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Modification of Cysteine 179 of I�B� Kinase by NimbolideLeads to Down-regulation of NF-�B-regulated Cell Survivaland Proliferative Proteins and Sensitization of Tumor Cells toChemotherapeutic Agents*

Received for publication, July 6, 2010, and in revised form, September 6, 2010 Published, JBC Papers in Press, September 9, 2010, DOI 10.1074/jbc.M110.161984

Subash C. Gupta‡, Sahdeo Prasad‡, Simone Reuter‡, Ramaswamy Kannappan‡, Vivek R. Yadav‡, Jayaraj Ravindran‡,Padmanabhan S. Hema§, Madan M. Chaturvedi‡1, Mangalam Nair§, and Bharat B. Aggarwal‡2

From the ‡Cytokine Research Laboratory, Department of Experimental Therapeutics, University of Texas M. D. Anderson CancerCenter, Houston, Texas 77030 and the §Organic Chemistry Section, National Institute for Interdisciplinary Science and Technology(Council for Scientific and Industrial Research), Trivandrum, 695 019 Kerala, India

Reverse pharmacology, also called the “bedside to bench”approach, that deals with new uses for a well known molecularentity has been used extensively in cancer drug development toidentify novel compounds and delineate their mechanisms ofaction. Here, we show that nimbolide, a triterpenoid isolatedfrom Azadirachta indica, enhanced the apoptosis induced byinflammatory cytokines and chemotherapeutic agents in tumorcells. This limonoid abrogated the expression of proteins asso-ciated with cell survival (Bcl-2, Bcl-xL, IAP-1, and IAP-2), pro-liferation (cyclin D1), invasion (MMP-9), and angiogenesis(VEGF), all regulated by nuclear factor (NF)-�B. Nimbolideinhibited the activation of NF-�B induced by carcinogens andinflammatory stimuli. Constitutively active NF-�B found inmost tumor cells was also inhibited.We found that suppressionof NF-�B activation by nimbolide was caused by inhibition ofI�B kinase (IKK), which led to suppression of I�B� phosphory-lation and degradation, nuclear translocation, DNA binding,and gene transcription. Reducing agent reversed the action ofthe limonoid, suggesting the involvement of a cysteine residue.Replacement of Cys179 of IKK-� with alanine abolished theeffect of nimbolide, suggesting that Cys179 plays a critical role ininhibiting the NF-�B activation. Overall, our results indicatethat nimbolide can sensitize tumor cells to chemotherapeuticagents through interaction with IKK, leading to inhibition ofNF-�B-regulated proteins.

Extensive research during the past 2 decades has made itclear that cancer is a disease in which multiple cell signalingpathways are dysregulated and thus mono-targeted therapiesare unlikely to provide a significant benefit (1). Natural prod-ucts derived from fruits, vegetables, spices, legumes, cereals,and traditional medicines are preferred as potential therapeu-

tics for cancer and other chronic diseases because of theirsafety, affordability, long term use, and ability to targetmultiplecell signaling pathways (2). Promiscuous drugs that interactwith multiple targets as compared with a single target arebecoming a virtue in drug development as in case of imatinib,sunitinib, or sorafenib (3). Neem (Azadirachta indica, a tree inthe Mahogany family; Azad Dirakht in Persian means “freetree”; calledMuarubaini in Swahili, whichmeans “treats 40 dif-ferent diseases”) is one such traditional medicinal plant, theextract of which has been used for thousands of years for mostacute and chronic diseases in India and Africa and thus hasbeen named “Heal All,” “Nature’s Drugstore,” “Village Phar-macy,” and “Panacea for all Diseases.” Over 300 bioactive com-ponents from this plant have been isolated, one-third of whichare limonoids (4–6). One of the components of this plant isnimbolide, which was first isolated from the leaves and flowersof neem (4, 7). Nimbolide, a tetranortriterpenoid, consists of aclassical limonoid skeletonwith an�,�-unsaturated ketone sys-tem and a �-lactonic ring (Fig. 1A) (8). This limonoid has beenshown to exhibit numerous biologic activities, including anti-feedant (inhibits normal feeding behavior) (9), anti-malarial(10), anti-microbial (11), and anti-cancer (7, 12–16) activities.This triterpenoid exhibits anti-proliferative activity in a wide

variety of tumor cells, including neuroblastoma cells (12),osteosarcoma cells, choriocarcinoma cells (17), macro-phages, leukemia cells, and melanoma cells (16). How nim-bolide exerts its antiproliferative effects is not fully under-stood. Nimbolide has been shown to induce G0/G1 cell cyclearrest (16), increase reactive oxygen species generation, acti-vate caspases, and modulate the activity of cyclins, cyclin-de-pendent kinase inhibitors, proliferating cell nuclear antigen,and p53 (18). In animal tumor models, nimbolide (10–100mg/kg) has been shown to exhibit chemopreventive activityagainst 7,12-dimethylbenz[�]anthracene (DMBA)3-inducedoral carcinogenesis (18, 19). Furthermore, the �,�-unsaturatedketone structural element of nimbolide has been linked to its* This work was supported, in whole or in part, by National Institutes of Health

Core Grant CA-16672 and Program Project Grant CA-124787-01A2. Thiswork was also supported by a grant from the Clayton Foundation forResearch (to B. B. A.) and a grant from the Center for Targeted Therapy ofMD Anderson Cancer Center.

1 Present address: Dept. of Zoology, University of Delhi, 110007 Delhi, India.2 Ransom Horne, Jr., Professor of Cancer Research. To whom correspondence

should be addressed. Tel.: 713-794-1817; Fax: 713-745-6339; E-mail: [email protected].

