CHEMOKINES, CYTOKINES, AND INTERLEUKINS

Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histoneacetyltransferase, suppresses expression of nuclear factor-�B–regulated geneproducts involved in cell survival, proliferation, invasion, and inflammationthrough inhibition of the inhibitory subunit of nuclear factor-�B� kinase, leadingto potentiation of apoptosisBokyung Sung,1 Manoj K. Pandey,1 Kwang Seok Ahn,1 Tingfang Yi,2 Madan M. Chaturvedi,1 Mingyao Liu,2 andBharat B. Aggarwal1

1Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston; and 2Institute ofBiosciences and Technology, Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston

Anacardic acid (6-pentadecylsalicylic acid)is derived from traditional medicinal plants,such as cashew nuts, and has been linkedto anticancer, anti-inflammatory, and radio-sensitization activities through a mecha-nism that is not yet fully understood. Be-cause of the role of nuclear factor-�B (NF-�B) activation in these cellular responses,we postulated that anacardic acid mightinterfere with this pathway. We found thatthis salicylic acid potentiated the apoptosisinduced by cytokine and chemotherapeuticagents, which correlated with the down-regulation of various gene products that

mediate proliferation (cyclin D1 andcyclooxygenase-2), survival (Bcl-2, Bcl-xL,cFLIP, cIAP-1, and survivin), invasion (ma-trix metalloproteinase-9 and intercellular ad-hesion molecule-1), and angiogenesis (vas-cular endothelial growth factor), all knownto be regulated by the NF-�B. We found thatanacardic acid inhibited both inducible andconstitutive NF-�B activation; suppressedthe activation of I�B� kinase that led toabrogation of phosphorylation and degrada-tion of I�B�; inhibited acetylation andnuclear translocation of p65; and sup-pressed NF-�B–dependent reporter gene ex-

pression. Down-regulation of the p300 his-tone acetyltransferase gene by RNAinterference abrogated the effect of anac-ardic acid on NF-�B suppression, suggest-ing the critical role of this enzyme. Overall,our results demonstrate a novel role foranacardic acid in potentially preventingor treating cancer through modulationof NF-�B signaling pathway. (Blood.2008;111:4880-4891)

© 2008 by The American Society of Hematology

Introduction

Traditional medicine that has been practiced for thousands of yearsis generally considered safe, but what the medicine consists of andhow it mediates its effects may not be understood. For instance,Amphipterygium adstringens (family Anacardiaceae) is a tree, thebark of which (locally called “cuachalate”) is widely used inMexico for treatment of gastric ulcers, gastritis, and stomachcancers.1,2 The active and possibly anti-inflammatory component inthis plant has been identified as 6-pentadecylsalicylic acid, oranacardic acid (Figure 1A).1 The same compound has also beenidentified in Ozoroa insignis (an African medicinal plant3), Anacar-dium occidentale (the cashew nut4), and Ginkgo biloba (Gink-goaceae; an Asian medicine5). The active principle has beenassociated with molluscicidal activity6 and antimicrobial activity.7,8

How anacardic acid mediates these effects is not fully understood,but it has been shown to have antioxidant activity9 and to inhibit theactivity of numerous enzymes, including tyrosinase,4 xanthineoxidase,10 phosphatidylinositol-specific phospholipase C,11 histoneacetyltransferase (HAT),12-14 tissue factor VIIa,15 lipoxygenase, andcyclooxygenase (COX).16-18 This compound has also been shownto be a mitochondrial uncoupler of oxidative phosphorylation.19

Anacardic acid exhibits antitumor activity3,5 and sensitizes tumor

cells to ionizing radiation.13 However, it is not fully understoodhow this salicylic acid mediates antitumor, radiosensitization, andanti-inflammatory activities.

One possible mechanism by which anacardic acid exerts itseffects is by modulating the nuclear factor-�B (NF-�B) pathwaycommonly involved in tumorigenesis, inflammation, and radiosen-sitization. The transcription factor NF-�B resides in the cytoplasmin its resting stage and then translocates to the nucleus and mediatestranscription of various gene products when it is activated. Variousinflammatory agents induce NF-�B activation, including cytokines(eg, tumor necrosis factor [TNF]), carcinogens, tumor promoters,cigarette smoke, environmental pollutants, ionizing radiation, andstress. NF-�B activation has been shown to control the expressionof more than 400 different gene products that have been linked withinflammation, tumor cell transformation, survival, proliferation,invasion, angiogenesis, metastasis, chemoresistance, and radioresis-tance. Thus, suitable inhibitors of NF-�B activation are activelybeing searched.

Because of the critical role of NF-�B in tumorigenesis,20

radiosensitization,21 and inflammation,22 in this study, we tested thehypothesis that anacardic acid may mediate its effects through

Submitted October 11, 2007; accepted March 13, 2008. Prepublished online asBlood First Edition paper, March 18, 2008; DOI 10.1182/blood-2007-10-117994.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2008 by The American Society of Hematology

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modulation of the NF-�B pathway. Our results demonstrated thatanacardic acid could suppress NF-�B activated by inflammatorycytokines, growth factors, and tumor promoters through the

inhibition of inhibitory subunit of NF-�B (I�B�) kinase, leadingto suppression of NF-�B–regulated gene products and potentiationof apoptosis.

Figure 1. Anacardic acid potentiates apoptosis induced by TNF and chemotherapeutic agents. (A) Structure of anacardic acid (AA). (B) Anacardic acid potentiatesTNF-induced apoptosis. KBM-5 cells were pretreated with 25 �mol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. The cells were stained withthe Live/Dead assay reagent for 30 minutes and then analyzed under a fluorescence microscope. The results shown are representative of 3 independent experiments.(C) Cells were pretreated with 25 �mol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for 16 hours. Cells were incubated with an anti–annexin V antibodyconjugated with FITC and then analyzed by flow cytometry for early apoptotic effects. (D) Cells were pretreated with 25 �mol/L anacardic acid for 4 hours and then incubatedwith 1 nmol/L TNF for 16 hours. Cells were fixed, stained with TUNEL assay reagent, and then analyzed by flow cytometry for apoptotic effects. (E) KBM-5 cells were incubatedwith 25 �mol/L anacardic acid for 4 hours and then treated with 1 nmol/L TNF for 24 hours. Whole-cell extracts were prepared and analyzed by Western blotting using theindicated antibodies. (F) Cells were pretreated with 25 �mol/L anacardic acid for 4 hours and then incubated with 1 nmol/L TNF for the indicated times. Whole-cell extracts wereprepared and subjected to Western blot analysis using an anti-PARP antibody.

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Methods

Reagents

A 25-mmol/L solution of anacardic acid (Calbiochem, San Diego, CA) wasprepared in 100% dimethyl sulfoxide, stored at-20°C, and then diluted asneeded in cell culture medium. Bacteria-derived recombinant humanTNF-� was kindly provided by Genentech (South San Francisco, CA).Penicillin, streptomycin, Iscove modified Dulbecco medium (IMDM),Dulbecco modified Eagle medium (DMEM), RPMI 1640 medium, and fetalbovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA).Phorbol 12-myristate 13-acetate (PMA), okadaic acid (OA), lipopolysaccha-ride (LPS), interleukin-1� (IL-1�), epidermal growth factor (EGF), andanti–�-actin antibody were purchased from Sigma-Aldrich (St Louis, MO).HAT p300 siRNA was purchased from Dharmacon (Lafayette, LA).Antibodies against p65, p50, inhibitory subunit of NF-�B (I�B�), cyclinD1, matrix metalloproteinase-9 (MMP-9), poly(ADP-ribose) poly-merase (PARP), inhibitor-of-apoptosis protein 1 (IAP1), Bcl-2, Bcl-xL,c-myc, caspase-3, caspase-8, caspase-9 and intercellular adhesion molecule1 (ICAM-1), and the annexin V staining kit were purchased from SantaCruz Biotechnology (Santa Cruz, CA). Anti-vascular endothelial growthfactor (VEGF) was purchased from NeoMarkers (Fremont, CA). Anti-survivin antibody was purchased from R&D Systems (Minneapolis, MN).Anti-COX-2 and anti–X-linked inhibitor of apoptosis (XIAP) antibodieswere purchased from BD Biosciences (San Jose, CA). Anti–phospho-I�B� (serine 32/36) and anti–phospho-p65 (serine 536), cleavedcaspase-3, cleaved caspase-9, and acetylated-lysine (Ac-K-103) antibod-ies were purchased from Cell Signaling Technology (Danvers, MA).Anti-p300 antibody was purchased from Millipore (Billerica, MA).Anti-I�B� kinase (IKK)�, anti-IKK�, and anti-cellular caspase-8(FLICE)–like inhibitory protein (c-FLIP) antibodies were kindlyprovided by Imgenex (San Diego, CA).

