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

Crotepoxide Chemosensitizes Tumor Cells through Inhibitionof Expression of Proliferation, Invasion, and AngiogenicProteins Linked to Proinflammatory Pathway*□S

Received for publication, March 7, 2010, and in revised form, June 22, 2010 Published, JBC Papers in Press, June 23, 2010, DOI 10.1074/jbc.M110.121061

Sahdeo Prasad‡, Vivek R. Yadav‡, Chitra Sundaram‡, Simone Reuter‡, Padmanabhan S. Hema§, Mangalam S. Nair§,Madan M. Chaturvedi‡, and Bharat B. Aggarwal‡1

From the ‡Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson CancerCenter, Houston, Texas 77030 and §Organic Chemistry Section, National Institute for Interdisciplinary Science and Technology(CSIR), Trivandrum, Kerala 695019, India

Crotepoxide (a substituted cyclohexane diepoxide), iso-lated from Kaempferia pulchra (peacock ginger), althoughlinked to antitumor and anti-inflammatory activities, themechanism by which it exhibits these activities, is not yetunderstood. Because nuclear factor �B (NF-�B) plays a criti-cal role in these signaling pathways, we investigated theeffects of crotepoxide on NF-�B-mediated cellular responsesin human cancer cells. We found that crotepoxide potenti-ated tumor necrosis factor (TNF), and chemotherapeuticagents induced apoptosis and inhibited the expression ofNF-�B-regulated gene products involved in anti-apoptosis(Bcl-2, Bcl-xL, IAP1,2 MCl-1, survivin, and TRAF1), apopto-sis (Bax, Bid), inflammation (COX-2), proliferation (cyclinD1 and c-myc), invasion (ICAM-1 and MMP-9), and angio-genesis (VEGF). We also found that crotepoxide inhibitedboth inducible and constitutive NF-�B activation. Crotep-oxide inhibition of NF-�B was not inducer-specific; it inhib-ited NF-�B activation induced by TNF, phorbol 12-myristate13-acetate, lipopolysaccharide, and cigarette smoke. Crotep-oxide suppression of NF-�B was not cell type-specificbecause NF-�B activation was inhibited in myeloid, leuke-mia, and epithelial cells. Furthermore, we found that crotep-oxide inhibited TAK1 activation, which led to suppression ofI�B� kinase, abrogation of I�B� phosphorylation and degra-dation, nuclear translocation of p65, and suppression ofNF-�B-dependent reporter gene expression. Overall, ourresults indicate that crotepoxide sensitizes tumor cells tocytokines and chemotherapeutic agents through inhibition of

NF-�B and NF-�B-regulated gene products, and this mayprovide the molecular basis for crotepoxide ability to sup-press inflammation and carcinogenesis.

Several chemotherapeutic, cytotoxic, and immunomodulat-ing agents are commonly used to treat cancer. However, mostmodern medicines tend to target only one gene product orpathway at a given time. This is perhaps one of the major rea-sons that some of the recently discovered medicines are lesseffective. Besides being prohibitively expensive, many of thesedrugs are also associated with serious side effects and morbid-ity. Still, the search continues for an ideal treatment that hasminimal side effects and is cost-effective. Therefore, traditionalmedicine, usually derived from plants is one of the alternativesto treat the chronic diseases including cancer. Although plant-derived products have been used for centuries to treat variousailments, their active components and mechanisms are notfully understood. Identifying the active chemical entities andtheir molecular targets would facilitate the discovery of newclinical uses for such products.Crotepoxide (Fig. 1A), a highly substituted cyclohexane diep-

oxide linked with anticancer activity, was first isolated from thefruit of Croton macrostachys (1, 2). More recently, crotepoxidewas identified as amain component inKaempferia rotunda (3),a member of the Zingiberaceae, or ginger family whose tuberhas traditionally been used to treat pneumonia, bronchitis,abdominal pain, dysentery, diarrhea, cold, and obesity (4, 5).Crotepoxide was also identified in various other medicinalplants including Piper kadsura (6–8), Monanthotaxis caffra(9), Friesodielsia obovata (Annonaceae) (10), Kaempferiaangustifolia (11), and Kaempferia pulchra (peacock ginger)(12). Although crotepoxide has been reported to have tumor-inhibiting (2, 13) and anti-inflammatory properties (6), theexact mechanisms through which crotepoxide exhibits theseproperties are not understood.Nuclear factor-�B (NF-�B), a transcription factor that has a

critical role in inflammation, is responsible for regulation ofgenes involved in cell survival, adhesion, differentiation, andgrowth. These genes include antiapoptotic (e.g. c-IAP, survivin,tumor necrosis factor receptor (TNFR)-associated factor(TRAF), cellular FLICE inhibitory protein, Bcl-2, and Bcl-xL),inflammatory (cyclooxygenase-2 (COX-2)), or invasive (matrix

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA-124787-01A2 (a program project grant) and CA-16 672 (a coregrant). This work was also supported by grants from the Clayton Founda-tion for Research (to B. B. A.) and the Center for Targeted Therapy of the MDAnderson Cancer Center.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Fig. 1.

1 The Ransom Horne, Jr. Professor of Cancer Research. To whom correspond-ence should be addressed: The University of Texas MD Anderson CancerCenter, 1515 Holcombe Blvd., Box 143, Houston, TX 77030. Tel.: 713-794-1817; Fax: 713-745-6339; E-mail: [email protected].

2 The abbreviations used are: IAP, inhibitor of apoptosis; TNFR, TNF recep-tor; TRAF, TNFR-associated factor; MMP-9, matrix metalloproteinase-9;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;TAK1, TGF-�-activated kinase 1; TRADD, TNFR-associated deathdomain; NIK, NF-�B-inducing kinase; TAB1, TAK1-binding protein 1;SEAP, secretory alkaline phosphatase; ICAM-1, intracellular adhesionmolecule-1; IKK, I�B kinase.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 35, pp. 26987–26997, August 27, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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metalloproteinase-9 (MMP-9) and vascular endothelial growthfactor (VEGF)) and can encode adhesion molecules, chemo-kines, and cell-cycle regulation (e.g. cyclin D1 and c-myc) (14).Most carcinogens, inflammatory agents, and tumor promoters,including cigarette smoke, phorbol ester, okadaic acid, hydro-gen peroxide, and tumor necrosis factor (TNF), have beenshown to activate NF-�B (15). However, several cancer celllines including human multiple myeloma (16), breast cancer(17), and prostate cancer (18) express constitutively activeNF-�B (19).

