IKK activation. IKK is required for TNF-induced phosphorylation of I{kappa}B{alpha} (13), and the phosphorylation of p65 requires IKK activation (30). Because ESI inhibited the phosphorylation of both I{kappa}B{alpha} and p65, we sought to determine the effect of ESI on TNF-induced IKK activation. Immune complex kinase assay showed that ESI suppressed the activation of IKK by TNF (Fig. 3G). However, neither TNF nor ESI had an effect on the expression of IKK-{alpha} or IKK-ß proteins. To evaluate whether ESI suppresses IKK activity directly by binding to the IKK protein or by suppressing the activation of IKK, we incubated whole-cell extracts from untreated and TNF-treated cells with ESI at various concentrations. Immune complex kinase assay showed that ESI did not directly affect the activity of IKK, suggesting that ESI modulates TNF-induced IKK activation (Fig. 3H).

ESI represses the TNF-induced NF-{kappa}B-dependent gene products involved in cell proliferation. COX-2 is overexpressed in tumor cells and mediates proliferation (31). In addition, cyclin D1 is overexpressed in a wide variety of tumors and mediates the progression of cells from G1 to S phase of the cell cycle (32). Furthermore, the role of c-Myc in the proliferation of tumors is well established (33). The expression of all three genes of COX-2, cyclin D1, and c-Myc is regulated by NF-{kappa}B (34–36). We found that ESI blocked the expression of these gene products induced by TNF (Fig. 4A ). This result further strengthened our postulate that ESI blocks TNF-induced expression of NF-{kappa}B-regulated gene products.


Figure 4
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  		Fig. 4. ESI inhibits the TNF-induced expression of NF-{kappa}B-dependent antiproliferative, antimetastatic, and antiapoptotic proteins. A, ESI inhibited the TNF-induced expression of COX-2, cyclin D1, and c-Myc. B, ESI inhibited the TNF-induced expression of MMP9 and ICAM-1. C, ESI inhibited the TNF-induced expression of antiapoptotic proteins. KBM-5 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared and analyzed using Western blot analysis with the indicated antibodies. For these experiments, we used four separate gels; based on molecular weight, each gel was sliced into two, and each slice was stripped two to three times and reprobed with different antibodies.

 
ESI represses the TNF-induced NF-{kappa}B-dependent gene products involved in angiogenesis and metastasis. The role of both MMP-9 and ICAM-1 in tumor angiogenesis and metastasis is well established. Both of these gene products are regulated by NF-{kappa}B (37, 38), so we investigated the effect of ESI on this regulation. Western blot analysis showed that ESI blocked TNF-induced ICAM-1 and MMP-9 protein expression in a time-dependent manner (Fig. 4B). This result suggests that ESI plays a role in suppression of both angiogenesis and metastasis.

ESI represses TNF-induced NF-{kappa}B-dependent antiapoptotic gene products. NF-{kappa}B regulates the expression of the antiapoptotic proteins IAP1/2, Bcl-2, Bcl-xL, Bfl-1/A1, TRAF1, FLIP, and survivin (39–47), so we examined whether ESI can modulate the TNF-induced expression of these proteins. As shown in Fig. 4C, ESI blocked the expression of all of these proteins.

ESI potentiates apoptosis induced by TNF. Activation of NF-{kappa}B can inhibit TNF-induced apoptosis (48–52), so we sought to determine the potential of ESI for enhancement of apoptosis induced by TNF using the LIVE/DEAD assay, PARP cleavage assay, Annexin V staining, and TUNEL staining. The LIVE/DEAD assay indicated that ESI up-regulated the rate of TNF-induced cytotoxicity from 7% to 39% (Fig. 5A ). In addition, as shown in Fig. 5B, the PARP cleavage assay indicated that ESI potentiated the TNF-induced activation caspases. Furthermore, Annexin V staining indicated that ESI up-regulated early TNF-induced apoptosis (Fig. 5C). Finally, TUNEL staining showed that TNF-induced apoptosis was enhanced by incubation with ESI (Fig. 5D). In this assay, ESI alone exhibited slight toxicity. Taken together, the results of all of these tests suggest that ESI enhanced the apoptotic effects of TNF.


