Deubiquitinase Inhibition of 19S Regulatory Particles …...Small Molecule Therapeutics Deubiquitinase Inhibition of 19S Regulatory Particles by 4-Arylidene Curcumin Analog AC17 Causes

Small Molecule Therapeutics

Deubiquitinase Inhibition of 19S Regulatory Particles by4-Arylidene Curcumin Analog AC17 Causes NF-kB Inhibitionand p53 Reactivation in Human Lung Cancer Cells

Binhua Zhou1, Yinglin Zuo1, Baojian Li3, Hao Wang1, Hao Liu1, Xianfeng Wang1, Xu Qiu1, Yun Hu1,Shijun Wen1,2, Jun Du1, and Xianzhang Bu1

AbstractProteasome inhibitors have been suggested as potential anticancer agents in many clinical trials. Recent

evidence indicates that proteasomal deubiquitinase (DUB) inhibitors, bearing a differentmechanism from that

of traditional proteasome inhibitors, would be appropriate candidates for new anticancer drug development.

In the present study, we describe the deubiquitinase inhibition of 19S regulatory particles (19S RP) by AC17, a

4-arylidene curcumin analog synthesized in our laboratory. Although 4-arylidene curcumin analogs were

reported to act as inhibitory kB (IkB) kinase (IKK) inhibitors, AC17 instead induced a rapid and marked

accumulation of ubiquitinated proteins without inhibiting proteasome proteolytic activities. In contrast to

its parent compound, curcumin, which is a proteasome proteolytic inhibitor, AC17 serves as an irreversible

deubiquitinase inhibitor of 19S RP, resulting in inhibition of NF-kB pathway and reactivation of proapoptotic

protein p53. In addition, in a murine xenograft model of human lung cancer A549, treatment with AC17

suppresses tumor growth in a manner associated with proteasome inhibition, NF-kB blockage, and p53

reactivation. These results suggest that 4-arylidene curcumin analogs are novel 19S deubiquitinase inhibitors

with great potential for anticancer drug development. Mol Cancer Ther; 12(8); 1381–92. �2013 AACR.

IntroductionThe ubiquitin-proteasome pathway (UPS) is responsi-

ble for the nonlysosomal degradation ofmost intracellularproteins and plays a crucial role in the regulation ofnumerous cellular and physiologic functions, includingprotein quality control, cell-cycle progression, prolifera-tion, differentiation, angiogenesis, and apoptosis (1, 2). InUPS, proteins targeted for degradation are first covalentlymodified by a polyubiquitin chain. Three classes ofenzymes are involved: ubiquitin-activating enzyme E1(3), ubiquitin-conjugating enzyme E2 (4), and ubiquitin-protein ligase E3 (5). E1 activates ubiquitinmonomers andtransfers them to E2, which thenwork in conjunctionwithE3 to specifically recognize and recruit substrates of theubiquitination reaction. The polyubiquitinated substratesare then recognized by 26S proteasome (a large multi-

subunit and ATP-dependent proteolytic complex) andrapidly degraded into small peptides (6).

Thus 26S proteasome is the key component of UPS. Onthe basis of both structure and function, 26S proteasomeconsists of a hollow cylindrical 20S proteolytic core par-ticle (20S CP) and one or two 19S regulatory particles (19SRP; ref. 7). At the middle of 26S proteasome is 20S CP,which is formed by 4 stacked rings made of different a(structural) and b (catalytic) subunits, with the b1, b2, andb5 subunits accounting for peptidylglutamyl peptidehydrolyzing-like (PGPH-like), trypsin-like (T-like), andchymotrypsin-like (CT-like) activities, respectively (8).Capping at either end of 20S CP is 19S RP that controlsubiquitin-tagged substrates for proteolysis in the catalyticchamber. In human body, 19S RP is associated with 3deubiquitinating enzymes (DUB): ubiquitin C-terminalhydrolase 5 (UCHL5), ubiquitin-specific proteases 14(USP14), and proteasomal deubiquitinase 1 (POH1;refs. 9–11). Though the physiologic functions of 19S deu-biquitinases are not completely clear, all of them havebeen proved to dynamically regulate 26S proteasomeactivity (12, 13).

