Butein, a Tetrahydroxychalcone, Inhibits Nuclear Factor (NF)-κB and NF-κB-regulated Gene Expression through Direct Inhibition of IκBα Kinase β on Cysteine 179 Residue*

Although butein (3,4,2′,4′-tetrahydroxychalcone) is known to exhibit anti-inflammatory, anti-cancer, and anti-fibrogenic activities, very little is known about its mechanism of action. Because numerous effects modulated by butein can be linked to interference with the NF-κB pathway, we investigated in detail the effect of this chalcone on NF-κB activity. As examined by DNA binding, we found that butein suppressed tumor necrosis factor (TNF)-induced NF-κB activation in a dose- and time-dependent manner; suppressed the NF-κB activation induced by various inflammatory agents and carcinogens; and inhibited the NF-κB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-β. We also found that butein blocked the phosphorylation and degradation of IκBα by inhibiting IκBα kinase (IKK) activation. We found the inactivation of IKK by butein was direct and involved cysteine residue 179. This correlated with the suppression of phosphorylation and the nuclear translocation of p65. In this study, butein also inhibited the expression of the NF-κB-regulated gene products involved in anti-apoptosis (IAP2, Bcl-2, and Bcl-xL), proliferation (cyclin D1 and c-Myc), and invasion (COX-2 and MMP-9). Suppression of these gene products correlated with enhancement of the apoptosis induced by TNF and chemotherapeutic agents; and inhibition of cytokine-induced cellular invasion. Overall, our results indicated that antitumor and anti-inflammatory activities previously assigned to butein may be mediated in part through the direct inhibition of IKK, leading to the suppression of the NF-κB activation pathway.

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Because butein exhibits anti-inflammatory and anti-proliferative effects and suppresses the expression of adhesion molecules, iNOS, Bcl-2, tissue inhibitor of metalloproteinase-1, and 12-lipooxygenase, all of which are known to be regulated by the transcription factor NF-B, we postulated that butein must mediate its effects by modulating the NF-B activation pathway, which has been closely linked to inflammation, tumorigenesis, proliferation, invasion, angiogenesis, and metastasis, and is activated in response to various inflammatory agents, carcinogens, tumor promoters, and growth factors (37)(38)(39). Therefore, the effect of butein on the regulation of this pathway was investigated in detail. We found that butein suppressed NF-B activation pathways activated by a variety of agents through the direct inhibition of IB␣ kinase (IKK), which led to the suppression of NF-B-regulated gene products and the enhancement of apoptosis induced by inflammatory cytokines.
Cell Lines-Cell lines H1299 (human lung adenocarcinoma), KBM-5 (human myeloid), Jurkat (human T cell leukemia) cells and A293 (human embryonic kidney) were obtained from American Type Culture Collection (Manassas, VA). The H1299 and Jurkat cells were cultured in RPMI 1640 medium, KBM-5 cells were cultured in Iscove's modified Dulbecco's medium with 15% fetal bovine serum, and A293 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. All culture media were supplemented with 100 units/ml penicillin and 100 g/ml streptomycin.
Electrophoretic Mobility Shift Assay-To determine NF-B activation, we prepared nuclear extracts and performed electrophoretic mobility shift assay (EMSA) as described previously (42). For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with antibodies against either p50 or p65 of NF-B for 30 min at 37°C before the complex was analyzed by EMSA. Preimmune serum was included as the negative control. The dried gels were visualized, and the radioactive bands were quantitated with a Storm 820 imaging system running ImageQuant software (Amersham Biosciences).
Western Blot Analysis-To determine the effect of butein on TNF-dependent IB␣ phosphorylation, IB␣ degradation, p65 translocation, and p65 phosphorylation, cytoplasmic and nuclear extracts were prepared as previously described (43,44). For the detection of cleavage products of PARP, caspases, antiapoptotic, and angiogenesis markers, whole cell extracts were prepared by subjecting TNF and TNF plus butein-treated cells to lysis in lysis buffer (20 mmol/liter Tris (pH 7.4), 250 mmol/ liter NaCl, 2 mmol/liter EDTA (pH 8.0), 0.1% Triton X-100, 0.01 g/ml aprotinin, 0.005 g/ml leupeptin, 0.4 mmol/liter phenylmethylsulfonyl fluoride, and 4 mmol/liter NaVO 4 ). Lysates were spun at 14,000 ϫ g for 10 min to remove insoluble material. Supernatants were collected and kept at Ϫ80°C. Either cytosolic, or nuclear extracts and whole cell lysates were resolved by SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with the relevant antibodies, and detected by an enhanced chemiluminescence reagent (Amersham Biosciences ECL TM ). The bands obtained were quantified using NIH Image analyzer (NIH, Bethesda, MD). For detection of precapases and cleaved caspases, specific antibodies against each were combined for detection.
