3-Formylchromone Interacts with Cysteine 38 in p65 Protein and with Cysteine 179 in IκBα Kinase, Leading to Down-regulation of Nuclear Factor-κB (NF-κB)-regulated Gene Products and Sensitization of Tumor Cells*

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.

It is now generally accepted that most chronic diseases, including cancer, are caused by dysregulation of inflammatory pathways. Thus, agents that can suppress proinflammatory pathways safely and effectively have potential in the prevention and treatment of cancer and other diseases. While searching for such agents, we focused on chromones, which have been linked with antibacterial, antifungal, and antitumor activities (1). In particular, we focused on 3-formylchromone (3-FC), 3 which has been shown to exhibit antibacterial (2), anti-inflammatory (3), and antitumor (2, 4 -6) activities. How this agent exhibits all these activities is not fully understood, but inhibition of protein-tyrosine phosphatase 1B (7), chelation of bivalent cations (8), multidrug resistance (5), p56 lck tyrosine kinase (9), and thymidine phosphorylase (10) have been implicated.
Because of the critical role of the NF-B pathway in inflammation and tumorigenesis, we postulated that 3-FC mediates its effects through modulation of this pathway. Under normal conditions, NF-B is present in the cytoplasm as an inactive heterotrimer consisting of p50, p65, and IB␣. However, when activated by various carcinogens, tumor promoters, or proinflammatory agents, IB␣ kinase (IKK) is activated, thus causing the phosphorylation, ubiquitination, and degradation of IB␣ by the 26 S proteasome. The p65/p50 subunit is then released from the cytoplasm and translocated to the nucleus, where it binds to a specific DNA sequence and activates the transcription of Ͼ500 genes (11,12). Several of the NF-B-regulated genes are linked to inflammation, cellular transformation, tumor cell survival, proliferation, invasion, angiogenesis, and metastasis (12). Although NF-B is active only in the immune system under physiological conditions, most tumor cells express constitutive NF-B, and these cells are addicted to it (13). Thus, suppression of the NF-B pathway is an important therapeutic target for the prevention and treatment of cancer.
We hypothesized that 3-FC modulates the NF-B activation pathway. To test this hypothesis, we assayed the effects of 3-FC on constitutive and inducible NF-B activation induced by various carcinogens, tumor promoters, and inflammatory agents. For most studies, TNF-␣ was used to activate NF-B, as NF-B activation by this cytokine is best understood. We examined the effects of 3-FC on NF-B-regulated gene products. We found that 3-FC inhibited NF-B activation by interacting with spe-cific proteins in the pathway, leading to suppression of NF-Bregulated gene products and chemosensitization of tumor cells.
Electrophoretic Mobility Shift Assay-To assess NF-B activation, we isolated nuclei from cells and performed EMSA essentially as described previously (14). In brief, nuclear extracts prepared from cancer cells were incubated with 32 P end-labeled 45-mer double-stranded NF-B oligonucleotide (15 g of protein with 16 fmol of DNA) from the HIV long terminal repeat (5Ј-TTGTTACAAGGGACTTTC CGCTG GGGACTTTC CAGGGA GGCGT GG-3Ј, with NF-B-binding sites shown in boldface type) for 30 min at 37°C. The resulting protein-DNA complex was separated from free oligonucleotides on 6.6% native polyacrylamide gels. The dried gels were visualized, and radioactive bands were quantified using a PhosphorImager imaging device (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. The EMSA for Oct-1 was performed as described for NF-B using 32 P end-labeled double-stranded oligonucleotides.
Immunocytochemistry for NF-B p65 Localization-To determine the effect of 3-FC on the nuclear translocation of p65, we performed immunocytochemical analysis as described previously (16). Briefly, cells were plated on a poly-L-lysinecoated glass slide with a Cytospin 4 centrifuge (ThermoShendon, Pittsburgh, PA), air-dried, and fixed with 4% paraformaldehyde. The slides were washed with PBS, blocked with 5% normal goat serum for 1 h, and then incubated with rabbit anti-p65 polyclonal antibody at a 1:200 dilution overnight at 4°C. The slides were washed, incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Invitrogen) at a 1:200 dilution for 1 h, and counterstained for nuclei with Hoechst 33342 (50 ng/ml) for 5 min. The stained slides were mounted with mounting medium and analyzed under a Labophot-2 fluorescence microscope (Nikon, Melville, NY). Photographs were taken using a Photometrics CoolSNAP cf color camera (Nikon) and analyzed using MetaMorph software (version 4.6.5, Universal Imaging, Sunnyvale, CA).