Because NF-�B is known to regulate inflammation andtumorigenesis, we hypothesized that the anti-inflammatoryand anticancer effects ascribed to crotepoxidemay be due to itsinhibition of NF-�B and NF-�B-regulated gene expression.Indeed we demonstrate that this crotepoxide can block NF-�Bpathway and potentiate the anticancer effects of various che-motherapeutic drugs.

EXPERIMENTAL PROCEDURES

Reagents—A 50-mM solution of crotepoxide, isolated fromK. pulchra as described below, was prepared in 100% dimethylsulfoxide, stored as small aliquots at �20 °C, and diluted in cellculture medium as needed. Bacteria-derived recombinanthuman TNF, purified to homogeneity with a specific activity of5� 107 units/mg, was provided byGenentech (South San Fran-cisco, CA). We obtained 5-flurouracil, cisplatin, thalidomide,velacade, and �-actin antibody from Sigma; antibodies againstp65, p50, I�B�, cyclin D1, COX-2, MMP-9, anti-poly(ADP-ri-bose) polymerase, IAP1, TRAF1, Bcl-2, and Bcl-xL were fromSanta Cruz Biotechnology (Santa Cruz, CA); phospho-specificanti-I�B� (Ser-32/36) and phospho-specific anti-p65 (Ser536)antibodies were fromCell Signaling Technology (Beverly,MA);anti-IKK-� and anti-IKK-� antibodies were from Imgenex (SanDiego, CA); anti-VEGF was from NeoMarkers (Fremont, CA);survivin antibody from R&D Systems (Minneapolis, MN).Isolation and Characterization—The fresh rhizomes of

K. pulchra were collected from a certified medicinal plantgrower in Trivandrum, Kerala, India. A voucher specimen(TBGT 20270) has been deposited in the Tropical BotanicalGarden and Research Institute Herbarium in Palode, Kerala,India. The air-dried powdered rhizome of K. pulchra (360 g)was extracted with acetone at room temperature (27 °C), whichafter removal of solvent under reduced pressure yielded theextract (9.17 g). The extract was subjected to gradient elutionsilica gel (100–200 mesh) column chromatography using thesolvents hexane:ethyl acetate (100:0–40:60) to give 145 frac-tions, which were grouped into six fraction pools based on sim-ilarities on thin layer chromatography. After further purifica-tion by silica gel column chromatography and elution withhexane:ethyl acetate (90:10–80:20) and crystallization fromdichloromethane-hexanemixture, the fifth fraction pool (1.4 g)yielded crotepoxide (1.2 g) as pure white crystals. We con-firmed the structure of crotepoxide ([(1R,2R,4R,5S,6R,7R)-4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]-octane-5,6-diol-diacetate]) on the basis of a comparison of the spectralvalues, viz. 1H,13C of nuclearmagnetic resonance, infrared, andmass spectra with those reported earlier (20).

Cell Lines—Chronic myelogenous leukemia KBM-5, humanmultiple myeloma MM1, human prostate cancer DU145,human head and neck cancer SCC4, human embryonic kidneycancer A293, human non-small-cell lung carcinoma H1299,and human colon cancer Caco2 cell lines were obtained fromAmerican Type Culture Collection (Manassas, VA). KBM-5cells were cultured in Iscove’s modified Dulbecco’s modifiedEagle’s medium with 15% FBS; SCC4 and A293 cells were cul-tured in DMEM with 10% FBS; MM1, DU145, H1299, andCaco2 cells were cultured in Roswell Park Memorial Institute1640 (RPMI) medium with 10% FBS. All media were supple-mented with 100 units/ml penicillin and 100 �g/mlstreptomycin.Electrophoretic Mobility Shift Assay (EMSA)—To determine

the effect of crotepoxide on TNF-activated NF-�B, we performedNF-�B-DNA binding using EMSA as previously described (21).Briefly, nuclear extracts prepared from treated cells (1� 106 cells/ml) were incubated with 32P-end-labeled, 45-mer, double-strandedNF-�B oligonucleotide (15�g of protein with 16 fmol ofDNA) from a human immunodeficiency virus long terminalrepeat, 5�-TTGTTACAAGGGACTTTCCGCTGGGGACTTT-CCAGGGAGGCGTGG-3� (boldface and underlines indicateNF-�B binding sites) for 30 min at 37 °C. The resultant DNA-proteincomplexwas separated fromfreeoligonucleotideson6.6%native polyacrylamide gels. We used a double-strandedmutated oligonucleotide, 5�-TGTTACAACTCACTTTCCG-CTGCTCACTTTCCAGGGAGGCGTGG-3�, to examine thespecificity of NF-�B-DNA binding. The specificity of bindingwas also examined by competition with the unlabeled oligonu-cleotide. For supershift assays, we incubated nuclear extractsprepared from TNF-treated cells with antibodies against thep50 or p65 subunits of NF-�B for 30 min at 37 °C before usingEMSA to analyze the complex. Preimmune serumwas includedas a negative control. The dried gels were visualized with aStorm 820 PhosphorImager, and radioactive bands were quan-titated using ImageQuant software (Amersham Biosciences).Western Blot Analysis—To determine the effect of crotep-

oxide on TNF-dependent I�B� phosphorylation, I�B� degra-dation, p65 translocation, and p65 phosphorylation, we pre-pared 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 crotepoxide-treated 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), andsodium orthovanadate (4 mM). Lysates were centrifuged at20,817 � g for 10 min to remove insoluble material. Superna-tant was collected and kept at �80 °C. Whole-cell lysates andcytosolic or nuclear extracts were resolved by SDS-PAGE.Afterelectrophoresis, proteins were electrotransferred to nitrocellu-lose membranes, blotted with the relevant antibody, anddetected by an enhanced chemiluminescence reagent (Amer-sham Biosciences ECL). The bands obtained were quantitatedusing NIH Image (National Institutes of Health).Cytotoxicity Assay—To determine the anti-proliferative and