3 The abbreviations used are: DMBA, 7,12-dimethylbenz[�]anthracene; IKK,I�B kinase; PMA, phorbol myristate acetate; OA, okadaic acid; PARP, poly-(ADP-ribose) polymerase; NIK, NF-�B-inducing kinase; TRADD, TNF recep-tor-associated death domain; SEAP, secretory alkaline phosphatase; PS,phosphatidylserine; CSC, cigarette smoke condensate; MTT, 3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 46, pp. 35406 –35417, November 12, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

35406 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 46 • NOVEMBER 12, 2010

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anticancer activity (20). Amide derivatives modified on the lac-tone ring were also found to enhance the cytotoxic activity ofnimbolide (15). Because of the critical role of NF-�B in apopto-sis, tumor cell survival, proliferation, invasion, and angiogene-sis and its activation by various carcinogens, including DMBA,we hypothesized that nimbolide may modulate this cell signal-ing pathway.NF-�B is a ubiquitous and evolutionarily conserved tran-

scription factor that is activated in response to a number ofcarcinogens and inflammatory stimuli, including cytokines (e.g.tumor necrosis factor (TNF)), tumor promoters, cigarettesmoke, environmental pollutants, ionizing radiation, andstress. In resting cells, NF-�B is kept in an inactive state in thecytoplasm as a heterotrimer consisting of p50, p65, and I�Bproteins, including I�B�, I�B�, and I�B� (21). In response toactivation signals, the I�B� subunit is phosphorylated at serineresidues 32 and 36 and ubiquitinated at lysine residues 21 and22, which target them for proteasome-mediated degradation.The p65 subunit is then phosphorylated and translocated to thenucleuswhere it binds to a specificDNAsequence and activatesthe transcription of over 500 genes involved in immunoregula-tion, growth regulation, inflammation, carcinogenesis, andapoptosis. The phosphorylation of I�B� is catalyzed by I�B�kinase (IKK), which consists of three subunits, IKK-�, IKK-�,and IKK-� (also called NEMO). Some natural compounds havebeen reported to inhibit NF-�B activation through modifica-tion of a specific cysteine residue (Cys179) in the activation loopof IKK-� (22–26).In this study, we investigated whether nimbolide modulates

the NF-�B signaling pathway in tumor cells. Our results dem-onstrate that this limonoid inhibits the NF-�B activation path-way induced by carcinogens, tumor promoters, inflammatorystimuli, and growth factors through direct interaction withCys179 of IKK-�, leading to suppression of I�B� phosphoryla-tion and degradation, inhibition of p65 nuclear translocation,down-regulation of NF-�B-regulated gene products, inhibitionof cell proliferation, and potentiation of apoptosis induced byTNF-� and chemotherapeutics in tumor cells.

EXPERIMENTAL PROCEDURES

Reagents—Nimbolide (Fig. 1A) was isolated from Aza-dirachta indica leaves as reported previously (27). A 50 mM

solution of this limonoid was prepared in dimethyl sulfoxideand stored in small aliquots at �20 °C. Nimbolide was dilutedin the culture medium just before use. Human recombinantTNF-� purified from bacterial cells to homogeneity with a spe-cific activity of 5 � 107 units/mg was provided by Genentech(South San Francisco, CA). Cigarette smoke condensate (CSC)was provided by Dr. C. Gary Gairola (University of Kentucky,Lexington). Penicillin, streptomycin, RPMI 1640 medium,Iscove’s modified Dulbecco’s medium, Dulbecco’s modifiedEagle’s medium, and fetal bovine serum (FBS) were obtainedfrom Invitrogen. Phorbol myristate acetate (PMA), lipopoly-saccharide (LPS), okadaic acid (OA), and antibodies againstFLAG and �-actin were obtained from Sigma. Antibodiesagainst p65, p50, cyclin D1, cyclooxygenase-2 (COX-2), matrixmetalloproteinase-9 (MMP-9), poly(ADP-ribose) polymerase(PARP), inhibitor of apoptosis protein-1 (IAP-1), IAP-2, Bcl-2,

Bcl-xL, intercellular adhesion molecule-1 (ICAM-1), c-Myc,caspase-3, -8, and -9, and the annexin V staining kit wereobtained from Santa Cruz Biotechnology (Santa Cruz, CA).Phospho-specific anti-I�B� (Ser32/36) and anti-p65 (Ser536)were purchased from Cell Signaling (Danvers, MA). An anti-body against p65, which was used for immunocytochemicalanalyses, was obtained from Abcam (Cambridge, MA). Thevascular endothelial growth factor (VEGF) antibody was pur-chased from NeoMarkers (Fremont, CA). Anti-I�B�, -IKK-�,and -IKK-� antibodies were obtained from Imgenex (SanDiego, CA).Cell Lines—The cell lines KBM-5 (human chronic myeloid

leukemia), U937 (human leukemic monocyte lymphoma),HL-60 (human promyelocytic leukemia), Jurkat (T-cell leuke-mia), A293 (human embryonic kidney), H1299 (human lungadenocarcinoma), U266 (human multiple myeloma), MCF-7(breast cancer), SCC-4 (human squamous cell carcinoma), andRPMI-8226 and MM.1S (human multiple myeloma) wereobtained from the American Type Culture Collection (Manas-sas, VA). K-562 (human chronic myeloid leukemia) was a giftfrom Dr. Hesham Amin (University of Texas M. D. AndersonCancer Center, Houston). KBM-5 cells were cultured inIscove’s modified Dulbecco’s medium with 15% FBS; K-562,HL-60, Jurkat, H1299, MCF-7, U937, RPMI-8226, MM.1S, andU266 cells were cultured in RPMI 1640 medium with 10% FBS;and A293 cells were cultured in Dulbecco’s modified Eagle’smedium supplemented with 10% FBS. SCC-4 cells were cul-tured in Dulbecco’s modified Eagle’s medium containing 10%FBS, nonessential amino acids, pyruvate, glutamine, and vita-mins. All culture media were supplemented with 100 units/mlpenicillin and 100 �g/ml streptomycin.Electrophoretic Mobility Shift Assay—To determine NF-�B

activation,we preparednuclear extracts andperformed an elec-trophoreticmobility shift assay (EMSA) as described previously(28).Western Blot Analysis—The cytoplasmic, nuclear, and

whole-cell extracts were prepared, and Western blot analysiswas performed as described previously (29).Enzyme-linked Immunosorbent Assay—Human ELISA sys-

tems kits (eBioscience, San Diego) were used for the detectionof interleukin-6 (IL-6) and TNF-�. The U-266 cells weretreated with different concentrations of nimbolide, and cell-free supernatants were collected after 24 h. The cytokine levelwas determined by following the manufacturer’s protocol.Invasion Assay—A membrane invasion culture system was

used to assess cell invasion. We used the BD BioCoat tumorinvasionsystem(BDBiosciences),whichcontainsan8-�mpoly-ethylene terephthalate membrane with a thin layer of reconsti-tuted Matrigel basement membrane matrix. The H1299 cells(2.5 � 104) were suspended in serum-free medium and seededinto the upper wells. After incubation overnight, cells weretreated with 2.5 �M nimbolide for 4 h and then stimulated with1 nM TNF in the presence of 1% FBS. After 24 h of incubation,the invaded cells were fixed, stained with Diff-Quik stain, andcounted in five random microscopic fields.IKKAssay—Weperformed IKK assay to determine the effect

of nimbolide onTNF-�-induced IKK activation using amethoddescribed previously (30).

