Suberoylanilide Hydroxamic Acid Potentiates Apoptosis,Inhibits Invasion, and Abolishes Osteoclastogenesis bySuppressing Nuclear Factor-�B Activation*

Received for publication, July 5, 2005, and in revised form, November 18, 2005 Published, JBC Papers in Press, December 23, 2005, DOI 10.1074/jbc.M507213200

Yasunari Takada‡1, Ann Gillenwater§, Haruyo Ichikawa‡, and Bharat B. Aggarwal‡2

From the Cytokine Research Laboratory, Departments of ‡Experimental Therapeutics and §Head and Neck Surgery, The Universityof Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Because of its ability to suppress tumor cell proliferation, angio-genesis, and inflammation, the histone deacetylase inhibitor sub-eroylanilide hydroxamic acid (SAHA) is currently in clinical trials.HowSAHAmediates its effects is poorly understood.We found thatin several human cancer cell lines, SAHA potentiated the apoptosisinduced by tumor necrosis factor (TNF) and chemotherapeuticagents and inhibited TNF-induced invasion and receptor activatorofNF-�B ligand-induced osteoclastogenesis, all ofwhich are knownto requireNF-�B activation. These observations correspondedwiththe down-regulation of the expression of anti-apoptotic (IAP1,IAP2, X chromosome-linked IAP, Bcl-2, Bcl-xL, TRAF1, FLIP, andsurvivin), proliferative (cyclin D1, cyclooxygenase 2, and c-Myc),and angiogenic (ICAM-1, matrix metalloproteinase-9, and vascularendothelial growth factor) gene products. Because several of thesegenes are regulated by NF-�B, we postulated that SAHA mediatesits effects by modulating NF-�B and found that SAHA suppressedNF-�B activation induced by TNF, IL-1�, okadaic acid, doxorubi-cin, lipopolysaccharide, H2O2, phorbol myristate acetate, and ciga-rette smoke; the suppressionwas not cell type-specific because bothinducible and constitutive NF-�B activation was inhibited.We alsofound that SAHA had no effect on direct binding of NF-�B to theDNA but inhibited sequentially the TNF-induced activation ofI�B� kinase, I�B� phosphorylation, I�B� ubiquitination, I�B�

degradation, p65 phosphorylation, and p65 nuclear translocation.Furthermore, SAHA inhibited the NF-�B-dependent reporter geneexpression activated by TNF, TNFR1, TRADD, TRAF2, NF-�B-in-ducing kinase, I�B� kinase, and the p65 subunit of NF-�B. Overall,our results indicated thatNF-�BandNF-�B-regulated gene expres-sion inhibited by SAHA can enhance apoptosis and inhibit invasionand osteoclastogenesis.

One of the modes of cancer therapy involves the manipulation oftranscription regulated by histone acetylation by histone acetylase

transferases and deacetylation by histone deacetylases (1). Severalinhibitors of histone deacetylases, including simple compounds such asbutyrate, cyclic tetrapeptides, benzamides (e.g. MS-275), and hydrox-amic acids (e.g. suberoylanilide hydroxamic acid; SAHA),3 are beingconsidered as potential therapeutic agents with which to treat cancer.SAHA has been shown to induce differentiation, growth arrest, andapoptosis of transformed human cells in culture at micromolar concen-trations. SAHA was originally identified based on its ability to inducedifferentiation ofmurine erythroleukemia cells (2). Subsequently, it wasfound to induce differentiation of human breast adenocarcinoma cells(3) and growth arrest in human prostate carcinoma (4), rhabdomyosar-coma (5), and bladder transitional cell carcinoma cells (6). SAHA hasalso been shown to induce apoptosis in a wide variety of cells, includingT cell leukemia (7, 8), acute promyelocytic leukemia (9), chronic mye-loid leukemia (10, 11), promyelomonocytic leukemia (12), multiplemyeloma (13), prostate carcinoma (4), and melanoma (14) cells. Fur-thermore, SAHA has been shown to suppress angiogenesis (15).How SAHA mediates its many effects is poorly understood. It has

been shown to up-regulate the expression of the cyclin-dependentkinase inhibitor p21 (6, 16) and down-regulate the expression ofcaspase inhibitors (12, 14, 16), c-Myc (11, 17), cyclin D1 (10), daxx(9), and thioredoxin (18). SAHA also down-regulates the productionof mediators of inflammation, including nitric oxide, tumor necrosisfactor (TNF), interleukin (IL)-1�, and IL-12 (19).Because genes involved in the regulation of apoptosis, proliferation,

angiogenesis, and inflammation are regulated by the transcription fac-tor NF-�B, we postulated that SAHAmediates its effects bymodulatingNF-�B activation and the associated gene expression. NF-�B activationis regulated by I�B� kinase (IKK) and leads to I�B� phosphorylation,ubiquitination, and degradation and, sequentially, the release of NF-�Bsubunits p50 and p65 for translocation to the nucleus, binding to theirconsensus sequences, and induction of gene transcription.The present study was designed to determine whether SAHA medi-

ates its effects by suppressing theNF-�Bpathway. Specifically, we inves-tigated the effects of SAHAon the cellular responses that requireNF-�Bactivation, on the NF-�B activation pathway, and on NF-�B-regulatedgene expression. Because TNF is a potent activator of the NF-�B acti-vation pathway and because how TNF activates NF-�B is well charac-terized (20), we used this inducer to examine themechanismof action of

* This work was supported in part by a grant from the Clayton Foundation for Research,Department of Defense U. S. Army Breast Cancer Research Program Grant BC010610,National Institutes of Health PO1 Grant CA91844 on lung chemoprevention, andNational Institutes of Health P50 Head and Neck SPORE Grant P50CA97007 (all toB. B. A.) and grants from the Odyssey Program and the Theodore N. Law Award forScientific Achievement Fund from The University of Texas M. D. Anderson CancerCenter (to Y. T.). The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 An Odyssey Program Special Fellow at The University of Texas M. D. Anderson CancerCenter.

2 A Ransom Horne, Jr., Distinguished Professor of Cancer Research. To whom corre-spondence should be addressed: Cytokine Research Laboratory, Dept. of Experimen-tal Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Hol-combe Blvd., Houston, TX 77030. Tel.: 713-792-3503/6459; Fax: 713-794-1613; E-mail:aggarwal@mdanderson.org.

3 The abbreviations used are: SAHA, suberoylanilide hydroxamic acid; NF-�B, nuclearfactor-�B; I�B, inhibitory subunit of NF-�B; IKK, I�B� kinase; SEAP, secretory alkalinephosphatase; PMA, phorbol myristate acetate; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling; IAP, inhibitor-of-apoptosis protein; COX, cyclooxygenase; TNF, tumor necrosis factor; TRAF, TNF recep-tor-associated factor; IL, interleukin; FBS, fetal bovine serum; EMSA, electrophoreticmobility shift assay; TSA, trichostatin A; NaB, sodium butyrate; RANKL, receptor acti-vator of NF-�B ligand; NIK, NF-�B-inducing kinase; PARP, poly(adenosine diphos-phate-ribose)polymerase; MTT, 3-(4,5-dimethyltriazole)-2,5-diphenyl tetrazoliumbromide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 9, pp. 5612–5622, March 3, 2006© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

5612 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

SAHA. The results showed that SAHA potentiated apoptosis inducedby TNF and chemotherapeutic agents and inhibited cell invasion andosteoclastogenesis. These effects were associated with the inhibition ofNF-�B activation and NF-�B-regulated gene expression. The results ofour studies provide a description of mechanisms through which SAHAcould mediate its effects. The ability of SAHA to enhance apoptosis,suppress invasion, and inhibit osteoclastogenesis provides novel targetsfor cancer therapy.