Cell lines

Human myeloid KBM-5 cells, human T-cell lymphoma Jurkat cells, humanlung adenocarcinoma H1299 cells, human embryonic kidney A293 cells,human prostate cancer Du145 cells, and human squamous cell carcinomaSCC4 cells were purchased from the American Type Culture Collection(Manassas, VA). KBM-5 cells were cultured in IMDM supplemented with15% FBS. H1299, Du145, and Jurkat cells were cultured in RPMI 1640medium, and A293 cells were cultured in DMEM supplemented with 10%FBS. SCC4 cells were cultured in DMEM containing 10% FBS, nonessen-tial amino acids, pyruvate, glutamine, and vitamins. The mouse embryonicfibroblast (MEF) derived from p65�/� C57BL/6J mice and its wild typewere kindly provided by Dr David Baltimore (California Institute ofTechnology, Pasadena, CA). Cells were cultured in DMEM supplementedwith 10% FBS. All media were also supplemented with 100 U/mLpenicillin and 100 �g/mL streptomycin.

Electrophoretic mobility shift assay

To assess NF-�B activation by TNF, we performed electrophoretic mobilityshift assay (EMSA) essentially as described previously.23

Western blot analysis

To determine the levels of protein expression in the cytoplasm or nucleus,we prepared extracts and fractionated them by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, theproteins were electrotransferred to nitrocellulose membranes, blotted withthe relevant antibody, and detected with an electrogenerated chemilumines-cence reagent (GE Healthcare, Chalfont St Giles, United Kingdom). Next,to determine the expression of gene products in whole-cell extracts oftreated cells (2 � 106 cells in 1 mL medium), 40 �g whole-cell lysate wasresolved by SDS-PAGE, electrotransferred to membrane, and then probedwith antibodies against various proteins.

IKK assay

To determine the effect of anacardic acid on TNF-induced IKK activation,we analyzed IKK essentially as described previously.24

NF-�B–dependent reporter gene expression assay

The effect of anacardic acid on TNF-induced NF-�B–dependent reportergene transcription in A293 cells was measured as described previously.24

Down-regulation of p300 histone acetyltransferase by siRNA

A293 cells (2 � 105) were plated in each well of 6-well plates and allowedto adhere for 24 hours. On the day of transfection, 12 �L HiPerFecttransfection reagent (Qiagen, Valencia, CA) was added along with 50 and100 nmol/L siRNA or control scrambled siRNA in 100 �L culture medium.After 48 hours of transfection, cells were recovered and used for appropri-ate determinations.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assay was performed as described previ-ously25 with some modification. KBM-5 (2 � 107 cells) were incubatedwith 25 �mol/L anacardic acid for 4 hours and then treated with 1 nmol/LTNF for the indicated times. Cells were then cross-linked with formalde-hyde, quenched with glycine, resuspended in SDS lysis buffer (1% SDS,10 mmol/L ethylenediaminetetraacetic acid [EDTA], and 50 mmol/Ltris(hydroxymethyl)aminomethane [Tris]-HCl, pH 8.0, with protease inhibi-tors, pH 8.0), sonicated on ice and centrifuged at 4°C. Supernatants(400 �L) were diluted to a final volume of 4 mL in a mixture of 9 partsdilution buffer (1% Triton X-100, 150 mmol/L NaCl, 2 mmol/L EDTA, and20 mmol/L Tris-HCl, with protease inhibitors, pH 8.0) and 1 part lysisbuffer. Mixtures were incubated with 4 �g anti-p65 antibody per samplewith rotation at 4°C overnight and then incubated with 100 �L protein Abeads at 4°C for 4 hours. After gentle centrifugation (2000 rpm), beads wereresuspended in 1 mL wash buffer (1% Triton X-100, 0.1% SDS,150 mmol/L NaCl, 2 mmol/L EDTA, and 20 mmol/L Tris-HCl, withprotease inhibitors, pH 8.0), washed 3 times. Finally immunocomplexeswere washed with buffer (1% Triton X-100, 0.1% SDS, 500 mmol/L NaCl,2 mmol/L EDTA, and 20 mmol/L Tris-HCl, pH 8.0, with proteaseinhibitors). The immune complexes were eluted with elution buffer (1%SDS, 100 mmol/L NaHCO3) followed by incubation with proteinase K andRNase A (500 �g/mL each) at 37°C for 30 minutes. Reverse cross-linkswere performed by placing the tubes at 65°C overnight. Immunoprecipi-tated DNA was extracted and dissolved in sterile water. PCR analyses werecarried out for 39 cycles with primers: 5�-TCTGGCGGAAACCTGT-GCGCTGG-3� (forward) and 5�-AAATTGCGTAAGCCCGGTGGG-3� (re-verse) for human COX-2 (the amplified fragment (�291� �120) containsthe NF-�B binding sites of �221-GGGACTACCC-�211),26,27 and5�-CAGTGGAATTCCCCAGCCTTGCCT-3� (forward, boldface indicatesNF-�B binding sites), 5�-CCACACTCCAGGCTCTGTC CTC-3� (reverse) forthe DNA fragment (��604� �489) of human MMP-9.28 Real-time polymer-ase chain reaction (PCR) was performed with RT2 Real-time SYBR Green/RoxPCR master mix (SuperArray, Frederick, MD).

Immunocytochemical analysis of NF-�B p65 localization

The effect of anacardic acid on the TNF-induced nuclear translocation ofp65 was examined by using an immunocytochemical method with anepifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan) and aPhotometrics Coolsnap CF color camera (Nikon, Lewisville, TX) asdescribed previously.29

Luciferase assay

The effect of anacardic acid on COX-2 promoter activity induced by TNFwas analyzed using a luciferase assay. A293 cells (2.5 � 105 cells/well)were seeded in 6-well plates. After overnight culture, the cells in each wellwere transfected with 0.5 �g of DNA consisting of COX-2 promoter-luciferase reporter by the calcium phosphate method. The COX-2 promoter(�375 59) was provided by Dr Xiao-Chun Xu (The University of Texas

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M. D. Anderson Cancer Center, Houston, TX). After 24 hours of transfec-tion, the cells were incubated with anacardic acid for 4 hours, then exposedto 1 nmol/L TNF for 20 hours and harvested. Luciferase activity wasmeasured using the luciferase assay system (Promega, Madison, WI) anddetected using the Victor3 microplate reader. (PerkinElmer Life andAnalytical Sciences, Waltham, MA).

Cytotoxicity assay

Cytotoxicity was assayed by the modified tetrazolium salt 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay asdescribed previously.29

Live/Dead assay

To assess cytotoxicity, we used the Live/Dead assay (Invitrogen), whichdetermines intracellular esterase activity and plasma membrane integrity. Inbrief, 2 � 105 cells were incubated with 25 �mol/L anacardic acid for4 hours and then treated with 1 nmol/L TNF for 16 hours at 37°C. Cellswere stained with the live and dead reagent (5 �mol/L ethidium homodimerand 5 �mol/L calcein-AM) and incubated at 37°C for 30 minutes. Cellswere analyzed under a fluorescence microscope (Labophot-2; Nikon).