Kinase Assay-To determine the effect of 3-FC on TNF-␣induced IKK and TAK1 activity, we used the kinase assay as described previously (17). In brief, the IKK-TAK1 complex from whole-cell extracts was precipitated with antibody against IKK␤ and TAK1, respectively. The complex was then treated with protein A/G-agarose beads (Pierce). After 2 h, the beads were washed with whole-cell extraction buffer and then resuspended in kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl 2 , 2 mM dithiothreitol, 20 Ci of [␥-32 P]ATP, 10 M unlabeled ATP, and 2 g of substrate (GST-IB␣ for IKK and His-MKK6 for TAK1). 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 by 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized using a Storm 820 imaging system.
ChIP Assay-The effect of 3-FC on binding of NF-B to the COX-2 promoter was analyzed by ChIP assay (Millipore, Temecula, CA) following the manufacturer's instructions. In brief, KBM-5 cells were incubated for 10 min with 1% formaldehyde, harvested, centrifuged, and resuspended in lysis buffer. After lysis and sonication, the lysate was collected by centrifugation and mixed with ChIP dilution buffer. The protein-DNA complex was immunoprecipitated using anti-p65 antibody. The complex was then washed, incubated with 5 M NaCl, and heated at 65°C for 4 h in the presence of proteinase K to reverse the cross-links of the protein-DNA complex. The DNA was recovered by extraction with phenol/chloroform and precipitation with ethanol. The DNA was then amplified by PCR using human COX-2 promoter primer sequences (5Ј-AAAGA-CATCTGGCGGAAACCT-3Ј (forward) and 5Ј-AGGAAGCT-GCCCCAATTTG-3Ј (reverse)). These primers correspond to sequences Ϫ434/Ϫ414 and Ϫ319/Ϫ337 of the human COX-2 promoter.
Apoptosis Assay-To measure apoptosis, we used the LIVE/ DEAD assay kit (Invitrogen). We stained the cells according to the manufacturer's instructions. In this assay, calcein acetoxymethyl ester, a non-fluorescent 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. Live and dead cells were counted under the Labophot-2 fluorescence microscope.
An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phosphatidylserine (PS) from the cytoplasmic interface of the membrane to the extracellular surface. We examined PS externalization using FITC-conjugated annexin V. Briefly, 5 ϫ 10 5 cells were pretreated with 3-FC (50 M) for 12 h, treated with TNF-␣ for 24 h, and analyzed for annexin V-positive cells following the manufacturer's instructions (Santa Cruz Biotechnology).
We also analyzed sub-G 1 fraction of the cell cycle using propidium iodide staining. This method is based on the fact that cells undergoing apoptosis will lose part of their DNA (due to DNA fragmentation), and those cells may be detected as a sub-G 1 population after propidium iodide staining.
Cell Proliferation Assay-We used a clonogenic assay to measure tumor growth because it produces results that are considered closer to the in vivo situation. Treated and untreated cells were seeded in 100-mm Petri dishes, allowed to form colonies for 14 days, and then stained as described previously (19).
Western Blot Analysis-Cytoplasmic, nuclear, and whole-cell extracts of untreated and treated cells were used in Western blot analysis. The protein extracts were resolved by SDS-PAGE. After electrophoresis, proteins were electrotransferred to nitrocellulose membranes, blotted with the relevant antibody, and detected using an enhanced chemiluminescence reagent (GE Healthcare).
Enzyme-linked Immunosorbent Assay-Human ELISA system kits (eBioscience, San Diego, CA) were used for the detection of IL-6 and TNF-␣. The U266 cells were treated with different concentrations of 3-FC, and cell-free supernatants were collected after 12 h. The cytokine level was determined following the manufacturer's protocol.
3-FC Molecular Docking with p65-DNA Complex-The crystal structure of the p65 domain was retrieved from the Protein Data Bank (code 1VKX). The docking program Glide (20,21) was used to study the interactions of 3-FC with p65. The protein was prepared using the default parameters of the protein preparation workflow in the Schrödinger suite. The docking grid was centered around Arg 33 , Arg 35 , Tyr 36 , Glu 39 , and Arg 187 , as they were deemed critical for ligand and DNA binding (22).