MATERIALS AND METHODS

Reagents—SAHA was purchased from Midwest Research Institute,Kansas, MO (other suppliers of SAHA include Aton Pharma, Inc., Ter-rytown, NY; BioVision, Mountain View, CA; Alexis Biochemicals,Grunberg, Germany). A 50-mM solution of SAHAwas prepared in 100%Me2SO, stored as small aliquots at�20 °C, and then diluted as needed incell culture medium. Bacteria-derived recombinant human TNF, puri-fied to homogeneity with a specific activity of 5 � 107 units/mg, waskindly provided by Genentech (South San Francisco, CA). Cigarettesmoke condensate, prepared as previously described (21), was kindlysupplied by Dr. Chandra Gairola (University of Kentucky, Lexington,KY). Penicillin, streptomycin, Iscove’s modified Dulbecco medium, andFBSwere obtained from Invitrogen. PMA, okadaic acid, H2O2, and anti-�-actin antibody were obtained from Sigma. Antibodies against p65,p50, I�B�, ICAM-1, c-Myc, cyclin D1, matrix metalloproteinase-9,poly(adenosine diphosphate-ribose)polymerase (PARP), IAP1, IAP2,Bcl-2, Bcl-xL, and TRAF1 and the annexin V staining kit were obtainedfrom Santa Cruz Biotechnology (Santa Cruz, CA). Anti-COX-2 andanti-X chromosome-linked IAP antibodies were obtained fromBDBio-sciences (San Diego, CA). Phospho-specific anti-I�B� (serine 32) andphospho-specific anti-p65 (serine 529) antibodies were purchased fromCell Signaling (Beverly, MA). Anti-IKK-�, anti-IKK-�, and anti-FLIPantibodies were kindly provided by Imgenex (San Diego, CA).

Cell Lines—Human myeloid KBM-5 cells, mouse macrophage Raw264.7 cells, human lung adenocarcinoma H1299 cells, human embry-onic kidneyA293 cells, and human squamous cell carcinomaMDA1986and SCC-4 cells were obtained fromAmerican Type Culture Collection(Manassas, VA). KBM-5 cells were cultured in Iscove’s modified Dul-becco medium supplemented with 15% FBS. Raw 264.7 cells were cul-tured in Dulbecco’s modified Eagle’s medium/F-12 medium, H1299cells were cultured in RPMI 1640medium, andA293 cells were culturedin Dulbecco’s modified Eagle’s medium supplemented with 10% FBS.MDA1986 and SCC-4 cells were cultured in Dulbecco’s modifiedEagle’s medium containing 10% FBS, nonessential amino acids, pyru-vate, glutamine, and vitamins. All media were also supplemented with100 units/ml of penicillin and 100 �g/ml of streptomycin.

Live and Dead Assay—To assess cytotoxicity, we used the Live andDead assay (Molecular Probes, Eugene, OR), which determines intra-cellular esterase activity and plasma membrane integrity as previouslydescribed (22).

Cytotoxicity Assay—The effect of SAHA on the cytotoxic effects ofTNF and chemotherapeutic reagents was determined by the 3-(4,5-dimethyltriazole)-2,5-diphenyl tetrazolium bromide (MTT) uptakemethod as previously described (22).

Annexin V Staining Assay—To determine the early effect of apopto-sis, we used annexin V staining assay as previously described (22).

TUNEL Assay—We also assayed cytotoxicity by the terminaldeoxynucleotidyl transferase-mediated deoxyuridine triphosphate nickend-labeling (TUNEL) method as previously described (22).

Invasion Assay—Themembrane invasion culture systemwas used toassess cell invasion because invasion through the extracellular matrix is

a crucial step in tumor metastasis. The BD BioCoat Tumor Invasionsystem is a chamber that has a light-tight polyethylene terephthalatemembrane with 8-�m-diameter pores and is coated with a reconsti-tuted basement membrane gel (BD Biosciences). A total of 2.5 � 104

H1299 cells were suspended in serum-free medium and seeded into theupper wells. After incubation overnight, cells were treated with 3 �M

SAHA for 12 h and then stimulated with 1 nM TNF for a further 24 h inthe presence of 1% FBS and the SAHA. The cells that invaded throughthe Matrigel were stained with 4 �g/ml of Calcein AM (MolecularProbes) in phosphate-buffered saline for 30 min at 37 °C and subjectedto scan fluorescence with a Victor 3 luminometer (PerkinElmerLife Sciences).

Osteoclast Differentiation Assay—To determine whether SAHAcould suppress RANKL-induced osteoclastogenesis, we culturedRAW 264.7 cells, which can differentiate into osteoclasts by RANKLin vitro (23).

Electrophoretic Mobility Shift Assay (EMSA)—To assess NF-�B acti-vation, we performed EMSA as described previously (24).

Western Blot Analysis—Todetermine the levels of protein expressionin the cytoplasmor nucleus, we prepared extracts and fractionated themby SDS-polyacrylamide gel electrophoresis (PAGE) as previouslydescribed (25).

IKK Assay—To determine the effect of SAHA on TNF-induced IKKactivation, IKK assay was performed by a method we described previ-ously (26).

NF-�B-dependent Reporter Gene Expression Assay—The effect ofSAHA on NF-�B-dependent reporter gene transcription induced byTNF and various genes was analyzed by secretory alkaline phosphatase(SEAP) assay as previously described (22).

Immunocytochemical Analysis of NF-�B p65 Localization—Theeffect of SAHA on the nuclear translocation of p65 was examined byimmunocytochemistry as previously described (27).

Luciferase Assay—The effect of SAHA on COX-2 promoter activ-ity induced by TNF was analyzed by luciferase assay as previouslydescribed (28).

RT-PCR Assay—The effect of SAHA on TNF-induced expression ofCOX-2 mRNA was analyzed using RT-PCR with �-actin as an internalcontrol as previously described (28).

Chromatin Immunoprecipitation Assay—The effect of SAHA onNF-�B-binding to COX-2 promoter was analyzed by chromatin immu-noprecipitation assay as described previously (29).

RESULTS

SAHA Potentiated Apoptosis Induced by TNF and ChemotherapeuticAgents—NF-�B activation inhibits apoptosis induced by TNF and che-motherapeutic agents (30).We investigatedwhether SAHAaffects suchapoptosis in KBM-5 cells by using the Live and Dead, PARP cleavage,annexin V staining, TUNEL staining, and MTTmethods. The Live andDead assay indicated that SAHA up-regulated TNF-induced cytotoxic-ity from 5 to 59% (Fig. 1A). Whether the enhanced cytotoxicity was dueto apoptosis was investigated. TNF-induced caspase activation, as indi-cated by PARP cleavage, was potentiated by SAHA (Fig. 1B). The resultsof annexin V staining indicated that SAHA up-regulated TNF-inducedearly apoptosis (Fig. 1C), and the results of TUNEL staining also showedthat TNF-induced apoptosis was enhanced by incubation with SAHA(Fig. 1D).Moreover, SAHAenhancedTNF-induced cytotoxicity as ana-lyzed by the MTT method (Fig. 1E). This histone deacetylase inhibitoralso enhanced the cytotoxic effects of cisplatin, 5-FU, doxorubicin, andtaxol (Fig. 1, F–I, respectively). The results from all these assays together

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5613

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

suggest that SAHA enhances the apoptotic effects of TNF and chemo-therapeutic agents.