Nimbolide Inhibits NF-�B-mediated Cellular Response

NOVEMBER 12, 2010 • VOLUME 285 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35407


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Immunocytochemical Analysis for NF-�B p65 Localization—We performed immunocytochemical analysis to determine theeffect of nimbolide on TNF-�-induced p65 nuclear transloca-tion, as described previously (24). Stained slides were analyzedusing a Labophot-2 fluorescence microscope (Nikon, Tokyo,Japan). Imageswere capturedwith a PhotometricsCoolsnapCFcolor camera (Nikon, Lewisville, TX) and analyzed with Meta-Morph version 4.6.5 software (Universal Imaging, Sunnyvale,CA).NF-�B-dependent Reporter Gene Expression Assay—The ef-

fect of nimbolide on the induction of NF-�B-dependent re-porter gene transcription by TNF-�, TNF receptor-1 (TNFR1),TNF receptor-associated death domain (TRADD), TNF recep-tor-associated factor 2 (TRAF2), NF-�B-inducing kinase (NIK),TAK1/TAB1-�, and IKK-�was analyzed using a secretory alka-line phosphatase (SEAP) assay, as described previously (31).Apoptosis by Live/Dead Assay—To determine plasma mem-

brane integrity and intracellular esterase activity, we performeda live/dead assay. It is a two-color fluorescence assay that simul-taneously determines live cells and dead cells. Intracellularesterases from live cells convert nonfluorescent cell-permeablecalcein acetoxymethyl (calceinAM) to the intensely fluorescentcalcein. Cleaved calcein is retainedwithin cells. It also examinesdead cells that have damaged membranes; the ethidiumhomodimer-1 (EthD-1) enters damaged cells and is fluorescentwhen bound to nucleic acids. EthD-1 produces a bright redfluorescence in damaged or dead cells. This assay was per-formed as described previously (32).Apoptosis by Phosphatidylserine Externalization Assay—The

annexin V assay provides a simple and effective method todetect apoptosis at a very early stage. This assay takes advan-tage of the fact that phosphatidylserine (PS) is translocatedfrom the inner (cytoplasmic) leaflet of the plasma membraneto the outer (cell surface) leaflet soon after the induction ofapoptosis and that the annexin V protein has a strong, spe-cific affinity for PS. PS on the outer leaflet is available to bindlabeled annexin V, providing the basis for a simple stainingassay. We measured the loss of membrane asymmetry thatoccurs when PS moves to the extracellular surface of themembrane using an annexin V staining kit (Santa Cruz Bio-technology). The assay was performed by following the man-ufacturer’s instructions.Measurement of Apoptosis and Cell Proliferation by the

3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl Tetrazolium Bro-mide Method—The effects of nimbolide on the proliferation ofleukemic cells and on the cytotoxic potential of TNF-� andother chemotherapeutic agents were determined bymeasuringmitochondrial dehydrogenase activity, using 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) as thesubstrate (33).Statistical Analysis—Different parameters were monitored

in normal and treated cells. Experiments were repeated a min-imum of three times. Data are given as the mean � S.D. Thestatistical analysis was carried out using a two-tailed unpairedStudent’s t test. A value of p � 0.05 was considered statisticallysignificant.

RESULTS

Our goal in this study was to determine whether nimbolideaffects the NF-�B activation pathway, NF-�B-regulated geneproducts, and NF-�B-mediated cellular responses in tumorcells. We focused primarily on TNF-�-induced cellular effectsbecause the role of TNF-� in the NF-�B activation pathwayis relatively well defined. When nimbolide or NF-�B activatorswere added alone, at the concentrations and the exposure timesemployed in our study, cell viability was not affected.Nimbolide Suppresses Cell Proliferation in Tumor Cells—Be-

cause NF-�B has been implicated in the survival and prolif-eration of various tumor cells, we examined the effect ofnimbolide on cell proliferation. The limonoid suppressed theproliferation of various myeloid leukemic (KBM-5, HL-60,U-937, and K-562), T-cell lymphoma (Jurkat), and myeloma(U266, MM.1S, and RPMI-8226) cells in a dose- and time-de-pendentmanner. The suppression of cell proliferation could beobserved at nimbolide concentrations as low as 1 �M (Fig. 1B).No significant inhibition of cell growth was noted after 1 day.These results indicate that nimbolide exhibits potent anti-pro-liferative effects on both leukemic and myeloid cells.Nimbolide Potentiates the Apoptosis Induced by TNF-� in

Tumor Cells—Among the cytokines, TNF-� is a well knownapoptosis-inducing agent (34). We investigated whether nim-bolide could potentiate TNF-�-induced apoptosis. When apo-ptosis was examined by measuring intracellular esteraseactivity, we found that nimbolide treatment increased TNF-�-induced apoptosis in KBM-5 and U266 cells in a dose-depen-dent manner. The TNF-�-induced apoptosis increased from 4to 59% in KBM-5 cells and from 5 to 50% in U-266 cells (Fig.2A). The results of theMTT assay also indicated that nimbolideenhanced the cytotoxic effects of TNF-� in a dose-dependentmanner (Fig. 2B).When apoptosis was examined by a PS externalization assay,

significantly more annexin V-positive cells were found aftertreatmentwith nimbolide andTNF-� than after treatmentwiththe cytokine alone (Fig. 2C). TNF-� binds to TNFR1 andsequentially recruits TRADD, FADD, and the FADD-like inter-leukin-1�-converting enzyme (caspase-8), leading to activationof caspase-9 and -3. Activated caspase-3 then induces PARPcleavage, a sign of apoptosis. Thus, we examined whether nim-bolide could enhance the TNF-�-induced activation ofcaspase-3, -8, and -9. We found that TNF-� alone had littleeffect on caspase activation, but nimbolide sensitized the cellsto caspase activation (Fig. 2D) and subsequent PARP cleavage(Fig. 2E). Overall, these results indicate that nimbolide signifi-cantly increases the apoptotic potential of TNF-� in tumorcells.Nimbolide Potentiates the Apoptosis Induced by Chemother-

apeutic Agents in Tumor Cells—Althoughmost tumor cells areinitially sensitive to chemotherapeutic agents, gradually theydevelop chemoresistance in part because of induction ofNF-�B-mediatedmultidrug resistance.Whether nimbolide canpotentiate the apoptotic effects of chemotherapeutic agentswas examined. We found that this limonoid potentiated theapoptotic effects of both 5-fluorouracil and thalidomide (Fig.2B) in KBM-5 cells. These results indicate that nimbolide sig-