Glide docking software in the standard precision mode was used to generate a set of receptor-ligand complexes. The top scoring complex was selected as the starting point to generate an ensemble of p65-3-FC complexes to simulate induced fit effects upon ligand binding. The exact modeling protocol was also applied for the generation of C38S mutant protein. The simulations were performed with our in-house tool ORELI (Optimized Receptor Ensembles using Ligand Information), 4 which was developed based on the Rosetta suite of programs (23,24). Briefly, an ensemble of 100 complexes was generated with a Monte-Carlo minimization method. For every iteration, the ligand 3-FC was randomly translated and rotated (1.0 Å and 1.5°) from the initial starting position, and the receptor residues within 6.5 Å of the ligand were optimized using low energy side chain rotamers. The resulting complex was scored using the all-atom Rosetta ligand scoring function and retained/rejected based on the Metropolis criterion. On the basis of our modeling and experimental data, we selected five top-ranked complexes. 3-FC was then redocked using Glide in the extra precision mode (20,21) into the optimized binding sites, and the best scored pose was retained.
Statistical Analysis-Different parameters were monitored in normal and treated cells. Experiments were repeated a minimum of three times. Data are given as the mean Ϯ S.D. Statistical analysis was carried out using Student's two-tailed unpaired t test. A value of p Ͻ 0.05 was considered statistically significant.

3-FC Inhibits NF-B Activation Induced by Carcinogens and
Inflammatory Agents-Numerous agents, including tumor promoters (e.g. okadaic acid and phorbol 12-myristate 13-acetate) and inflammatory agents (such as TNF-␣ and LPS), are known to activate NF-B. Various studies show that the mechanisms by which these agents activate NF-B differ significantly (25). When we investigated whether 3-FC affects NF-B activation induced by these agents, we found that all of them activated NF-B in human myeloid KBM-5 cells and that pretreatment with 3-FC suppressed the activation (Fig. 1B), suggesting that 3-FC acts at a step in the NF-B activation pathway that is common to all of these agents.

3-FC Suppresses TNF-␣-induced NF-B Activation in Doseand Time-dependent
Manner-We first examined the dose and time required to suppress TNF-␣-induced NF-B activation by 3-FC. EMSA results revealed that 3-FC inhibited TNF-␣-mediated NF-B activation in a dose-dependent (Fig. 1C, left panel) and time-dependent (right panel) manner.
3-FC Inhibits Constitutive NF-B Expression-Several tumor cell types are known to constitutively express NF-B through a mechanism yet to be fully defined (13). Human multiple myeloma RPMI-8226 and U266 cells in particular are known to have constitutively active NF-B. To determine whether 3-FC affects NF-B expression in these cells, we exposed multiple myeloma cells to 3-FC and then analyzed the nuclear extracts using EMSA. As shown in Fig. 1E, 3-FC completely suppressed constitutive NF-B activation in these cells, indicating that 3-FC can suppress both inducible and constitutive NF-B activation.

3-FC Does Not Inhibit Oct-1 Activation in Tumor Cells-Whether 3-FC inhibits activation of Oct-1 under the conditions it suppresses NF-B was investigated.
The results showed that 3-FC had no affect on constitutive Oct-1 (Fig. 1F).
3-FC Inhibits IB␣ Degradation and Phosphorylation-The translocation of NF-B to the nucleus is preceded by the phosphorylation and proteolytic degradation of IB␣. To determine whether the inhibition of TNF-␣-induced NF-B activation is due to the inhibition of IB␣ degradation, we pretreated cells with 3-FC and then exposed the cells to TNF-␣ for various times. We examined the cells for NF-B activation in the nucleus by EMSA and for IB␣ degradation in the cytoplasm by Western blot analysis. EMSA revealed that although TNF-␣ activated NF-B in control cells in a time-dependent manner, the cytokine had no effect on cells pretreated with 3-FC ( Fig.  2A). Western blot analysis revealed that although TNF-␣ induced IB␣ degradation in the control cells within 15 min, TNF-␣ had no effect on IB␣ degradation in 3-FC-treated cells (Fig. 2B). These results indicate that 3-FC inhibited both TNF-␣-induced NF-B activation and IB␣ degradation.