SAHA Suppressed TNF-induced Invasion Activity—The mechanismof tumor metastasis has been studied, and it is known that matrix met-alloproteinases, cyclooxygenases, and adhesion molecules play a major

role in it (31). It is also known that TNF can induce tumor metastasis-related genes such as matrix metalloproteinase-9, COX-2, and ICAM-1(30). To investigate the effect of SAHA on TNF-induced metastaticactivity, we examined the invasive activity in vitro. For this study, weseeded theH1299 cells into the upperwells of aMatrigel invasion cham-

FIGURE 1. SAHA potentiates apoptosis induced by TNF and chemotherapeutic agents. A, KBM-5 cells were pretreated with 3 �M SAHA for 12 h and then incubated with 1 nM TNFfor 16 h. Cells were stained with Live and Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. B, cells were pretreated with 3 �M SAHA for 12 h and thenincubated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared and subjected to Western blot analysis using anti-PARP antibody. C, cells were pretreated with 3�M SAHA for 12 h and then incubated with 1 nM TNF for 16 h. Cells were incubated with anti-annexin V antibody conjugated with fluorescein isothiocyanate and then analyzed witha flow cytometer for early apoptotic effects. D, cells were pretreated with 3 �M SAHA for 12 h and then incubated with 1 nM TNF for 16 h. Cells were fixed, stained with TUNEL assayreagent, and then analyzed with a flow cytometer for apoptotic effects. E–I, 5,000 cells/well were seeded in triplicate onto 96-well plates. Cells were pretreated with 3 �M SAHA for 12 hand then incubated with 1 nM TNF (E), 3 �g/ml of cisplatin (F), or 0.1 �M 5-FU (G) for 24 h, or they were pretreated with 1 �M SAHA for 12 h and then incubated with 0.1 �M doxorubicin(H) or 5 nM taxol (I) for 24 h. Thereafter, cell viability was analyzed by the MTT method. Data in panels E–I are presented as means � S.D.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

5614 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

ber in the absence of serum. The cells were pretreated with SAHA andthen treated with TNF in the presence of 1% serum and the SAHA. TNFinduced 3.3-fold higher invasive activity, and SAHA suppressed it(Fig. 2A).

SAHA Suppressed RANKL-induced Osteoclastogenesis—It has beenshown that RANKL can induce osteoclastogenesis through the activa-tion of NF-�B (32, 33). We examined whether SAHA can suppressRANKL-induced osteoclastogenesis. For this study, we pretreatedRAW 264.7 cells with SAHA and then treated them with RANKL for 4or 5 days. We found that RANKL induced osteoclast differentiation, asindicated by the expression of tartrate resistance acid phosphatase-pos-itive, and that SAHA suppressed it (Fig. 2B).

SAHA Repressed TNF-induced NF-�B-dependent Anti-apoptoticGene Products—We found that SAHA potentiated the apoptotic effectsof TNF. Because NF-�B regulates the expression of the anti-apoptoticproteins survivin, inhibitor-of-apoptosis protein 1/2 (IAP1/2), X chro-mosome-linked IAP, Bcl-2, Bcl-xL, TRAF1, and FLIP (30), we examinedwhether SAHA can modulate the expression of these anti-apoptoticgene products induced by TNF in KBM-5 cells. The results of Westernblot analysis showed that TNF induced these anti-apoptotic proteins ina time-dependent manner and that SAHA suppressed it (Fig. 3A).

SAHA Repressed Expression of TNF-induced NF-�B-dependent GeneProducts Involved in Cell Proliferation and Invasion—TNF has beenshown to induce ICAM-1, matrix metalloproteinase-9, c-Myc, COX-2,

cyclin D1, and vascular endothelial growth factor, which have NF-�B-binding sites in their promoters (30). We pretreated KBM-5 cells withSAHA and treated themwith TNF for up to 24 h, and then we preparedwhole-cell extracts and analyzed protein expression by Western blotanalysis. TNF induced the expression of these proteins in a time-de-pendent manner, and SAHA suppressed it (Fig. 3B). These results fur-ther suggest a role for SAHA in blocking the TNF-induced NF-�B acti-vation pathway.

SAHA Blocked NF-�B Activation Induced by IL-1�, Okadaic Acid,Doxorubicin, Lipopolysaccharide, H2O2, PMA, and Cigarette SmokeCondensate—IL-1�, okadaic acid, doxorubicin, lipopolysaccharide,H2O2, PMA, and cigarette smoke condensate are potent activators ofNF-�B, but themechanisms bywhich these agents activateNF-�Bdiffer(30).We used EMSA to examine the effect of SAHAon the activation ofNF-�B by these agents. Pretreatment of KBM-5 cells with SAHA sup-pressed the activation of NF-�B induced by all agents, including TNF(Fig. 4A). These results suggest that SAHA acts at a step in the NF-�Bactivation pathway that is common to all eight agents.

Inhibition of NF-�B Activation by SAHA Was Not CellType-specific—Distinct signal transduction pathways can mediateNF-�B induction in different cell types (34, 35). Because TNF is oneof the most potent activators of NF-�B and because the mechanismof activation of NF-�B is relatively well established (20), we exam-ined the effect of SAHA on TNF-induced NF-�B activation inhuman myeloid KBM-5 cells (Fig. 4B) and lung adenocarcinomaH1299 cells (Fig. 4C). These cells were pretreated with differentconcentrations of SAHA, treated with TNF, and subjected to EMSA.TNF activated NF-�B in both cell types, and SAHA strongly inhib-ited most of this activation. SAHA alone did not activate NF-�B.

Whether SAHA could suppress constitutive NF-�B activation wasalso examined by EMSA. Human squamous cell carcinoma MDA1986cells and SCC-4 cells are known to express constitutively active NF-�B(36). Treatment of these cells with different concentrations of SAHAsuppressed constitutive NF-�B activation (Fig. 4,D and E, respectively).

SAHA Inhibited TNF-dependent NF-�B Activation—Previous stud-ies from our laboratory have shown that at a high concentration (1 nM),TNF can activate NF-�B within 5 min and that this induction is moreintense than that obtained using a 10-fold lower concentration of TNFfor a longer time (37). To determine the effect of SAHA on NF-�Bactivation at a high TNF concentration, KBM-5 cells were pretreatedwith SAHA, treated with up to 1,000 pM TNF, and then analyzed forNF-�B activation by EMSA. TNF at a concentration of 1 nM activatedNF-�B activity strongly; however, cells pretreated with SAHAmarkedlyinhibited this activation (Fig. 5A). These results show that SAHA is apotent inhibitor of TNF-induced NF-�B activation.