Annexin V assay

To detect apoptosis, we used annexin V antibody conjugated with thefluorescent dye fluorescein isothiocyanate (FITC). In brief, 106 cells werepretreated with 25 �mol/L anacardic acid for 12 hours, treated with1 nmol/L TNF for 24 hours, and then subjected to annexin V staining. Cellswere washed, stained with FITC-conjugated anti–annexin V antibody, andthen analyzed with a flow cytometer (FACSCalibur; BD Biosciences).

Terminal deoxynucleotidyl transferase dUTP nick-end labelingassay

We assayed cytotoxicity by the terminal deoxynucleotidyl transferasedUTP nick-end labeling (TUNEL) method using an in situ cell death-detection reagent (Roche Pharmaceuticals, Belleville, NJ). In brief, 106

cells were pretreated with 25 �mol/L anacardic acid for 4 hours and with1 nmol/L TNF for 24 hours at 37°C. Thereafter, cells were incubated withreaction mixture for 60 minutes at 37°C. Stained cells were quantified byflow cytometry (FACSCalibur; BD Biosciences).

Results

Anacardic acid potentiated apoptosis induced by TNF andchemotherapeutic agents

Because the activation of NF-�B has been shown to inhibitapoptosis induced by TNF and chemotherapeutic agents,30,31 weinvestigated whether anacardic acid affects TNF- and chemo-therapeutic agent–induced apoptosis. As determined by theMTT method, anacardic acid enhanced cytotoxicity induced byTNF, cisplatin, and doxorubicin in variety of human cancer cell

lines (Table 1). To determine whether the enhancement ofcytotoxicity was due to an increase in apoptosis, we used theLive/Dead assay to detect intracellular esterase activity andplasma membrane integrity. This assay indicated that anacardicacid up-regulated TNF-induced apoptosis from 4% to 25%(Figure 1B). We also used annexin V staining to detect an earlystep in apoptosis, in which the membrane phospholipid phospha-tidylserine moves from the cell cytoplasmic interface to theextracellular surface. These results also indicated enhancementof TNF-induced apoptosis by anacardic acid (Figure 1C).Similar results were obtained with TUNEL staining, whichdetects DNA strand breaks (Figure 1D). Results from all theseassays together suggest that anacardic acid enhances the apopto-tic effects of TNF and chemotherapeutic agents.

Anacardic acid potentiated the TNF-induced caspase activation

TNF binds to the TNF receptor (TNFR1), which then sequen-tially recruits TNFR-associated death domain protein (TRADD),Fas-associated death domain (FADD), and FADD-like IL-1�–converting enzyme (FLICE; also called caspase-8), leading toactivation of caspase-9 and caspase-3.32 Whether anacardic acidaffects TNF-induced activation of caspases was investigated.We found that TNF alone had a minimal effect on activation ofcaspase-8, caspase-9, or caspase-3, whereas treatment withanacardic acid potentiated the activation as indicated by thecleaved products (Figure 1E).

The activated caspase-3 is thus known to induce PARP cleav-age. Whether anacardic acid activates TNF-induced PARP cleav-age was also examined. Results in Figure 1F show that whereasTNF and anacardic acid alone had minimal effect on PARPcleavage; 2 together were very effective in inducing cleavage ofPARP. These results suggest that anacardic acid enhances theapoptotic effects of TNF.

Anacardic acid suppressed the expression of antiapoptoticgene products

We investigated whether potentiation of TNF-induced apoptosis byanacardic acid is mediated through the down-regulation of cell-survival gene products. Western blot analysis showed that TNFinduced these antiapoptotic proteins in a time-dependent mannerand that anacardic acid suppressed this increase (Figure 2A). Thus,the enhancement of apoptosis by anacardic acid could be due todown-regulation of these antiapoptotic proteins.

Anacardic acid suppressed expression of gene productsinvolved in cell proliferation

Various gene products, including cyclin D1, c-myc, and COX-2,are induced by TNF and have been linked with proliferation of

Table 1. Anacardic acid potentiates apoptosis induced by TNF and chemotherapeutic agents

Cell lines

Apoptosis, %

Medium TNF Cisplatin Doxorubicin

Anacardic acid, �mol/L 0 25 0 25 0 25 0 25

KBM-5 0 0.6 3.5 1.9 1.7 1.2 32.8 1.0 12.3 3.4 33.8 5.5 9.3 2.8 31.8 5.4

Jurkat 0 0.6 4.1 1.1 3.1 0.8 26.7 2.7 6.1 1.0 24.6 1.8 3.2 0.8 29.6 2.7

H1299 0 0.5 4.4 1.2 5.4 0.9 35.8 5.7 8.9 2.1 34.9 4.8 5.6 1.4 29.7 4.7

Du145 0 0.9 7.6 2.0 2.5 1.1 29.3 1.1 3.2 0.9 31.7 2.6 7.2 3.1 27.3 0.8

SCC4 0 0.8 3.8 1.7 6.7 4.1 30.7 5.4 7.5 3.1 34.6 4.2 6.4 1.2 33.6 4.1

Cells (5 � 103/well) were seeded in triplicate in 96-well plates; 12 hours later, cells were pre-exposed to anacardic acid (25 �mol/L) for 4 hours and then to TNF (1 nmol/L),cisplatin (3 �g/mL), or doxorubicin (0.1 �mol/L) for 24 hours. Apoptosis was analyzed by the MTT method.

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tumor cells.20 We investigated whether antitumor effects linked toanacardic acid were mediated through the suppression of thesegene products’ expression. Western blot analysis showed that TNFinduced expression of these proteins and that anacardic acidsuppressed the expression (Figure 2B). These results indicate themolecular mechanism by which anacardic acid suppresses tumorcell proliferation.

Anacardic acid suppressed expression of gene productsinvolved in invasion and angiogenesis

Invasion and angiogenesis are critical for tumor metastasis and areinduced by TNF. ICAM-1, MMP-9, and VEGF have been impli-cated in invasion and angiogenesis; therefore, we examinedwhether this salicylic acid can suppress the expression of thesegene products. Western blot analysis showed that TNF inducedexpression of these proteins and that anacardic acid suppressedtheir expression (Figure 2B). These results suggest the potential

mechanism by which how this compound suppresses invasion andangiogenesis.

Anacardic acid inhibited TNF-dependent NF-�B activation

Because apoptosis and the various gene products noted above areregulated by NF-�B, we investigated whether this salicylate couldmodulate the NF-�B activation pathway. EMSA showed thatanacardic acid suppressed TNF-induced NF-�B activation in both adose-dependent manner (Figure 3A) and a time-dependent manner(Figure 3B). Anacardic acid alone did not activate NF-�B.

Because NF-�B is a complex of protein in which variouscombinations of the Rel/NF-�B protein constitute active NF-�Bheterodimers that bind specific DNA sequences,33 we decided thatit was important to show that the band visualized by EMSA inTNF-treated cells was indeed NF-�B. When nuclear extracts fromTNF-stimulated cells were treated with antibodies against the p50(NF-�B1) or p65 (RelA) subunits of NF-�B, the major band wasshifted to a higher molecular mass (Figure 3C), suggesting that theTNF-activated complex consisted of p50 and p65 subunits. Preim-mune serum had no effect on this band, excess (100-fold) unlabeledNF-�B caused complete disappearance of the band, and a mutantoligonucleotide of NF-�B did not affect NF-�B bindingactivity (Figure 3C).

Anacardic acid did not interfere with formation of theTNF-induced NF-�B complex directly

We next sought to determine whether anacardic acid directlymodified the binding of NF-�B complex to the DNA. EMSAshowed that anacardic acid did not modify the DNA-binding abilityof the NF-�B complex (Figure 3D). Therefore, we concludedthat anacardic acid inhibits NF-�B activation indirectly ratherthan directly.