To determine whether the inhibition of TNF-␣-induced IB␣ degradation is due to the inhibition of IB␣ phosphorylation, we blocked IB␣ degradation with the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal. Western blotting with an antibody that recognizes the serine-phosphorylated (Ser 32 / Ser 36 ) form of IB␣ revealed that 3-FC strongly suppressed TNF-␣-induced IB␣ phosphorylation (Fig. 2C).
3-FC Inhibits p65 Translocation into Nucleus-We then examined whether 3-FC affects TNF-␣-induced nuclear translocation. Western blot analysis showed that TNF-␣ induced nuclear translocation of p65 in a time-dependent manner in KBM-5 cells (Fig. 2D, left panel). When the cells were pretreated with 3-FC, TNF-␣ failed to induce nuclear translocation of p65.
TNF-␣ induces the phosphorylation of p65 at Ser 536 , which is required for its transcriptional activity (11). Western blot analysis showed that TNF-␣ induced the phosphorylation of p65 and that 3-FC strongly suppressed it (Fig. 2D, left panel).
To confirm the results observed by Western blot analysis, we performed immunocytochemistry to localize p65 inside the cells. The results indicated that in untreated or 3-FC-treated KBM-5 cells, p65 was localized in the cytoplasm. Treatment with TNF-␣ induced p65 nuclear translocation, and 3-FC pretreatment suppressed TNF-␣-induced nuclear translocation (Fig. 2D, right panel).
3-FC Inhibits TNF-␣-induced IKK Activation-IKK is required for TNF-␣-induced IB␣ phosphorylation and p65 phosphorylation (26,27). Because 3-FC inhibited the phosphorylation of IB␣ and p65, we determined the effect of 3-FC on TNF-␣-induced IKK activation in KBM-5 cells. An immune complex kinase assay showed that TNF-␣ activated IKK as early as 2 min after TNF-␣ treatment and that treatment with 3-FC strongly suppressed this activation (Fig. 2E). Neither TNF-␣ nor 3-FC affected the expression of the IKK␣ or IKK␤ protein.
3-FC Directly Inhibits IKK Activation-To determine whether 3-FC suppresses IKK activity directly or indirectly by suppressing its activation, we incubated the immune complexes with 3-FC at various concentrations and then examined the kinase activity. The results showed that 3-FC directly inhibited the activity of IKK in a dose-dependent manner (Fig. 2F). This indicated that 3-FC can directly modulate TNF-␣-induced IKK activation.
Because the IKK␤ subunit of the IKK complex contains various cysteine residues, we hypothesized that 3-FC may inhibit IKK through direct modification of one or more of these cysteine residues. We used a reducing agent, DTT, to determine whether the modulation of IKK activity by 3-FC is caused by the oxidation of critical cysteine residues. The addition of DTT to the kinase reaction mixture reversed the 3-FC-mediated inhibition of TNF-␣-induced IKK activity (Fig. 2G), suggesting that a cysteine residue is involved in the pathway.
IKK␤ contains a cysteine at position 179 in its activation loop that is critical for its activity (28). To determine whether this residue is involved in 3-FC-mediated IKK inhibition, we transfected A293 cells with wild-type FLAG-IKK␤ or FLAG-IKK␤ with a C179A mutation. 3-FC inhibited wild-type IKK␤. In contrast, 3-FC had no apparent effect on the activity of the IKK␤ mutant (Fig. 2H). These findings suggest that 3-FC inhibits IKK␤ activity by modifying Cys 179 . (29), we sought to determine whether the inhibitory effect of 3-FC on NF-B is mediated through TAK1. The results of the TAK1 kinase assay revealed that 3-FC suppressed TNF-␣-induced TAK1 activation (Fig. 2I).

3-FC Directly Blocks Binding of NF-B p65
Subunit to DNA-Previous studies have shown that certain agents suppress NF-B activation by directly blocking the binding of NF-B to DNA (30,31). We determined whether 3-FC mediates suppression of NF-B activation through a similar mechanism using nuclear extracts from TNF-␣-treated cells. The nuclear extracts were incubated with 3-FC for different times and then assayed for NF-B binding to the DNA. EMSA showed that the chromone inhibited NF-B binding to the DNA in a time-dependent manner (Fig. 3A).