We also investigated the length of incubation required for SAHA tosuppress TNF-induced NF-�B activation. KBM-5 cells were incubatedwith SAHA for up to 12 h, exposed to TNF, and subjected to EMSA.SAHA by itself did not activate NF-�B, and TNF-induced NF-�B acti-vation was inhibited by SAHA at 12 h (Fig. 5B). Treatment of cells with30�M SAHA for 12 h had no effect on the cell viability as determined bythe MTT method (see Table 1).NF-�B is a complex of proteins in which various combinations of

Rel/NF-�B protein constitute activeNF-�B heterodimers that bind spe-cific DNA sequences (35). To show that the band visualized by EMSA inTNF-treated cells was indeed NF-�B, we incubated nuclear extractsfromTNF-stimulated KBM-5 cells with antibodies against the p50 (NF-�B1) or p65 (RelA) subunit of NF-�B. Both antibodies shifted the majorband to a higher molecular mass (Fig. 5C), thus suggesting that the

FIGURE 2. SAHA suppresses TNF-induced invasive activity and RANKL-inducedosteoclastogenesis. A, H1299 cells (2.5 � 104) were seeded into the upper wells of aMatrigel invasion chamber overnight in the absence of serum, pretreated with 3 �M

SAHA for 12 h, treated with 1 nM TNF for 24 h in the presence of 1% serum, and thensubjected to invasion assay. The value for no-SAHA and no-TNF was set to 1.0. B, RAW264.7 cells (1 � 104) were plated overnight, pretreated with 0.3 �M SAHA for 12 h, andthen treated with 5 nM RANKL. 4 and 5 days later, cells were stained for tartrate resistanceacid phosphatase-positive and evaluated for osteoclastogenesis. Photographs weretaken after 5 days of incubation with RANKL.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5615

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

TNF-activated complex consisted of p50 and p65 subunits. Preimmuneserum had no effect on this band, excess (100-fold) unlabeled NF-�Bcaused complete disappearance of the band, and a mutant oligonucleo-tide of NF-�B did not affect NF-�B binding activity.

To determine whether the inhibitory activity of SAHA was due toinhibition of I�B� degradation, we pretreated KBM-5 cells with SAHA,exposed them to TNF for up to 60 min, and examined them for NF-�Bactivation by EMSA and for I�B� status in the cytoplasm by Westernblot analysis. Activation of NF-�B was detected with increased TNFincubation times, and SAHA-pretreated cells showed dramaticallydecreased TNF-induced activation of NF-�B (Fig. 5D). The transloca-tion of NF-�B to the nucleus is preceded by the proteolytic degradationof I�B� (35). In our study, TNF induced I�B� degradation in controlcells within 5 min, but in SAHA-pretreated cells, TNF had no effect onI�B� degradation (Fig. 5E). These results indicate that SAHA inhibitsboth TNF-induced NF-�B activation and I�B� degradation.

SAHA Inhibited TNF-dependent I�B� Phosphorylation andUbiquitination—To determine whether the inhibition of TNF-inducedI�B degradation was due to inhibition of I�B� phosphorylation andubiquitination, we used the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal to block degradation of I�B� (38). KBM-5 cells werepretreated with SAHA, treated with 50 �g/ml N-acetyl-leucyl-leucyl-norleucinal for 30 min, exposed to TNF, and then examined for I�B�

phosphorylation and ubiquitination status by Western blot analysisusing an antibody that recognizes the serine-phosphorylated form ofI�B�. TNF-induced I�B� phosphorylation was strongly suppressed bySAHA (Fig. 5F). The same membrane was reprobed with anti-I�B�

antibody. The results show TNF-induced I�B� ubiquitination was alsosuppressed by SAHA (Fig. 5F).

SAHA Inhibited TNF-induced IKK Activation—IKK is required forTNF-induced phosphorylation of I�B� (35). Because SAHA inhibitedthe phosphorylation of I�B�, we determined its direct effect on TNF-

FIGURE 3. SAHA represses TNF-induced NF-�B-dependent expression of anti-apoptosis-, pro-liferation-, and metastasis-related gene prod-ucts. SAHA inhibits the expression of TNF-inducedanti-apoptotic proteins (A) and of cyclin D1, c-Myc,COX-2, matrix metalloproteinase 9 (MMP-9),ICAM-1, and vascular endothelial growth factor(VEGF) (B). KBM-5 cells were incubated with 3 �M

SAHA for 12 h and then treated with 1 nM TNF forthe indicated times. Whole-cell extracts were pre-pared and subjected to Western blot analysisusing the relevant antibodies.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

5616 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

induced IKK activation in KBM-5 cells. Results from the immune com-plex kinase assay showed that TNF activated IKK as early as 5 min afterTNF treatment but that SAHA strongly suppressed this activation (Fig.5G). Neither TNF nor SAHA affected the expression of IKK-� or IKK-�proteins.To evaluate whether SAHA suppressed IKK activity directly by bind-

ing the IKK protein or indirectly by suppressing the activation of IKK,we incubated whole-cell extracts from untreated and TNF-treated

KBM-5 cells with up to 30 �M SAHA. Results from the immune com-plex kinase assay showed that SAHA did not directly affect the activityof IKK, suggesting that SAHA modulates TNF-induced IKK activation(data not shown).

SAHA Inhibited TNF-induced Nuclear Translocation of p65—Todeterminewhether SAHAdirectlymodified the binding of NF-�B com-plex to the DNA, we incubated nuclear extracts from TNF-treatedKBM-5 cells with up to 30 �M SAHA and then analyzed DNA binding

FIGURE 4. A, SAHA blocks NF-�B activation induced by IL-1�, okadaic acid, doxorubicin, lipopolysaccharide, H2O2, PMA, and cigarette smoke condensate. KBM-5 cells were preincu-bated with 30 �M SAHA for 12 h and then treated with 0.1 nM TNF, 100 ng/ml of IL-1� or 10 �g/ml of lipopolysaccharide (LPS) for 30 min, 50 nM okadaic acid (OA) or 5 �M doxorubicin(Dox) for 4 h, 250 �M H2O2 for 2 h, or 15 �g/ml of phorbol 12-myristate 13-acetate (PMA) or 10 �g/ml of cigarette smoke condensate (CSC) for 1 h. The cells were then analyzed forNF-�B activation by EMSA. B and C, inhibition of NF-�B activation by SAHA is not cell type-specific. SAHA suppresses TNF-induced NF-�B in a dose-dependent manner in KBM-5 cells(B) and H1299 cells (C). Cells were incubated with different concentrations of SAHA for 12 h and then incubated with 0.1 nM TNF for 30 min. Nuclear extracts were then prepared andassayed for NF-�B activation by EMSA. D and E, SAHA inhibits constitutive NF-�B activation. MDA1986 cells (D) and SCC-4 cells (E) were incubated with different concentrations ofSAHA for 12 h. Nuclear extracts were then prepared and assayed for NF-�B activation by EMSA.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5617

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

activity by EMSA. Our results showed that SAHA did not modify theDNA binding ability of the NF-�B complex (Fig. 6A), and we concludedthat SAHA inhibits NF-�B activation indirectly rather than directly.