Suppression of NF-�B activation by anacardic acid was notunique to TNF

A wide variety of stimuli have been shown to activate NF-�B,including IL-1�, LPS, PMA, OA, and EGF, through mechanismsthat may differ. We investigated whether anacardic acid abrogatesNF-�B activation by all these agents. EMSA showed that all theseagents activated NF-�B and that anacardic acid suppressed activa-tion (Figure 3E).

Inhibition of NF-�B activation by anacardic acid was not celltype–specific

Because distinct signal transduction pathways can mediate NF-�Binduction in different cell types,34 we examined the effect ofanacardic acid on TNF-induced NF-�B activation in human lungadenocarcinoma H1299 cells and human T-cell leukemia Jurkatcells. EMSA showed that anacardic acid inhibited TNF-activatedNF-�B in both cell types (Figure 3F,G).

Anacardic acid inhibited constitutive NF-�B activation intumor cells

A wide variety of tumor cells have been shown to expressconstitutive NF-�B activation through mechanisms that are notfully understood. Using EMSA, we examined whether anacardicacid could suppress constitutive NF-�B activation in humanprostate cancer Du145 cells and squamous cell carcinoma SCC4cells. Treatment with various concentrations of anacardic acid

Figure 2. Anacardic acid represses TNF-induced NF-�B–dependent expressionof antiapoptosis-, proliferation-, and metastasis-related gene products.(A) Antiapoptotic proteins. (B) Proliferative and metastatic proteins. KBM-5 cells wereincubated with 25 �mol/L anacardic acid for 4 hours and then treated with 1 nmol/LTNF for the indicated times. Whole-cell extracts were prepared and subjected toWestern blot analysis using the relevant antibodies.

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Figure 3. Anacardic acid inhibits TNF-dependent NF-�B activation. (A) Effect of anacardic acid per dose. KBM-5 cells were preincubated with indicated concentrations ofanacardic acid for 4 hours, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA to test for NF-�B activation. (B) Effect of anacardic acid according toexposure duration. Cells were preincubated with 25 �mol/L anacardic acid for the indicated times, treated with 0.1 nmol/L TNF for 30 minutes, and then subjected to EMSA totest for NF-�B activation. (C) NF-�B induced by TNF is composed of p65 and p50 subunits. Nuclear extracts from untreated or TNF-treated cells were incubated with theindicated antibody, preimmune serum, unlabeled NF-�B oligoprobe, or mutant oligoprobe and then assayed for NF-�B activation by EMSA. (D) The direct effect of anacardicacid on NF-�B complex was investigated. Nuclear extracts were prepared from untreated cells or cells treated with 0.1 nmol/L TNF and incubated for 30 minutes with theindicated concentrations of anacardic acid. They were then assayed for NF-�B activation by EMSA. (E) Anacardic acid blocks NF-�B activation induced by TNF, IL-1�, LPS,PMA, OA, and EGF. KBM-5 cells were preincubated with 25 �mol/L anacardic acid for 4 hours and then treated with 0.1 nmol/L TNF, 100 ng/mL IL-1�, or 10 �g/mL LPS for30 minutes; 500 nmol/L OA for 4 hours or 25 �g/mL PMA or 100 ng/mL EGF for 2 hours. The cells were then analyzed for NF-�B activation by EMSA. (F-I) Inhibition of NF-�Bactivation by anacardic acid is not cell type–specific. H1299, Jurkat, Du145, and SCC4 cells were incubated with 25 �mol/L anacardic acid for 4 hours and then incubated with0.1 nmol/L TNF for 30 minutes. Nuclear extracts were then prepared and assayed for NF-�B activation by EMSA.

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suppressed constitutive NF-�B activation in both cell types(Figure 3H,I).

Anacardic acid inhibited TNF-dependent I�B� phosphorylationand degradation

The translocation of NF-�B to the nucleus is preceded by theproteolytic degradation of I�B�,33 so we next sought to determinewhether anacardic acid inhibitory activity was due to inhibition ofI�B� degradation. EMSA showed that NF-�B was activated withincreasing TNF incubation times and that anacardic acid pretreat-ment dramatically decreased this activation (Figure 4A). Westernblot analysis showed that TNF induced I�B� degradation incontrol cells within 5 minutes but that anacardic acid inhibitedthis degradation (Figure 4B). These results indicate thatanacardic acid inhibited both TNF-induced NF-�B activation andI�B� degradation.

To determine whether the inhibition of TNF-induced I�Bdegradation was due to inhibition of I�B� phosphorylation andubiquitination, we used the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) to block degradation of I�B�.35 West-ern blot analysis using an antibody that recognizes the serine-phosphorylated form of I�B� showed that TNF induced I�B�phosphorylation and that anacardic acid suppressed phosphoryla-tion (Figure 4C).

Anacardic acid inhibited TNF-induced activation of IKK

Because IKK is required for TNF-induced phosphorylation ofI�B� and because anacardic acid inhibited the phosphorylation ofI�B�, we determined the effect of anacardic acid on TNF-inducedIKK activation. Results from the immune complex kinase assayshowed that TNF induced the activation of IKK in a time-dependent manner and that anacardic acid suppressed TNF-activated IKK (Figure 4D). Neither TNF nor anacardic acidaffected the expression of IKK� or IKK� proteins (Figure 4D).

Anacardic acid did not directly inhibit TNF-induced IKK

We and others have previously shown that certain agents suppressNF-�B activation by direct interaction with IKK.29,36 We examinedwhether anacardic acid suppressed IKK activity directly by bindingwith the IKK protein. The immune complex kinase assay ofwhole-cell extracts from untreated and TNF-treated cells showedthat anacardic acid did not directly affect the activity of IKK,suggesting that anacardic acid modulated TNF-induced IKK activa-tion indirectly (Figure 4E).

Anacardic acid inhibited nuclear translocation of p65

p65 is a subunit of NF-�B that has nuclear localization signals andretained in the cytoplasm by I�B�. We examined whether thedegradation of I�B� leads to nuclear translocation of p65. Wefound that TNF induced the nuclear translocation of p65 in as littleas 5 minutes of incubation and that anacardic acid suppressed p65translocation (Figure 4B). The immunocytochemical assay alsoconfirmed that anacardic acid suppressed the translocation of p65from the cytoplasm to the nucleus (Figure 4H).

Anacardic acid inhibited acetylation of p65

Because anacardic acid has been shown to inhibit HAT,13 which hasbeen linked to acetylation of p65,37 we investigated whetheranacardic acid could also inhibit acetylation of p65. Western blot

analysis showed that TNF induced the acetylation of p65 and thatanacardic acid blocked the TNF-induced acetylation (Figure 4F).

Anacardic acid suppressed TNF-induced HAT activity

Whether anacardic acid directly inhibits HAT activity was alsoinvestigated. To determine this, whole-cell extracts from cellstreated with TNF, anacardic acid, or both were resolved onSDS-PAGE and then analyzed by Western blot using anti-acetyllysine antibody. As shown Figure 4G, TNF induced acetylation ofseveral proteins, whereas anacardic acid suppressed the acetylationof these proteins.

Anacardic acid repressed TNF-induced NF-�B–dependentreporter gene expression

Although EMSA showed that anacardic acid blocked NF-�Bactivation, DNA binding alone does not always correlate withNF-�B–dependent gene transcription, suggesting that there areadditional regulatory steps. We investigated whether anacardic acidcould suppress the TNF-induced NF-�B reporter activity. TNFinduced a NF-�B–regulated secretory alkaline phosphatase (SEAP)reporter gene’s expression in a dose-dependent manner, andanacardic acid suppressed the expression (Figure 5A).