As theUS Food andDrugAdministration approved the20S proteolytic inhibitor bortezomib (VELCADE) fortreatment ofmultiple myeloma in 2003 (14), UPS has beenconsidered an appropriate therapeutic target for cancertherapy and caught increasing academic. In the pastseveral decades, many structurally diverse inhibitors of20S CP have been discovered from both synthetic and

Authors' Affiliations: 1School of Pharmaceutical Sciences, Sun Yat-senUniversity; 2TheStateKey Laboratory ofOncology inSouthChina, SunYat-sen University Cancer Center, Guangzhou; and 3Department of Chemistry,The University of Hong Kong, Hong Kong, China

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Xianzhang Bu, School of PharmaceuticalSciences, Sun Yat-sen University, Guangzhou 510275, China. Phone:86-020-39943054; Fax: 86-020-39943054; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-1057

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on July 7, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 21, 2013; DOI: 10.1158/1535-7163.MCT-12-1057

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natural product, in which at least 5 are at different stagesof clinical development (15). However, not all patientswith multiple myeloma respond to bortezomib treatment(16, 17). The molecular mechanisms of resistance to bor-tezomib are multiple, involving increased expression ofantiapoptotic proteins or mutated status of proapoptoticfactors, such as overexpression of Bcl-2 (18–20) or disrup-tion of p53 (21). Therefore, the development of novelproteasome inhibitors for second-line treatment isrequired.

AC17, a 4-arylidene curcumin analog, was initiallysynthesized and identified as an inhibitorykB (IkB) kinase(IKK) inhibitor in our preliminary study (22). Comparedwith the parent compound curcumin, AC17 showsimproved oral bioavailability,metabolic stability (23), andmoderately potent anticancer activities against severaldifferent cancer cell lines, including lung cancer, coloncancer, breast cancer, and hepatocellular carcinomacells (Supplementary Fig. S1). As we have previouslydescribed, IKK blockage by 4-arylidene curcumin analogsonly partly reveals the mechanism of the anticancer activ-ity of AC17 (22). Although the exact mode of action ofAC17 remains unclear, AC17 most likely is a pleiotropicmolecule such as curcumin, which modulates numeroustargets (24–26). In the present study, AC17 serves asa nonclassical proteasome inhibitor that blocks the deu-biquitinase activity of 19S RP without inhibiting theproteolytic activities of 20S CP, inducing a rapid andnoticeable accumulation of protein-ubiquitin conjugates,resulting in inhibition of NF-kB pathway and reactivationof p53 function. Furthermore, treatment of human lungcancer-bearing BALB/c-nu mice with AC17 resulted intumor growth suppression, correlating with in vivo ubi-quitinated proteins accumulation, transcription factorNF-kB inhibition, and proapoptotic protein p53 reactiva-tion. These results show thatAC17 represents anovel classof proteasome inhibitors through inhibiting 19S deubi-quitinase activity but not 20S proteolytic activities, whichcould potentially be used for the treatment of humancancers.

Materials and MethodsCell culture, chemical reagents, and enzymes

Human lung carcinoma cell lines A549 and NCI-H1299were obtained from the cell bank of the Shanghai Instituteof Biochemistry and Cell Biology, in which they weretested and authenticated for genotypes by DNA-finger-printing analysis. The cell lines were not passaged over 6months, and thus no authentication was done by theauthors. Cells were grown in RPMI-1640medium (Gibco)supplemented with 10% FBS (Gibco), 100 U/mL penicil-lin, and 100 mg/mL streptomycin. All cells were culturedand maintained at 37�C under humidified atmospherewith 5% CO2.

4-Arylidene curcumin analogAC17, (1E,6E)-1,7-bis(3,4-dimethoxyphenyl)-4-(4-hydroxy-3-methoxybenzylidene)hepta-1,6-diene-3,5-dione), was synthesized in an analo-gous manner as previously reported (22). Other reagents

were obtained from the following sources: TNFa, NEM(N-ethylmaleimide), MG-132, Nutlin-3a, and dimethylsulfoxide (DMSO; Sigma); Ub-AMC (ubiquitin-7-amido-4-methylcoumarin), human 19S proteasome, human 20Sproteasome, human 26S proteasome, Suc-LLVY-AMC,and Z-LLE-AMC (BostonBiochem); Z-ARR-AMC (Cal-biochem); and Vectors (pRL-TK and pNF-kB-luc;Promega).