It is possible that 3-FC inhibits the binding of NF-B to the DNA through modification of NF-B proteins (31). We found that co-incubation of nuclear extracts with 3-FC in the presence of DTT reversed the effect of 3-FC completely (Fig. 3B). It has been shown that the p65 subunit of NF-B has a sensitive cysteine that is highly reactive (22). Thus, we transfected p65 Ϫ/Ϫ cells with the p65 plasmid and examined whether DTT can reverse the inhibitory effect of 3-FC on p65-DNA binding. Fig. 3C shows that 3-FC inhibited p65 binding to the DNA and that DTT reversed the effect.
It has been shown that Cys 38 in p65 is highly susceptible to various agents (15,22). To determine whether Cys 38 is a target for 3-FC, we transfected p65 Ϫ/Ϫ cells with a wild-type or mutant (C38S) p65 plasmid. EMSA results showed that 3-FC inhibited the DNA binding of wild-type p65 but not mutant p65 (Fig. 3D). These results demonstrate that Cys 38 in p65 is a target of 3-FC.

3-FC Suppresses Expression of NF-B-regulated Tumor Cell Survival and Proliferative, Invasive, and Angiogenic Gene
Products-Because 3-FC inhibits NF-B activation, we examined whether 3-FC can modulate the expression of NF-B-regulated gene products. We found that TNF-␣ induced expression of cell survival proteins (Bcl-2, Bcl-x L , survivin, and cIAP-1) in a time-dependent manner, whereas pretreatment with 3-FC down-regulated the expression (Fig. 5A).
We also examined the effects of 3-FC on NF-B-regulated cell proliferative proteins. The results indicated that 3-FC inhibited the expression of cyclin D 1 and COX-2 induced by TNF-␣ (Fig. 5A). The results also indicated that TNF-␣ induced the expression of invasive (MMP-9 and ICAM-1) and angiogenic (VEGF) proteins in a time-dependent manner, and 3-FC was found to suppress the expression of these proteins (Fig. 5A).
3-FC Inhibits TNF-␣-induced COX-2 Promoter Activity-TNF-␣ induces the expression of the COX-2 gene, which has NF-B-binding sites in its promoter region (34). Because 3-FC suppressed the expression of COX-2, we examined the effect of 3-FC on TNF-␣-induced COX-2 promoter activity. A293 cells were transfected with a plasmid containing full-length COX-2 promoters. The results indicated that TNF-␣ induced COX-2 promoter activity, whereas 3-FC suppressed the activity in a dose-dependent manner (Fig. 5B). These results suggest that the suppression of COX-2 promoter activity by 3-FC may be due to its inhibitory effects on NF-B activity.
3-FC Inhibits Binding of p65 to COX-2 Promoter-Whether the inhibitory effects of 3-FC on COX-2 promoter activity are due to the inability of p65 to bind to COX-2 was examined. A293 cells were transfected with full-length or mutant COX-2 promoter (pGL2COX-2-luc(Ϫ449/Ϫ225)(B1/B2)) plasmids, and COX-2 reporter activity was examined. The results indicated that TNF-␣ was not able to induce COX-2 reporter activity and that 3-FC did not affect the activity in cells transfected with the mutant COX-2 promoter plasmid (Fig. 5C). These results suggested that 3-FC inhibited COX-2 expression by suppressing NF-B binding to the COX-2 promoter.
To further confirm that the decrease in COX-2 promoter activity by 3-FC is due to the reduction in the binding of p65 to the COX-2 promoter, we performed ChIP assay. The results indicated that TNF-␣ enhanced binding of p65 to COX-2, whereas pretreatment with 3-FC decreased the binding (Fig.  5D).
3-FC Down-regulates Proinflammatory Cytokine Production in Multiple Myeloma Cells-We investigated whether 3-FC has an effect on the production of IL-6 and TNF-␣ in U266 cells  p65 mt (C38S)). The p65 Ϫ/Ϫ cells were transfected with wild-type and mutant p65 plasmids. Nuclear extracts of transfected cells were incubated with 3-FC for 30 min and then assayed for DNA binding by EMSA.

3-FC Interacts with Cys 38 in p65 and Cys 179 in IB␣ Kinase
that are regulated by NF-B. The production of IL-6 and TNF-␣ in U266 cells was suppressed by 3-FC in a concentration-dependent manner (Fig. 5E).
To confirm the results of esterase activity, the sub-G 1 fraction, an indicator of apoptosis, was analyzed. The results indicated that apoptosis was induced at 23.2% by TNF-␣, at 11.2% by 3-FC, and at 39.6% by TNF-␣ plus 3-FC (Fig. 6B).
