Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of Proliferation, Invasion, and Angiogenic Proteins Linked to Proinflammatory Pathway*

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor κB (NF-κB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-κB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-κB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,2 MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-κB activation. Crotepoxide inhibition of NF-κB was not inducer-specific; it inhibited NF-κB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-κB was not cell type-specific because NF-κB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IκBα kinase, abrogation of IκBα phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-κB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-κB and NF-κB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.

Several chemotherapeutic, cytotoxic, and immunomodulating agents are commonly used to treat cancer. However, most modern medicines tend to target only one gene product or pathway at a given time. This is perhaps one of the major reasons that some of the recently discovered medicines are less effective. Besides being prohibitively expensive, many of these drugs are also associated with serious side effects and morbidity. Still, the search continues for an ideal treatment that has minimal side effects and is cost-effective. Therefore, traditional medicine, usually derived from plants is one of the alternatives to treat the chronic diseases including cancer. Although plantderived products have been used for centuries to treat various ailments, their active components and mechanisms are not fully understood. Identifying the active chemical entities and their molecular targets would facilitate the discovery of new clinical uses for such products. Crotepoxide (Fig. 1A), a highly substituted cyclohexane diepoxide linked with anticancer activity, was first isolated from the fruit of Croton macrostachys (1,2). More recently, crotepoxide was identified as a main component in Kaempferia rotunda (3), a member of the Zingiberaceae, or ginger family whose tuber has traditionally been used to treat pneumonia, bronchitis, abdominal pain, dysentery, diarrhea, cold, and obesity (4,5). Crotepoxide was also identified in various other medicinal plants including Piper kadsura (6 -8), Monanthotaxis caffra (9), Friesodielsia obovata (Annonaceae) (10), Kaempferia angustifolia (11), and Kaempferia pulchra (peacock ginger) (12). Although crotepoxide has been reported to have tumorinhibiting (2,13) and anti-inflammatory properties (6), the exact mechanisms through which crotepoxide exhibits these properties are not understood.
Because NF-B is known to regulate inflammation and tumorigenesis, we hypothesized that the anti-inflammatory and anticancer effects ascribed to crotepoxide may be due to its inhibition of NF-B and NF-B-regulated gene expression. Indeed we demonstrate that this crotepoxide can block NF-B pathway and potentiate the anticancer effects of various chemotherapeutic drugs.

Cell Lines-Chronic
Electrophoretic Mobility Shift Assay (EMSA)-To determine the effect of crotepoxide on TNF-activated NF-B, we performed NF-B-DNA binding using EMSA as previously described (21). Briefly, nuclear extracts prepared from treated cells (1 ϫ 10 6 cells/ ml) were incubated with 32 P-end-labeled, 45-mer, doublestranded NF-B oligonucleotide (15 g of protein with 16 fmol of DNA) from a human immunodeficiency virus long terminal repeat, 5Ј-TTGTTACAAGGGACTTTCCGCTGGGGACTTT-CCAGGGAGGCGTGG-3Ј (boldface and underlines indicate NF-B binding sites) for 30 min at 37°C. The resultant DNAprotein complex was separated from free oligonucleotides on 6.6% native polyacrylamide gels. We used a double-stranded mutated oligonucleotide, 5Ј-TGTTACAACTCACTTTCCG-CTGCTCACTTTCCAGGGAGGCGTGG-3Ј, to examine the specificity of NF-B-DNA binding. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, we incubated nuclear extracts prepared from TNF-treated cells with antibodies against the p50 or p65 subunits of NF-B for 30 min at 37°C before using EMSA to analyze the complex. Preimmune serum was included as a negative control. The dried gels were visualized with a Storm 820 PhosphorImager, and radioactive bands were quantitated using ImageQuant software (Amersham Biosciences).
Western Blot Analysis-To determine the effect of crotepoxide on TNF-dependent IB␣ phosphorylation, IB␣ degradation, p65 translocation, and p65 phosphorylation, we prepared cytoplasmic or nuclear extracts as described previously (22). To detect the cleavage products of anti-poly(ADP-ribose) polymerase and any antiapoptotic and angiogenesis markers, we prepared whole-cell extracts by subjecting crotepoxidetreated cells to lyses in lysis buffer containing Tris (pH 7.4; 20 mM), sodium chloride (250 mM), EDTA (pH 8.0; 2 mM), Triton X-100 (0.1%), aprotinin (0.01 g/ml), leupeptin (0.005 g/ml), phenylmethylsulfonyl fluoride (0.4 mM), and sodium orthovanadate (4 mM). Lysates were centrifuged at 20,817 ϫ g for 10 min to remove insoluble material. Supernatant was collected and kept at Ϫ80°C. Whole-cell lysates and cytosolic or nuclear extracts were resolved by SDS-PAGE. After electrophoresis, proteins were electrotransferred to nitrocellulose membranes, blotted with the relevant antibody, and detected by an enhanced chemiluminescence reagent (Amersham Biosciences ECL). The bands obtained were quantitated using NIH Image (National Institutes of Health).