cytotoxicity-potentiating effects of crotepoxide on leukemic

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cells, we used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) dye uptake method as described pre-viously (23).Immunocytochemistry for p65 Localization—To examine the

effect of crotepoxide on the nuclear translocation of p65, we per-formed immunocytochemical analysis as previously described(24). Briefly, cells were plated on a poly-L-lysine-coated glass slidewith a Cytospin 4 centrifuge (ThermoShendon, Pittsburgh, PA),air dried, and fixed with 4% paraformaldehyde. The slideswere washed in PBS, blocked with 5% normal goat serum for1 h, and then incubated with rabbit polyclonal p65 antibodyat a 1:200 dilution overnight at 4 °C. The slides were thenwashed, incubated with goat anti-rabbit IgG-Alexa Fluor 594(Invitrogen) at a 1:200 dilution for 1 h and counterstained fornuclei with Hoechst 33342 (50 ng/ml) stain for 5 min.Stained slides were mounted with mounting medium pur-chased from Sigma and analyzed under a Labophot-2 fluo-rescence microscope (Nikon, Melville, NY). Photographswere taken using a Photometrics Coolsnap CF color camera(Nikon) and MetaMorph software (Version 4.6.5, UniversalImaging, Sunnyvale, CA).Immune Kinase Complex Assay for TGF-�-activated Kinase 1

(TAK1)—To determine the effect of crotepoxide on TNF-in-duced I�B kinase (IKK) and TAK1 activation, we analyzed IKKand TAK1 as previously described (25). Briefly, the IKK andTAK1 complex from whole-cell extracts was precipitated withantibodies against IKK-� or TAK1 and then treated with pro-tein A/G-Sepharose beads (Pierce). After 2 h, the beads werewashed with lysis buffer and then resuspended in a kinase assaymixture containing HEPES (pH 7.4; 50 mmol/liter), magne-sium chloride (20 mmol/liter), dithiothreitol (2 mmol/liter),[�-32P]adenosine triphosphate (ATP; 20 �Ci), unlabeled ATP(10 �mol/liter), and substrate glutathione S-transferase-I�B�(amino acids 1–54; 2 �g) or His-MKK6. After incubation at30 °C for 30 min, we terminated the reaction by boiling withSDS sample buffer for 5 min. Finally, the protein was resolvedon 10% SDS-PAGE, the gel was dried, and the radioactive bandswere visualized using a Storm 820 PhosphorImager. To deter-mine the total amounts of IKK-� and IKK-� or TAK1 in eachsample, we resolved whole-cell proteins (40 �g) on 7.5% SDS-PAGE, electrotransferred the proteins to a nitrocellulosemem-brane, 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 ester-ase activity and plasma membrane integrity. Briefly, 2 � 105cells were incubated with crotepoxide (50 �mol/liter) for 2 hand then treated with TNF (1 nmol/liter) for 16 h at 37 °C. Cellswere stained with the live and dead reagents (ethidiumhomodimer (5 �mol/liter) and calcein acetoxymethyl ester (5�mol/liter)) and incubated at 37 °C for 30 min. Cells were ana-lyzed under a fluorescence microscope (Labophot-2; Nikon).NF-�B-dependent Reporter Gene Expression Assay—To

determine the effect of crotepoxide on TNF-, TNFR-, TNFR-associated death domain (TRADD)-, TRAF2-, NF-�B-inducingkinase (NIK)-, TAK-1/TAK1-binding protein-1 (TAB1)-, andIKK-NF-�B-dependent reporter gene transcription, we per-formed a secretory alkaline phosphatase (SEAP) assay as previ-

ously described (26) with the following exceptions. Briefly,A293 cells (5 � 105 cells/well) were plated in 6-well plates andtransiently transfected by the calcium phosphate method withpNF-�B-SEAP (0. 5 �g). To examine TNF-induced reportergene expression, we transfected the cells with SEAP expressionplasmid (0.5 �g) and control plasmid pCMV-FLAG1 (1.5 �g)DNA for 24 h.We then treated the cells with crotepoxide for 2 hand stimulated them with TNF (0.1 nM). The cell culturemedium was harvested after 24 h of TNF treatment. To exam-ine reporter gene expression induced by various genes, A293cells were transfected with pNF-�B-SEAP plasmid (0.5 �g)with expressing plasmid (0.5 �g) and pCMV-FLAG1 controlplasmid (1.5 �g) for 24 h, treated with crotepoxide, and thenharvested from cell culture medium after an additional 24 hof incubation. The culture medium was analyzed for SEAPactivity as recommended by the manufacturer (ClontechLaboratories, Mountain View, CA) with a Victor 3 micro-plate reader (PerkinElmer Life and Analytical Sciences, Bos-ton, MA).AnnexinVAssay—Anearly indicator of apoptosis is the rapid

translocation and accumulation of themembrane phospholipidphosphatidylserine from the cytoplasmic interface of themem-brane to the extracellular surface. This loss ofmembrane asym-metry can be detected using the binding properties of annexinV. To identify apoptosis, we used an annexin V antibody con-jugated with fluorescein isothiocyanate fluorescence dye.Briefly, 5 � 105 cells were pretreated with crotepoxide (50 �M),treated with TNF for 24 h at 37 °C, and subjected to annexin Vstaining. The cells were washed in PBS, resuspended in 100 �lof binding buffer containing a fluorescein isothiocyanate-con-jugated anti-annexin V antibody, and then analyzed with aFACSCalibur flow cytometer (BD Biosciences).