One of the early events of apoptosis is externalization of the membrane PS on the cell surface. Because of the affinity of annexin V for PS, annexin V staining can be used to detect early apoptotic cells. The effect of 3-FC on PS externalization induced by TNF-␣ was investigated. The number of annexin V-positive cells was significantly increased when cells were pretreated with 3-FC before TNF-␣ treatment (Fig. 6C).
Whether 3-FC enhances the TNF-␣-induced activation of caspase-3, caspase-8, and caspase-9 and cleavage of PARP was investigated. We found that TNF-␣ alone had little effect on PARP cleavage and caspase activation. However, pretreatment of the cells with 3-FC increased caspase activation and PARP cleavage (Fig. 6D). We also found that 3-FC treatment completely suppressed the colony-forming ability of tumor cells (Fig. 6E).
3-FC Docking Studies-Finally, we employed molecular docking studies to confirm the experimental observations of 3-FC binding to p65. 3-FC was docked just below the L1 DNAbinding loop consisting of Arg 33 , Cys 38 , and Arg 41 . Upon optimization with ORELI, the above loop region was moved upwards slightly compared with the crystal structure (Fig. 7). The distance between the Cys 38 and Cys 120 changed from 7.7 Å in the crystal structure to 10.2 Å in the model. This altered the 1 angle of Tyr 36 from Ϫ66.3°(in the crystal structure) to Ϫ67.5°such that the phenol moiety in the residue is oriented toward the DNA-binding region (Fig. 7). We believe that this conformational change presents steric hindrance to DNA, thus preventing its binding to p65. However, for the C38S mutant protein, the loop region seems to maintain a conformation similar to the crystal structure of p65 (Fig. 7), maintaining p65-DNA binding. These modeling results are in agreement with experimental observations.

DISCUSSION
This is the first report to suggest that a chromone can suppress the NF-B activation pathway and expression of NF-Bregulated gene products. In this study, 3-FC inhibited constitutive NF-B activation, a critical element in the survival and proliferation of various tumor cell types (35). The inhibition of NF-B activation by 3-FC was specific, as it failed to inhibit Oct-1 activation. We found that 3-FC acted at two different steps in the NF-B signaling pathway. First, it interacted directly with the p65 subunit of NF-B, and second, it suppressed TNF-␣-induced IKK activation.
We found that 3-FC could inhibit the binding of reconstituted p65 to the DNA in vitro, which suggests that p65 is the direct target of 3-FC. These results are consistent with findings that 3-FC also suppressed the p65-induced NF-B reporter activity. The reversal of the effects of 3-FC by a reducing agent suggests that a cysteine residue in p65 is modified by this agent. These results are consistent with those reported previously from our laboratory with caffeic acid phenethyl ester (31) and with those reported by another laboratory with sesquiterpene lactone parthenolide (22). A cysteine residue (Cys 38 ) has been identified in p65 subunits of NF-B that is crucial for DNA binding (22). The results showed that when Cys 38 was replaced with serine in p65, 3-FC failed to inhibit the DNA-binding ability of p65. This result suggests that 3-FC modifies Cys 38 , leading to NF-B suppression. Whole-cell extracts were prepared and analyzed by Western blot analysis using the indicated antibodies. The results shown are representative of three independent experiments. B, 3-FC inhibits the COX-2 promoter activity induced by TNF-␣. Cells were transiently transfected with a COX-2 promoter linked to the luciferase reporter gene plasmid for 24 h and then treated with the indicated concentrations of 3-FC for 12 h. Cells were treated with 1 nM TNF-␣ for an additional 24 h, lysed, and subjected to a luciferase assay. Variations in transfection efficiency were normalized by measuring ␤-galactosidase activity. The luciferase activity was estimated as luciferase count/␤-galactosidase count. The values are the mean Ϯ S.D. for three independent replicates. * and #, significance of difference compared with the control and TNF-␣-alone groups, respectively (p Ͻ 0.05). C, COX-2 promoter with mutant NF-B-binding elements is resistant to 3-FC treatment. A293 cells were transfected with a luciferase expression construct ligated to the full-length (white bars) or mutant (black bars) COX-2 promoter. Cells were treated with 3-FC for 12 h, followed by TNF-␣ for an additional 24 h, and then lysed and subjected to a luciferase assay. The values are the mean Ϯ S.D. for three independent replicates. * and #, significance of difference compared with the control and TNF-␣-alone groups, respectively (p Ͻ 0.05). D, effect of 3-FC on binding of NF-B to the COX-2 promoter. Cells were treated with 100 M 3-FC for 12 h, followed by 1 nM TNF-␣ for the indicated times, and the proteins were cross-linked with DNA by formaldehyde and subjected to ChIP assay using anti-p65 antibody and the COX-2 primer. Reaction products were resolved by electrophoresis. IP, immunoprecipitate. E, 3-FC downregulates IL-6 and TNF-␣ production in U266 cells. Cells were treated with the indicated concentrations of 3-FC, and cell free supernatants were harvested after 12 h. The levels of IL-6 and TNF-␣ were detected by ELISA. The values are the mean Ϯ S.D. for three independent replicates. *, significance of difference compared with the control (p Ͻ 0.05).