Immunocytochemistry for p65 Localization-To examine the effect of crotepoxide on the nuclear translocation of p65, we performed immunocytochemical analysis as previously described (24). Briefly, cells were plated on a poly-L-lysine-coated glass slide with a Cytospin 4 centrifuge (ThermoShendon, Pittsburgh, PA), air dried, and fixed with 4% paraformaldehyde. The slides were washed in PBS, blocked with 5% normal goat serum for 1 h, and then incubated with rabbit polyclonal p65 antibody at a 1:200 dilution overnight at 4°C. The slides were then washed, incubated with goat anti-rabbit IgG-Alexa Fluor 594 (Invitrogen) at a 1:200 dilution for 1 h and counterstained for nuclei with Hoechst 33342 (50 ng/ml) stain for 5 min. Stained slides were mounted with mounting medium purchased from Sigma and analyzed under a Labophot-2 fluorescence microscope (Nikon, Melville, NY). Photographs were taken using a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph software (Version 4.6.5, Universal Imaging, Sunnyvale, CA).
Immune Kinase Complex Assay for TGF-␤-activated Kinase 1 (TAK1)-To determine the effect of crotepoxide on TNF-induced IB kinase (IKK) and TAK1 activation, we analyzed IKK and TAK1 as previously described (25). Briefly, the IKK and TAK1 complex from whole-cell extracts was precipitated with antibodies against IKK-␣ or TAK1 and then treated with protein A/G-Sepharose beads (Pierce). After 2 h, the beads were washed with lysis buffer and then resuspended in a kinase assay mixture containing HEPES (pH 7.4; 50 mmol/liter), magnesium chloride (20 mmol/liter), dithiothreitol (2 mmol/liter), [␥-32 P]adenosine triphosphate (ATP; 20 Ci), unlabeled ATP (10 mol/liter), and substrate glutathione S-transferase-IB␣ (amino acids 1-54; 2 g) or His-MKK6. After incubation at 30°C for 30 min, we terminated the reaction 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 using a Storm 820 PhosphorImager. To determine the total amounts of IKK-␣ and IKK-␤ or TAK1 in each sample, we resolved whole-cell proteins (40 g) on 7.5% SDS-PAGE, electrotransferred the proteins to a nitrocellulose membrane, and then blotted the proteins with either anti-IKK-␣/ anti-IKK-␤ or anti-TAK1 antibodies.
Live/Dead Assay-To assess cytotoxicity, we used the live/ dead assay (Invitrogen), which determines intracellular esterase activity and plasma membrane integrity. Briefly, 2 ϫ 10 5 cells were incubated with crotepoxide (50 mol/liter) for 2 h and then treated with TNF (1 nmol/liter) for 16 h at 37°C. Cells were stained with the live and dead reagents (ethidium homodimer (5 mol/liter) and calcein acetoxymethyl ester (5 mol/liter)) and incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon).
NF-B-dependent Reporter Gene Expression Assay-To determine the effect of crotepoxide on TNF-, TNFR-, TNFRassociated death domain (TRADD)-, TRAF2-, NF-B-inducing kinase (NIK)-, TAK-1/TAK1-binding protein-1 (TAB1)-, and IKK-NF-B-dependent reporter gene transcription, we performed a secretory alkaline phosphatase (SEAP) assay as previ-ously described (26) 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 TNF-induced reporter gene expression, we transfected the cells with SEAP expression plasmid (0.5 g) and control plasmid pCMV-FLAG1 (1.5 g) DNA for 24 h. We then treated the cells with crotepoxide for 2 h and stimulated them with TNF (0.1 nM). 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 pNF-B-SEAP plasmid (0.5 g) with expressing plasmid (0.5 g) and pCMV-FLAG1 control plasmid (1.5 g) for 24 h, treated with crotepoxide, 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 Laboratories, Mountain View, CA) with a Victor 3 microplate reader (PerkinElmer Life and Analytical Sciences, Boston, MA).