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nificantly enhances the apoptotic potential of chemotherapeu-tic agents.Nimbolide Suppresses the Expression of NF-�B-regulated

Anti-apoptotic Gene Products—We investigated in detail hownimbolide potentiates the effect of cytokines or chemothera-peutic agents. The anti-apoptotic proteins Bcl-2, Bcl-xL, IAP-1,and IAP-2 are all known to be induced by TNF-� and to play amajor role in cell survival. We found that nimbolide down-regulated the expression of all these TNF-�-induced proteins(Fig. 3A).Nimbolide Suppresses the Expression of Proliferation Proteins

in Tumor Cells—We next examined whether suppression ofcell proliferation by nimbolide is due to down-regulation of

proteins such as cyclin D1, c-Myc, and COX-2. We found thatthe limonoid inhibited the expression of all three of these TNF-�-induced proteins (Fig. 3B).Nimbolide Suppresses the Expression of Proteins Linked to

Invasion and Metastasis—MMP-9, by virtue of its ability todegrade the extracellularmatrix, has been implicated in cellularinvasion (35). ICAM-1, an adhesion molecule, has also beenshown to be required for tumor metastasis (36), and VEGFplays a critical role in angiogenesis by promoting vascular endo-thelial cell growth and enhancing vascular permeability (37).Thus, we examined whether nimbolide can modulate theexpression of these gene products. Our results revealed thatnimbolide down-regulated the expression of all these proteins(Fig. 3C).Nimbolide Down-regulates Pro-inflammatory Cytokine Pro-

duction in Multiple Myeloma Cells—Both IL-6 and TNF-�have been linked with the proliferation of leukemic cells. Weinvestigatedwhether nimbolide has an effect on the productionof IL-6 and TNF-� in U266 cells. The production of IL-6 andTNF-� in U266 cells was suppressed by nimbolide in a concen-tration-dependent manner (Fig. 3D).Nimbolide Suppresses TNF-�-induced Tumor Cell Invasion—

We next examined whether nimbolide can affect tumor cellinvasion. As shown in Fig. 3E, TNF-� increased tumor cell inva-sion by almost 2.4-fold, and pretreating cells with nimbolidesuppressed the TNF-�-induced cell invasion.Nimbolide Suppresses TNF-�-induced NF-�B Activation—

Whether enhancement of apoptosis or down-regulation ofanti-apoptotic, proliferative, and metastatic gene products, allrequire the activation of NF-�B. Therefore, we investigatedwhether the TNF-�-induced activation of NF-�B is modulatedby nimbolide.We found that TNF-� induces NF-�B activation,whereas nimbolide alone did not. However, pretreatment ofcells with nimbolide inhibited the TNF-�-induced NF-�B acti-vation in a dose- (Fig. 4A) and time-dependent (Fig. 4B) man-ner. Treatment of cells with 10 �M nimbolide for 4 h was opti-mal for inhibiting the TNF-�-induced NF-�B activationwithout affecting cell viability.NF-�B is a complex of proteins in which various combina-

tions of Rel or NF-�B proteins constitute active NF-�B het-erodimers that bind to a specific DNA sequence. In our study,when nuclear extracts from TNF-�-activated cells were incu-bated with p50 (NF-�B) or p65 (RelA) antibodies, the resultingbands were shifted to higher molecular masses, suggesting thatthe TNF-�-activated complex consisted of p50 and p65. Preim-mune serum had no effect onDNAbinding, and the addition ofexcess unlabeled NF-�B (cold oligonucleotide; 100-fold excess)caused a decrease in the intensity of the band, whereas the addi-tion of a mutated oligonucleotide had no effect on DNA bind-ing (Fig. 4C). These results indicated that the band visualized inTNF-�-treated cells was indeed NF-�B.Nimbolide Inhibits NF-�B Activation Induced by Carcino-

gens and Inflammatory Stimuli—Numerous agents, includingcigarette smoke, OA, PMA, and LPS, are potent activators ofNF-�B, but they work by different mechanisms. We deter-minedwhether nimbolide affects theNF-�B activation inducedby these agents using EMSA. All of these agents activatedNF-�B in KBM-5 cells, and nimbolide suppressed this activa-

FIGURE 1. Nimbolide suppresses cell proliferation of tumor cells. A, chem-ical structure of nimbolide. B, cells (3000 cells/100 �l) were plated in fourreplicates, treated with the indicated concentrations of nimbolide, and sub-jected to an MTT assay on days 0, 1, 3, and 5. Cell proliferation was analyzed bymeasuring the absorbance at 570 nm.




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tion in a dose-dependent manner (Fig. 4D). These results sug-gest that nimbolide acts at a step in the NF-�B activation path-way that is common to all four agents.Nimbolide Inhibits Constitutive NF-�BActivation inMyeloid

Cells—Human multiple myeloma cells (U266, MM.1S, andRPMI-8226) are known to constitutively express NF-�B. Wefound that nimbolide inhibited constitutive NF-�B activation

in these cells (Fig. 4E), indicatingthat it can suppress not only induc-ible but also constitutively activeNF-�B in tumor cells. Nimbolidealso inhibited constitutive NF-�B inSCC-4 cells (Fig. 4E).Inhibition of NF-�B Activation by

Nimbolide Is Not Cell-specific—Wenext determined whether the nim-bolide-mediated inhibition in TNF-�-induced NF-�B activation is celltype-specific. The inhibitory effectof nimbolide on TNF-�-inducedNF-�B activation was observed notonly inKBM-5 cells but also in otherleukemic cells (U-937, HL-60, Jur-kat, K-562; Fig. 4F) and in MCF-7,H-1299, and A293 cells (Fig. 4G).Nimbolide Does Not Directly Af-

fect the Binding of NF-�B to DNA—Some NF-�B inhibitors, such asplumbagin (38), L-phenylalaninechloromethyl ketone (a serine pro-tease inhibitor) (25), and caffeic acidphenethyl ester (39), directlymodifyNF-�B to suppress its DNAbinding.We determined whether nimbolidemediates suppression of NF-�Bactivation through a similar mecha-nism using EMSA. Nimbolide didnotmodify the DNA-binding abilityof NF-�B proteins prepared fromTNF-�-treated cells (Fig. 5A), sug-gesting that it inhibits NF-�B acti-vation using a mechanism differentfrom that of plumbagin, L-phenyl-alanine chloromethyl ketone, andcaffeic acid phenethyl ester.Nimbolide Inhibits TNF-�-de-

pendent I�B� Phosphorylation andDegradation—I�B� is the inhibi-tory subunit present in the NF-�Bcomplex. The translocation of NF-�B to the nucleus is preceded bythe phosphorylation, ubiquitina-tion, and proteolytic degradation ofI�B�. To determine whether theinhibition of TNF-�-induced NF-�B activation was due to suppres-sion of I�B� degradation, we pre-treated KBM-5 cells with nimbolide

and exposed them to TNF-� for 0–60 min. We then analyzedthe DNA binding of NF-�B in the nuclear fraction by EMSAand I�B� degradation in the cytoplasmic fraction by Westernblotting. TNF-� induced NF-�B in control cells within 5 min,and the level plateaued after 30 min. However, in the cells pre-treated with nimbolide, TNF-� was unable to induce NF-�Bactivation (Fig. 5B). TNF-� induced I�B� degradation in