Anacardic acid repressed NF-�B–dependent reporter geneexpression induced by TNFR1, TRADD, TRAF2, NIK, and IKK

TNF has been shown to activate NF-�B activation throughsequential interaction with the TNF receptor (TNFR), TRADD,TNFR-associated factor 2 (TRAF2), NF-�B–inducing kinase (NIK),and IKK, resulting in phosphorylation of I�B�.38,39 We examinedwhere in this pathway the salicylic acid acts. To determine theeffect of anacardic acid on NF-�B–dependent reporter geneexpression, cells were transiently transfected with TNFR1-,TRADD-, TRAF2-, NIK-, IKK-, and p65-expressing plasmids andthen monitored for NF-�B–dependent SEAP expression. We foundthat cells transfected with any of these plasmids expressed theNF-�B–regulated reporter gene and that for all except the p65plasmid, expression was suppressed by anacardic acid (Figure 5B).These results suggest that the anacardic acid effect occurs at a stepupstream from p65.

Anacardic acid repressed NF-�B–dependent reporter geneexpression induced by TAK1

Tumor growth factor (TGF)-activated kinase 1 (TAK1), a memberof the mitogen-activated protein kinase (MAPK) family, wasoriginally identified as a key regulator of MAPK activation inTGF-�–induced signaling pathways. It is activated by variousinflammatory stimuli, including TNF, IL-1, and LPS.40 Recentstudies indicate that TAK1 plays a major role in TNF-inducedNF-�B activation through its interaction with TAK1-bindingprotein (TAB) 1 and TAB2.41 We examined whether anacardic acidsuppressed TNF-induced NF-�B activation through the inhibitionof TAK1. As shown in Figure 5B, NF-�B–dependent reporter geneexpression was induced in cells transfected with TAK1/TAB1, andanacardic acid inhibited this activation.

Anacardic acid inhibited TNF-induced COX-2 promoter activity

TNF induces COX-2, which has NF-�B binding sites in itspromoter.42 Because down-regulation of NF-�B by anacardic

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Figure 4. Anacardic acid inhibits TNF-dependent I�B� phosphorylation, I�B� degradation, p65 phosphorylation, and p65 nuclear translocation. (A) Anacardic acidinhibits TNF-induced activation of NF-�B. KBM-5 cells were incubated with 25 �mol/L anacardic acid for 4 hours, treated with 0.1 nmol/L TNF for the indicated times, and thenanalyzed for NF-�B activation by EMSA. (B) Effect of anacardic acid on TNF-induced I�B� degradation, p65 phosphorylation, and p65 nuclear translocation. Cells wereincubated with 25 �mol/L anacardic acid for 4 hours and treated with 0.1 nmol/L TNF for the indicated times. Cytoplasmic extracts (CE) and nuclear extracts (NE) wereprepared, fractionated on SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blot analysis was performed using the indicated antibody. An anti–�-actinantibody was the loading control. (C) Effect of anacardic acid on the phosphorylation of I�B� by TNF. Cells were preincubated with 25 �mol/L anacardic acid for 4 hours,incubated with 50 �g/mLALLN for 30 minutes, and then treated with 0.1 nmol/L TNF for 10 minutes. Cytoplasmic extracts were fractionated and then subjected to Western blotanalysis using a phospho-specific anti-I�B� antibody. The same membrane was reblotted with anti-I�B� antibody. (D) Anacardic acid inhibits TNF-induced I�B� kinase activity.Whole-cell extracts were immunoprecipitated with antibody against IKK� and analyzed by an immune complex kinase assay. To examine the effect of anacardic acid on thelevel of expression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK� and anti-IKK� antibodies.(E) Direct effect of anacardic acid on IKK activation induced by TNF. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNF and immunoprecipitatedwith anti-IKK� antibody. The immunocomplex kinase assay was performed in the absence or presence of the indicated concentration of anacardic acid. (F) Effect of anacardicacid on TNF-induced acetylation of p65. Cells were treated with 25 �mol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF. Whole-cell extracts were prepared,immunoprecipitated with an anti-p65 antibody, and subjected to Western blot analysis using an anti–acetyl-lysine antibody. The same blots were reprobed with anti-p65 antibody. (G) Effectof anacardic acid on TNF-induced protein acetylation. Cells were treated with 25 �mol/L anacardic acid for 4 hours and then exposed to 1 nmol/L TNF for 20 minutes. Whole cell extractswere prepared and subjected to Western blot analysis using an anti–acetyl-lysine antibody. (H) Immunocytochemical analysis of p65 localization. Cells were incubated with 25 �mol/Lanacardic acid for 4 hours and then treated with 1 nmol/LTNF for 15 minutes. Cells were subjected to immunocytochemical analysis.

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acid suppressed the expression of NF-�B–regulated gene prod-ucts, including COX-2, we examined the effect of anacardic acidon TNF-induced COX-2 promoter activity by using a COX-2

promoter-luciferase reporter plasmid. We found that TNF in-duced COX-2 promoter activity and that anacardic acid sup-pressed this activity in a dose-dependent manner (Figure 5C).This result suggests that anacardic acid inhibited NF-�B–regulated gene expression by suppressing NF-�B binding to theCOX-2 promoter.

NF-�B was needed for the effects of anacardic acid onsuppression of gene products

To determine whether presence of NF-�B is essential for the effectsof anacardic acid, we used cells in which NF-�B gene (p65) hasbeen deleted. Results showed that TNF induced the expression ofantiapoptotic proteins (such as survivin, XIAP, and Bcl-2) inwild-type (Figure 6A left panel), whereas anacardic acid sup-

Figure 5. Anacardic acid represses NF-�B–dependent reporter gene expres-sion induced by TNF and various plasmids. (A) Anacardic acid inhibits theNF-�B–dependent reporter gene expression induced by TNF. A293 cells weretransiently transfected with a NF-�B–containing plasmid for 24 hours. After transfec-tion, the cells were incubated with the indicated concentrations of anacardic acid for4 hours and then treated with 1 nmol/L TNF for an additional 24 hours. Thesupernatants of the culture media were assayed for SEAP activity. Data arepresented as mean ( SD). (B) Anacardic acid inhibits the NF-�B–dependentreporter gene expression induced by TNF, TNFR1, TRADD, TRAF2, NIK, IKK, p65,and TAK1/TAB1. Cells were transiently transfected with a NF-�B–containing plasmidalone or with the indicated plasmids. After transfection, cells were incubated with25 �mol/L anacardic acid for 4 hours and then incubated with the relevant plasmid foran additional 24 hours. TNF-treated cells were incubated with 25 �mol/L anacardicacid for 4 hours and then treated with 1 nmol/L TNF for an additional 24 hours. Thesupernatants of the culture media were assayed for SEAP activity. Data arepresented as mean ( SD). (C) Anacardic acid inhibits the COX-2 promoter activityinduced by TNF. Cells were transiently transfected with a COX-2 promoter linked tothe luciferase reporter gene plasmid for 24 hours and treated with the indicatedconcentrations of anacardic acid for 4 hours. Cells were then treated with 1 nmol/LTNF for an additional 24 hours, lysed, and subjected to a luciferase assay. Data arepresented as mean ( SD).

Figure 6. Down-regulation of p300 HAT abrogates the effect of anacardic acid.(A) TNF regulates antiapoptotic gene expression. The wild-type and p65�/� MEFcells were pretreated with 25 �mol/L anacardic acid for 4 hours and then incubatedwith 1 nmol/L TNF for the indicated times. Whole-cell extracts were prepared andsubjected to Western blot analysis using the relevant antibodies. (B) Anacardic acidinhibits binding of NF-�B to the COX-2 and MMP-9 promoter. KBM-5 cells werepretreated with 25 �mol/L anacardic acid for 4 hours and treated with 1 nmol/L TNFfor the indicated times, and the proteins were cross-linked with DNA by formaldehydeand then subjected to chromatin immunoprecipitation (ChIP) assay using an anti-p65antibody with the COX-2 and MMP-9 primers. Reaction products were resolved byelectrophoresis. (C) Down-regulation of p300 by RNA interference reverses the effectof anacardic acid. A293 cells were transfected with indicated concentration of p300siRNA or scrambled (SC) control. After 48 hours, cells were harvested, and whole-cellextracts were prepared and analyzed by Western blotting with an anti-p300 antibody.(D) Transfected cells were preincubated with 25 �mol/L anacardic acid for 4 hoursand then treated with 0.1 nmol/L TNF for 30 minutes. The nuclear extracts wereprepared and assayed for NF-�B activation by EMSA.