Annexin V Assay-An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface of the membrane to the extracellular surface. This loss of membrane asymmetry can be detected using the binding properties of annexin V. To identify apoptosis, we used an annexin V antibody conjugated with fluorescein isothiocyanate fluorescence dye. Briefly, 5 ϫ 10 5 cells were pretreated with crotepoxide (50 M), treated with TNF for 24 h at 37°C, and subjected to annexin V staining. The cells were washed in PBS, resuspended in 100 l of binding buffer containing a fluorescein isothiocyanate-conjugated anti-annexin V antibody, and then analyzed with a FACSCalibur flow cytometer (BD Biosciences).

RESULTS
This study was undertaken to investigate the anti-inflammatory and antitumor effects of crotepoxide. For this we first investigated the effect of crotepoxide alone on the survival of various tumor cells, and then we examined in combination with chemotherapeutic agents. We also determined its effects on the NF-B activation pathway as induced by various carcinogens and inflammatory stimuli as well as expression of NF-B-regulated gene products and apoptosis in leukemic cells. The duration of exposure and concentration of crotepoxide used to examine its effect on NF-B pathway had a minimal effect on the viability of these cells as determined by trypan blue dye exclusion test (data not shown).
Crotepoxide Inhibits Proliferation and Potentiates TNF-and Chemotherapeutic Agent-induced Apoptosis-The results of MTT assay showed that crotepoxide inhibited the proliferation of leukemic cells, KBM-5, MM1, and U266 cells in a dose-and time-dependent manner (Fig. 1B). Whether this compound can enhance the apoptosis induced by apoptotic cytokine TNF, was investigated by an annexin V staining method. We found that crotepoxide up-regulated TNF-induced apoptosis from 9 to 28% (Fig. 1C, left panels). However, crotepoxide alone showed only 9% cell death. To confirm the result of annexin V assay, we used esterase staining (i.e. the live/dead assay) and found that crotepoxide increased TNF-induced apoptosis from 16 to 40% (Fig. 1C, middle panels). When we examined crotepoxide for caspase activation by Western blotting, a classical hallmark of apoptosis, we found activation of caspase-9 and caspase-3 and subsequent anti-poly(ADP-ribose) polymerase cleavage, indi-cating that crotepoxide potentiates the TNF-induced caspase activation (Fig. 1C, right panels). Crotepoxide alone induced apoptosis, albeit at a low extent, and the mechanism of this effect is not known at this point.
Next, we investigated the effect of crotepoxide on apoptosis induced by chemotherapeutic drugs. Results of MTT uptake showed that crotepoxide up-regulates cell death induced by 5-flurouracil, cisplatin, thalidomide, and velacade (Fig. 1D, left panel). This result was further confirmed by live dead assay. According to this assay, crotepoxide potentiated apoptosis induced by 5-fluorouracil (from 21 to 44%), cisplatin (from 23 to 43%), thalidomide (from 18 to 56%), and velacade (from 28 to 65%) (Fig. 1D, right panel). These results together indicate that crotepoxide potentiates the apoptotic effects of TNF and chemotherapeutic agents.
Crotepoxide Inhibits Expression of Cell Proliferative Gene Products-Because crotepoxide inhibits the proliferation of various tumor cell lines, we investigated whether this effect is mediated through the regulation of NF-B-mediated expression of cell proliferation gene products such as c-myc (27) and cyclin D1 (28). Our results revealed that crotepoxide inhibited cyclin D1 and c-myc expression in a dose-dependent fashion induced by TNF. Under these conditions, crotepoxide alone showed minimal cytotoxicity ( Fig. 2A).
Crotepoxide Suppresses the Expression of Proinflammatory Gene Products-Because crotepoxide exhibits anti-inflammatory activity (6), we investigated whether this effect is mediated through the regulation of NF-B mediated expression of COX2. Results revealed that crotepoxide blocked TNF-induced expression of this proinflammatory protein in a time-dependent manner. Under these conditions crotepoxide alone showed minimal cytotoxicity ( Fig. 2A).
Crotepoxide Modulates Expression of Pro-apoptotic Gene Products-Next, we determined whether crotepoxide regulates the expression of proapoptotic and tumor suppressor gene products. We found that crotepoxide induced the expression of Bax and cleavage of Bid, which was inhibited by TNF (Fig. 2B).