Western blotting and immunoprecipitation analysisWhole-cell lysates were prepared by using cell lysis

buffer (Beyotime) and were boiled in 1� Laemmli reduc-ing sample buffer. Equal protein amounts were electro-phoresed on SDS-PAGE gels, transferred to membranes,and immunoblotted. For immunoprecipitation, cellularlysate (500 mg)was used to immunoprecipitate, andWest-ern blotting was conducted to examine ubiquitination orinteraction of proteins.

Antibodies were purchased from the following sources:anti-b-actin, anti-mouse double minute 2 homolog (anti-MDM2), anti-glyceraldehyde 3-phosphate dehydrogenase(anti-GAPDH), anti-ubiquitin, and goat anti-mouse/rabbitIgG-conjugated horseradish peroxidase (Santa Cruz Bio-technology); anti-IkBa, anti-p-IkBa (Ser32), anti-p65, anti-p53, and anti-p21 (Cell Signaling Technology).

Levels of mRNA by real-time reverse transcriptasePCR

To determine mRNA expression, 500 ng of total RNAextracted from each sample were used for reverse tran-scription (RT) reaction in 10 mL of reaction volume using areverse transcription system (TaKaRa) according to themanufacturer’s instructions. The SYBR Premix Ex Taq Kit(TaKaRa) was used for real-time PCR reaction. Afternormalization to GAPDH gene, expression levels for eachtarget genewere calculated using the comparative thresh-old cycle method.

Sequences of PCR primers were as follows:

p53, forward, 50- GCGCACAGAGGAAGAATCTCG-30,and reverse, 50- TTTGGCTGGGGAGAGGAGCTG-30;p21, forward, 50- TGTACCCTTGTGCCTCGCTC-30,and reverse, 50- TGGAGAAGATCAGCCGGCGT-30;IkBa, forward, 50- CTGAGCTCCGAGACTTTCGAGG-30,and reverse, 50- CGTCCTCTGTGAACTCCGTG-30;MDM2, forward, 50- ATCTTGGCCAGTATATTATG-30,and reverse, 50-GTTCCTGTAGATCATGGTAT-30;GAPDH, forward, 50-CACCCAGAAGACTGTGGATG-

G-30,and reverse, 50-GTCTACATGGCAACTGTGAGG-30.

Reporter gene assayCells at approximately 70% confluency were trans-

fected with 0.2 mg DNA/cm2 per pNF-kB-luc plasmidusing Lipofectamine 2000 (Invitrogen). pRL-TK wascotransfected as a control for transfection efficiency.Twenty-four hours after transfection, AC17 was added

Zhou et al.

Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics1382




for 1 hour before exposure to TNFa for another 2 hours.Then cells were lysed and luciferase activity was deter-mined with a dual-luciferase assay kit (Promega).

Proteasome activity assayFluorogenic peptide substrates Suc-LLVY-AMC, Z-LLE-

AMC, and Z-ARR-AMCwere used to assay for the protea-somal CT-like, PGPH-like, and T-like activities. To assesscellular proteasome activity, 20mgwhole-cell extractswereincubated at 37�C with 40 mmol/L fluorogenic substratesin 200 mL assay buffer containing 50 mmol/L Tris-HCl(pH 8.0), 150 mmol/L NaCl, 10% (v/v) glycerol, and0.03% (w/v) SDS. For the evaluation of T-like activity, SDSwas omitted from the assay buffer. To assess proteasomeactivity in vitro, purified proteasome was incubated withcompounds for 30 minutes at 37�C before addition ofsubstrates. Fluorescence intensity was measured using aFlex Station 3 microplate reader (Molecular Devices) atlex¼ 380 nm and lem¼ 460 nm after compensation for thebuffer background and compoundquenchingfluorescenceaccording to the Stern–Volmer equation.