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pressed these gene expression. In p65�/� cells that lack functionalNF-�B, TNF failed to induce the expression of these antiapoptoticgene products (Figure 6A right panel). Thus, these results suggestthat NF-�B was needed for the expression of these gene products.

Anacardic acid inhibited TNF-induced COX-2 and MMP-9through NF-�B

Whether the lack of TNF-induced COX-2 and MMP-9 expressionin anacardic acid–treated cells was due to suppression of NF-�Bactivation in vivo was examined by using chromatin immunopre-cipitation assay targeting NF-�B binding in the COX-2 andMMP-9 promoter. For this, KBM-5 cells were pretreated withanacardic acid and then treated with TNF for indicated times.Thereafter, the cross-linked reaction was carried out in situ withDNA-protein complexes, and the chromatin was isolated andsheared. Subsequently, the chromatin was immunoprecipitatedwith anti-p65 antibody, and the DNA was purified and subjected toPCR using COX-2 or MMP-9 promoter-specific primer. We foundthat TNF induced NF-�B binding to both COX-2 and MMP-9promoters in a time-dependent manner and that anacardic acidsuppressed it (Figure 6B). Overall, these results suggest thatanacardic acid inhibited NF-�B–regulated gene expression bysuppressing NF-�B binding to the promoter of these genes.

Down-regulation of p300 HAT abrogates the effect of anacardicacid

Anacardic acid has been shown to inhibit the activity of histoneacetyltransferase p300.12 Whether the effect of anacardic acid onNF-�B is mediated by p300 HAT was investigated. The results inFigure 6C show that siRNA but not scrambled control RNAdown-regulated the expression of p300 protein. Furthermore,depletion of p300 abrogated the effect of anacardic acid onTNF-induced NF-�B activation (Figure 6D). These results clearlydemonstrate that p300 HAT is a target of anacardic acid forsuppression of NF-�B activation.

Discussion

The goal of this study was to investigate the effect of anacardic acidon the NF-�B activation pathway and NF-�B–regulated geneproducts that regulate apoptosis. We found that anacardic acidsuppressed NF-�B activated by carcinogens, growth factors, andinflammatory stimuli through inhibition of IKK activation, I�B�phosphorylation, I�B� degradation, p65 phosphorylation, andNF-�B–dependent reporter gene expression. Under identical condi-tions, anacardic acid alone had no effect. As a result, anacardic aciddown-regulates the expression of NF-�B–dependent gene productsinvolved in cell proliferation, antiapoptosis, invasion, and angiogen-esis. We also demonstrated that anacardic acid potentiates apopto-sis induced by TNF and chemotherapeutic agents and thus mayhave potential as an anticancer agent.

To our knowledge, this is the first report of an investigation intothe effect of anacardic acid on NF-�B activation by a variety ofstimuli. Our results show that anacardic acid suppressed NF-�Bactivated by a variety of stimuli, suggesting that anacardic acidmust act at a step common to all these activators. In addition, ourresults showed that anacardic acid blocked NF-�B activationwithout directly interfering with its DNA binding. NF-�B activa-tion in response to different stimuli requires IKK activation, whichphosphorylates I�B� at serine 32 and 36, leading to degradation of

I�B�.33 We found that this inhibition was mediated through theinhibition of IKK by anacardic acid, which led to the suppression ofphosphorylation and the degradation of I�B�.

We investigated how anacardic acid suppressed IKK activationby examining its effects on several kinases that function upstreamof IKK, such as mitogen-activated protein kinase kinase kinase(MEKK) 1,43 MEKK3,44 protein kinase C,45 glycogen synthasekinase-3�,46 TAK1,47 phosphoinositide-dependent kinase 1,48 andAkt.49 Recent studies indicate that TAK1 plays a major role in thecanonical pathway activated by cytokines through its interactionwith TAB1 and TAB2.47 TAK1 also has been shown to be recruitedby the TNFR1 through TRADD, TRAF2, and receptor-interactingprotein. Indeed, our study shows for the first time that TAK1-induced NF-�B activation is inhibited by anacardic acid, therebysuggesting that TAK1 is the main upstream stimulatory kinasemodulated by anacardic acid.

Besides inducible NF-�B activation, we found that anacardicacid inhibited constitutive NF-�B activation. Constitutively activeNF-�B has been found in a wide variety of leukemic and tumorepithelial cells50 and is needed for these cells’ proliferation.51,52 It isnot fully understood why tumor cells express constitutively activeNF-�B, but IKK has been implicated.51,52 Thus, it is possible thatanacardic acid’s inhibition of IKK in tumor cells is linked to itsability to suppress constitutive NF-�B activation.

Anacardic acid has been reported to inhibit HAT, p300, andp300/cAMP response element-binding protein–binding protein–associated factor.12,14,53 Anacardic acid has been shown to inhibitHAT-dependent transcription of histone H3 but to have no effect ontranscription from naked DNA.12 In addition, acetylation of RelA(p65) at lysine 310, one step in the TNF-induced NF-�B activationpathway, is regulated by prior phosphorylation of serine 276 and536. Such phosphorylated and acetylated forms of RelA displayenhanced transcriptional activity.37 Our data suggest that anacardicacid may suppress TNF-induced p65 acetylation through inhibitionof HAT activity.

It is also possible that anacardic acid could suppress TNF-induced NF-�B activation through inhibition of reactive oxygenspecies (ROS) production. That anacardic acid could exhibitantioxidant role has been well demonstrated.9,10,17 The antioxidantcapacity of anacardic acid is more related to inhibition of superox-ide generation (IC50 0.04 mmol/L) and of xanthine oxidase(IC50 0.30 mmol/L) than to scavenging of hydroxyl radicals.Antioxidant effects of anacardic acid could also be due to itsactivity as mitochondrial uncoupler of oxidative phosphorylationdescribed previously.19 Hayakawa et al, however, reported thatproduction of ROS is unrelated to the NF-�B activation.54 ThusNF-�B inhibitory activity of anacardic acid may not be linked to itsability to quench ROS.

We also found that anacardic acid down-modulated expressionof antiapoptotic survivin, XIAP, Bcl-2, Bcl-xL, and FLIP. Thesegene products are regulated by NF-�B, and their overexpression innumerous tumors has been associated with tumor survival, chemore-sistance, and radioresistance. Previous reports show that anacardicacid could sensitize tumor cells to ionizing radiation13; it is possiblethat these effects are mediated through the mechanism describedhere. We also observed that anacardic acid potentiates the apoptoticeffects of TNF, cisplatin, and doxorubicin, suggesting that ana-cardic acid could be used to enhance this effect in chemotherapeu-tic regimens.

Anacardic acid also suppressed gene products that have beenimplicated in angiogenesis and invasion. We found that theexpression of NF-�B–regulated gene products involved in invasion

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(eg, COX-2, MMP-9, and ICAM-1) and proliferation (eg, cyclinD1 and c-Myc) were abrogated by anacardic acid. Likewise,Paramashivappa et al (2003) showed that anacardic acid inhibitshuman COX-2 activity18 and Granzzini et al (1991) found thiseffect on lipoxygenase,16 which also is regulated by NF-�B.These results are in agreement also with results showing thatcelecoxib also inhibits COX-2 expression through inhibition ofNF-�B activation.55

We found that anacardic acid also suppressed the expression ofVEGF, a growth factor critical for tumor angiogenesis. Bevaci-zumab (Avastin), an antibody against VEGF, has been approved forthe treatment of cancer and macular degeneration,56 and our resultsdemonstrate the potential of anacardic acid in these diseases.