Crotepoxide Suppresses the Expression of Tumor Cell Survival Gene Products-Because crotepoxide enhances TNF-and chemotherapy-induced apoptosis, we investigated whether this effect is mediated through the regulation of NF-B-mediated expression of the antiapoptotic proteins c-IAP1 (29), Bcl-2 (30), Bcl-xL (31), mcl-1, TRAF-1 (32), and survivin. We found that crotepoxide blocked the TNF-induced expression of these tumor cell survival proteins in a time-dependent manner (Fig. 2C).
Crotepoxide Suppresses the Expression of TNF-induced Metastatic Gene Products-TNF has been shown to induce the expression of ICAM-1 (33), VEGF (34), and MMP-9 (35), all of which have NF-B-binding sites in their promoters. We investigated whether crotepoxide can modulate NF-B-regulated gene products involved in metastasis and found that crotepoxide abolished TNF-induced expression of ICAM-1, MMP-9, and VEGF (Fig. 2D).  Crotepoxide Suppresses TNF-induced NF-B Activation-Because NF-B regulates various cellular responses including proliferation, apoptosis, inflammation, and chemosensitization, all regulated by NF-B, we reasoned that crotepoxide must modulate the NF-B cell-signaling pathway. Therefore, we investigated whether crotepoxide inhibits NF-B activation. The experimental condition that was used to study the mechanism of NF-B inhibition involved short duration of exposure, and under this condition crotepoxide alone did not show any cell death. EMSA revealed that although crotepoxide alone had no effect on NF-B activation, but crotepoxide inhibited TNFmediated NF-B activation in a dose (Fig. 3A, left panel)and time-dependent manner (Fig. 3A, right panel).
NF-B is a complex of proteins in which various combinations of Rel or NF-B proteins constitute active NF-B heterodimers that bind specific DNA sequences. To confirm that the band visualized by EMSA in TNF-treated cells was NF-B, we incubated nuclear extracts from TNF-activated cells with antibodies to the p50 (NF-B) and p65 (RelA) subunits of NF-B. The resulting bands that were shifted to higher molecular masses (Fig. 3B) suggested that the TNF-activated complex consisted of p50 and p65. Preimmune serum (PIS) 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 mutated oligonucleotide had no effect on the DNA binding.
Crotepoxide Inhibits Robust Activation of NF-B-Our previous studies have shown that a high concentration of TNF (1 nM) induces more robust and rapid (5 min) NF-B activation (21). To determine whether crotepoxide could inhibit the NF-B robust response to TNF, we challenged crotepoxide-treated cells with increasing concentrations of TNF (up to 1 nM) for 30 min and then examined for NF-B activation (Fig. 3C). Although NF-B activation by 1 nM TNF was very strong, crotepoxide inhibited NF-B activation regardless of whether NF-B was activated with 0.01 or 1 nM TNF, suggesting that crotepoxide is a very potent inhibitor of NF-B activation.
Crotepoxide Does Not Directly Affect Binding of NF-B to the DNA-Some NF-B inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (a serine protease inhibitor), caffeic acid phenethyl ester, and plumbagin (36 -39) directly modify the NF-B protein so that the protein can no longer bind to DNA. We investigated whether crotepoxide mediates suppression of NF-B activation through a similar mechanism. Incubating nuclear extract from TNF-treated cells with crotepoxide revealed that crotepoxide did not modify the DNA binding ability of NF-B proteins (Fig. 3D), suggesting that crotepoxide inhibits NF-B activation by a mechanism different from direct modification.
Crotepoxide Inhibited NF-B Activation Induced by Various Agents-In addition to TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS), okadaic acid, cigarette smoke con- densate, and H 2 O 2 are potent activators of NF-B (40 -44). Therefore, we investigated the effects of crotepoxide on NF-B activated by these agents. DNA binding assays revealed that all these agents activated NF-B in human KBM-5 cells; incubating KBM-5 cells with crotepoxide suppressed this activation to a variable degree (Fig. 4A). H 2 O 2 -induced NF-B activation was completely suppressed with crotepoxide. Cells were viable at this concentration and exposure time. These results suggest that crotepoxide acts at a step in the NF-B activation pathway that is common to all these agents.