Figure 5
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  		Fig. 5. ESI enhances TNF-induced cytotoxicity. A, KBM-5 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours. The cells were stained with LIVE/DEAD assay reagent for 30 minutes and then analyzed under a fluorescence microscope as described in Materials and Methods. B, KBM-5 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared, subjected to SDS-PAGE, and blotted with an anti-PARP antibody. C, KBM-5 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours. Cells were then incubated with a FITC-conjugated Annexin V antibody and analyzed using a flow cytometer as described in Materials and Methods. D, KBM-5 cells were coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 16 hours. Cells were then fixed, stained with TUNEL assay reagent, and analyzed using a flow cytometer as described in Materials and Methods.

 
ESI suppresses TNF-induced invasion activity. MMPs, COXs, and adhesion molecules that are regulated by NF-{kappa}B have been shown to mediate tumor invasion (53), and TNF can induce expression of genes involved in tumor metastasis (54). We examined whether ESI modulates TNF-induced invasive activity in vitro. For this study, we used H1299 cells seeded in the top chamber of a Matrigel invasion chamber in the absence of serum. We coincubated cells with TNF in the presence or absence of ESI for 24 hours. As shown in Fig. 6A , TNF induced invasive activity, whereas ESI suppressed it.


Figure 6
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  		Fig. 6. ESI suppresses TNF-induced invasive activity. A, H1299 cells were seeded in the top chamber of a Matrigel invasion chamber overnight in the absence of serum, coincubated with 2 µmol/L ESI and 1 nmol/L TNF for 24 hours in the presence of 1% serum, and then subjected to invasion assay as described in Materials and Methods. B, ESI inhibited RANKL-induced osteoclastogenesis. RAW 264.7 cells were incubated either alone or in the presence of 5 nmol/L RANKL with 0.03 µmol/L ESI for 5 days and stained for TRAP expression. TRAP-positive cells were photographed. Original magnification, x100. C, RAW 264.7 cells were incubated either alone or in the presence of 5 nmol/L RANKL with ESI at the indicated concentrations for 5 days and stained for TRAP expression. Multinucleated (three nuclei) osteoclasts were counted. D, the proposed mechanism by which ESI inhibits NF-{kappa}B activation and NF-{kappa}B-regulated gene expression involved in cell proliferation and invasion.

 
ESI inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells. A recent study implicated RANKL, a member of the TNF superfamily, as a major mediator of bone resorption (55). Thus, agents that can suppress RANKL signaling have the potential to inhibit bone resorption and osteoclastogenesis. To determine the effect of ESI on osteoclastogenesis, we coincubated RAW 264.7 cells with ESI at different concentrations in the presence of RANKL and allowed them to grow and differentiate into osteoclasts. Figure 6B and C shows that ESI significantly decreased the RANKL-induced differentiation of osteoclasts. An ESI concentration of 0.03 µmol/L was sufficient to reduce the rate of osteoclastogenesis by >50%. Under these conditions, the cells remained fully viable (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The anti-inflammatory activities of ESI suggest that it must mediate its effects by suppressing NF-{kappa}B activation. In the present study, we found that ESI indeed inhibited activation of NF-{kappa}B by a variety of inflammatory agents and in a variety of cell lines. Specifically, we found that NF-{kappa}B activity was inhibited because ESI suppressed IKK activation, resulting in inhibition of I{kappa}B{alpha} phosphorylation and degradation. Consequently, ESI also blocked p65 phosphorylation and p65 nuclear translocation. Furthermore, it suppressed expression of gene products involved in cell proliferation, antiapoptosis, and invasion. Suppression of NF-{kappa}B by ESI enhanced the apoptosis induced by TNF and inhibited TNF-induced cellular invasion and RANKL-induced osteoclastogenesis (Fig. 6D).

This is the first study to investigate the mechanism by which ESI and ESD mediate their effects. Our results indicate that ESI inhibits NF-{kappa}B activation instantly, as we found suppression even when we added it after initiation of NF-{kappa}B activation by TNF. In this respect, suppression of NF-{kappa}B activation induced by ESI differs from that induced by curcumin (56), flavopiridol (30), and farnesyl transferase inhibitors (57). The latter requires preincubation for several hours before activating the cells for NF-{kappa}B. Similar to ESI, we showed previously that 1'-acetoxychavicol acetate instantly inhibits TNF-induced NF-{kappa}B activation (58). Apparently, ESI acts at same mechanism as ACA (58). How ESI is taken up by the cells, is unclear. Helenarin, which has structural similarity to ESI, is a cell-permeable sesquiterpene lactone and is known to inhibit NF-{kappa}B activation (59). It is possible that ESI enters the cells through a mechanism similar to that of helenarin.