TNF induces the phosphorylation of p65, which is required for itstranscriptional activity (39). After phosphorylation, theNF-�B p65 sub-unit is translocated to the nucleus. In our study, Western blot analysis

FIGURE 5. A–C, SAHA inhibits TNF-dependent NF-�B activation in KBM-5 cells. A, effect of SAHA on the activation of NF-�B induced by different concentrations of TNF. Cells were incubatedwith 30 �M SAHA for 12 h, treated with different concentrations of TNF for 30 min, and then subjected to EMSA to assay for NF-�B activation. B, cells were preincubated with 30 �M SAHA forthe indicated times, treated with 0.1 nM TNF for 30 min, and then subjected to EMSA to assay for NF-�B activation. C, NF-�B induced by TNF is composed of p65 and p50 subunits. Nuclearextracts from untreated or TNF-treated cells were incubated with the indicated antibodies, preimmune serum, unlabeled NF-�B oligo-probe, or mutant oligo-probe and then assayed forNF-�B activation by EMSA. D–G, SAHA inhibits TNF-dependent I�B� degradation. D, SAHA inhibits TNF-induced activation of NF-�B. Cells were incubated with 30 �M SAHA for 12 h, treatedwith 0.1 nM TNF for the indicated times, and then analyzed for NF-�B activation by EMSA. E, effect of SAHA on TNF-induced degradation of I�B�. Cells were incubated with 30 �M SAHA for 12 hand treated with 0.1 nM TNF for the indicated times. Cytoplasmic extracts were prepared, fractionated on 10% SDS-PAGE, and electrotransferred to nitrocellulose membrane. Western blotanalysis was performed using anti-I�B� antibody. Anti-�-actin antibody was the loading control. F, effect of SAHA on the phosphorylation and ubiquitination of I�B� by TNF. Cells werepreincubated with 30 �M SAHA for 12 h, incubated with 50 �g/ml of N-acetyl-leucyl-leucyl-norleucinal (ALLN) for 30 min, and then treated with 0.1 nM TNF for 10 min. Cytoplasmic extractswere fractionated and then subjected to Western blot analysis using phospho-specific anti-I�B� antibody. The same membrane was reblotted with anti-I�B� antibody. G, effect of SAHA onthe activation of IKK by TNF. KBM-5 cells were preincubated with 30 �M SAHA for 12 h, incubated with 50 �g/ml of ALLN for 30 min, and then treated with 1 nM TNF for the indicated times.Whole-cell extracts were immunoprecipitated with antibody against IKK-� and analyzed by an immune complex kinase assay. To examine the effect of SAHA on the expression level of IKKproteins, whole-cell extracts were fractionated on SDS-PAGE and examined by Western blot analysis using anti-IKK-� and anti-IKK-� antibodies.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

5618 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

TABLE 1Effect of SAHA on cell viability of human myeloid cellsHumanmyeloid KBM-5 cells (5,000 cells/well) were treated in triplicate with different concentrations of SAHA for the indicated times, and then cell viability was examinedby the MTT method.

DoseTreatment

12 h 24 hMean S.D. Percent Mean S.D. Percent

�M

0 0.59 0.01 100 0.72 0.01 1001 0.66 0.00 111 0.70 0.01 983 0.66 0.00 112 0.69 0.01 95

10 0.67 0.01 113 0.68 0.01 9530 0.65 0.01 111 0.66 0.00 92

100 0.63 0.00 107 0.69 0.03 95

FIGURE 6. SAHA inhibits TNF-induced nucleartranslocation of p65. A, direct effect of SAHA onthe NF-�B complex. Nuclear extracts were pre-pared from untreated KBM-5 cells or cells treatedwith 0.1 nM TNF for 30 min, incubated for 30 minwith the indicated concentrations of SAHA, andthen assayed for NF-�B activation by EMSA. B,Western blot analysis of p65 using nuclearextracts. Cells were incubated with 30 �M SAHA for12 h and treated with 0.1 nM TNF for the indicatedtimes. Nuclear extracts were prepared and sub-jected to Western blot analysis using anti-p65 anti-body. For loading control of nuclear protein, themembrane was blotted with anti-PARP antibody.C, Western blot analysis of p65 and phosphoryl-ated p65 using cytoplasmic extracts. Cells wereincubated with 30 �M SAHA for 12 h and treatedwith 0.1 nM TNF for the indicated times. Cytoplas-mic extracts were prepared and subjected toWestern blot analysis using anti-p65 antibody andphospho-specific anti-p65 antibody. D, immuno-cytochemical analysis of p65 localization. Cellswere incubated with 30 �M SAHA for 12 h and thentreated with 1 nM TNF for 15 min. Cells were sub-jected to immunocytochemical analysis asdescribed under “Materials and Methods.”

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5619

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

showed that TNF induced nuclear translocation of p65 in a time-de-pendentmanner in KBM-5 cells, as early as 5min after TNF stimulation(Fig. 6B). When the cells were pretreated with SAHA, TNF failed toinduce nuclear translocation of p65. We also determined the effect ofSAHA on TNF-induced phosphorylation of p65 in cytoplasm.Westernblot analysis showed that TNF induced the phosphorylation of p65within 5min but that this phosphorylation gradually decreased and thatSAHA strongly suppressed this phosphorylation (Fig. 6C).

An immunocytochemical assay confirmed the effect of SAHA on thesuppression of nuclear translocation of p65. In untreated KBM-5 cells,p65 localized in the cytoplasm, TNF induced its nuclear translocation,and SAHA suppressed its nuclear translocation to the nucleus (Fig. 6D).

SAHA Repressed TNF-induced NF-�B-dependent Reporter GeneExpression—Although we have shown by EMSA that SAHA blockedNF-�B activation, DNA binding alone does not always correlate withNF-�B-dependent gene transcription, suggesting that there are addi-tional regulatory steps (40). TNF-inducedNF-�B activation is mediatedthrough sequential interaction with the TNF receptor. To determinethe effect of SAHA on TNF-induced NF-�B-dependent reporter geneexpression, we transiently transfected A293 cells with the NF-�B-regu-lated SEAP reporter construct, incubated them with up to 3 �M SAHA,and then stimulated them with TNF. We found that TNF induced

NF-�B-regulated reporter gene expression and that SAHA suppressedit in a dose-dependent manner (Fig. 7A).TNF-inducedNF-�B activation ismediated through sequential inter-

action of the TNF receptor with TRADD, TRAF2, NIK, and IKK, result-ing in phosphorylation of I�B� (41, 42). To determine the effect ofSAHA on TNF-induced NF-�B-dependent reporter gene expression,A293 cells were transiently transfected with TNFR1-, TRADD-,TRAF2-, NIK-, IKK-, and p65-expressing plasmids and thenmonitoredfor NF-�B-dependent SEAP expression. Our results revealed that cellstransfected with these plasmids showedNF-�B-regulated reporter geneexpression and that all were suppressed by SAHA (Fig. 7B). Theseresults suggest that the effect of SAHA occurs at a step downstreamfrom p65.