Kinase Assay-To determine the effect of butein on TNFinduced IKK activation, we performed an immunocomplex kinase assay using GST-IB␣ as the substrate as described previously (45). Briefly, the IKK complex from whole cell extracts was precipitated with antibody against IKK-␣ and treated with protein A/G-Sepharose beads (Pierce). After 2 h, the beads were washed with whole cell extract buffer and then resuspended in a kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl 2 , 2 mM DTT, 20 Ci of [␣-32 P]ATP, 10 M unlabeled ATP, and 2 g of substrate GST-IB␣ (amino acids . After incubation at 30°C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized with the Storm 820 imaging system. To determine the total amounts of IKK-␣ and IKK-␤ in each sample, 30 g of whole cell proteins were resolved on 10% SDS-PAGE, electrotransferred to a nitrocellu-lose membrane, and then blotted with either anti-IKK-␣ or anti-IKK-␤ antibody. For JNK assay, whole cell extracts were precipitated with antibody against JNK1, and performed kinase assay using GSTc-Jun (amino acids 1-79) as described (46). To determine the amount of JNK1 in each sample, Western blotting was performed against JNK1 antibody.
NF-B-dependent Reporter Gene Expression Assay-To determine the effect of butein on TNF-, TNF receptor (TNFR-), TNFR-associated death domain (TRADD-), TNFR-associated factor 2 (TRAF2-), NF-B-inducing kinase (NIK), TAK1/ TAB1-, and IKK-NF-B-dependent reporter gene transcription, we performed the secretory alkaline phosphatase (SEAP) assay as previously described (47), with the following exceptions. Briefly, A293 cells (5 ϫ 10 5 cells/well) were plated in 6-well plates and transiently transfected by the calcium phosphate method with pNF-B-SEAP (0.5 g). To examine TNFinduced reporter gene expression, we transfected the cells with 0.5 g of the SEAP expression plasmid and 1.5 g of the control plasmid pCMV-FLAG1 DNA for 24 h. We then treated the cells for 4 h with butein and stimulated them with 1 nM TNF. The cell culture medium was harvested after 24 h of TNF treatment. To examine reporter gene expression induced by various genes, A293 cells were transfected with 0.5 g of pNF-B-SEAP plasmid with 0.5 g of an expressing plasmid and 1.5 g of the control plasmid pCMV-FLAG1 for 24 h, treated with butein, and then harvested from cell culture medium after an additional 24 h of incubation. The culture medium was analyzed for SEAP activity as recommended by the manufacturer (Clontech) using a Victor 3 microplate reader (Perkin-Elmer Life Sciences).
Immunocytochemistry for NF-B p65 Localization-Immunocytochemistry was used to examine the effect of butein on the nuclear pools of p65 as previously described (48). Briefly, treated cells were plated on a poly-L-lysine-coated glass slide by centrifugation (Cytospin 4; Thermoshendon), air dried, and fixed with 4% paraformaldehyde. After being washed in phosphate-buffered saline, the slides were blocked with 5% normal goat serum for 1 h and incubated with rabbit polyclonal antihuman p65 antibody at a 1/200 dilution. After overnight incubation at 4°C, the slides were washed, incubated with goat antirabbit IgG-Alexa Fluor 594 antibody (Molecular Probes, Eugene, OR) at a 1/200 dilution for 1 h, and counterstained for nuclei with Hoechst 33342 (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma) and analyzed under a fluorescence microscope (Labophot-2; Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph version 4.6.5 software (Universal Imaging).