We also found that ESI inhibited NF-{kappa}B activation induced by highly diverse inflammatory stimuli (TNF, LPS, IL-1ß, and PMA). Most of these agents activate NF-{kappa}B through different pathways (11–13). Because ESI inhibited NF-{kappa}B activation induced by all of the agents tested, it must suppress activation at a common step. Various tumor cells express a constitutively activated form of NF-{kappa}B through a mechanism that is not fully understood (11). ESI also suppressed constitutive NF-{kappa}B activation in our study. Unlike some other inhibitors (27–29), however, ESI did not modify the NF-{kappa}B proteins to prevent their binding to DNA. Because ESI also inhibited TNF-induced phosphorylation and degradation of I{kappa}B{alpha}, ESI may mediate its inhibitory effect through IKK, the kinase required for I{kappa}B{alpha} phosphorylation. How ESI suppresses TNF-induced IKK activation is not clear at the present time. In addition, the kinase that causes TNF-induced activation of IKK is not known. Some reports have suggested that autoactivation of IKK (60–62). Our data also suggest that ESI had no effect on the expression of IKK protein.

In our study, ESI down-regulated the expression of NF-{kappa}B-regulated gene products involved in cell proliferation (COX-2, cyclin D1, and c-Myc), invasion (MMP-9 and ICAM-1), and antiapoptosis (IAP1, IAP2, Bcl-2, Bcl-xL, Bfl-1/A1, TRAF1, FLIP, and survivin). We also found that ESI potentiated the apoptotic effects of TNF. It is very likely that this potentiation is mediated through the suppression of antiapoptotic gene products regulated by NF-{kappa}B. ESI also suppressed TNF-induced tumor invasion. Invasion and metastasis require the expression of MMP-9, COX-2, and ICAM-1, all of which were modulated by ESI. Thus, these results suggest that ESI is effective not only as a chemopreventive agent but also as a therapeutic agent through regulation of various mechanisms as indicated above.

Overall, our results showed that ESI is an effective blocker of the NF-{kappa}B pathway and thus may have potential in treatment of a wide variety of NF-{kappa}B-linked proinflammatory diseases (63). However, further studies are needed in animals to validate these findings for the therapeutic use of ESI and ESD.


   Acknowledgments
 
We thank Donald R. Norwood for his careful review of the article and Dr. Bryant Darnay for supplying the RANKL protein.


   Footnotes
 
Grant support: Clayton Foundation for Research (B.B. Aggarwal), NIH PO1 grant CA91844 on lung chemoprevention (B.B. Aggarwal), and a NIH P50 Head and Neck Specialized Programs of Research Excellence grant P50CA97007 (B.B. Aggarwal).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: B.B. Aggarwal is a Ransom Horne, Jr., Professor of Cancer Research.