Ub-AMC protease assayCells were lysed in an assay buffer of 50 mmol/L Tris-

HCl (pH7.5), 5mmol/LMgCl2, 1mmol/LDTT, 2mmol/LATP, and250mmol/Lsucrose.Tenmicrogramsof clarifiedlysate was incubated with 400 nmol/L Ub-AMC at 37�C,and fluorescence intensity was recorded at excitation/emission of 380/460 nm using a spectrofluorometer.Purified proteasome was incubated in assay buffer

containing AC17 (indicated concentration), vehicle(DMSO), or positive control (NEM) at 37�C for 30minutes,following addition of 400 nmol/L Ub-AMC. The reactionwas quantified at excitation/emission of 380/460 nmaftercompensation for the buffer background and compoundquenching fluorescence according to the Stern–Volmerequation.

Cell-cycle analysisBoth adherent and floating cells were harvested and

fixedwith 75% ethanol at�20�C overnight. Then the cellswere incubated with 5 mg/mL propidium iodide (PI) and5 mg/mL RNase A at 37�C for 30 minutes. Cell-cycledistribution and sub-G1 DNA content cells were mea-sured with an EPICS XL analyzer (Beckman Coulter).

Cellular and nuclear morphology analysisA Nikon Eclipse Ti-S microscope was used for all

microscopic imaging with either phase contrast for cellu-lar morphology or fluorescence for nuclear morphology.For fluorescent nuclear morphology analysis, cells werewashed with ice-cold PBS, fixed in 4% paraformaldehydefor 1 hour, stained with 50 mmol/L Hoechst 33342 in thedark at 4�C for 10 minutes, and then observed.

Animal experimentsAll animal experiments complied with the Zhongshan

School of Medicine Policy on the Care and Use of Labo-

ratory Animals. Female BALB/c-nu mice (5 weeks old)were purchased from Shanghai SLACLaboratory AnimalCo. Ltd andmaintained inpathogen-free conditions.A549cells were harvested during log-phase growth and resus-pended in RPMI-1640 medium at 5 � 107 cells/mL. Eachmousewas injected subcutaneously in the right flankwith1 � 107 cells. When the tumor volume reached approxi-mate 120 mm3, mice were randomized into 3 groups andtreatment was initiated (at day 7). AC17 (1 and 5 mg/kgbody weight) dissolved in 200 mL solution (polyethyleneglycol 400: Cremophor EL: Physiological Saline, 8:1:11)was administered intraperitoneally every fifth day for 4times. Control groupwas treatedwith an equal volume ofvehicle. The tumor volumewas estimated according to theformula: tumor volume¼ L�W2/2, where L is the lengthand W is the width of tumor. One day after the lastinjection, tumors and organswere rapidly frozen in liquidnitrogen and stored at �70�C for protein extraction, andanother portion was fixed in formalin for histology.

ImmunohistochemistryFormalin-fixed tissue sectionswere evaluated by hema-

toxylin and eosin (H&E) staining and immunohistochem-istry. Immunofluorescence staining of formalin-fixed par-affin-embedded tissue was conducted as previouslydescribed (27).

Statistical analysisCell data were derived from at least 3 independent

experiments, and animal data were derived from thexenograft study described above. Student t test was usedto assess the differences between sets of data. Probabilityvalues below 0.05 were considered significant.

ResultsCompared with curcumin, AC17 displayed much

greater growth inhibitory activities against several typesof cancer cell lines (Fig. 1A, Supplementary Fig. S1).Notably, AC17 was more toxic to lung cancer cell linessuch as A549 and NCI-H1299 cells compared with mouseprimary hepatocytes (Supplementary Fig. S2). Our pre-vious study showed that AC17 inhibited TNFa-inducedNF-kB activation in an NF-kB translocation assay with anIC50 of 1.0 � 0.55 mmol/L (Fig. 1B; ref. 22). Dual-Gloluciferase analysis affirmed that AC17 blocked TNFa-induced transcriptional activation of NF-kB (Fig. 1C).Although NF-kB pathway inhibition is partially respon-sible for the potent anticancer activity of AC17 (Supple-mentary Fig. S3), the underlying mechanism remainsunclear. The effects of AC17 both in vitro and in vivoweretherefore investigated in details.