Whether anacardic acid has any effect on normal cells is notclear. A recent report suggests that anacardic acid and its analogsinhibit the growth of tumor cells but had no effect on nonmalignantcells.57 The pharmacokinetic and pharmacodynamic effects ofanacardic acid in animals need to be investigated to fully realize itsclinical potential.

Overall, our results demonstrate that anacardic acid is a potentinhibitor of NF-�B activation, which may explain its anti-angiogenic, antiproliferative, proapoptotic, antimetastatic, anti-

inflammatory, and immunomodulatory effects. Further studies areneeded to explore its therapeutic potential against cancer andcardiovascular and neurologic diseases.

Acknowledgments

We thank Alyson Todd for careful comments on the manuscript.This work was supported financially by the Clayton Foundation

(to B.B.A.) and by a National Cancer Institute core grant.

Authorship

Contribution: B.S., M.K.P., K.S.A., and T.Y. conducted all theexperiments. M.M.C. analyzed the data. B.B.A. and M.L. super-vised and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no compet-ing financial interests.

Correspondence: Prof Bharat B. Aggarwal, The University ofTexas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,Box 143, Houston, TX 77030; e-mail: aggarwal@mdanderson.org.

References

1. Olivera Ortega AG, Soto Hernandez M, MartinezVazquez M, Terrazas Salgado T, Solares ArenasF. Phytochemical study of cuachalalate (Am-phiptherygium adstringens, Schiede ex Schlecht).J Ethnopharmacol. 1999;68:109-113.

2. Acevedo HR, Rojas MD, Arceo SD, et al. Effect of6-nonadecyl salicylic acid and its methyl ester onthe induction of micronuclei in polychromaticerythrocytes in mouse peripheral blood. MutatRes. 2006;609:43-46.

3. Rea AI, Schmidt JM, Setzer WN, Sibanda S, Tay-lor C, Gwebu ET. Cytotoxic activity of Ozoroa in-signis from Zimbabwe. Fitoterapia. 2003;74:732-735.

4. Kubo I, Kinst-Hori I, Yokokawa Y. Tyrosinase in-hibitors from Anacardium occidentale fruits. J NatProd. 1994;57:545-551.

5. Itokawa H, Totsuka N, Nakahara K, Takeya K,Lepoittevin JP, Asakawa Y. Antitumor principlesfrom Ginkgo biloba L. Chem Pharm Bull. 1987;35:3016-3020.

6. Sullivan JT, Richards CS, Lloyd HA, Krishna G.Anacardic acid: molluscicide in cashew nut shellliquid. Planta Med. 1982;44:175-177.

7. Muroi H, Kubo I. Antibacterial activity of anacardicacid and totarol, alone and in combination withmethicillin, against methicillin-resistant Staphylo-coccus aureus. J Appl Bacteriol. 1996;80:387-394.

8. Kubo J, Lee JR, Kubo I. Anti-Helicobacter pyloriagents from the cashew apple. J Agric FoodChem. 1999;47:533-537.

9. Trevisan MT, Pfundstein B, Haubner R, et al.Characterization of alkyl phenols in cashew(Anacardium occidentale) products and assay oftheir antioxidant capacity. Food Chem Toxicol.2006;44:188-197.

10. Masuoka N, Kubo I. Characterization of xanthineoxidase inhibition by anacardic acids. BiochimBiophys Acta. 2004;1688:245-249.

11. Lee JS, Cho YS, Park EJ, et al. PhospholipaseCgamma1 inhibitory principles from the sarcotes-tas of Ginkgo biloba. J Nat Prod. 1998;61:867-871.

12. Balasubramanyam K, Swaminathan V, Ranga-nathan A, Kundu TK. Small molecule modulatorsof histone acetyltransferase p300. J Biol Chem.2003;278:19134-19140.

13. Sun Y, Jiang X, Chen S, Price BD. Inhibition ofhistone acetyltransferase activity by anacardicacid sensitizes tumor cells to ionizing radiation.FEBS Lett. 2006;580:4353-4356.

14. Mai A, Rotili D, Tarantino D, et al. Small-moleculeinhibitors of histone acetyltransferase activity:identification and biological properties. J MedChem. 2006;49:6897-6907.

15. Wang D, Girard TJ, Kasten TP, LaChance RM,Miller-Wideman MA, Durley RC. Inhibitory activityof unsaturated fatty acids and anacardic acidstoward soluble tissue factor-factor VIIa complex.J Nat Prod. 1998;61:1352-1355.

16. Grazzini R, Hesk D, Heininger E, et al. Inhibitionof lipoxygenase and prostaglandin endoperoxidesynthase by anacardic acids. Biochem BiophysRes Commun. 1991;176:775-780.

17. Ha TJ, Kubo I. Lipoxygenase inhibitory activity ofanacardic acids. J Agric Food Chem. 2005;53:4350-4354.

18. Paramashivappa R, Phani Kumar P, Subba RaoPV, Srinivasa Rao A. Design, synthesis and bio-logical evaluation of benzimidazole/benzothiazoleand benzoxazole derivatives as cyclooxygenaseinhibitors. Bioorg Med Chem Lett. 2003;13:657-660.

19. Toyomizu M, Okamoto K, Ishibashi T, Chen Z,Nakatsu T. Uncoupling effect of anacardic acidsfrom cashew nut shell oil on oxidative phosphory-lation of rat liver mitochondria. Life Sci. 2000;66:229-234.

20. Kumar A, Takada Y, Boriek AM, Aggarwal BB.Nuclear factor-kappaB: its role in health and dis-ease. J Mol Med. 2004;82:434-448.

21. Garg AK, Buchholz TA, Aggarwal BB. Chemosen-sitization and radiosensitization of tumors byplant polyphenols. Antioxid Redox Signal. 2005;7:1630-1647.

22. Aggarwal BB, Shishodia S, Sandur SK, PandeyMK, Sethi G. Inflammation and cancer: how hot isthe link? Biochem Pharmacol. 2006;72:1605-1621.

23. Chaturvedi MM, Mukhopadhyay A, Aggarwal BB.Assay for redox-sensitive transcription factor.Methods Enzymol. 2000;319:585-602.

24. Sung B, Pandey MK, Aggarwal BB. Fisetin, aninhibitor of cyclin-dependent kinase 6, down-regulates nuclear factor-�B-regulated cell prolif-eration, antiapoptotic and metastatic gene prod-

ucts through the suppression of TAK-1 andreceptor-interacting protein-regulated I�B� ki-nase activation. Mol Pharmacol. 2007;71:1703-1714.

25. Peters AH, Kubicek S, Mechtler K, et al. Partition-ing and plasticity of repressive histone methyl-ation states in mammalian chromatin. Mol Cell.2003;12:1577-1589.

26. Zabel U, Schreck R, Baeuerle PA. DNA binding ofpurified transcription factor NF-�B. Affinity, speci-ficity, Zn2� dependence, and differential half-siterecognition. J Biol Chem. 1991;266:252-260.

27. Nakao S, Ogtata Y, Shimizu E, Yamazaki M, Fu-ruyama S, Sugiya H. Tumor necrosis factor alpha(TNF-alpha)-induced prostaglandin E2 release ismediated by the activation of cyclooxygenase-2(COX-2) transcription via NFkappaB in humangingival fibroblasts. Mol Cell Biochem. 2002;238:11-18.

28. Ogawa K, Chen F, Kuang C, Chen Y. Suppres-sion of matrix metalloproteinase-9 transcriptionby transforming growth factor-beta is mediated bya nuclear factor-kappaB site. Biochem J. 2004;381:413-422.

29. Pandey MK, Sandur SK, Sung B, Sethi G, Kunnu-makkara AB, Aggarwal BB. Butein, a tetrahy-droxychalcone, inhibits nuclear factor (NF)-�Band NF-�B-regulated gene expression throughdirect inhibition of I�B� kinase � on cysteine 179residue. J Biol Chem. 2007;282:17340-17350.