Received 4/17/06; revised 7/ 6/06; accepted 7/21/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lin CC, Tsai CC, Yen MH. The evaluation of hepatoprotective effects of Taiwan folk medicine ‘teng-khia-u’. J Ethnopharmacol 1995;45:113–23.[CrossRef][Medline]
  2. Rajesh MG, Latha MS. Hepatoprotection by Elephantopus scaber Linn. in CCl4-induced liver injury. Indian J Physiol Pharmacol 2001;45:481–6.[Medline]
  3. Tsai CC, Lin CC. Anti-inflammatory effects of Taiwan folk medicine ‘Teng-Khia-U’ on carrageenan- and adjuvant-induced paw edema in rats. J Ethnopharmacol 1999;64:85–9.[Medline]
  4. Poli A, Nicolau M, Simoes CM, et al. Preliminary pharmacologic evaluation of crude whole plant extracts of Elephantopus scaber. Part I: in vivo studies. J Ethnopharmacol 1992;37:71–6.[CrossRef][Medline]
  5. Lee KH, Cowherd CM, Wolo MT. Antitumor agents. XV: deoxyelephantopin, an antitumor principle from Elephantopus carolinianus Willd. J Pharm Sci 1975;64:1572–3.[Medline]
  6. Kurosawa T, Nakanishi K, Wu W, et al. Deoxyelephantopin and its interrelation with elephantopin. Tetrahedron Lett 1970;11:2863–6.
  7. de Silva LB, Herath WHMW, Jennings RC, et al. A novel sesquiterpene lactone from Elephantopus scaber. Phytochemistry 1982;21:1173–5.[CrossRef]
  8. But PP, Hon P-M, Cao H, et al. Sesquiterpene lactones from Elephantopus scaber. Phytochemistry 1997;44:113–6.[CrossRef]
  9. Zhang D, Haruna M, McPhail AT, et al. Cytotoxic germacronolides of Elephantopus carolinianus and the structure and stereochemistry of isodeoxyelephantopin. Phytochemistry 1986;25:899–904.[CrossRef]
  10. Govindachari TR, Vishvanathan N, Fuhrer H. Isodeoxyelephantopin, a new germacranediolide from Elephantopus scaber Linn. Indian J Chem 1972;10:272.
  11. Aggarwal BB. Nuclear factor {kappa}B: the enemy within. Cancer Cell 2004;6:203–8.[CrossRef][Medline]
  12. Ghosh S, May MJ, Kopp EB. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998;16:225–60.[CrossRef][Medline]
  13. Ducut Sigala JL, Bottero V, Young DB, et al. Activation of transcription factor NF{kappa}B requires ELKS, an I{kappa}B kinase regulatory subunit. Science 2004;304:1963–7.[Abstract/Free Full Text]
  14. Chaturvedi MM, Mukhopadhyay A, Aggarwal BB. Assay for redox-sensitive transcription factor. Methods Enzymol 2000;319:585–602.[Medline]
  15. Takada Y, Mukhopadhyay A, Kundu GC, et al. Hydrogen peroxide activates NF-{kappa}B through tyrosine phosphorylation of I{kappa}B{alpha} and serine phosphorylation of p65: evidence for the involvement of I{kappa}B{alpha} kinase and Syk protein-tyrosine kinase. J Biol Chem 2003;278:24233–41.[Abstract/Free Full Text]
  16. Manna SK, Mukhopadhyay A, Aggarwal BB. IFN-{alpha} suppresses activation of nuclear transcription factors NF-{kappa} B and activator protein 1 and potentiates TNF-induced apoptosis. J Immunol 2000;165:4927–34.[Abstract/Free Full Text]
  17. Takada Y, Singh S, Aggarwal BB. Identification of a p65 peptide that selectively inhibits NF-{kappa} B activation induced by various inflammatory stimuli and its role in down-regulation of NF-{kappa}B-mediated gene expression and up-regulation of apoptosis. J Biol Chem 2004;279:15096–104.[Abstract/Free Full Text]
  18. Shevde NK, Bendixen AC, Dienger KM, et al. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc Natl Acad Sci U S A 2000;97:7829–34.[Abstract/Free Full Text]
  19. Anto RJ, Mukhopadhyay A, Shishodia S, et al. Cigarette smoke condensate activates nuclear transcription factor-{kappa}B through phosphorylation and degradation of I{kappa}B({alpha}): correlation with induction of cyclooxygenase-2. Carcinogenesis 2002;23:1511–8.[Abstract/Free Full Text]
  20. Shishodia S, Majumdar S, Banerjee S, et al. Ursolic acid inhibits nuclear factor{kappa}B activation induced by carcinogenic agents through suppression of I{kappa}B{alpha} kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Res 2003;63:4375–83.[Abstract/Free Full Text]
  21. Garg AK, Aggarwal BB. Reactive oxygen intermediates in TNF signaling. Mol Immunol 2002;39:509–17.[CrossRef][Medline]