Inhibition of NF-B Activation by Crotepoxide Was Not Cell Type-specific-Because the signal transduction pathway mediated by NF-B may be distinct in different cell types, we also investigated whether crotepoxide blocked TNF-induced NF-B activation in human embryonic kidney A293 cells, lung cancer H1299 cells, and colon cancer Caco2 cells (Fig. 4B). Crotepoxide inhibited TNF-induced NF-B activation in these cells, indicating that crotepoxide-induced suppression of NF-B activation is not cell type-specific.
Crotepoxide Suppressed Constitutive NF-B Activation-Most tumor cells express constitutively active NF-B (19); however, the mechanism of constitutive activation is not well understood. Prostate cancer DU145, multiple myeloma MM1, and squamous cell carcinoma SCC4 cells are known to express constitutively active NF-B. Treating DU145, MM1, and SCC4 cells with crotepoxide suppressed constitutive NF-B activation (Fig. 4C).
Crotepoxide Inhibited 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 crotepoxide and then exposed the cells to TNF for various times. We then examined the cells for NF-B 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 (Fig. 5A) as early as 5 min with peak activation at 30 min, TNF had no effect on cells pretreated with crotepoxide. Moreover, Western blot analysis revealed that although TNF induced IB␣ degradation in the control cells in 10 min, TNF had no effect on IB␣ degradation in crotepoxide-treated cells (Fig. 5B). In addition, crotepoxide inhibited TNF-induced phosphorylation of IB␣ and its subsequent degradation (Fig. 5B). These results indicate that crotepoxide 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 to block IB␣ degradation. Western blotting with an antibody that recognizes the serine-phosphorylated (Ser-32) form of IB␣ revealed that crotepoxide strongly suppressed TNF-induced IB␣ phosphorylation (Fig. 5C).
Crotepoxide Inhibited TNF-induced IB␣ Kinase Activation-IKK is required for TNF-induced phosphorylation of IB␣ and for the phosphorylation of p65 (45). Because crotepoxide inhibited IB␣ phosphorylation, we investigated the effects of crotepoxide on TNF-induced IKK activation. Immune complex kinase assays showed that crotepoxide suppressed TNF-induced IKK activation (Fig. 5D). Neither TNF nor crotepoxide affected the expression of IKK proteins.
Crotepoxide Did Not Directly Inhibit TNF-induced IKK-Certain agents suppress NF-B activation by directly interacting with IKK (24,46). We investigated whether crotepoxide binds with the IKK protein to directly suppress IKK activity. The immune complex kinase assay of whole-cell extracts from untreated and TNF-treated cells showed that crotepoxide did not directly affect IKK activity, suggesting that crotepoxide indirectly modulated TNF-induced IKK activation (Fig. 5E).
Crotepoxide Inhibited TNF-induced Phosphorylation of IKK␣/␤-Next we investigated whether crotepoxide suppresses activation of IKK␣/␤ induced by TNF. We observed that it inhibited phosphorylation of IKK␣/␤ and activation. IKK proteins were unchanged either by TNF or crotepoxide treatment (Fig. 5F).
Next, we investigated how crotepoxide inhibited TNF-induced TAK1 activation. We assayed whether crotepoxide binds with the TAK1 protein to directly suppress TAK1 activity. The immune complex kinase assay of whole-cell extracts from untreated and TNF-treated cells showed that crotepoxide directly affected TAK1 activity. Results indicate that crotepoxide directly modulated TNF-induced TAK1 activation (Fig. 6B).
Crotepoxide Inhibited Nuclear Translocation of p65-p65 is a subunit of NF-B that has nuclear localization signals and is retained in the cytoplasm by IB␣. We investigated whether IB␣ degradation leads to the nuclear translocation of p65. Western blotting revealed that TNF induced the nuclear translocation of p65 in as few as 10 min of incubation and that crotepoxide suppressed p65 translocation (Fig. 6C). The immunocytochemical assay confirmed that crotepoxide suppressed the translocation of p65 (Fig. 6D).
Crotepoxide Did Not Modulate STAT3 and MAPK Activation-We further investigated whether crotepoxide also regulates signaling pathways other than NF-B. Therefore, we studied the effect of crotepoxide on STAT3 and MAPK pathways. We found that crotepoxide did not inhibit constitutive phosphorylation (data not shown) nor did IL-6 induce STAT3 phosphorylation (data not shown). We also observed that crotepoxide failed to suppress TNF-induced MAPK phosphorylation, suggesting that crotepoxideinduced apoptosis is not associated with STAT3 or MAPK pathway (data not shown).