We found that 3-FC targeted IKK to suppress the TNF-␣induced phosphorylation and degradation of IB␣ that were concomitant with the inhibition of nuclear translocation and phosphorylation of p65. We showed that 3-FC directly inhibited TNF-␣-activated IKK. Furthermore, the addition of a reducing agent reversed the effects of 3-FC on IKK activation, suggesting the involvement of cysteine residues. The mutation of Cys 179 of IKK␤ to alanine abolished the inhibitory effect of 3-FC on IKK activation. In addition to direct effects of 3-FC on IKK, inhibition of TAK1 may also be responsible for the inhibition of IKK by 3-FC. However, how this chromone inhibits TAK1 remains to be elucidated.
There are two possibilities by which this chromone can modify cysteine residues in p65 and IKK: 1) by redox cycling and 2) by direct interaction. Redox cycling results in the generation of reactive oxygen species and depletes cellular glutathione levels. A previous study demonstrated that the depletion of cellular GSH by diethyl maleate prevented NF-B induction in rat hepatocytes (36). Because 3-FC is a free radical scavenger (37), it is very unlikely that the effects of 3-FC are mediated through reactive oxygen species generation. It is also unlikely that reactive oxygen species are produced under the in vitro conditions used for the modification of IKK and p65 by 3-FC. This eliminates the first possibility. From molecular docking studies, we found that, in the presence 3-FC, Cys 38 underwent alkylation and that the tyrosine molecule at position 36 moved from its position, which in turn inhibited p65 binding to DNA. However, the DNA binding of p65 in which

3-FC Interacts with Cys 38 in p65 and Cys 179 in IB␣ Kinase
Cys 38 had been mutated was not affected. These results suggest that 3-FC interacts with the cysteine residue directly. A similar mechanism has been reported for cyclopentenone prostaglandins (38), arsenite (28), butein (16), nitric oxide (39), nimbolide (40), and bharangin (41). However, other sophisticated techniques, such as mass spectrometry, circular dichroism, x-ray crystallography, and NMR spectroscopy, are needed to provide more conclusive information on such binding interactions.
We found that 3-FC inhibited NF-B activation induced by inflammatory stimuli (TNF-␣ and LPS) and tumor promoters (phorbol ester and okadaic acid), suggesting that 3-FC must act at a step common to all these activators. The IKK complex is critical for the NF-B activation by all these inducers (42). It is likely that the inhibition of IKK activity is the common step for the inhibition of NF-B by 3-FC.
We found that 3-FC down-regulated the expression of NF-B-regulated gene products, such as survivin, Bcl-x L , Bcl-2, and cIAP-1, all of which are known to suppress apoptosis. We also found that 3-FC inhibited the expression of cyclin D 1 and COX-2, involved in cell proliferation. The potentiation of TNF-␣-induced apoptosis by 3-FC could be accounted for by the inhibition of anti-apoptotic proteins as described herein. Beside these, 3-FC abrogated TNF-␣-induced proteins involved in invasion (ICAM-1 and MMP-9) and angiogenesis (e.g. VEGF). The down-regulation of the expression of ICAM-1 and VEGF by 3-FC suggests that this chromone may have a role in the inhibition of invasion and angiogenesis of tumor cells.
Overall, in this study, we have demonstrated that 3-FC is a potent inhibitor of NF-B activation, which may explain its anti-inflammatory and anticancer effects. Further studies using animal models are needed to explore its therapeutic potential against cancer and other diseases.