SAHA Inhibited TNF-induced COX-2 Expression—TNF inducesCOX-2, which has NF-�B-binding sites in its promoter (30). Becausedown-regulation of NF-�B by SAHA suppressed the expression ofNF-�B-regulated gene products, we examined the effect of SAHA onTNF-induced COX-2 promoter activity by using a COX-2 promoter-luciferase reporter plasmid. A293 cells were transiently transfected withthis plasmid, pretreated with up to 3 �M SAHA, exposed to TNF, andthen analyzed for luciferase activity by using whole-cell lysate. Wefound that TNF induced COX-2 promoter activity, which was sup-

FIGURE 7. SAHA represses NF-�B-dependentreporter gene expression induced by TNF. A,SAHA inhibits the NF-�B-dependent reportergene expression induced by TNF. A293 cells weretransiently transfected with an NF-�B-containingplasmid for 24 h. After transfection, the cells wereincubated with the indicated concentrations ofSAHA for 12 h and then treated with 1 nM TNF foran additional 24 h. The supernatants of the culturemedium were assayed for SEAP activity. B, SAHAinhibits the NF-�B-dependent reporter geneexpression induced by TNF, TNFR1, TRADD, TRAF2,NIK, IKK, and p65. Cells were transiently trans-fected with an NF-�B-containing plasmid alone orwith the indicated plasmids. After transfection,cells were incubated with 3 �M SAHA for 12 h andthen incubated with the relevant plasmid for anadditional 24 h. For TNF-treated cells, cells wereincubated with 3 �M SAHA for 12 h and thentreated with 1 nM TNF for an additional 24 h. Thesupernatants of the culture medium were assayedfor SEAP activity. C, SAHA inhibits the COX-2 pro-moter activity induced by TNF. Cells were tran-siently transfected with a COX-2 promoter linkedto the luciferase reporter gene plasmid for 24 hand treated with the indicated concentrations ofSAHA for 12 h. Cells were then treated with 1 nM

TNF for an additional 24 h, lysed, and subjected toa luciferase assay. Experiments in panels A–C arepresented as means � S.D. D, SAHA inhibits COX-2mRNA expression induced by TNF. KBM-5 cellswere pretreated with 3 �M SAHA for 12 h, treatedwith 1 nM TNF for 4 h, and then analyzed for COX-2mRNA expression by RT-PCR. E, SAHA inhibitsbinding of NF-�B to the COX-2 promoter. Cellswere pretreated with 3 �M SAHA for 12 h andtreated with 1 nM TNF for the indicated times. Theproteins were cross-linked with DNA by formalde-hyde and then subjected to chromatin immuno-precipitation assay using an anti-p65 antibodywith the COX-2 primer. Reaction products wereresolved by electrophoresis.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

5620 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

pressed by SAHA in a dose-dependentmanner (Fig. 7C).We also exam-ined the effect of SAHA on induction of COX-2 mRNA transcript.KBM-5 cells were pretreated with SAHA and exposed to TNF for up to4 h, and the total RNA was purified and subjected to RT-PCR. TNFinduced COX-2 mRNA in a time-dependent manner, and treatmentwith SAHA inhibited it (Fig. 7D).Whether the lack of TNF-induced COX-2 expression in SAHA-

treated cells was due to suppression of NF-�B activation in vivo wasexamined by chromatin immunoprecipitation assay targeting NF-�Bbinding in the COX-2 promoter. KBM-5 cells were pretreated withSAHA, treated with TNF for up to 2 h, and cross-linked in situ withDNA-protein complexes, and the chromatin was isolated and sheared.Subsequently, the chromatin was immunoprecipitated with anti-p65antibody, and theDNAwas purified and subjected to PCR using COX-2promoter-specific primer. We found that TNF induced NF-�B bindingto COX-2 promoters in a time-dependent manner and that SAHA sup-pressed it (Fig. 7E). Overall, these results suggest that SAHA inhibitsNF-�B-regulated gene expression by suppressing NF-�B binding to theportion of the COX-2 promoter.

DISCUSSION

This study was designed to determine whether SAHA mediates itseffects by suppressing theNF-�Bpathway.We found that SAHApoten-tiated the apoptosis induced by TNF and chemotherapeutic agents,inhibited TNF-induced invasion and RANKL-induced osteoclastogen-esis, and down-regulated the expression of anti-apoptotic, proliferative,and angiogenic gene products. We also demonstrated that SAHA sup-pressed NF-�B activation induced by a wide variety of agents, inhibitedboth inducible and constitutive NF-�B activation, and inhibited IKKactivation, I�B� phosphorylation, I�B� ubiquitination, I�B� degrada-tion, p65 phosphorylation, p65 nuclear translocation, and NF-�B-de-pendent reporter gene expression.Our observation that SAHA potentiated the apoptotic effects of TNF

and chemotherapeutic agents agrees with results from other studies.Nakata et al. (43) reported that SAHA sensitized human malignanttumor cells to apoptosis induced by TNF-related apoptosis-inducingligand (TRAIL)/APO2-L. Ruefli et al. (7, 44) found that SAHA sensi-tized cells to various chemotherapeutic agents. Kim et al. (45) showedthat pretreatment of cells with SAHA increased the killing efficiency ofVP-16, ellipticine, doxorubicin, and cisplatin in cancer cell lines. Ruefliet al. (7) implicated the role of P-glycoprotein and BID cleavage in cellchemosensitization to SAHA. Nimmanapalli et al. (46) found thatSAHA enhanced imatinib-induced apoptosis of Bcr-Abl-positivehuman acute leukemia cells. Our own results implicate the role ofNF-�B activation induced by cytokines and chemotherapeutic agentsand are consistent with published reports that NF-�B activation cansuppress apoptosis induced by cytokines (47–49) and chemotherapeu-tic agents (50–52).We also found that SAHAmodulated the expressionof anti-apoptotic genes regulated by NF-�B.Our studies showed for the first time that SAHA inhibited TNF-

induced invasion of tumor cells. The role of matrix metalloproteinase-9and adhesion molecules in invasion has been demonstrated (53–55),and we found that the expression of these gene products was down-regulated by SAHA.Our studies also demonstrated for the first time that SAHA abolished

the RANKL-induced differentiation ofmonocytes into osteoclasts. Thisresult is consistent with those of Rahman et al. (56), who observed thattrichostatin A (TSA) and sodium butyrate (NaB), both inhibitors ofhistone deacetylase, suppressed the differentiation of monocytes into

osteoclasts. This suppression was linked to the suppression of TNF-induced NF-�B activation by TSA and NaB.

We also showed for the first time that SAHA inhibited NF-�B acti-vation induced by agents (e.g. TNF, IL-1�, PMA, lipopolysaccharide,and cigarette smoke condensate) that activate NF-�B through differentmechanisms (21, 26, 57, 58). These results suggest that SAHA acts at astep common to all of these activators. In response to most of thesestimuli, NF-�B activation proceeds through sequential activation ofIKK, phosphorylation at serines 32 and 36 of I�B�, and ubiquitination atlysines 21 and 22 of I�B�, leading finally to degradation of I�B� and therelease of NF-�B (35). We found that SAHA blocked NF-�B activationby inhibiting IKK. Whether other histone deacetylase inhibitors couldsuppress NF-�B activation is highly controversial. Some researchershave observed thatNaB andTSA can suppressNF-�B activation (56, 59,60), whereas Chen et al. (61) found that TSA enhanced TNF-inducedNF-�B activation in human embryonic kidney 293 cells, andMayo et al.(62) found no effect of TSA and NaB onNF-�B activation.Why there issuch a discrepancy in results is not clear. Whether cell type, inhibitordose, exposure length, type of NF-�B activator, or method used todetermine NF-�B activation (e.g. reporter assay or DNA binding assay)affects the outcome requires investigation.We clearly showed that SAHA alone did not affect DNA binding of

NF-�B or NF-�B-dependent reporter gene expression and that it did notdirectly inhibit IKK activity. SAHAdid, however, block TNF-induced acti-vation of IKK, which led to suppression of I�B� phosphorylation, ubiquiti-nation, and subsequently degradation. The phosphorylation and nucleartranslocation of p65were also abolishedby SAHA.HowSAHA inhibits theactivation of IKK, which has been shown to phosphorylate p65 (63–66), isunclear. Numerous kinases, including Akt, have been implicated in theactivation of IKK.NaB andTSAhave been shown to inhibitAkt (62). Thus,SAHA might inhibit TNF-induced IKK activation by suppressing Akt,which has also been shown to phosphorylate p65 (41). Therefore, the sup-pressionofp65phosphorylationbySAHAcouldalsobedue to inhibitionofAkt. Whether SAHA interferes with the TNF-induced NF-�B activationpathway by suppressing other, unknown kinases is uncertain. Using a pho-toaffinity labeling technique, Webb et al. (67) identified ribosomal proteinS3 as a potential target of SAHA. Whether this protein has any role inNF-�B activation is unknown at present.