FIGURE 2. Nimbolide potentiates the apoptotic effects of TNF-� and chemotherapeutic agents in chronicmyeloid leukemic and multiple myeloma cells. A, nimbolide enhances TNF-�-induced apoptosis. KBM-5and U266 cells were pretreated with the indicated concentrations of nimbolide for 4 h and then incubated with1 nM TNF-� for 24 h. The cells were stained with a live/dead assay reagent for 30 min and analyzed under afluorescence microscope. Values below each photomicrograph represent percent apoptotic cells. B, nimbolideenhances TNF-�-, 5-fluorouracil-, and thalidomide-induced cytotoxicity. We seeded 5000 cells in four repli-cates, pretreated them with the indicated concentrations of nimbolide, and then incubated them with che-motherapeutic agents (1 nM TNF-�, 0.1 �M 5-fluorouracil, and 10 �g/ml thalidomide) for 24 h. Cell viability wasanalyzed by the MTT assay. C, nimbolide potentiates TNF-�-induced early apoptosis in KBM-5 cells, as deter-mined by the annexin V assay. Cells were pretreated with 2.5 �M nimbolide for 4 h and then incubated with 1nM TNF-� for 24 h. The cells were incubated with an fluorescein isothiocyanate-conjugated annexin V antibodyand then analyzed using flow cytometry. D, nimbolide induces TNF-�-induced caspase activation. Cells wereincubated with 2.5 �M nimbolide for 4 h and then treated with 1 nM TNF-� for 24 h. Whole-cell extracts wereprepared and analyzed by Western blotting, using the indicated antibodies. E, nimbolide induces PARP cleav-age. Cells were pretreated with 2.5 �M nimbolide for 4 h and then incubated with 1 nM TNF-� for the indicatedtimes. Whole-cell extracts were prepared and analyzed by Western blotting using an anti-PARP antibody.Figures are representative of one of three independent experiments. The values in the histograms representthe means � S.D. of three independent replicates. * indicates the significance of difference compared withTNF-� group; p � 0.05. NL, nimbolide; 5-FU, 5-fluorouracil.




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KBM-5 cells after 10 min, and the maximum level of degrada-tionwas seen after 30min; I�B�was resynthesized after 60min.In nimbolide-pretreated cells, the TNF-�-induced I�B� degra-dation was completely suppressed (Fig. 5C). These results indi-cate that nimbolide inhibits both TNF-�-induced NF-�B acti-vation and I�B� degradation.To determine whether the inhibition of TNF-�-induced

I�B� degradation was due to inhibition of I�B� phosphoryla-tion, we used the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal to block I�B� degradation. Western blotting withan antibody that recognized the phosphorylated form of I�B�(Ser32 and Ser36) revealed that TNF-� induced the phosphor-

ylation of I�B�, and nimbolidecompletely suppressed this phos-phorylation (Fig. 5D). These resultsindicate that nimbolide inhibitsTNF-�-induced I�B� degradationby inhibiting I�B� phosphorylation.Nimbolide Directly Inhibits TNF-

�-induced IKK Activation—TheTNF-�-induced phosphorylationof I�B� is mediated through IKK.The inhibition in TNF-�-inducedI�B� phosphorylation by nimbolideprompted us to determine the effectof nimbolide on IKK activation. Akinase assay revealed that TNF-�activated IKK in a time-dependentmanner and that nimbolide com-pletely suppressed this activation(Fig. 5E). Neither TNF-� nor nim-bolide had any effect on the expres-sion of IKK-� or IKK-� proteins.To determine whether nimbolide

suppresses IKK activity directly orindirectly by suppressing its activa-tion, we incubated an immunecomplex prepared from TNF-�-stimulated cells with various con-centrations of nimbolide and thenassayed for IKK activity. Nimbolideinhibited IKK activity (Fig. 5F), sug-gesting that it directly modulatesTNF-�-induced IKK activation.Nimbolide Binds to and Inhibits

IKKActivation—Because the IKK-�subunit of the IKK complex con-tains various cysteine residues, wehypothesized that nimbolide mayinhibit IKK through direct modifi-cation of one or more of these cys-teine residues. We used a reducingagent, dithiothreitol (DTT), toinvestigate whether the modula-tion of IKK activity by nimbolidewas caused by the oxidation ofcritical cysteine residues. Theaddition of DTT to the kinase

reaction mixture reversed the nimbolide-mediated inhibi-tion of TNF-�-induced IKK activity (Fig. 5G), suggestingthat a cysteine residue is involved in the pathway. IKK-�contains a cysteine at position 179 in its activation loop thatis critical for its activity. To determine whether this residueis involved in nimbolide-mediated IKK inhibition, we trans-fected A293 cells with wild-type FLAG-IKK-� or FLAG-IKK-� with a C179A mutation. Nimbolide inhibited wild-type IKK-�. In contrast, nimbolide had no apparent effect onthe activity of the mutated IKK-� (Fig. 5H). These findingssuggest that nimbolide inhibits IKK-� activity by modifyingCys179.

FIGURE 3. Nimbolide inhibits TNF-�-induced expression of NF-�B-dependent anti-apoptotic, prolifera-tive, and metastatic proteins. KBM-5 cells were incubated with 2.5 �M nimbolide for 4 h and then treated with1 nM TNF-� for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting usingantibodies against anti-apoptotic (A), proliferative (B), and metastatic proteins (C). One of the three indepen-dent experiments is shown. D, nimbolide down-regulates IL-6 and TNF-� production in U266 cells. The cells(2 � 106) were treated with the indicated concentrations of nimbolide, and cell-free supernatants were har-vested after 24 h. The levels of IL-6 and TNF-� were detected by ELISA. E, nimbolide suppresses TNF-� inducedcell invasion. H1299 cells (2.5 � 104 cells) were seeded into the top chamber of a Matrigel invasion chambersystem overnight in the absence of serum and then treated with 2.5 �M nimbolide. After incubation for 4 h, thecells were treated with TNF-� in the presence of 1% serum and then assayed for invasion as described under“Experimental Procedures.” The value for control was set to 1.0. The values given in histograms represent themeans � S.D. of three independent replicates. * and # indicates the significance of difference compared withcontrol and TNF-� group, respectively; p � 0.05. NL, nimbolide.