Inhibition of IkBa degradation by AC17 suppressesNF-kB activation

Following treatment with the protein translationalinhibitor (cycloheximide, CHX), the decay rate of IkBawas much slower than that without AC17 pretreatment(Fig. 1D). No significant change of IkBa mRNA level in a

Deubiquitinase Inhibition of 19S RP by AC17

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time-dependent manner was observed, as indicated byRT-PCR analysis of IkBa mRNA expression in AC17-treated A549 cells (Fig. 1E). These results strongly implythat the regulation of IkBa expression by AC17 occurs atposttranslational level, not at transcriptional level. Thedegradation of IkB inUPS is required for the release ofNF-kB, which after translocation to the nucleus will activategenes involved in cell proliferation and survival. Todetermine whether AC17 inhibits IkB degradation, IkBawas immunoprecipitated from the extracted whole-cellproteins, and the ubiquitination state of IkBa was mea-sured by Western blotting. Compared with the well-known 20S proteolytic inhibitor MG-132, AC17 resultedin an accumulation of polyubiquitinated IkBa after treat-

ing A549 cells for 12 hours (Fig. 1F). The interactionbetween IkBa and NF-kB p65 subunit was detected inAC17- orMG-132–treatedA549 cells, indicating that therewas no dissociation of IkBa from p65 after AC17 or MG-132 exposure (Fig. 1G), and thus prevented NF-kB tran-scriptional activity. In short, NF-kB activation could beinhibited by AC17 by blocking UPS of IkB degradation.

Rapid ubiquitination is induced by AC17 by blockingthe deubiquitinase activity of 19S RP

Western blot analysis of whole-cell extracts fromAC17-treatedA549 cells showedamarked concentration-depen-dent accumulation of ubiquitinated proteins after 2 hoursof treatment (Fig. 2A, left). A similar increase in protein

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Figure 1. AC17 inhibits NF-kBactivation by inhibiting degradationof IkBa. A, the chemical structuraldesign of curcumin to AC17. B,A549 cells were treatedwith DMSO(control) or AC17 for 30 minutes,followed by stimulation with TNFa(10 ng/mL) for 30minutes, and thenNF-kB subcellular localization wasverified by immunofluorescencestainingwith p65 antibody. C, A549cells were cotransfected with pNF-kB-luc and pRL-TK and treated asindicated with TNFa (10 ng/mL),AC17 (1 mmol/L), and thecombination before determiningNF-kBtranscriptional activityby thedual-luciferase reporter assaysystem.The resultspresentmean�SD of 3 independent experiments.�, P < 0.05. D, A549 cells weretreated either with or without 4mmol/LAC17 for 1 hour, followedbyincubation with CHX (5 mmol/L) fordifferent time points as indicated,and then whole-cell extracts wereanalyzed by Western blotting forIkBa expression. The intensity ofimmunoblots digitized by ImageJsoftwarewas normalized tob-actin,and then arbitrarily normalized withthe intensity value for IkBa at time 0to 1.00. E, A549 cells were treatedwith AC17 for different times asindicated, and total RNA wasisolated and subjected to RT-PCRanalysis for IkBa mRNA levels.Columns, mean (n¼ 3); bars, SD. Fand G, A549 cells were treated witheither AC17 or MG-132 for 12hours, then the extracted wholeproteins were immunoprecipitatedwith IkBa or p65 antibody. Thelevels of ubiquitinated IkBa (F) andinteraction of p65 with IkBa (G)were detected by Western blotanalysis. TNFa and IgG were usedfor comparison. b-Actin proteinamounts were used to verify equalprotein for immunoprecipitation.

Zhou et al.





ubiquitination was also observed in AC17-treated NCI-H1299 cells (Fig. 2A, right). As shown in Fig. 2B, a rapid,time-dependent accumulation of ubiquitinated proteinswas induced in A549 cells by AC17 treatment. Similar toAC17, MG-132 induced an increase of protein ubiquitina-tion in A549 cells (Fig. 2C), suggesting similar down-stream effectors for AC17 and MG-132. However, furtherinvestigation of the effect of AC17 on 20S proteolyticactivities showed no significant decline of any of protea-

some proteolytic activities following AC17 incubation inA549 cells, purified human 20S or 26S proteasome,where-as MG-132 exhibited substantial inhibitory effects in allassays (Fig. 2D). These results show that, unlike MG-132,AC17 does not directly block proteasome proteolyticactivities in vitro.