We showed that SAHA inhibited NF-�B-regulated gene transcrip-tion and NF-�B-regulated gene products involved in cell proliferation(e.g. cyclin D1 and COX-2), anti-apoptosis (e.g. survivin, IAP1, IAP2, Xchromosome-linked IAP, Bcl-2, Bcl-xL, Bfl-1/A1, TRAF1, and FLIP),and invasion (matrix metalloproteinase-9 and vascular endothelialgrowth factor). To our knowledge, there is no other published report ofthe regulation of these gene products by SAHA. SAHA induction of cellcycle arrest (4, 68) may be due to down-regulation of cyclin D1, as wehave described. The down-regulation of inflammatory cytokines TNFand IL-1 and of NO (19), c-Myc (17), and thioredoxin (18) by SAHAcould also be due to suppression of NF-�B, as we have described. SAHAhas also been shown to reduce acute graft-versus-host disease and pre-serve the graft-versus-leukemia effect (69); these effects may also bemediated through NF-�B suppression, as we have described.Overall, the results of our studies provide a description of mecha-

nisms through which SAHA could mediate its effects. The ability ofSAHA to enhance apoptosis, suppress invasion, and inhibit osteoclas-togenesis provides novel targets for cancer therapy.

Acknowledgment—We thank Elizabeth L. Hess for carefully proofreading themanuscript and providing valuable comments. We also thank Dr. BryantDarnay for RANKL protein.

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

MARCH 3, 2006 • VOLUME 281 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5621

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

REFERENCES1. Marks, P., Rifkind, R. A., Richon, V. M., Breslow, R., Miller, T. & Kelly, W. K. (2001)

Nat. Rev. Cancer 1, 194–2022. Richon, V. M., Webb, Y., Merger, R., Sheppard, T., Jursic, B., Ngo, L., Civoli, F.,

Breslow, R., Rifkind, R. A. & Marks, P. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,5705–5708

3. Munster, P. N., Troso-Sandoval, T., Rosen, N., Rifkind, R., Marks, P. A. & Richon,V. M. (2001) Cancer Res. 61, 8492–8497

4. Butler, L.M., Agus, D. B., Scher, H. I., Higgins, B., Rose, A., Cordon-Cardo, C., Thaler,H. T., Rifkind, R. A., Marks, P. A. & Richon, V. M. (2000) Cancer Res. 60, 5165–5170

5. Kutko, M. C., Glick, R. D., Butler, L. M., Coffey, D. C., Rifkind, R. A., Marks, P. A.,Richon, V. M. & LaQuaglia, M. P. (2003) Clin. Cancer Res. 9, 5749–5755

6. Richon, V. M., Sandhoff, T. W., Rifkind, R. A. &Marks, P. A. (2000) Proc. Natl. Acad.Sci. U. S. A. 97, 10014–10019

7. Ruefli, A. A., Bernhard, D., Tainton, K.M., Kofler, R., Smyth,M. J. & Johnstone, R.W.(2002) Int. J. Cancer 99, 292–298

8. Rahmani, M., Yu, C., Reese, E., Ahmed, W., Hirsch, K., Dent, P. & Grant, S. (2003)Oncogene 22, 6231–6242

9. Amin, H. M., Saeed, S. & Alkan, S. (2001) Br. J. Haematol. 115, 287–29710. Yu, C., Rahmani, M., Almenara, J., Subler, M., Krystal, G., Conrad, D., Varticovski, L.,

Dent, P. & Grant, S. (2003) Cancer Res. 63, 2118–212611. Shao, Y., Gao, Z., Marks, P. A. & Jiang, X. (2004) Proc. Natl. Acad. Sci. U. S. A. 101,

18030–1803512. Gao, N., Dai, Y., Rahmani, M., Dent, P. & Grant, S. (2004) Mol. Pharmacol. 66,

956–96313. Mitsiades, C. S., Mitsiades, N. S., McMullan, C. J., Poulaki, V., Shringarpure, R.,

Hideshima, T., Akiyama, M., Chauhan, D., Munshi, N., Gu, X., Bailey, C., Joseph, M.,Libermann, T. A., Richon, V. M., Marks, P. A. & Anderson, K. C. (2004) Proc. Natl.Acad. Sci. U. S. A. 101, 540–545

14. Facchetti, F., Previdi, S., Ballarini, M., Minucci, S., Perego, P. & La Porta, C. A. (2004)Apoptosis 9, 573–582

15. Deroanne, C. F., Bonjean, K., Servotte, S., Devy, L., Colige, A., Clausse, N., Blacher, S.,Verdin, E., Foidart, J. M., Nusgens, B. V. & Castronovo, V. (2002) Oncogene 21,427–436

16. Gui, C. Y., Ngo, L., Xu,W. S., Richon,V.M.&Marks, P. A. (2004)Proc. Natl. Acad. Sci.U. S. A. 101, 1241–1246

17. Xu, Y., Voelter-Mahlknecht, S. &Mahlknecht, U. (2005) Int. J.Mol.Med.15, 169–17218. Butler, L. M., Zhou, X., Xu, W. S., Scher, H. I., Rifkind, R. A., Marks, P. A. & Richon,

V. M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11700–1170519. Leoni, F., Zaliani, A., Bertolini, G., Porro, G., Pagani, P., Pozzi, P., Dona, G., Fossati, G.,

Sozzani, S., Azam, T., Bufler, P., Fantuzzi, G., Goncharov, I., Kim, S. H., Pomerantz,B. J., Reznikov, L. L., Siegmund, B., Dinarello, C. A. & Mascagni, P. (2002) Proc. Natl.Acad. Sci. U. S. A. 99, 2995–3000

20. Aggarwal, B. B. (2003) Nat. Rev. Immunol. 3, 745–75621. Anto, R. J., Mukhopadhyay, A., Shishodia, S., Gairola, C. G. & Aggarwal, B. B. (2002)

Carcinogenesis 23, 1511–151822. Takada, Y., Khuri, F. R. & Aggarwal, B. B. (2004) J. Biol. Chem. 279, 26287–2629923. Bharti, A. C., Takada, Y., Shishodia, S. & Aggarwal, B. B. (2004) J. Biol. Chem. 279,

6065–607624. Takada, Y. & Aggarwal, B. B. (2003) J. Biol. Chem. 278, 23390–2339725. Takada, Y. & Aggarwal, B. B. (2003) J. Immunol. 171, 3278–328626. Takada, Y., Mukhopadhyay, A., Kundu, G. C., Mahabeleshwar, G. H., Singh, S. &

Aggarwal, B. B. (2003) J. Biol. Chem. 278, 24233–2424127. Takada, Y. & Aggarwal, B. B. (2004) J. Biol. Chem. 279, 4750–475928. Shishodia, S. & Aggarwal, B. B. (2004) Cancer Res. 64, 5004–501229. Takada, Y., Fang, X., Jamaluddin, M. S., Boyd, D. D. & Aggarwal, B. B. (2004) J. Biol.