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Nimbolide Inhibits TNF-�-induced p65 Nuclear Transloca-tion and Phosphorylation—I�B� degradation is required forthe nuclear translocation of p65. Thus, we determined whethernimbolide can affect TNF-�-induced p65 nuclear transloca-tion. An immunocytochemical analysis revealed that TNF-�induced the translocation of p65 to the nucleus in KBM-5 cells,whereas pretreatment of cells with nimbolide suppressed thistranslocation. In untreated cells and cells treated with nimbol-ide alone, p65 was localized to the cytoplasm. These resultsconfirmed that nimbolide inhibited the TNF-�-induced p65nuclear translocation (Fig. 6A).The effect of nimbolide on TNF-�-induced p65 nuclear

translocation was further confirmed by Western blot analysis.TNF-�-induced p65 translocation in a time-dependent man-ner, and nimbolide suppressed this translocation (Fig. 6B). Inaddition, phosphorylation of p65 is required for its transcrip-tional activity. Thus, wemeasured levels of phosphorylated p65in cells treated with and without nimbolide.We found that p65phosphorylation at Ser536 occurred in a time-dependent man-ner in TNF-�-treated cells but not in cells that had also beentreated with the limonoid (Fig. 6B).Nimbolide Represses TNF-�-induced NF-�B-dependent Re-

porter Gene Expression—Using DNA binding assays, we dem-onstrated that nimbolide inhibits NF-�B activation; however,DNA binding alone is not always associated with NF-�B-de-pendent gene transcription, suggesting that additional regula-tory steps are involved. Therefore, we determined whethernimbolide affects TNF-�-induced reporter gene transcription.We transiently transfected A293 cells with anNF-�B-regulatedSEAP reporter construct, pretreated the cells with nimbolide,and stimulated them with TNF-�. We observed 8.2 timeshigher SEAP activity than in cells expressing the vector control;this induction was completely abolished by the dominant neg-ative I�B�, indicating the specificity of the assay. When cellswere pretreated with nimbolide, the TNF-�-induced NF-�B-dependent SEAP expression was inhibited in a dose-dependentmanner (Fig. 6C). These results indicate that nimbolide inhibitsthe NF-�B-dependent reporter gene expression induced byTNF-�.

Because TNF-�-induced NF-�B activation is mediatedthrough the sequential interaction of TNFR1 with TRADD,TRAF2, TAK1, and IKK-�, we determined the site of action fornimbolide in the TNF-� signaling pathway. A SEAP assayrevealed that TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, andIKK-� plasmids significantly induced reporter gene expression,and nimbolide treatment substantially suppressed NF-�B-de-pendent gene expression (Fig. 6D).

DISCUSSION

The objective of this study was to determine whether nim-bolide, a triterpenoid isolated from a traditionally used plantcalled neem, exhibits its activities through modulation of theNF-�B activation pathway, which has been associated withinflammation, tumor cell survival, proliferation, invasion, andangiogenesis (40). We found that nimbolide inhibited the pro-liferation of various tumor cells; potentiated the apoptosisinduced by the inflammatory cytokine TNF-� and chemother-apeutic agents; suppressed the expression of NF-�B-dependentgene products involved in cell survival, cell proliferation,metastasis, and angiogenesis; and suppressed the production ofcytokines. These effects were mediated through the suppres-sion of constitutive and inducible NF-�B activation by inhibi-tion of IKK activation, leading to the suppression of I�B� phos-phorylation and degradation, and p65 nuclear translocationand phosphorylation.This is the first report to suggest that nimbolide can in-

hibit the NF-�B pathway. Among all the cytokines, TNF-� isperhaps the most potent activator of NF-�B, a cell survival fac-tor. TNF-�, however, is known to activate both the NF-�B andapoptosis pathways simultaneously. Thus, suppression of theTNF-�-induced NF-�B activation potentiated TNF-�-inducedapoptosis in tumor cells. Similarly, apoptosis induced by che-motherapeutic agents, such as 5-fluorouracil and thalidomide,was also enhanced by the limonoid.Although the pro-apoptoticeffects of the limonoid have been reported previously, to ourknowledge, this is the first report of its effects in combinationwith cytokines or chemotherapeutic agents in tumor cells.We also found that nimbolide suppressed the NF-�B-regu-

lated expression of anti-apoptotic genes, including Bcl-2, Bcl-xL, IAP-1, and IAP-2. Overexpression of these genes has beenassociated with tumor cell survival, chemoresistance, andradioresistance in numerous tumor cell types. Suppression ofNF-�B activation by nimbolide was concomitant with the sup-pression of these gene products, which may account for thepotentiation of apoptosis induced by TNF-� and chemothera-peutic agents. This is in agreement with the findings of a previ-ous report that demonstrated that nimbolide exhibits pro-ap-optotic effects through modulation of the cell survival proteinBcl-2 (18). Furthermore, an increase in caspase-3, -8, and -9cleavage products suggests that the flavonone potentiates apo-ptosis through both extrinsic and intrinsic pathways.Our results also show for the first time that nimbolide sup-

presses the expression of NF-�B-regulated genes involved incell proliferation and metastasis. Down-regulation of the