  22. Pahl HL. Activators and target genes of Rel/NF-{kappa}B transcription factors. Oncogene 1999;18:6853–66.[CrossRef][Medline]
  23. Bonizzi G, Piette J, Merville MP, et al. Distinct signal transduction pathways mediate nuclear factor-{kappa}B induction by IL-1ß in epithelial and lymphoid cells. J Immunol 1997;159:5264–72.[Abstract]
  24. Ghosh S, Karin M. Missing pieces in the NF-{kappa}B puzzle. Cell 2002;109 Suppl:S81–96.[CrossRef][Medline]
  25. Bharti AC, Shishodia S, Reuben JM, et al. Nuclear factor-{kappa}B and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood 2004;103:3175–84.[Abstract/Free Full Text]
  26. Aggarwal S, Takada Y, Singh S, et al. Inhibition of growth and survival of human head and neck squamous cell carcinoma cells by curcumin via modulation of nuclear factor-{kappa}B signaling. Int J Cancer 2004;111:679–92.[CrossRef][Medline]
  27. Natarajan K, Singh S, Burke TR, Jr., et al. Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-{kappa}B. Proc Natl Acad Sci U S A 1996;93:9090–5.[Abstract/Free Full Text]
  28. Mahon TM, O'Neill LA. Studies into the effect of the tyrosine kinase inhibitor herbimycin A on NF-{kappa}B activation in T lymphocytes. Evidence for covalent modification of the p50 subunit. J Biol Chem 1995;270:28557–64.[Abstract/Free Full Text]
  29. Finco TS, Beg AA, Baldwin AS, Jr. Inducible phosphorylation of I{kappa}B{alpha} is not sufficient for its dissociation from NF-{kappa}B and is inhibited by protease inhibitors. Proc Natl Acad Sci U S A 1994;91:11884–8.[Abstract/Free Full Text]
  30. Takada Y, Aggarwal BB. Flavopiridol inhibits NF-{kappa}B activation induced by various carcinogens and inflammatory agents through inhibition of I{kappa}B{alpha} kinase and p65 phosphorylation:abrogation of cyclin D1, cyclooxygenase-2, and matrix metalloprotease-9. J Biol Chem 2004;279:4750–9.[Abstract/Free Full Text]
  31. Chun KS, Surh YJ. Signal transduction pathways regulating cyclooxygenase-2 expression: potential molecular targets for chemoprevention. Biochem Pharmacol 2004;68:1089–100.[CrossRef][Medline]
  32. Polsky D, Cordon-Cardo C. Oncogenes in melanoma. Oncogene 2003;22:3087–91.[CrossRef][Medline]
  33. Schmidt EV. The role of c-myc in regulation of translation initiation. Oncogene 2004;23:3217–21.[CrossRef][Medline]
  34. Yamamoto K, Arakawa T, Ueda N, et al. Transcriptional roles of nuclear factor {kappa}B and nuclear factor-interleukin-6 in the tumor necrosis factor {alpha}-dependent induction of cyclooxygenase-2 in MC3T3-1 cells. J Biol Chem 1995;270:31315–20.[Abstract/Free Full Text]
  35. Guttridge DC, Albanese C, Reuther JY, et al. NF-{kappa}B controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 1999;19:5785–99.[Abstract/Free Full Text]
  36. Esteve PO, Robledo O, Potworowski EF, et al. Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway. Biochem Biophys Res Commun 2002;296:864–9.[CrossRef][Medline]
  37. Sanceau J, Boyd DD, Seiki M, et al. Interferons inhibit tumor necrosis factor-{alpha}-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with NF-{kappa}B. J Biol Chem 2002;277:35766–75.[Abstract/Free Full Text]
  38. Gorgoulis VG, Zacharatos P, Kotsinas A, et al. p53 activates ICAM-1 (CD54) expression in an NF-{kappa}B-independent manner. EMBO J 2003;22:1567–78.[CrossRef][Medline]
  39. Zhu L, Fukuda S, Cordis G, et al. Anti-apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning. FEBS Lett 2001;508:369–74.[CrossRef][Medline]
  40. Chu ZL, McKinsey TA, Liu L, et al. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-{kappa}B control. Proc Natl Acad Sci U S A 1997;94:10057–62.[Abstract/Free Full Text]
  41. You M, Ku PT, Hrdlickova R, et al. ch-IAP1, a member of the inhibitor-of-apoptosis protein family, is a mediator of the antiapoptotic activity of the v-Rel oncoprotein. Mol Cell Biol 1997;17:7328–41.[Abstract]
  42. Stehlik C, de Martin R, Kumabashiri I, et al. Nuclear factor (NF)-{kappa}B-regulated X-chromosome-linked iap gene expression protects endothelial cells from tumor necrosis factor {alpha}-induced apoptosis. J Exp Med 1998;188:211–6.[Abstract/Free Full Text]