Chem. 279, 39541–3955430. Kumar, A., Takada, Y., Boriek, A. M. & Aggarwal, B. B. (2004) J. Mol. Med. 82,

434–44831. Liotta, L. A., Thorgeirsson, U. P. & Garbisa, S. (1982) Cancer Metastasis Rev. 1,

277–28832. Abu-Amer, Y. & Tondravi, M. M. (1997) Nat. Med. 3, 1189–1190

33. Dai, S., Hirayama, T., Abbas, S. & Abu-Amer, Y. (2004) J. Biol. Chem. 279,37219–37222

34. Bonizzi, G., Piette, J., Merville, M. P. & Bours, V. (1997) J. Immunol. 159, 5264–527235. Ghosh, S. & Karin, M. (2002) Cell 109, (suppl.) S81-S9636. Nakayama, H., Ikebe, T., Beppu, M. & Shirasuna, K. (2001) Cancer 92, 3037–304437. Chaturvedi, M. M., LaPushin, R. & Aggarwal, B. B. (1994) J. Biol. Chem. 269,

14575–1458338. Vinitsky, A., Michaud, C., Powers, J. C. & Orlowski, M. (1992) Biochemistry 31,

9421–942839. Ghosh, S., May, M. J. & Kopp, E. B. (1998) Annu. Rev. Immunol. 16, 225–26040. Nasuhara, Y., Adcock, I.M., Catley,M., Barnes, P. J. &Newton, R. (1999) J. Biol. Chem.

274, 19965–1997241. Simeonidis, S., Stauber, D., Chen, G., Hendrickson, W. A. & Thanos, D. (1999) Proc.

Natl. Acad. Sci. U. S. A. 96, 49–5442. Hsu, H., Shu, H. B., Pan, M. G. & Goeddel, D. V. (1996) Cell 84, 299–30843. Nakata, S., Yoshida, T., Horinaka, M., Shiraishi, T., Wakada, M. & Sakai, T. (2004)

Oncogene 23, 6261–627144. Ruefli, A. A., Ausserlechner, M. J., Bernhard, D., Sutton, V. R., Tainton, K. M., Kofler,

R., Smyth, M. J. & Johnstone, R. W. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,10833–10838

45. Kim, M. S., Blake, M., Baek, J. H., Kohlhagen, G., Pommier, Y. & Carrier, F. (2003)Cancer Res. 63, 7291–7300

46. Nimmanapalli, R., Fuino, L., Stobaugh, C., Richon, V. & Bhalla, K. (2003) Blood 101,3236–3239

47. Samanta, A. K., Huang, H. J., Bast, R. C., Jr. & Liao, W. S. (2004) J. Biol. Chem. 279,7576–7583

48. Takada, Y., Singh, S. & Aggarwal, B. B. (2004) J. Biol. Chem. 279, 15096–1510449. Shishodia, S. & Aggarwal, B. B. (2004) J. Biol. Chem. 279, 47148–4715850. Tergaonkar, V., Pando, M., Vafa, O., Wahl, G. & Verma, I. (2002) Cancer Cell 1,

493–50351. Arlt, A., Vorndamm, J., Breitenbroich, M., Folsch, U. R., Kalthoff, H., Schmidt, W. E.

& Schafer, H. (2001) Oncogene 20, 859–86852. Bottero, V., Busuttil, V., Loubat, A.,Magne, N., Fischel, J. L.,Milano, G. & Peyron, J. F.

(2001) Cancer Res. 61, 7785–779153. Yamamoto, K., Arakawa, T., Ueda, N. & Yamamoto, S. (1995) J. Biol. Chem. 270,

31315–3132054. Esteve, P.O., Chicoine, E., Robledo,O., Aoudjit, F., Descoteaux, A., Potworowski, E. F.

& St-Pierre, Y. (2002) J. Biol. Chem. 277, 35150–3515555. van de Stolpe, A., Caldenhoven, E., Stade, B. G., Koenderman, L., Raaijmakers, J. A.,

Johnson, J. P. & van der Saag, P. T. (1994) J. Biol. Chem. 269, 6185–619256. Rahman, M. M., Kukita, A., Kukita, T., Shobuike, T., Nakamura, T. & Kohashi, O.

(2003) Blood 101, 3451–345957. Pahl, H. L. (1999) Oncogene 18, 6853–686658. Garg, A. & Aggarwal, B. B. (2002) Leukemia 16, 1053–106859. Yin, L., Laevsky, G. & Giardina, C. (2001) J. Biol. Chem. 276, 44641–4464660. Yu, Z., Zhang, W. & Kone, B. C. (2002) J. Am. Soc. Nephrol. 13, 2009–201761. Chen, L., Fischle, W., Verdin, E. & Greene, W. C. (2001) Science 293, 1653–165762. Mayo, M. W., Denlinger, C. E., Broad, R. M., Yeung, F., Reilly, E. T., Shi, Y. & Jones,

D. R. (2003) J. Biol. Chem. 278, 18980–1898963. Yang, F., Tang, E., Guan, K. & Wang, C. Y. (2003) J. Immunol. 170, 5630–563564. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young,

D. B., Barbosa, M., Mann, M., Manning, A. & Rao, A. (1997) Science 278, 860–86665. Sakurai, H., Chiba,H.,Miyoshi, H., Sugita, T.&Toriumi,W. (1999) J. Biol. Chem. 274,

30353–3035666. Sizemore, N., Lerner, N., Dombrowski, N., Sakurai, H. & Stark, G. R. (2002) J. Biol.

Chem. 277, 3863–386967. Webb, Y., Zhou, X., Ngo, L., Cornish, V., Stahl, J., Erdjument-Bromage, H., Tempst,

P., Rifkind, R. A., Marks, P. A., Breslow, R. & Richon, V. M. (1999) J. Biol. Chem. 274,14280–14287

68. Huang, L. & Pardee, A. B. (2000)Mol. Med. 6, 849–86669. Reddy, P., Maeda, Y., Hotary, K., Liu, C., Reznikov, L. L., Dinarello, C. A. & Ferrara,

J. L. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 3921–3926

SAHA Inhibits NF-�B Activation and Potentiates Apoptosis

5622 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 9 • MARCH 3, 2006

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from

Yasunari Takada, Ann Gillenwater, Haruyo Ichikawa and Bharat B. AggarwalB ActivationκAbolishes Osteoclastogenesis by Suppressing Nuclear Factor-

Suberoylanilide Hydroxamic Acid Potentiates Apoptosis, Inhibits Invasion, and

doi: 10.1074/jbc.M507213200 originally published online December 23, 20052006, 281:5612-5622.J. Biol. Chem. 

  10.1074/jbc.M507213200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/281/9/5612.full.html#ref-list-1

This article cites 69 references, 46 of which can be accessed free at

by guest on February 18, 2020http://w

ww

.jbc.org/D

ownloaded from