Live/Dead Assay-To measure apoptosis, we also used the Live/Dead assay (Molecular Probes), which determines intracellular esterase activity and plasma membrane integrity (49). Calcein-AM, a nonfluorescent polyanionic dye, is retained by live cells, in which it produces intense green fluorescence through enzymatic (esterase) conversion. In addition, the ethidium homodimer enters cells with damaged membranes and binds to nucleic acids, thereby producing a bright red fluorescence in dead cells. Briefly, 2 ϫ 10 5 cells were incubated with 25 M butein for 4 h and treated with 1 nM TNF up to 24 h at 37°C. Cells were stained with the Live/Dead reagent (5 M ethidium homodimer and 5 M calcein-AM) and incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon).
Cytotoxicity Assay-The effects of butein on the cytotoxic effects of TNF and other chemotherapeutic agents were determined by the MTT uptake method as previously described (48). Briefly, 5,000 cells were incubated with 25 M butein in triplicate in a 96-well plate and treated with the indicated concentrations of 1 nM TNF, 0.1 M 5-fluorouracil (5-FU), and 0.1 M doxorubicin for 24 h at 37°C. An MTT solution was added to each well and incubated for 2 h at 37°C. An extraction buffer (20% SDS, 50% dimethyl formamide) was added, and the cells were incubated overnight at 37°C. Then the optical density was measured at 570 nm using a 96-well multiscanner (MRX Revelation, Dynex Technologies, Chantilly, VA).
Annexin V Assay-The annexin V assay uses the binding properties of annexin V to detect the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic membrane interface to the extracellular surface, an indicator of early apoptosis. We detected this loss of membrane asymmetry using an annexin V antibody conjugated with the fluorescein isothiocyanate fluorescence dye. Briefly, 5 ϫ 10 5 cells were pretreated with butein (25 M), treated with 1 nM TNF up to 16 h at 37°C, and subjected to annexin V staining. The cells were washed in phosphate-buffered saline, resuspended in 100 l of binding buffer containing a fluorescein isothiocyanate-conjugated anti-annexin V antibody, and analyzed with a flow cytometer (FACSCalibur, BD Biosciences).
Invasion Assay-This assay was performed mostly as previously described (50). Because invasion through the extracellular matrix is a crucial step in tumor metastasis, a membrane invasion culture system was used to assess cell invasion. The BD BioCoat tumor invasion system consists of chambers with a lightproof polyethylene terephthalate membrane coated with a reconstituted basement membrane gel with 8-m diameter pores (BD Biosciences). We suspended 2.5 ϫ 10 4 non-small cell adenocarcinoma H1299 cells in serum-free medium and seeded the upper wells with them. After incubation overnight, the cells were treated with the indicated concentration of butein for 4 h and stimulated with 1 nM TNF for an additional 24 h in the presence of 1% fetal bovine serum. The cells that invaded the lower chamber by migrating through the Matrigel during incubation were stained with 4 g/ml calcein-AM in phosphate-buffered saline for 30 min at 37°C and scanned for fluorescence with a Victor 3 multiplate reader (PerkinElmer); fluorescent cells were counted.

RESULTS
We investigated the effects of butein on the NF-B activation pathway induced by various carcinogens and inflammatory stimuli, on NF-B-regulated gene expression, and on apoptosis induced by cytokines and chemotherapeutic agents. The concentration of butein used and the duration of exposure had minimal effect on the viability of different cell lines studied as determined by the trypan blue dye exclusion test (data not shown). We focused on TNF-induced NF-B activation because the NF-B activation pathway activated by TNF has been relatively well characterized (51).

Butein Suppresses TNF-induced NF-B Activation in a Dose-and
Time-dependent Manner-We first determined the dose and time of exposure to butein required to suppress TNF-induced NF-B activation. For this cells were first pretreated with butein, then exposed to TNF for NF-B activation. EMSA showed that butein alone had no effect on NF-B activation, but it inhibited TNF-mediated NF-B activation in a dose-and time-dependent manner (Fig. 1, B and C, respectively). Whether butein can suppress NF-B in cells pre-activated with TNF was also determined. We found that butein downregulated quite effectively TNF induced NF-B even when cells were treated with the inhibitor after NF-B activation (see lane 3 versus lane 5 in Fig. 1D).
NF-B is a complex of proteins, in which various combinations of Rel/ NF-B proteins constitute active NF-B heterodimers that bind to a specific DNA sequence. Thus, to show that the band visualized by EMSA in TNF-treated cells was indeed NF-B, nuclear extracts from TNF-activated cells were incubated with antibodies to the p50 (NF-B) and p65 (RelA) subunits of NF-B and analyzed by EMSA. The results in Fig. 1E, showed the bands had shifted to higher molecular masses suggesting that the TNF-activated complex consisted of p50 and p65. Preimmune serum had no effect on DNA binding. The addition of excess unlabeled NF-B (cold oligonucleotide; 100-fold) caused a complete disappearance of the band, whereas the addition of mutated oligonucleotide had no effect on the DNA binding.