  43. Catz SD, Johnson JL. BCL-2 in prostate cancer: a mini review. Apoptosis 2003;8:29–37.[CrossRef][Medline]
  44. Tamatani M, Che YH, Matsuzaki H, et al. Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF{kappa}B activation in primary hippocampal neurons. J Biol Chem 1999;274:8531–8.[Abstract/Free Full Text]
  45. Grumont RJ, Rourke IJ, Gerondakis S. Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis. Genes Dev 1999;13:400–11.[Abstract/Free Full Text]
  46. Zong WX, Edelstein LC, Chen C, et al. The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-{kappa}B that blocks TNF{alpha}-induced apoptosis. Genes Dev 1999;13:382–7.[Abstract/Free Full Text]
  47. Kreuz S, Siegmund D, Scheurich P, et al. NF-{kappa}B inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol 2001;21:3964–73.[Abstract/Free Full Text]
  48. Verma VN, Shenoy CN, Nadkarni JJ. Augmentation of cisplatin cytotoxicity using cytokines on cervical carcinoma cell lines. Cancer Biother Radiopharm 1996;11:349–54.[Medline]
  49. Wang D, Baldwin AS, Jr. Activation of nuclear factor-{kappa}B-dependent transcription by tumor necrosis factor-{alpha} is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem 1998;273:29411–6.[Abstract/Free Full Text]
  50. Giri DK, Aggarwal BB. Constitutive activation of NF-{kappa}B causes resistance to apoptosis in human cutaneous T cell lymphoma HuT-78 cells. Autocrine role of tumor necrosis factor and reactive oxygen intermediates. J Biol Chem 1998;273:14008–14.[Abstract/Free Full Text]
  51. Mayo MW, Wang CY, Cogswell PC, et al. Requirement of NF-{kappa}B activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 1997;278:1812–5.[Abstract/Free Full Text]
  52. Pomerantz JL, Baltimore D. Signal transduction. A cellular rescue team. Nature 2000;406:26–7, 29.[CrossRef][Medline]
  53. Liotta LA, Thorgeirsson UP, Garbisa S. Role of collagenases in tumor cell invasion. Cancer Metastasis Rev 1982;1:277–88.[CrossRef][Medline]
  54. van de Stolpe A, Caldenhoven E, Stade BG, et al. 12-O-tetradecanoylphorbol-13-acetate- and tumor necrosis factor {alpha}-mediated induction of intercellular adhesion molecule-1 is inhibited by dexamethasone. Functional analysis of the human intercellular adhesion molecular-1 promoter. J Biol Chem 1994;269:6185–92.[Abstract/Free Full Text]
  55. Kong YY, Yoshida H, Sarosi I, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development, and lymph-node organogenesis. Nature 1999;397:315–23.[CrossRef][Medline]
  56. Singh S, Aggarwal BB. Activation of transcription factor NF-{kappa}B is suppressed by curcumin (diferuloylmethane) [corrected]. J Biol Chem 1995;270:24995–5000.[Abstract/Free Full Text]
  57. Takada Y, Khuri FR, Aggarwal BB. Protein farnesyltransferase inhibitor (SCH 66336) abolishes NF-{kappa}B activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-{kappa}B-regulated gene expression and up-regulation of apoptosis. J Biol Chem 2004;279:26287–99.[Abstract/Free Full Text]
  58. Ichikawa H, Takada Y, Murakami A, et al. Identification of a novel blocker of I{kappa}B{alpha} kinase that enhances cellular apoptosis and inhibits cellular invasion through suppression of NF-{kappa}B-regulated gene products. J Immunol 2005;174:7383–92.[Abstract/Free Full Text]
  59. Lyss G, Knorre A, Schmidt TJ, et al. The anti-inflammatory sesquiterpene lactone helenalin inhibits the transcription factor NF-{kappa}B by directly targeting p65. J Biol Chem 1998;273:33508–16.[Abstract/Free Full Text]
  60. Delhase M, Hayakawa M, Chen Y, et al. Positive and negative regulation of I{kappa}B kinase activity through IKKß subunit phosphorylation. Science 1999;284:309–13.[Abstract/Free Full Text]
  61. Karin M. The beginning of the end: I{kappa}B kinase (IKK) and NF-{kappa}B activation. J Biol Chem 1999;274:27339–42.[Free Full Text]
  62. Karin M. How NF-{kappa}B is activated:the role of the I{kappa}B kinase (IKK) complex. Oncogene 1999;18:6867–74.[CrossRef][Medline]
  63. Kumar A, Takada Y, Boriek AM, et al. Nuclear factor-{kappa}B: its role in health and disease. J Mol Med 2004;82:434–48.[Medline]



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