30. Mayo MW, Wang CY, Cogswell PC, et al. Re-quirement of NF-kappaB activation to suppressp53-independent apoptosis induced by onco-genic Ras. Science. 1997;278:1812-1815.

31. Giri DK, Aggarwal BB. Constitutive activation ofNF-�B causes resistance to apoptosis in humancutaneous T cell lymphoma HuT-78 cells. Auto-crine role of tumor necrosis factor and reactiveoxygen intermediates. J Biol Chem. 1998;273:14008-14014.

32. Wang CY, Mayo MW, Korneluk RG, Goeddel DV,Baldwin AS Jr. NF-kappaB antiapoptosis: induc-tion of TRAF1 and TRAF2 and c-IAP1 and c-IAP2to suppress caspase-8 activation. Science. 1998;281:1680-1683.

33. Ghosh S, Karin M. Missing pieces in the NF-kappaB puzzle. Cell. 2002;109 Suppl:S81-S96.

34. Bonizzi G, Piette J, Merville MP, Bours V. Distinctsignal transduction pathways mediate nuclear

4890 SUNG et al BLOOD, 15 MAY 2008 � VOLUME 111, NUMBER 10

For personal use only.on July 16, 2015. by guest www.bloodjournal.orgFrom

factor-kappaB induction by IL-1beta in epithelialand lymphoid cells. J Immunol. 1997;159:5264-5272.

35. Vinitsky A, Michaud C, Powers JC, Orlowski M.Inhibition of the chymotrypsin-like activity of thepituitary multicatalytic proteinase complex. Bio-chemistry. 1992;31:9421-9428.

36. Yore MM, Liby KT, Honda T, Gribble GW, SpornMB. The synthetic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole blocksnuclear factor-kappaB activation through directinhibition of IkappaB kinase beta. Mol CancerTher. 2006;5:3232-3239.

37. Chen LF, Williams SA, Mu Y, et al. NF-kappaBRelA phosphorylation regulates RelA acetylation.Mol Cell Biol. 2005;25:7966-7975.

38. Simeonidis S, Stauber D, Chen G, HendricksonWA, Thanos D. Mechanisms by which IkappaBproteins control NF-kappaB activity. Proc NatlAcad Sci U S A. 1999;96:49-54.

39. Hsu H, Shu HB, Pan MG, Goeddel DV. TRADD-TRAF2 and TRADD-FADD interactions definetwo distinct TNF receptor 1 signal transductionpathways. Cell. 1996;84:299-308.

40. Yamaguchi K, Shirakabe K, Shibuya H, et al.Identification of a member of the MAPKKK familyas a potential mediator of TGF-beta signal trans-duction. Science. 1995;270:2008-2011.

41. Kanayama A, Seth RB, Sun L, et al. TAB2 andTAB3 activate the NF-kappaB pathway throughbinding to polyubiquitin chains. Mol Cell. 2004;15:535-548.

42. Yamamoto K, Arakawa T, Ueda N, Yamamoto S.Transcriptional roles of nuclear factor kappa Band nuclear factor-interleukin-6 in the tumor ne-crosis factor alpha-dependent induction of cyclo-

oxygenase-2 in MC3T3-E1 cells. J Biol Chem.1995;270:31315-31320.

43. Lee FS, Peters RT, Dang LC, Maniatis T. MEKK1activates both IkappaB kinase alpha and IkappaBkinase beta. Proc Natl Acad Sci U S A. 1998;95:9319-9324.

44. Yang J, Lin Y, Guo Z, et al. The essential role ofMEKK3 in TNF-induced NF-kappaB activation.Nat Immunol. 2001;2:620-624.

45. Lallena MJ, Diaz-Meco MT, Bren G, Paya CV,Moscat J. Activation of IkappaB kinase beta byprotein kinase C isoforms. Mol Cell Biol. 1999;19:2180-2188.

46. Hoeflich KP, Luo J, Rubie EA, Tsao MS, Jin O,Woodgett JR. Requirement for glycogen syn-thase kinase-3beta in cell survival and NF-kappaB activation. Nature. 2000;406:86-90.

47. Sakurai H, Miyoshi H, Toriumi W, Sugita T. Func-tional interactions of transforming growth factor�-activated kinase 1 with I�B kinases to stimulateNF-�B activation. J Biol Chem. 1999;274:10641-10648.

48. Tanaka H, Fujita N, Tsuruo T. 3-Phosphoinositide-dependent protein kinase-1-mediated I�B kinase� (IKKB) phosphorylation activates NF-�B signal-ing. J Biol Chem. 2005;280:40965-40973.

49. Gustin JA, Maehama T, Dixon JE, Donner DB.The PTEN tumor suppressor protein inhibits tu-mor necrosis factor-induced nuclear factor �Bactivity. J Biol Chem. 2001;276:27740-27744.

50. Jackson-Bernitsas DG, Ichikawa H, Takada Y, etal. Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-kappaB activation and proliferation in humanhead and neck squamous cell carcinoma. Onco-gene. 2007;26:1385-1397.

51. Bharti AC, Donato N, Singh S, Aggarwal BB. Cur-cumin (diferuloylmethane) down-regulates theconstitutive activation of nuclear factor-�B andI�B� kinase in human multiple myeloma cells,leading to suppression of proliferation and induc-tion of apoptosis. Blood. 2003;101:1053-1062.

52. Shishodia S, Amin HM, Lai R, Aggarwal BB. Cur-cumin (diferuloylmethane) inhibits constitutiveNF-kappaB activation, induces G1/S arrest, sup-presses proliferation, and induces apoptosis inmantle cell lymphoma. Biochem Pharmacol.2005;70:700-713.

53. Davidson SM, Townsend PA, Carroll C, et al. Thetranscriptional coactivator p300 plays a criticalrole in the hypertrophic and protective pathwaysinduced by phenylephrine in cardiac cells but isspecific to the hypertrophic effect of urocortin.Chembiochem. 2005;6:162-170.

54. Hayakawa M, Miyashita H, Sakamoto I, et al. Evi-dence that reactive oxygen species do not medi-ate NF-kappaB activation. EMBO J. 2003;22:3356-3366.

55. Shishodia S, Aggarwal BB. Cyclooxygenase(COX)-2 inhibitor celecoxib abrogates activationof cigarette smoke-induced nuclear factor (NF)-kappaB by suppressing activation of IkappaBal-pha kinase in human non-small cell lung carci-noma: correlation with suppression of cyclin D1,COX-2, and matrix metalloproteinase-9. CancerRes. 2004;64:5004-5012.

56. Ferrara N. Vascular endothelial growth factor:basic science and clinical progress. Endocr Rev.2004;25:581-611.

57. Eliseeva ED, Valkov V, Jung M, Jung MO. Char-acterization of novel inhibitors of histone acetyl-transferases. Mol Cancer Ther. 2007;6:2391-2398.

ANACARDIC ACID SUPPRESSES NF-�B PATHWAY 4891BLOOD, 15 MAY 2008 � VOLUME 111, NUMBER 10

For personal use only.on July 16, 2015. by guest www.bloodjournal.orgFrom

online March 18, 2008 originally publisheddoi:10.1182/blood-2007-10-117994

2008 111: 4880-4891  

and Bharat B. AggarwalBokyung Sung, Manoj K. Pandey, Kwang Seok Ahn, Tingfang Yi, Madan M. Chaturvedi, Mingyao Liu 

kinase, leading to potentiation of apoptosisαBκfactor-and inflammation through inhibition of the inhibitory subunit of nuclear regulated gene products involved in cell survival, proliferation, invasion,

−Bκacetyltransferase, suppresses expression of nuclear factor-Anacardic acid (6-nonadecyl salicylic acid), an inhibitor of histone 

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