RESULTS

This study was undertaken to investigate the anti-inflamma-tory and antitumor effects of crotepoxide. For this we firstinvestigated the effect of crotepoxide alone on the survival ofvarious tumor cells, and thenwe examined in combinationwithchemotherapeutic agents.We also determined its effects on theNF-�B activation pathway as induced by various carcinogensand inflammatory stimuli as well as expression of NF-�B-regu-lated gene products and apoptosis in leukemic cells. The dura-tion of exposure and concentration of crotepoxide used toexamine its effect on NF-�B pathway had a minimal effect onthe viability of these cells as determined by trypan blue dyeexclusion test (data not shown).Crotepoxide Inhibits Proliferation and Potentiates TNF- and

Chemotherapeutic Agent-induced Apoptosis—The results ofMTT assay showed that crotepoxide inhibited the proliferationof leukemic cells, KBM-5, MM1, and U266 cells in a dose- andtime-dependentmanner (Fig. 1B).Whether this compound canenhance the apoptosis induced by apoptotic cytokine TNF, wasinvestigated by an annexin V staining method. We found thatcrotepoxide up-regulated TNF-induced apoptosis from 9 to28% (Fig. 1C, left panels). However, crotepoxide alone showedonly 9% cell death. To confirm the result of annexin V assay, weused esterase staining (i.e. the live/dead assay) and found thatcrotepoxide increased TNF-induced apoptosis from 16 to 40%

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(Fig. 1C, middle panels). When we examined crotepoxide forcaspase activation by Western blotting, a classical hallmark ofapoptosis, we found activation of caspase-9 and caspase-3 andsubsequent anti-poly(ADP-ribose) polymerase cleavage, indi-

cating that crotepoxide potentiates the TNF-induced caspaseactivation (Fig. 1C, right panels). Crotepoxide alone inducedapoptosis, albeit at a low extent, and the mechanism of thiseffect is not known at this point.

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Next, we investigated the effect of crotepoxide on apoptosisinduced by chemotherapeutic drugs. Results of MTT uptakeshowed that crotepoxide up-regulates cell death induced by5-flurouracil, cisplatin, thalidomide, and velacade (Fig. 1D, leftpanel). This result was further confirmed by live dead assay.According to this assay, crotepoxide potentiated apoptosisinduced by 5-fluorouracil (from 21 to 44%), cisplatin (from 23to 43%), thalidomide (from 18 to 56%), and velacade (from 28 to65%) (Fig. 1D, right panel). These results together indicate thatcrotepoxide potentiates the apoptotic effects of TNF and che-motherapeutic agents.Crotepoxide Inhibits Expression of Cell Proliferative Gene

Products—Because crotepoxide inhibits the proliferation ofvarious tumor cell lines, we investigated whether this effect ismediated through the regulation of NF-�B-mediated expres-sion of cell proliferation gene products such as c-myc (27) and

cyclin D1 (28). Our results revealedthat crotepoxide inhibited cyclinD1and c-myc expression in a dose-de-pendent fashion induced by TNF.Under these conditions, crotep-oxide alone showed minimal cyto-toxicity (Fig. 2A).Crotepoxide Suppresses the Ex-

pression of Proinflammatory GeneProducts—Because crotepoxide ex-hibits anti-inflammatory activity(6), we investigated whether thiseffect is mediated through the regu-lation of NF-�B mediated expres-sion of COX2. Results revealed thatcrotepoxide blocked TNF-inducedexpression of this proinflammatoryprotein in a time-dependent man-ner. Under these conditions crotep-oxide alone showed minimal cyto-toxicity (Fig. 2A).Crotepoxide Modulates Expression

of Pro-apoptotic Gene Products—Next, we determined whether cro-tepoxide regulates the expression ofproapoptotic and tumor suppressorgene products.We found that crote-poxide induced the expression ofBax and cleavage of Bid, which wasinhibited by TNF (Fig. 2B).Crotepoxide Suppresses the Ex-

pression of Tumor Cell SurvivalGene Products—Because crotep-

oxide enhances TNF- and chemotherapy-induced apoptosis,we investigated whether this effect is mediated through theregulation of NF-�B-mediated expression of the antiapoptoticproteins c-IAP1 (29), Bcl-2 (30), Bcl-xL (31), mcl-1, TRAF-1(32), and survivin. We found that crotepoxide blocked theTNF-induced expression of these tumor cell survival proteinsin a time-dependent manner (Fig. 2C).Crotepoxide Suppresses the Expression of TNF-induced Met-

astatic Gene Products—TNF has been shown to induce theexpression of ICAM-1 (33), VEGF (34), and MMP-9 (35), all ofwhich have NF-�B-binding sites in their promoters. We inves-tigated whether crotepoxide can modulate NF-�B-regulatedgene products involved in metastasis and found that crotep-oxide abolished TNF-induced expression of ICAM-1, MMP-9,and VEGF (Fig. 2D).

FIGURE 1. Crotepoxide inhibits the proliferation of leukemic cells and potentiated the apoptotic effects of TNF and chemotherapeutic agents.A, shown is the chemical structure of crotepoxide. Ph, phenyl. B, crotepoxide inhibited the proliferation of KBM-5, MM1, and U266 cells. Cells were seeded in96-well plates and treated with the indicated concentrations of crotepoxide. Cell proliferation was analyzed by MTT assay on days 1, 3, and 5. C, crotepoxideenhanced TNF-induced apoptosis. KBM-5 cells were pretreated with crotepoxide (50 �M) for 2 h then treated with TNF (1 nM) for 24 h. Cell death wasdetermined by fluorescence-activated cell sorting using annexin V/propidium iodide staining (left panel) and by live/dead assay (middle panel). Cleavage ofcaspase-9 and �3, and poly(ADP-ribose) polymerase was determined by Western blotting in whole-cell extracts of crotepoxide- and TNF-treated cells (rightpanel). D, crotepoxide potentiated cytotoxicity induced by 5-flurouracil (5-FU), cisplatin (Cis), thalidomide (Thal), and velacade (Vel) is shown. Five thousandcells were seeded in triplicate in 96-well plates, pretreated with crotepoxide (50 �M) for 2 h, and then incubated with chemotherapeutic agents for 24 h. Cellviability was then analyzed by MTT assay (left panel). Crotepoxide also potentiated chemotherapy-induced apoptosis. KBM-5 cells (1 � 106) were pretreatedwith crotepoxide (50 �M) for 2 h then treated with TNF (1 nM) for 24 h. Cell death was analyzed by a live/dead assay (right panel).

FIGURE 2. Crotepoxide inhibits the TNF-induced expression of NF-�B-regulated gene products. Crotep-oxide inhibited the expression of TNF-induced cell-proliferative, pro-apoptotic, anti-apoptotic, metastatic, andangiogenic proteins. KBM-5 cells were incubated with crotepoxide (50 �M) for 2 h and then treated with TNF (1nM) for the indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis usingantibodies against cell proliferative cyclin D1, c-myc, and COX-2 proteins (A), proapoptotic Bax and Bid proteins(B), anti-apoptotic Bcl-2, Bcl-xL, survivin, Mcl-1, TRAF1, and c-IAP1 proteins (C), and metastatic and angiogenicVEGF, ICAM-1, and matrix metalloproteinase-9 (MMP-9) proteins (D).