Crotepoxide Repressed TNF-induced NF-B-dependent Reporter Gene Expression-Although EMSA showed that crotepoxide blocked NF-B activation, DNA binding alone does not always correlate with NF-B-dependent gene transcription, suggesting that there are additional regulatory steps. We investigated whether crotepoxide could suppress TNF-induced NF-B reporter activity. TNF induced the expression of an NF-Bregulated SEAP reporter gene in a dose-dependent manner, and crotepoxide suppressed the expression (Fig. 7A).

DISCUSSION
We investigated the effect of crotepoxide on TNF-and chemotherapy-induced apoptosis and on NF-B signaling pathway activation. We found that crotepoxide alone suppressed the proliferation of various types of tumor cells and potentiated TNF-and chemotherapeutic drugs-induced apoptosis. This correlated with the down-regulation of various gene products that mediate inflammation, cell proliferation, cell survival, invasion, and angiogenesis, all of which are regulated by NF-B. We also found that crotepoxide suppressed NF-B activated by various agents by inhibiting IKK activation, IB␣ phosphorylation, IB␣ degradation, p65 phosphorylation, and NF-B-dependent reporter gene expression.
Our study is the first to investigate the effect of crotepoxide on NF-B activation. Crotepoxide inhibited NF-B activation induced by carcinogens, cigarette smoke, and inflammatory stimuli, suggesting that crotepoxide must act at a step common to all these activators. Although crotepoxide has been reported to act as an anti-inflammatory agent (6), its mechanism of action has not been described. NF-B, which is known to play major role in inflammation, was inhibited by crotepoxide. Crotepoxide not only inhibits inducible NF-B activation but also inhibits constitutively active NF-B in tumor cells. Constitutive NF-B activation is critical to the survival and proliferation of various tumor cell types (19). NF-B activation in response to different stimuli requires IKK activation, which phosphorylates IB␣ at serine 32 and 36, leading to IB␣ degradation and p65 translocation to the nucleus (50). We found that crotepoxide suppressed IKK, which in turn suppressed IB␣ phosphorylation and degradation. IKK is also involved in constitutive activation of NF-B in tumor cells (51). Thus, it is possible that crotepoxide inhibition of IKK is linked to its ability to suppress constitutive NF-B activation.
We also investigated the ways in which crotepoxide inhibits IKK activation. Several studies have suggested that TAK1 plays a major role in TNF-induced NF-B activation by interacting with TAB1 and TAB2. For instance, TAK1 can bind to and activate IKK, leading to NF-B activation (52). We found that crotepoxide directly inhibited the activation of TAK1. TAK1 has also been shown to be recruited by TNFR1 through TRADD, TRAF2, and receptor-interacting protein (47). Indeed, our study showed for the first time that crotepoxide inhibits TAK1induced NF-B activation, which suggests that TAK1 is the main upstream stimulatory kinase modulated by crotepoxide. We were intrigued to find that crotepoxide also inhibited IKKinduced NF-B reporter gene activity. It is likely that the overexpressed IKK requires TAK1/TAB for its activation, which could be inhibited by crotepoxide. TAK1-dependent activation FIGURE 6. Crotepoxide inhibits TAK1 activation and nuclear translocation of p65. A, crotepoxide inhibited TNF-induced TAK1 activation. KBM-5 cells were preincubated with crotepoxide (50 M) for 2 h and then treated with TNF (1 nM) for the indicated times. Whole-cell extracts were immunoprecipitated with antibody against TAK1 and analyzed by an immune complex kinase assay. To examine the effect of crotepoxide on the level of expression of TAK1 proteins, Western blot analysis of whole-cell extracts (WCE) was performed with anti-TAK1 antibody. B, crotepoxide directly affected TNF-induced TAK1 activation. Whole cell extracts were prepared from KBM-5 cells treated with TNF (1 nM) and immunoprecipitated with anti-TAK1 antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentrations of crotepoxide. C, crotepoxide inhibited TNF-induced p65 phosphorylation. KBM-5 cells were left untreated or pretreated with crotepoxide (50 M) for 2 h at 37°C and then treated with TNF (0.1 nM) for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting with antibodies against p65 and phospho-specific p65. For loading control of nuclear protein, the membrane was blotted with anti-poly(ADP-ribose) polymerase antibody (PARP). D, crotepoxide inhibited the nuclear translocation of p65. KBM-5 cells were first treated with crotepoxide (50 M) for 2 h at 37°C and then exposed to TNF (0.1 nM) for 15 min. Cells were centrifuged and underwent immunocytochemical analysis.
of IKK␤ requires Lys-63 ubiquitinylation and interaction with IKK-␥ in the IKK-complex (53). Because crotepoxide inhibits TAK1-dependent IKK␤ phosphorylation in vivo, it is possible that it would also inhibit the IKK␤-dependent reporter gene activity. Alternatively, its ability to directly inhibit IKK in vivo cannot be ruled out based on our studies.