Molecular analysis has showed that a cross-conju-gated a,b-unsaturated dienone compound with 2 steri-cally accessible electrophilic b-carbons is a molecular

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Figure 2. AC17 induces a rapidaccumulation of polyubiquitinatedproteins with the 19S DUBinhibition. A, A549 cells (left) andNCI-H1299 cells (right) wereincubated with the indicatedconcentration of AC17 for 2 hoursbefore whole-cell extracts weresubjected to Western blot withubiquitin antibody. B, A549 cellswere treated with 4 mmol/L AC17for the different times, and theextractswere analyzed byWesternblot for the accumulation ofubiquitinated proteins. C, A549cells were treated with AC17 (4mmol/L) or MG-132 (10 mmol/L) for2 hours, then whole-cell lysateswere examined for ubiquitinatedproteins. GAPDH and b-actin wereused as a loading control. D,purified human 20S (left) and 26Sproteasome (middle) wereincubatedwithAC17 (50mmol/L) orMG-132 (10 mmol/L) for 30minutesbefore analysis of 3 proteasomalproteolytic activities; lysates (right)from the control (DMSO), AC17-treated, MG-132-treated A549cells (10 mmol/L, 2 hours) wereanalyzed for these activities.Columns, average of triplicateexperiments; bars, SD. E, A549cells (left) and NCI-H1299 cells(right) were treated with 4 mmol/LAC17 for 4 hours, and total DUBactivity was determined fromwhole-cell extracts by measuringUb-AMC cleavage. NEM(10 mmol/L) was a control. F,purified human 19S (left) and 26Sproteasome (right) were incubatedwith AC17 (50 mmol/L) or NEM(10 mmol/L) for 30 minutes,following DUB activity wasmeasured as described inMaterials and Methods. Theresults are from 1 representativein 3 independent experiments.Similar results are observed inadditional 2 assays.






determinant of deubiquitinase inhibitors (28), and theinhibition of cellular deubiquitinase activity leads to anincrease in ubiquitinated proteins. On the basis ofthe chemical structure of AC17 (Fig. 1A), the possibilityof deubiquitinase inhibition in AC17-treated cells wasassessed. However, no distinct reduction of total deubi-quitinase activity was detected using Ub-AMC as asubstrate in A549 or NCI-H1299 cells following AC17treatment (Fig. 2E).As apositive control,NEMcompletelysuppressed total deubiquitinase activity in both lungcarcinoma cell lines (Fig. 2E). Given the similar pharma-cologic effect of a novel class of proteasome inhibitors,which block the 19S deubiquitinase activity without inhi-biting the 20S proteolytic activities (29), we hypothesizethatAC17blocks 19Sdeubiquitinase activity indirectly forits proteasome inhibition. To assess the effect of AC17 onthe deubiquitinase activity of 19S RP, purified human 19Sor 26S proteasome was incubated with AC17 for 30 min-utes, and then the substrate Ub-AMC was added. Thefluorescence intensity change as a consequence of sub-strate cleavage showed that AC17 significantly inhibitedthe 19S deubiquitinase activity (Fig. 2F), and IC50 value is4.23 � 0.010 mmol/L (Supplementary Fig. S4). 19S deubi-quitinase inhibition by AC17 was further confirmed byK63-linked ubiquitin tetramer chain disassembly in vitro(Supplementary Fig. S5). In addition, AC17 was found tobe an irreversible 19S deubiquitinase inhibitor (Supple-mentary Fig. S6), and the inhibitory activity of AC17 wascompletely lost in the presence of glutathione (GSH;Supplementary Fig. S7). These together suggest thatAC17may covalently bind to cysteine residues of target pro-teins. These observations show that AC17 is not a generaldeubiquitinase inhibitor such as NEM; instead, AC17selectively inhibits 26S proteasome by blocking the 19Sdeubiquitinase activity (necessary for efficient cellularprotein degradation), resulting in the accumulation ofubiquitinated proteins.