Butein Inhibits Constitutive NF-B Activation-Whether butein alone could inhibit constitutive NF-B in tumor cells was also investigated. Multiple myeloma U266 cells are known to express constitu-  tive NF-B activation (52). U266 cells were treated with different concentrations of butein for 4 h and then analyzed for NF-B activation. Butein inhibited the constitutive NF-B activation in MM cells (Fig. 1F). These results indicate that butein can suppress not only inducible but also constitutively active NF-B in tumor cells.

Butein Inhibits NF-B Activation Induced by Carcinogens and Inflammatory
Stimuli-Cigarette smoke condensate (CSC), TNF, okadaic acid, LPS, and PMA are potent activators of NF-B, but they work by means of different mechanisms (40,(53)(54)(55)(56). The DNA binding assays we used to examine the effect of butein on the activation of the NF-B pathway by CSC, TNF, okadaic acid, LPS, and PMA revealed that all five agents activated NF-B in human myeloid leukemia KBM-5 cells and that butein suppressed this activation (Fig. 1G). These results suggest that butein acts at a step in the NF-B activation pathway that is common to all five agents.
Butein Does Not Directly Affect Binding of NF-B to the DNA-Some NF-B inhibitors such as plumbagin directly modify NF-B to suppress its DNA binding (57). We determined whether butein mediates suppression of NF-B activation through a similar mechanism. We incubated the nuclear extract from TNF-treated cells with butein, and we found that butein did not modify the DNA binding ability of NF-B proteins (Fig. 1H). These results suggested that butein inhibits NF-B activation by a mechanism different from that of plumbagin.
Butein Inhibits TNF-dependent IB␣ Degradation and Phosphorylation-The translocation of NF-B to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of IB␣. To determine whether the inhibition of TNF-induced NF-B activation was due to the inhibition of IB␣ degradation, we pretreated cells with butein and exposed them to TNF for various time periods. We then examined the cells for NF-B by EMSA and for IB␣ degradation in the cytoplasm by Western blot analysis. TNF activated NF-B in the control cells in a time-dependent manner ( Fig. 2A). TNF induced NF-B in control cells as early as 10 min and peaks at 30 min and declined in 60 min, but had no effect on buteinpretreated cells. Moreover, TNF induced IB␣ degradation in control cells as early as 10 min, but in butein-pretreated cells TNF had no effect on IB␣ degradation in butein-pretreated cells (Fig. 2B). These results indicated that butein inhibited both TNF-induced NF-B activation and IB␣ degradation.
To determine whether the inhibition of TNF-induced IB␣ degradation was due to the inhibition of IB␣ phosphorylation, we used the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) to block the degradation of IB␣. Western blotting with an antibody that recognized the serine-phosphorylated form of IB␣ showed that butein strongly suppressed TNF-induced IB␣ phosphorylation (Fig. 2C).
Butein Directly Inhibits TNF-induced IB␣ Kinase (IKK) Activation-Because butein inhibits the phosphorylation and degradation of IB␣, we tested the effect of butein on TNFinduced IKK activation, which is required for the TNF-induced phosphorylation of IB␣. TNF-activated IKK and butein completely suppressed the TNF-induced activation of IKK (Fig. 2D,  upper panel). Neither TNF nor butein had any effect on the expression of IKK-␣ or IKK-␤ proteins (Fig. 2D, second and  third panels).
To evaluate whether butein suppresses IKK activity directly by binding IKK or indirectly by suppressing its activation, we incubated whole cell extracts from untreated cells and TNF-stimulated cells with anti-IKK-␣ antibody. After being precipitated with protein A/G-Sephadex beads, the immunocomplexes were treated with various concentrations of butein. Results from the immune complex kinase assay showed that butein directly inhibited the activity of IKK (Fig. 2E). This finding suggested that butein directly modulates TNF-induced IKK activation.