We also found that crotepoxide can suppress TNF-induced IB␣ degradation, which is mediated through the inhibition of IB␣ phosphorylation. Crotepoxide inhibits TNF-induced IB␣ phosphorylation and, therefore, delays the degradation of IB␣, indicating that crotepoxide mediates its effects through mechanisms different from those of N-acetyl-leucyl-leucyl-norleucinal.
NF-B activation leads to the expression of genes that are involved in the proliferation, survival, angiogenesis, invasion, and metastasis of cancer (54). In the current study, we found that crotepoxide inhibited the expression of cyclin D1 and c-myc, both of which are regulated by NF-B. Crotepoxide inhibition of cyclin D1 and c-myc could be the mechanism of crotepoxide-induced inhibition of cancer cell proliferation. In addition, we found that crotepoxide suppressed the expression of various antiapoptotic gene products including TRAF1, Bcl-2, Bcl-xL, and IAP-1. These gene products are regulated by NF-B, and their overexpression in numerous tumors has been associated with tumor survival, chemoresistance, and radioresistance.
Besides NF-B, another transcription factor STAT3 is known to be involved in tumorigenesis (55). However, in the present study, we found that crotepoxide did not influence the activation of STAT3, indicating diepoxide-induced apoptosis of cancer cells is not due to the inhibition of STAT3 activation.
In addition, crotepoxide potentiated apoptosis induced by TNF and various chemotherapeutic agents including 5-fluorouracil, cisplatin, thalidomide, and velacade. Crotepoxide down-regulation of various antiapoptotic gene products can sensitize the cells to the apoptotic effects of TNF. Kupchan et al.
(2) stated that crotepoxide inhibits tumorigenesis, and our results showed that crotepoxide has anti-proliferative effects on various leukemic cells which could be due to down-regulation of these anti-proliferative gene products. Similarly, crotepoxide also suppressed gene products that have been implicated in metastasis and angiogenesis. We found that crotepoxide abrogated the expression of NF-B-regulated gene products involved in invasion (e.g. MMP-9 and ICAM-1) and angiogenesis (e.g. VEGF). Thus, the suppression of TNF-induced metastasis and angiogenesis could be due to the down-regulation of ICAM-1, MMP-9, and VEGF.
Overall, our results demonstrate that crotepoxide is a potent inhibitor of NF-B activation and mediates its anti-proliferative, proapoptotic, anti-angiogenic, anti-metastatic, and antiinflammatory activities through NF-B-regulated gene products. In the future, animal studies are needed to investigate whether crotepoxide can suppress tumor growth and further potentiate chemotherapy-induced apoptosis. The results of the current study suggest that crotepoxide is a potent anti-inflammatory agent with tumorigenesis-suppressing potential. Supplementary Fig. 1  Crotepoxide does not modulate STAT3 and MAPK pathways.
(A) Crotepoxidde did not suppress STAT3 activation. KBM-5 cells (2 x 10 6 cells/mL) was treated with crotepoxide with indicated concentration for 2 hours and then exposed with IL-6 for 15 min. Whole cell lysates were prepared and subjected to western blotting with antibody against pSTAT3. For loading control, the membrane was blotted with anti-STAT3 antibody.
(B) Crotepoxide did not inhibit constitutive STAT3 activation. U266 cells (2x10 6 cells/mL) were treated with crotepoxide with indicated concentration. After 2 h of incubation cells harvested, whole cell lysates were prepared and subjected to western blotting with antibody against pSTAT3. For loading control, the membrane was blotted with anti-STAT3 antibody.
(C) Crotepoxidde did not suppress MAPK activation. KBM-5 cells were pretreated with crotepoxide (50 μM) for 2 hours and then treated with TNF (0.1 nM) for the indicated times. Whole cell extracts were prepared and analyzed by western blotting with antibodies against phospho-MAPK. For equal loading of protein, the membrane was blotted with anti-MAPK antibody.