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Crotepoxide Suppresses TNF-induced NF-�B Activation—Be-cause NF-�B regulates various cellular responses includingproliferation, apoptosis, inflammation, and chemosensitiza-tion, all regulated by NF-�B, we reasoned that crotepoxidemust modulate the NF-�B cell-signaling pathway. Therefore,we investigatedwhether crotepoxide inhibitsNF-�B activation.The experimental condition that was used to study the mecha-nism of NF-�B inhibition involved short duration of exposure,and under this condition crotepoxide alone did not show anycell death. EMSA revealed that although crotepoxide alone hadno effect on NF-�B activation, but crotepoxide inhibited TNF-mediated NF-�B activation in a dose (Fig. 3A, left panel)- andtime-dependent manner (Fig. 3A, right panel).

NF-�B is a complex of proteins inwhich various combinations of Relor NF-�B proteins constitute activeNF-�B heterodimers that bind spe-cific DNA sequences. To confirmthat the band visualized by EMSA inTNF-treated cells was NF-�B, weincubated nuclear extracts fromTNF-activated cells with antibodiesto the p50 (NF-�B) and p65 (RelA)subunits of NF-�B. The resultingbands that were shifted to highermolecular masses (Fig. 3B) sug-gested that the TNF-activated com-plex consisted of p50 and p65. Pre-immune serum (PIS) had no effecton DNA binding. The addition ofexcess unlabeledNF-�B (cold oligo-nucleotide, 100-fold) caused a com-plete disappearance of the band,whereas mutated oligonucleotidehad no effect on the DNA binding.Crotepoxide Inhibits Robust Acti-

vationofNF-�B—Ourprevious stud-ies have shown that a high concen-tration of TNF (1 nM) induces morerobust and rapid (5 min) NF-�Bactivation (21). To determinewhether crotepoxide could inhibitthe NF-�B robust response to TNF,we challenged crotepoxide-treatedcells with increasing concentrationsof TNF (up to 1 nM) for 30 min andthen examined forNF-�B activation(Fig. 3C). Although NF-�B activa-tion by 1 nM TNF was very strong,crotepoxide inhibited NF-�B acti-vation regardless of whether NF-�Bwas activated with 0.01 or 1 nMTNF, suggesting that crotepoxide isa very potent inhibitor of NF-�Bactivation.Crotepoxide Does Not Directly Af-

fect Binding of NF-�B to the DNA—Some NF-�B inhibitors such as

N-tosyl-L-phenylalanine chloromethyl ketone (a serine prote-ase inhibitor), caffeic acid phenethyl ester, and plumbagin (36–39) directlymodify theNF-�Bprotein so that the protein cannolonger bind to DNA. We investigated whether crotepoxidemediates suppression of NF-�B activation through a similarmechanism. Incubating nuclear extract fromTNF-treated cellswith crotepoxide revealed that crotepoxide did not modify theDNA binding ability of NF-�B proteins (Fig. 3D), suggestingthat crotepoxide inhibits NF-�B activation by amechanism dif-ferent 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-

FIGURE 3. Crotepoxide inhibits TNF-induced NF-�B activation. A, crotepoxide inhibited TNF-induced NF-�Bactivation in a dose-dependent fashion (left panel). KBM-5 cells (2 � 106) were incubated with the indicatedconcentrations of crotepoxide for 2 h and then treated with TNF (0.1 nM) for 30 min. The nuclear extracts wereanalyzed for NF-�B activation by EMSA. Crotepoxide also inhibited NF-�B activation in a time-dependentmanner (right panel). KBM-5 cells were preincubated with crotepoxide (50 �M) for the indicated times and thentreated with TNF (0.1 nM) for 30 min. The nuclear extracts were prepared and analyzed for NF-�B activation byEMSA. B, TNF-induced NF-�B is composed of p65 and p50 subunits. Nuclear extracts from untreated cells orcells treated with TNF (0.1 nM) were incubated with the indicated antibodies, an unlabeled NF-�B oligoprobe,or a mutant oligoprobe and analyzed for NF-�B activation by EMSA. PIS, pre-immune serum. C, crotepoxideinhibited NF-�B activation induced by high doses of TNF. KBM-5 cells (2 � 106) were preincubated for 2 hat 37 °C with or without crotepoxide (50 �M) and then treated for 30 min with the indicated concentrationsof TNF. Nuclear extracts were prepared, and NF-�B was assayed. D, crotepoxide did not directly affectNF-�B-DNA binding. Nuclear extracts (NE) were prepared from untreated cells or cells treated with TNF(0.1 nM), incubated for 30 min with the indicated concentrations of crotepoxide, and then analyzed forNF-�B activation by EMSA.

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densate, and H2O2 are potent activators of NF-�B (40–44).Therefore, we investigated the effects of crotepoxide on NF-�Bactivated by these agents. DNA binding assays revealed that allthese agents activated NF-�B in human KBM-5 cells; incubat-ing KBM-5 cells with crotepoxide suppressed this activation toa variable degree (Fig. 4A). H2O2-inducedNF-�Bactivationwascompletely suppressed with crotepoxide. Cells were viable atthis concentration and exposure time. These results suggestthat crotepoxide acts at a step in the NF-�B activation pathwaythat is common to all these agents.Inhibition of NF-�B Activation by Crotepoxide Was Not Cell