IKK-␤ contains various cysteine residues. The modulation of IKK activity by butein through the modification of critical cysteine residues was investigated using reducing agents, such as DTT. We found that adding DTT to the kinase reactions and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic (C) and nuclear extracts (N) were prepared and analyzed by Western blotting using antibodies against p65. For loading control of nuclear and cytoplasmic protein, the membrane was reblotted with anti-PARP and ␤-actin antibody, respectively. B, immunocytochemical analysis of p65 localization. KBM-5 cells were first treated with 50 M butein for 4 h at 37°C and then exposed to 0.1 nM TNF for 15 min. After cytospin, immunocytochemical analysis was done as described under "Materials and Methods." C, butein inhibits TNF-induced phosphorylation of p65. KBM-5 cells were either untreated or pretreated with 50 M butein for 4 h at 37°C and then treated with 0.1 nM TNF for the indicated times. Cytoplasmic (C) and nuclear extracts (N) were prepared and analyzed by Western blotting using phospho-specific p65 antibodies. For loading control of nuclear and cytoplasmic protein, the membrane was reblotted with anti-PARP and ␤-actin antibody, respectively. reversed the butein-mediated inhibition of TNF-induced IKK activity (Fig. 2F).
At position 179 in its activation loop, IKK-␤ contains a cysteine that is critical for its biological activity. To determine whether this cysteine is involved in butein-mediated inhibition, we transfected A293 cells to express wild-type FLAG-IKK-␤ or FLAG-IKK-␤ with a C179A mutation. Butein treatment was associated with the inhibition of wild-type IKK-␤ (Fig. 2G). In contrast, butein had no apparent effect on IKK-␤(C179A) activity (Fig. 2G). These findings proved that butein inhibited IKK␤ activity by directly reacting with the Cys-179 residue.
Butein Has No Effect on TNF-induced JNK and p38 MAPK Activation-Whether the effect of butein on TNF-induced NF-B activation is specific or it also inhibits other signals transmitted by TNF, was investigated. We found that TNF activated p38 MAPK in a time-dependent manner, and butein had no effect on this activation (Fig. 2H). We also found by immunocomplex kinase assay that TNF activated JNK in a time-dependent manner, and butein had no effect on this activation (Fig. 2I). These results indicate that the effect of butein on IKK activation is specific.
Butein Inhibits TNF-induced Nuclear Translocation of p65-Whether butein modulates TNF-induced nuclear translocation of p65, was investigated. We found that on treatment of cells with the p65 subunit, NF-B disappeared from the cytoplasm and appeared in the nucleus (Fig. 3A). Treatment of cells with butein, however, suppressed the TNF-induced nuclear translocation of p65 in a time-dependent manner (Fig. 3A, top panel). An immunocytochemistry assay also confirmed that butein suppressed the translocation of p65 to the nucleus (Fig. 3B).
Butein Inhibits TNF-induced Phosphorylation of p65-We also investigated the effect of butein on the TNF-induced phos-phorylation of p65 at serine residue 536, because phosphorylation is required for the transcriptional activity of p65 (58). TNF-induced p65 phosphorylation in the cytoplasm in a time-dependent manner. p65 was phosphorylated as early as 10 min after TNF stimulation and increased up to 30 min (Fig. 3C,  upper panel). In cells treated with butein, TNF failed to induce p65 phosphorylation. Similar results were obtained with nuclear p65 phosphorylation (Fig. 3C, lower  panel).
Butein Represses TNF-induced NF-B-dependent Reporter Gene Expression-Although we observed by EMSA that butein blocked NF-B activation, DNA binding alone does not always correlate with NF-B-dependent gene transcription, suggesting that additional regulatory steps exist (59). After transiently transfecting butein-pretreated and untreated cells with the NF-B-regulated SEAP reporter construct and stimulating the cells with TNF to determine the effect of butein on TNF-induced NF-B-dependent reporter gene expression, we determined that stimulation with TNF caused almost 16 times more SEAP activity than it did in the vector control (Fig. 4A). Also, most of the TNF-induced SEAP activity was abolished by dominant-negative IB␣, indicating specificity. When the cells were pretreated with butein, TNF-induced NF-B-dependent SEAP expression was inhibited. These results proved that butein also inhibits NF-B-dependent reporter gene expression induced by TNF.