FIGURE 4. Nimbolide down-regulates constitutive and TNF-�-induced NF-�B activation in different cell lines. Dose-dependent (A) and time-dependent(B) effects of nimbolide on TNF-�-induced NF-�B activation. KBM-5 cells were incubated with the indicated concentrations of nimbolide for 4 h (A) or with 10�M nimbolide for the indicated times (B) and then treated with 0.1 nM TNF-� for 30 min. Nuclear extracts were prepared and assayed for NF-�B activation usingEMSA. The fold activation of NF-�B as compared with control and percentage of viable cells (CV) are shown at the bottom. C, TNF-�-induced NF-�B is composedof p65 and p50 subunits. Nuclear extracts from untreated cells or cells treated with 0.1 nM TNF-� were incubated with the indicated antibodies, an unlabeledNF-�B oligoprobe, or a mutant oligoprobe. They were then assayed for NF-�B activation by EMSA. D, nimbolide inhibits NF-�B activation induced by CSC, OA,PMA, and LPS. KBM-5 cells were incubated with 5 and 10 �M nimbolide for 4 h and then treated with 10 �g/ml CSC for 1 h, 500 nM OA for 4 h, 100 ng/ml PMAfor 2 h, or 10 �g/ml LPS for 30 min. Nuclear extracts were analyzed for NF-�B activation using EMSA. The fold activation of NF-�B as compared with control isshown at the bottom. E, effect of nimbolide on constitutive NF-�B activation. U266, MM.1S, RPMI-8226, and SCC-4 cells were incubated with nimbolide at theindicated concentrations for 4 h. Nuclear extracts were prepared and analyzed for NF-�B activation by EMSA. F and G, effect of nimbolide on TNF-�-inducedNF-�B activation in other types of cells. U-937, HL-60, Jurkat, K-562, MCF-7, H-1299, and A293 cells were incubated with 10 �M nimbolide for 4 h and then treatedwith 0.1 nM TNF-� for 30 min. Nuclear extracts were assayed for NF-�B activation using EMSA. The fold activation of NF-�B as compared with control is shownat the bottom. One of three independent experiments is shown. NL, nimbolide.




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expression of the gene products involved in cell proliferation(c-Myc, cyclin D1, and COX-2) by nimbolide was concomitantwith an inhibition of cell proliferation in various tumor cells.This result is further supported by a previous report thatshowed the ability of nimbolide to suppress the proliferation of

a wide variety of cancer cells (12, 18, 19). In addition, the abilityof nimbolide to suppress the TNF-�-induced expression ofICAM-1 and MMP-9 suggests that it has a role in preventingmetastasis and tumor invasion. This result was further con-firmed by an earlier study showing that the nimbolide sup-

FIGURE 5. Down-regulation of TNF-�-induced NF-�B activation by nimbolide involves cysteine 179 of IKK. A, direct effect of nimbolide on NF-�B-DNAbinding. Nuclear extracts prepared from untreated cells or cells treated with 0.1 nM TNF-� were incubated for 30 min with the indicated concentrations ofnimbolide in vitro. The effect of nimbolide on NF-�B-DNA binding was then analyzed by EMSA. B, nimbolide inhibits the TNF-�-induced activation of NF-�B.KBM-5 cells were preincubated with 10 �M nimbolide for 4 h, treated with 0.1 nM TNF-� for the indicated times, and then analyzed for NF-�B activation by EMSA.The fold activation of NF-�B as compared with control is indicated at the bottom. C, nimbolide inhibits TNF-�-induced degradation of I�B�. KBM-5 cells wereincubated with 10 �M nimbolide for 4 h and treated with 0.1 nM TNF-� for the indicated times. Cytoplasmic extracts were prepared and analyzed for I�B� byWestern blotting. D, nimbolide inhibits TNF-�-induced phosphorylation of I�B�. KBM-5 cells were treated with 10 �M nimbolide for 4 h, incubated with 50�g/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 min, and then treated with 0.1 nM TNF-� for 10 min. Cytoplasmic extracts were analyzed by Westernblotting using a phospho-specific I�B� antibody (Ser32/36). The same membrane was reprobed with I�B� and �-actin antibody. E, effect of nimbolide onTNF-�-induced IKK activation. KBM-5 cells were preincubated with 10 �M nimbolide for 4 h and then treated with 1 nM TNF-� for the indicated times. Whole-cellextracts were immunoprecipitated with an antibody against IKK-� and analyzed using an immune complex kinase assay. The effect of nimbolide on IKK proteinexpression was determined by Western blot analysis using anti-IKK-� and anti-IKK-� antibodies. F, direct effect of nimbolide on IKK activation induced byTNF-�. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nM TNF-� and immunoprecipitated with an anti-IKK-� antibody. The immunopre-cipitated complex was incubated with the indicated concentrations of nimbolide, and an immunocomplex kinase assay was performed. G, nimbolide-inducedsuppression of TNF-�-induced IKK activation was reversed by the reducing agent DTT. Assays were performed as indicated in Fig. 4F, except that IKK activitywas also determined in the presence of DTT (100 �M). H, kinase activity of mutated IKK (C179A) is unaffected by nimbolide. A293 cells were transfected withwild-type FLAG-IKK-� (IKK-� WT) or mutated FLAG-IKK-� (IKK-� MT (C179A)). Whole-cell extracts were prepared, immunoprecipitated, incubated with nimbol-ide, and subjected to an IKK assay. One of three independent experiments is shown. NL, nimbolide.




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presses the TNF-�-induced invasion activity of cancer cells. Inaddition, we showed that nimbolide inhibits TNF-�-inducedVEGF expression. VEGF is a specific mitogen for vascular

endothelial cells and is involved incarcinogenesis as an angiogenic fac-tor. These results are in agreementwith those of a recent report thatdemonstrated that this limonoidhas anti-invasive and anti-angio-genic activities in cancer cells,although the mechanism by whichthis occurs has not been described(19).Nimbolide has been shown to

suppress DMBA-induced carcino-genesis in an animal model of oraloncogenesis (18). However, theauthors of that study did not reportits mechanism of suppression.Because DMBA has been shown toactivate NF-�B (41), the inhibitoryeffects of nimbolide on the NF-�Bactivation pathway could alsoaccount for its anticarcinogenicactivity.We found that nimbolide targets

IKK to suppress the TNF-�-in-duced phosphorylation and degra-dation of I�B� that is concomitantwith the inhibition of nuclear trans-location and phosphorylation ofp65. We showed that nimbolidedirectly binds to and inhibits TNF-�-activated IKK. Furthermore, theaddition of a reducing agent (DTT)reversed the effects of nimbolide onIKK activation, suggesting theinvolvement of cysteine residues.Indeed, the mutation of residueCys179 of IKK-� to alanine abol-ished the inhibitory effect of nim-bolide on IKK activation. Theseresults indicate that despite theinvolvement of more than 50 differ-ent proteins in theNF-�B activationpathway (42), nimbolide exerts itsinhibitory effect by modifyingCys179 in the IKK-� activation loop.