Type-specific—Because the signal transduction pathway medi-ated by NF-�B may be distinct in different cell types, we alsoinvestigated whether crotepoxide blocked TNF-inducedNF-�B activation in human embryonic kidney A293 cells, lungcancer H1299 cells, and colon cancer Caco2 cells (Fig. 4B).Crotepoxide inhibited TNF-induced NF-�B activation in these

cells, indicating that crotepoxide-induced suppression ofNF-�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 under-stood. Prostate cancer DU145, multiple myeloma MM1, andsquamous cell carcinoma SCC4 cells are known to express con-stitutively active NF-�B. Treating DU145, MM1, and SCC4cells with crotepoxide suppressed constitutive NF-�B activa-tion (Fig. 4C).Crotepoxide Inhibited TNF-dependent I�B� Degradation

and Phosphorylation—The translocation of NF-�B to thenucleus is preceded by the phosphorylation, ubiquitination,and proteolytic degradation of I�B�. To determinewhether theinhibition of TNF-induced NF-�B activation was due to theinhibition of I�B� degradation, we pretreated cells with crote-poxide and then exposed the cells to TNF for various times.Wethen examined the cells for NF-�B in the nucleus by EMSA andfor I�B� degradation in the cytoplasmbyWestern blot analysis.EMSA revealed that although TNF activated NF-�B in controlcells in a time-dependent manner (Fig. 5A) as early as 5 minwith peak activation at 30 min, TNF had no effect on cells pre-treated with crotepoxide. Moreover, Western blot analysisrevealed that although TNF induced I�B� degradation in thecontrol cells in 10 min, TNF had no effect on I�B� degradationin crotepoxide-treated cells (Fig. 5B). In addition, crotepoxideinhibited TNF-induced phosphorylation of I�B� and its subse-quent degradation (Fig. 5B). These results indicate that crotep-oxide inhibited both TNF-induced NF-�B activation and I�B�degradation.To determine whether the inhibition of TNF-induced I�B�

degradation was due to the inhibition of I�B� phosphorylation,we used the proteasome inhibitor N-acetyl-leucyl-leucyl-nor-leucinal to block I�B� degradation. Western blotting with anantibody that recognizes the serine-phosphorylated (Ser-32)form of I�B� revealed that crotepoxide strongly suppressedTNF-induced I�B� phosphorylation (Fig. 5C).Crotepoxide Inhibited TNF-induced I�B� Kinase Activation—

IKK is required for TNF-induced phosphorylation of I�B� andfor the phosphorylation of p65 (45). Because crotepoxide inhib-ited I�B� phosphorylation, we investigated the effects of crote-poxide on TNF-induced IKK activation. Immune complexkinase assays showed that crotepoxide suppressed TNF-in-duced IKK activation (Fig. 5D). Neither TNF nor crotepoxideaffected the expression of IKK proteins.Crotepoxide Did Not Directly Inhibit TNF-induced IKK—Cer-

tain agents suppress NF-�B activation by directly interactingwith IKK (24, 46). We investigated whether crotepoxide bindswith the IKK protein to directly suppress IKK activity. Theimmune complex kinase assay of whole-cell extracts fromuntreated and TNF-treated cells showed that crotepoxide didnot directly affect IKK activity, suggesting that crotepoxideindirectly modulated TNF-induced IKK activation (Fig. 5E).Crotepoxide Inhibited TNF-induced Phosphorylation of

IKK�/�—Next we investigated whether crotepoxide sup-presses activation of IKK�/� induced by TNF. We observedthat it inhibited phosphorylation of IKK�/� and activation. IKK

FIGURE 4. Crotepoxide inhibits NF-�B activation induced by differentstimuli. A, crotepoxide blocked NF-�B activation induced by phorbol 12-my-ristate 13-acetate (PMA), LPS, okadaic acid (OA), cigarette smoke condensate(CSC), and hydrogen peroxide (H2O2). Human myeloid leukemia KBM-5 cellswere preincubated with crotepoxide (50 �M) for 2 h and then treated withokadaic acid (500 nM) for 4 h, phorbol 12-myristate 13-acetate (25 ng/ml) for2 h, LPS (10 �g/ml) for 2 h, and cigarette smoke condensate (40 �g/ml) andhydrogen peroxide (H2O2; 250 �M) for 1 h each. Nuclear extracts were ana-lyzed for NF-�B activation by EMSA. B, crotepoxide suppressed TNF-inducedNF-�B in different cell types. A293, H1299, and Caco2 cells were incubatedwith crotepoxide (50 �M) for 2 h and then incubated with TNF (0.1 nM) for 30min. Nuclear extracts were then prepared and analyzed for NF-�B activationby EMSA. C, crotepoxide inhibited constitutive NF-�B activation. DU145,MM1, and SCC-4 cells were incubated with crotepoxide (50 �M) for 2 h.Nuclear extracts were then prepared and analyzed for NF-�B activation byEMSA.

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proteins were unchanged either by TNF or crotepoxide treat-ment (Fig. 5F).Crotepoxide Inhibited TNF-induced TAK1 Activation—TAK1

plays an essential role in the TNF-induced IKK andNF-�B acti-vation (47). Because crotepoxide inhibited IKK activation, weinvestigated whether crotepoxide suppresses TNF-inducedTAK1 activation. Results of immune complex kinase assaysshowed that crotepoxide suppressed TNF-induced TAK1 acti-vation (Fig. 6A). Neither TNF nor crotepoxide affected theexpression of TAK1 proteins.Next, we investigated how crotepoxide inhibited TNF-in-

duced TAK1 activation. We assayed whether crotepoxide binds

with the TAK1 protein to directlysuppress TAK1 activity. The im-mune complex kinase assay ofwhole-cell extracts from untreatedand TNF-treated cells showed thatcrotepoxide directly affected TAK1activity. Results indicate that crote-poxide directly modulated TNF-in-duced TAK1 activation (Fig. 6B).Crotepoxide Inhibited Nuclear

Translocation of p65—p65 is a sub-unit ofNF-�B that has nuclear local-ization signals and is retained in thecytoplasm by I�B�.We investigatedwhether I�B� degradation leads tothe nuclear translocation of p65.Western blotting revealed that TNFinduced the nuclear translocation ofp65 in as few as 10min of incubationand that crotepoxide suppressedp65 translocation (Fig. 6C). Theimmunocytochemical assay con-firmed that crotepoxide suppressedthe translocation of p65 (Fig. 6D).Crotepoxide Did Not Modulate

STAT3 andMAPK Activation—Wefurther investigated whether crote-poxide also regulates signaling path-ways other than NF-�B. Therefore,we studied the effect of crotepoxideon STAT3 and MAPK pathways.We found that crotepoxide did notinhibit constitutive phosphoryla-tion (data not shown) nor did IL-6induce STAT3 phosphorylation(data not shown). We also observedthat crotepoxide failed to suppressTNF-induced MAPK phosphoryla-tion, suggesting that crotepoxide-induced apoptosis is not associatedwith STAT3 or MAPK pathway(data not shown).Crotepoxide Repressed TNF-in-

duced NF-�B-dependent ReporterGene Expression—Although EMSAshowed that crotepoxide blocked