Butein Represses the Expression of TNF-induced NF-B-dependent Antiapoptotic Gene Products-Because NF-B regulates the expression of the antiapoptotic proteins IAP2 (62), Bcl-2 (63), and Bcl-xL (64), we investigated whether butein could modulate the TNF-induced expression of these antiapoptotic genes. In this study, TNF induced the expression of antiapoptotic proteins IAP2, Bcl-2, and Bcl-xL, and butein blocked that expression in a time-dependent manner (Fig. 5A).

Butein Suppresses the Expression of TNF-induced NF-B-dependent Gene Products Involved in the Proliferation and
Metastasis of Tumor Cells-We also investigated whether butein could modulate the NF-B-regulated gene products involved in the proliferation, invasion, and angiogenesis of tumor cells. Because TNF has been shown to induce the expression of c-Myc, Cyclin D1, COX-2, ICAM-1, MMP-9, and VEGF, all of which have NF-B binding sites in their promoters (65-68), we investigated whether butein inhibits the TNF-in-duced expression of these proteins. Untreated and butein-pretreated cells were examined for TNF-induced gene products by Western blot analysis using specific antibodies. Western blotting showed that TNF induced the expression of c-Myc, Cyclin D1, COX-2, ICAM-1, MMP-9 and VEGF gene products, and that butein abolished it (Fig. 5, B and C).
Butein Potentiates the TNF-induced Caspase Activation-TNF-␣ binds to the TNF receptor (TNFR1), which then sequentially recruits TRADD and TRAF2 leading to activation of NF-B and caspases (69). Whether butein affects TNFinduced activation of caspase 8 (also called FLICE) and caspase-3 was investigated. We found that TNF alone had a minimal effect on activation of caspase-8 or caspase-3, whereas treatment with butein potentiated the activation as indicated by the cleaved caspases (Fig. 5D).
Butein Does Not Alter Expression of RIP and TRADD-Whether butein affects the regulatory protein involved in TNF signaling was examined. We found that butein had no effect on expression of TRADD or RIP, two most critical proteins linked to TNF signaling (Fig. 5E).
Butein Potentiates Apoptosis Induced by TNF and Chemotherapeutic Agent-Because the activation of NF-B has been shown to inhibit apoptosis induced by TNF and chemotherapeutic agents (69,70). We investigated whether butein affects apoptosis induced by TNF and chemotherapeutic agents in several ways. According to results from the Live/Dead assay, butein up-regulated TNF-induced apoptosis from 15 to 65% in KBM-5 cells (Fig. 6A). Whether butein modulates TNF-induced apoptosis in other cell types was also investigated. Our results show that butein also up-regulated TNF-induced apoptosis from 5 to 70% also in human Jurkat T cells (Fig. 6B). The cytotoxicity assay using MTT also showed that butein enhanced the cytotoxicity induced by TNF, doxorubicin, and 5-fluorouracil (Fig. 6C). In addition, annexin V staining showed that butein up-regulated TNF-induced early apoptosis (Fig.  6D), and caspase-mediated PARP cleavage showed that butein substantially enhanced the apoptotic effects of TNF (Fig. 6E). Butein alone at this concentration had minimal effect. These results together indicated that butein potentiated the apoptotic effects of TNF and chemotherapeutic agents.
Butein Suppresses TNF-induced Invasion Activity-The expression of gene products (e.g. COX-2 and MMP-9) has been shown to mediate tumor cell invasion (71). The ability of butein to modulate TNF-induced tumor cell invasion activity in vitro was examined using a Matrigel invasion assay. Analysis of the Matrigel invasion assay indicated that butein suppressed TNFinduced invasion (Fig. 6F).

DISCUSSION
The goal of this study was to determine whether the antiinflammatory and anti-proliferative effects of butein were mediated through modulation of NF-B and its regulated gene products. We found that butein suppressed the NF-B activation induced by various carcinogens and inflammatory agents and that the inhibition of NF-B was due to the inhibition of IKK activation, leading to the suppression of IB␣ phosphorylation and degradation. Butein also down-regulated NF-B-dependent gene products involved in cell proliferation, anti-apo- ptosis, invasion, and angiogenesis. This down-regulation led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents and to a suppression of invasion.