Previous reports have shown thatCys179 of IKK-� is prone to redoxrecycling (43), which results in thegeneration of reactive oxygen spe-cies. Indeed, nimbolide has beenknown to induce the production ofreactive oxygen species (17). How-ever, because nimbolide inhibitedIKK both in vitro and in vivo, it isunlikely that its effects are mediated

through the generation of reactive oxygen species. Our resultssuggest that nimbolide modifies Cys179. An �- and �-unsatur-ated ketone group of nimbolide has been linked to its antican-

FIGURE 6. Nimbolide inhibits TNF-�-induced phosphorylation and nuclear translocation of p65 andNF-�B-dependent reporter gene expression induced by TNF-� and different molecules in the NF-�B-signaling pathway. A, immunocytochemical analysis of p65 localization. KBM-5 cells were treated with 10 �M

nimbolide for 4 h, exposed to 0.1 nM TNF-� for 15 min, and analyzed for p65 localization. B, KBM-5 cells wereuntreated or pretreated with 10 �M nimbolide for 4 h and then treated with 0.1 nM TNF-� for the indicatedtimes. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against p65 andphospho-p65 (Ser536). PARP was used as an internal control. Figures are representative of one of three inde-pendent experiments. C, nimbolide inhibits TNF-�-induced NF-�B-dependent SEAP expression. A293 cellswere transiently transfected with an NF-�B-containing plasmid linked with the SEAP gene. Cells were treatedwith nimbolide for 4 h at the indicated concentrations, followed by 1 nM TNF-� for 24 h. Cell supernatants werecollected and assayed for SEAP activity. The results are expressed as the change in activity relative to the vectorcontrol. DN, dominant negative. D, nimbolide inhibits NF-�B-dependent reporter gene expression induced byTNF-�, TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-�. A293 cells were transfected with pNF-�B-SEAPplasmid, expression plasmid, and control plasmid for 24 h and treated with nimbolide. The cell supernatantswere then assayed for SEAP activity. Where indicated, the cells were exposed to 1 nM TNF-� for 12 h. The resultsare expressed as the change in activity relative to the vector control. Values given are the mean � S.D. of threeindependent replicates. * and # indicate the significance of difference compared with control and TNF-�/plasmid alone group respectively; p � 0.05. NL, nimbolide.




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cer activity (20). Thus, it is possible that this ketone group inter-acts with the cysteine residue of IKK. In addition, nimbolideappears to specifically target Cys179 of IKK-�, as it did not inter-fere with the DNA binding ability of NF-�B through directinteraction with the cysteine residue present in the p65 subunit(39).The IKK complex is an important site for the integration

of signals that regulate the NF-�B pathway. Several agents,including aspirin, sulindac, cyclopentenone prostaglandins,cytochrome P450 epoxygenase-derived eicosanoids, andviral proteins, have been shown to inhibit the NF-�B path-way through IKK (44–46), but their mechanisms of inhibi-tion remain elusive. Our results reveal the critical role of acysteine residue in the activation loop of IKK-�. This same res-idue is targeted by nitric oxide (22), butein (24), cobrotoxin(23), and xanthohumol (26). Given the relevance of NF-�B incancer, our data suggest that IKK is a highly attractive target for

therapeutic development. Some ofthe selective small molecule inhibi-tors of the IKK complex that havebeen recently developed by the phar-maceutical industry include PS-1145,BMS-345541, and AS602828 (47,48).We also found that nimbolide

inhibited constitutive and inducibleNF-�B activation in myeloid andleukemic cells. Constitutively acti-vated NF-�B has been found to becritical for the survival and prolifer-ation of various tumor cell types(49). Some of the potential mecha-nisms of constitutive NF-�B activa-tion are I�B� overexpression, I�B�gene mutations, enhanced I�B�degradation, constitutive IKKactivation, and constitutive ex-pression of inflammatory cyto-kines (33, 49, 50). We found thatnimbolide inhibits the constitu-tive secretion of IL-6 and TNF-�by U-266 cells, which couldaccount for its inhibition of con-stitutive NF-�B activation andsuppression of cell proliferation.The suppression ofNF-�B activa-

tion induced by CSC, TNF-�, OA,PMA, and endotoxin suggests thatnimbolide may act at a step com-mon to all these activators. TNF-�activates NF-�B through thesequential recruitment of TNFR1,TRADD, TRAF2, NIK, TAK1/TAB1, and IKK. Nimbolide sup-pressed the activation of NF-�Binduced by overexpression of theseintermediates.In summary, our results suggest

that nimbolide exhibits anti-carcinogenic, anti-proliferative,anti-inflammatory, and apoptotic effects through the sup-pression of the IKK-induced NF-�B pathway, which is acti-vated by a wide variety of carcinogens and inflammatoryagents. On the basis of our findings, we propose that nim-bolide inhibits IKK by modifying Cys179, which blocksNF-�B activation and the transcription of NF-�B-regulatedgene products (Fig. 7). This limonoid may exhibit activitiesagainst human myeloid, leukemia, lymphoma, and other typesof cancer. However, further studies in animals and humans willbe required to determine fully the potential of this fascinatingmolecule for cancer prevention or treatment.

Acknowledgments—We thankKateNewberry for carefully proofread-ing the manuscript and Drs. Veera Baladandayuthapani and WeiQiao for statistical advice.

FIGURE 7. A schematic diagram showing nimbolide (NL) activity in the TNF-�-induced NF-�B signalingpathway.




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Nair and Bharat B. AggarwalYadav, Jayaraj Ravindran, Padmanabhan S. Hema, Madan M. Chaturvedi, Mangalam Subash C. Gupta, Sahdeo Prasad, Simone Reuter, Ramaswamy Kannappan, Vivek R.

Sensitization of Tumor Cells to Chemotherapeutic AgentsB-regulated Cell Survival and Proliferative Proteins andκDown-regulation of NF-

Kinase by Nimbolide Leads toαBκModification of Cysteine 179 of I

doi: 10.1074/jbc.M110.161984 originally published online September 9, 20102010, 285:35406-35417.J. Biol. Chem.

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VOLUME 285 (2010) PAGES 35406 –35417DOI 10.1074/jbc.A110.161984

Modification of cysteine 179 of I�B� kinase bynimbolide leads to down-regulation of NF-�B-regulatedcell survival and proliferative proteins and sensitizationof tumor cells to chemotherapeutic agents.Subash C. Gupta, Sahdeo Prasad, Simone Reuter, Ramaswamy Kannappan,Vivek R. Yadav, Jayaraj Ravindran, Padmanabhan S. Hema, Madan M. Chaturvedi,Mangalam Nair, and Bharat B. Aggarwal

PAGE 35409:

It has been brought to our attention that the nimbolide structurepresented in Fig. 1A of the above manuscript was incorrectly repre-sented. A methyl group at C4 is missing. The correct structure of nim-bolide is shown below. The authors regret and apologize for any confu-sion and inconvenience due to this error.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 15, p. 12152, April 6, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

12152 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 15 • APRIL 6, 2012

ADDITIONS AND CORRECTIONS

Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

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