NF-�B activation, DNA binding alone does not always corre-late with NF-�B-dependent gene transcription, suggestingthat there are additional regulatory steps. We investigatedwhether crotepoxide could suppress TNF-induced NF-�Breporter activity. TNF induced the expression of an NF-�B-regulated SEAP reporter gene in a dose-dependent manner,and crotepoxide suppressed the expression (Fig. 7A).Crotepoxide Suppressed NF-�B-dependent Reporter Gene

Expression Induced by TNFR1, TRADD, TRAF2, NIK, TAK1,and IKK—TNF has been shown to activate NF-�B activationthrough sequential interaction with TNFR1, TRADD, TRAF2,NIK, TAK1, and IKK, resulting in I�B� phosphorylation (48,

FIGURE 5. Crotepoxide inhibits TNF-induced I�B� degradation, I�B� phosphorylation, and IKK activa-tion. Crotepoxide inhibited TNF-induced NF-�B activation and I�B� degradation. KBM-5 cells were incubatedwith crotepoxide (50 �M) for 2 h and then treated with TNF (0.1 nM) for the indicated times. A, nuclear extractswere analyzed for NF-�B activation by EMSA. B, cytoplasmic extracts were analyzed for I�B� degradation byWestern blotting with antibodies against anti-phospho-I�B� and anti-I�B�. Equal protein loading was evalu-ated by �-actin. C, shown is the effect of crotepoxide on TNF-induced I�B� phosphorylation. Cells were prein-cubated with crotepoxide (50 �M) for 2 h, incubated with N-acetyl-leucyl-leucyl-norleucinal (ALLN; 50 �g/ml)for 30 min, and then treated with TNF (0.1 nM) for 10 min. Cytoplasmic extracts were fractionated and thensubjected to Western blotting with phospho-specific I�B� antibody. The same membrane was reblotted with�-actin. Ub, ubiquitin. D, crotepoxide inhibited TNF-induced IKK activation. KBM-5 cells were preincubatedwith crotepoxide (50 �M) for 2 h and then treated with TNF (1 nM) for the indicated times. Whole-cell extractswere immunoprecipitated with antibody against IKK-� and analyzed by an immune complex kinase assay. Toexamine the effect of crotepoxide on the level of expression of IKK proteins, we fractionated whole-cell extractson sodium dodecyl sulfate-polyacrylamide electrophoresis gels and examined by Western blot analysis withanti-IKK-� and anti-IKK-� antibodies. E, crotepoxide directly affected TNF-induced IKK activation. Whole-cellextracts (WCE) were prepared from KBM-5 cells treated with TNF (1 nM) and immunoprecipitated with anti-IKK�antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicatedconcentrations of crotepoxide. F, crotepoxide inhibited the phosphorylation of IKK�/�. TNF (1 nM) wasexposed for an indicated time period in 2 h before crotepoxide (50 �M)-pretreated KBM-5 cells. Whole-cellextracts were prepared and then subjected to Western blotting with phospho-specific anti-IKK�/� antibody.The same membrane was reblotted with anti-IKK� and anti-IKK� antibodies.

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49). To determine the effect of crotepoxide on NF-�B-depen-dent reporter gene expression, we transiently transfected thecells with TNFR1-, TRADD-, TRAF2-, NIK-, IKK-, and TAK1/TAB1-expressing plasmids and then monitored the cells forNF-�B-dependent SEAP expression. Transiently transfect-ing cells with TNFR1-, TRADD-, TRAF2-, NIK-, IKK-, andTAK1/TAB1-expressing plasmids revealed that the plasmid-transfected cells expressed the NF-�B-regulated reportergene and that crotepoxide suppressed the expression, sug-gesting that the target for crotepoxide action is upstream toIKK (Fig. 7B).

DISCUSSION

We investigated the effect of crotepoxide on TNF- and che-motherapy-induced apoptosis and onNF-�B signaling pathwayactivation. We found that crotepoxide alone suppressed theproliferation of various types of tumor cells and potentiatedTNF- and chemotherapeutic drugs-induced apoptosis. Thiscorrelated with the down-regulation of various gene products

thatmediate inflammation, cell pro-liferation, cell survival, invasion,and angiogenesis, all of which areregulated by NF-�B. We also foundthat crotepoxide suppressed NF-�Bactivated by various agents by in-hibiting IKK activation, I�B� phos-phorylation, I�B� degradation, p65phosphorylation, and NF-�B-depen-dent reporter gene expression.Our study is the first to investi-

gate the effect of crotepoxide onNF-�B activation. Crotepoxide in-hibited NF-�B activation inducedby carcinogens, cigarette smoke,and inflammatory stimuli, suggest-ing that crotepoxide must act at astep common to all these activators.Although crotepoxide has been re-ported to act as an anti-inflamma-tory agent (6), its mechanism ofaction has not been described.NF-�B, which is known to playmajor role in inflammation, wasinhibited by crotepoxide. Crotep-oxide not only inhibits inducibleNF-�B activation but also inhibitsconstitutively active NF-�B intumor cells. Constitutive NF-�Bactivation is critical to the survivaland proliferation of various tumorcell types (19). NF-�B activation inresponse to different stimuli re-quires IKK activation, which phos-phorylates I�B� at serine 32 and 36,leading to I�B� degradation andp65 translocation to the nucleus(50). We found that crotepoxidesuppressed IKK, which in turn sup-

pressed I�B� phosphorylation and degradation. IKK is alsoinvolved in constitutive activation of NF-�B in tumor cells (51).Thus, it is possible that crotepoxide inhibition of IKK is linkedto its ability to suppress constitutive NF-�B activation.