The inhibition of the NF-B activation induced by TNF, okadaic acid, CSC, LPS, and PMA suggest that butein acts at a step common to all of these activators. In response to most of these agents, NF-B activation required IKK activation. We found that this chalcone suppressed the activation of IKK. In investigations of the inhibition of IKK activation on butein, we found that butein can directly inhibit IKK activity and that the suppression of IKK activation by butein could be reversed by reducing agents. This suggests that butein alters the redox status of IKK. Because redox-sensitive cysteine residues have been identified in IKK (Cys-179) (72), we also explored the possibility that butein mediates its effects by oxidizing the critical cysteine residue present in IKK. We found that butein modulated IKK activity but had no direct effect on the DNA binding ability of p65. Our results further indicate that Cys-179 is involved in the modification of IKK activity by butein. Moreover, the lack of effect of butein on JNK and p38 MAPK activation indicates specificity.
The pattern of activity modulation we found butein to exhibit is similar to that of other agents. For example, N-ethylmaleimide and p-hydroxymercuribenzoate directly suppressed IKK activity in vitro, but IKK was reactivated by the addition of thiol-reducing agents (e.g. DTT and GSH) (73,74). The inhibitory effects of N-ethylmaleimide on IKK activity were abolished by the expression of mutant IKK␤, which contains alanine at residue 179 rather than cysteine (75,76). Like butein, triterpenoid CDDO-Me has also been found to inhibit the NF-B pathway by directly inhibiting IKK-␤ on Cys-179 (77). In addition to its presence in IKK-␤, cysteine thiols have been found in several transcription factors, such as AP-1, p53, and NF-E2-related factor-2 (Nrf-2), as redox sensors in the transcriptional regulations of many genes essential for the regulation of cellular homeostasis (78,79). The critical cysteine residues that are modified by the inducers have been identified in Keap1, a component of Nrf-2 that regulates phase 2 genes (79).
TNF activates NF-B through the sequential recruitment of TNFR1, TRAF2, RIP, TAK1, and IKK (41,60), and we found that butein suppressed NF-B activation by several of these plasmids. IKK has also been implicated in the phosphorylation of p65 (80). We found that butein suppressed the phosphorylation of p65.
In the present study, butein suppressed the TNF-induced expression of anti-apoptotic gene products including Bcl-2, Bcl-xL, and cIAP, all known to be regulated by NF-B. These results are in agreement with previous studies indicating that butein can suppress Bcl-2 expression (16,17). We also found that butein down-regulated the expression of cell proliferative gene products c-Myc, COX-2, and Cyclin D1, which could explain the previously reported ability of butein to suppress the proliferation of a wide variety of tumor cells, including breast carcinoma (9, 10), colon carcinoma (11,12), osteosarcoma (13), lymphoma (14,15), acute myelogenous leukemia (16), melanoma (17), and hepatic stellate (18). In this study, butein also suppressed the expression of ICAM-1, MMP-9, and VEGF, which have been linked with the invasion and metastasis of tumors. The ability of butein to suppress the expression of adhesion molecules is consistent with previous reports. Although we did not examine the suppression of butein for iNOS expression, which was reported previously (23), that ability may be explained by the suppression of butein for NF-B activation as described in this report. Similarly, the anti-inflammatory effects previously attributed to butein (32) could also be due to the inhibition of COX-2 and various other gene products, as reported here. Butein has been shown to suppress phorbol ester-induced skin tumor formation (2), and this effect may also be mediated through the abrogation of phorbol ester-induced NF-B activation, also as shown in this study. In addition, we found that butein suppressed MMP-9-mediated tumor cell invasion. These effects are similar to those reported with a specific inhibitor of NF-B (49).
Our studies also showed that butein can potentiate the apoptotic effects of TNF, 5-fluorouracil, and doxorubicin. NF-Bregulated gene products have been associated with resistance to both TNF and chemotherapeutic agents. Thus the down-regulation of these genes by butein may account for the sensitization (6,20). We also found that butein potentiated the cleavage of caspase-8 and -3, which is consistent with the previous report that butein increases apoptosis through cleavage of caspase-3 (16). Moreover, butein did not alter the expression of RIP and TRADD involved in TNF signaling.
Overall, our results suggest that the anti-carcinogenic, antiinflammatory, and apoptotic effects of butein are mediated through the inhibition of the IKK-induced NF-B pathway, which is activated by a wide variety of carcinogens and inflammatory agents. Based on these results, we conclude that butein is a direct inhibitor of IKK, through which it blocks NF-Band NF-B-regulated gene products. Further studies in animals and humans will be required to recognize the full potential of butein in the prevention or treatment of cancer.