We also investigated the ways in which crotepoxide inhibitsIKK activation. Several studies have suggested that TAK1 playsa major role in TNF-induced NF-�B activation by interactingwith TAB1 and TAB2. For instance, TAK1 can bind to andactivate IKK, leading to NF-�B activation (52). We found thatcrotepoxide directly inhibited the activation of TAK1. TAK1has also been shown to be recruited by TNFR1 through TRADD,TRAF2, and receptor-interacting protein (47). Indeed, ourstudy showed for the first time that crotepoxide inhibits TAK1-induced NF-�B activation, which suggests that TAK1 is themain upstream stimulatory kinase modulated by crotepoxide.We were intrigued to find that crotepoxide also inhibited IKK-induced NF-�B reporter gene activity. It is likely that the over-expressed IKK requires TAK1/TAB for its activation, whichcould be inhibited by crotepoxide. TAK1-dependent activation

FIGURE 6. Crotepoxide inhibits TAK1 activation and nuclear translocation of p65. A, crotepoxide inhibitedTNF-induced TAK1 activation. KBM-5 cells were preincubated with crotepoxide (50 �M) for 2 h and then treatedwith TNF (1 nM) for the indicated times. Whole-cell extracts were immunoprecipitated with antibody againstTAK1 and analyzed by an immune complex kinase assay. To examine the effect of crotepoxide on the level ofexpression of TAK1 proteins, Western blot analysis of whole-cell extracts (WCE) was performed with anti-TAK1antibody. B, crotepoxide directly affected TNF-induced TAK1 activation. Whole cell extracts were preparedfrom KBM-5 cells treated with TNF (1 nM) and immunoprecipitated with anti-TAK1 antibody. The immunocom-plex 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 withcrotepoxide (50 �M) for 2 h at 37 °C and then treated with TNF (0.1 nM) for the indicated times. Nuclear extractswere prepared and analyzed by Western blotting with antibodies against p65 and phospho-specific p65. Forloading control of nuclear protein, the membrane was blotted with anti-poly(ADP-ribose) polymerase anti-body (PARP). D, crotepoxide inhibited the nuclear translocation of p65. KBM-5 cells were first treated withcrotepoxide (50 �M) for 2 h at 37 °C and then exposed to TNF (0.1 nM) for 15 min. Cells were centrifuged andunderwent immunocytochemical analysis.

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of IKK� requires Lys-63 ubiquitinylation and interaction withIKK-� in the IKK-complex (53). Because crotepoxide inhibitsTAK1-dependent IKK� phosphorylation in vivo, it is possiblethat it would also inhibit the IKK�-dependent reporter geneactivity. Alternatively, its ability to directly inhibit IKK in vivocannot be ruled out based on our studies.We also found that crotepoxide can suppress TNF-in-

duced I�B� degradation, which is mediated through theinhibition of I�B� phosphorylation. Crotepoxide inhibitsTNF-induced I�B� phosphorylation and, therefore, delaysthe degradation of I�B�, indicating that crotepoxide medi-ates its effects through mechanisms different from those ofN-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 foundthat crotepoxide inhibited the expression of cyclin D1 andc-myc, both of which are regulated by NF-�B. Crotepoxideinhibition of cyclin D1 and c-myc could be the mechanism ofcrotepoxide-induced inhibition of cancer cell proliferation. In

addition, we found that crotepoxide suppressed the expressionof various antiapoptotic gene products includingTRAF1, Bcl-2,Bcl-xL, and IAP-1. These gene products are regulated byNF-�B, and their overexpression in numerous tumors hasbeen associated with tumor survival, chemoresistance, andradioresistance.Besides NF-�B, another transcription factor STAT3 is

known to be involved in tumorigenesis (55). However, in thepresent study, we found that crotepoxide did not influence theactivation of STAT3, indicating diepoxide-induced apoptosisof 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-flu-orouracil, cisplatin, thalidomide, and velacade. Crotepoxidedown-regulation of various antiapoptotic gene products cansensitize the cells to the apoptotic effects of TNF.Kupchan et al.(2) stated that crotepoxide inhibits tumorigenesis, and ourresults showed that crotepoxide has anti-proliferative effects onvarious leukemic cells which could be due to down-regulationof these anti-proliferative gene products. Similarly, crotepoxidealso suppressed gene products that have been implicated inmetastasis and angiogenesis. We found that crotepoxideabrogated the expression of NF-�B-regulated gene productsinvolved in invasion (e.g.MMP-9 and ICAM-1) and angiogen-esis (e.g.VEGF). Thus, the suppression of TNF-induced metas-tasis and angiogenesis could be due to the down-regulation ofICAM-1, MMP-9, and VEGF.Overall, our results demonstrate that crotepoxide is a potent

inhibitor of NF-�B activation and mediates its anti-prolifera-tive, proapoptotic, anti-angiogenic, anti-metastatic, and anti-inflammatory activities through NF-�B-regulated gene prod-ucts. In the future, animal studies are needed to investigatewhether crotepoxide can suppress tumor growth and furtherpotentiate chemotherapy-induced apoptosis. The results of thecurrent study suggest that crotepoxide is a potent anti-inflam-matory agent with tumorigenesis-suppressing potential.

Acknowledgments—We thank Joe Munch for carefully editing themanuscript. We also thank Dr. Bryant Darnay for supplying the His-MKK6 protein.

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Crotepoxide Suppresses NF-�B and Potentiates Apoptosis

AUGUST 27, 2010 • VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 26997

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Supplementary Fig. 1

pSTAT3

0 0 10 25 50 100 CrotepoxideIL-6

STAT3

pSTAT3

STAT3

0 5 10 15 30 60 0 5 10 15 30 60 Time (Min)

Medium Crotepoxide

p38 MAPK

Phospho-p38 MAPK

(A)

(B)

(C)

0 10 25 50 100 Crotepoxide

TNF TNF

Legend of Supplementary Fig. 1 Crotepoxide does not modulate STAT3 and MAPK pathways. (A) Crotepoxidde did not suppress STAT3 activation. KBM-5 cells (2 x 106 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 (2x106 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.

Hema, Mangalam S. Nair, Madan M. Chaturvedi and Bharat B. AggarwalSahdeo Prasad, Vivek R. Yadav, Chitra Sundaram, Simone Reuter, Padmanabhan S.

PathwayProliferation, Invasion, and Angiogenic Proteins Linked to Proinflammatory

Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of

doi: 10.1074/jbc.M110.121061 originally published online June 23, 20102010, 285:26987-26997.J. Biol. Chem. 

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