AC17 reactivates wild-type p53 by inhibiting p53degradation

For tumors expressing wild-type p53, p53 reactivationcan be achieved in the context of a general disruption ofUPS as a therapeutic goal (30). Our previous studiesindicated that A549 cells were more sensitive to AC17treatment than NCI-H1299 cells (Supplementary Fig. S2).As p53 is partially responsible for the cytotoxicity ofAC17(Supplementary Fig. S3) and there is an obviousdifferencebetween A549 cells and NCI-H1299 cells in the p53 status(wild-type p53 in A549 cells and null p53 in NCI-H1299cells), it is hypothesized that wild-type p53 is reactivatedby AC17 through inhibiting 19S deubiquitinase activityin A549 cells. Exposing A549 cells to AC17 for 12 hoursnoticeably increased p53 expression, as well as its down-stream targets (MDM2 and p21) expression in a dose-dependent manner (Fig. 3A). MDM2 is a key negativeregulator of p53, which promotes p53 ubiquitination andtargets p53 forproteasomal degradation (31). p21 is awell-characterized cyclin-dependent kinase inhibitor that

plays an important role in cell-cycle control. p21 over-expression results in cell-cycle arrest,which contributes toits tumor suppressor function. Cell-cycle analysis showedthat sub-G1 cell populations of A549 cells increased in aconcentration-dependent manner after a 24-hour incuba-tion with AC17 (Fig. 3B), indicating that apoptosis wasinduced by AC17 treatment. AC17-induced apoptosiswas further shown by apoptosis-associated cellular andnuclearmorphologic changes. Comparedwith the controlgroup, cellular morphology changes (spherical shape anddetachment) were observed in AC17-treated A549 cells(Fig. 3C, top). After Hoechst staining, apoptotic nuclearchanges (punctate or granular and bright nuclei) werevisualized only in cells treatedwithAC17, not cells treatedwith DMSO (Fig. 3C, bottom). These results support thehypothesis that AC17 reactivates wild-type p53 andinduces apoptosis in a concentration-dependent mannerin A549 cells.

To investigate whether 19S deubiquitinase inhibitoryactivity of AC17 is associated with wild-type p53 reacti-vation, p53 mRNA expression and the interactionbetween MDM2 and p53 were studied. RT-PCR analysisrevealed that after incubation, A549 cells with AC17, p53mRNA decreased, whereas MDM2 and p21 mRNAincreased in a concentration-dependent manner (Fig.3D). Presumably, the reactivation of wild-type p53 byAC17 occurs at posttranslational level. As the tumorsuppressor activity of p53 is effectively inhibited by itscellular inhibitor MDM2 through direct interaction withp53, blocking MDM2-p53 interaction is an importantstrategy for anticancer drug design (32). To determinewhether the MDM2–p53 interaction is inhibited by AC17treatment, aliquots of A549 cells after AC17, MG-132, andpositive control (Nutlin-3a) treatment were subjected tocoimmunoprecipitation analysis. As shown in Fig. 3E,treatment with AC17 or MG-132 did not dissociate p53from MDM2, whereas Nutlin-3a caused the dissociationof p53-MDM2 complex. If 19S deubiquitinase inhibitionby AC17 is responsible for wild-type p53 reactivation, thelevels of ubiquitinatedp53 should be increased afterAC17treatment of A549 cells. Comparedwith the control, accu-mulation of ubiquitinated p53was detected after 12 hoursof AC17 treatment (Fig. 3F). MG-132 treatment had asimilar effect, but Nutlin-3a treatment prevented theaccumulation of polyubiquitinated p53 (Fig. 3F). Collec-tively, these results show that, as a 19S deubiquitinaseinhibitor, AC17 reactivates endogenous wild-type p53 byinhibiting UPS of p53 degradation.

Suppression of human lung adenocarcinoma A549xenograft proliferation by AC17 is associated withproteasome inhibition, p53 reactivation, and NF-kBblockage

AC17 is a novel proteasomal deubiquitinase inhibitorthat blocks NF-kB pathway and reactivates wild-type p53in the cultured lung cancer cell line A549. Therefore, wefurther investigated the antitumor effects of AC17 in vivo.To do so, A549 cells were subcutaneously injected into the

Zhou et al.





flanks of female BALB/c nude mice. When the tumorsreached approximately 120 mm3, mice were randomizedinto 3 groups and treated by intraperitoneal injectionwitheither vehicle control or AC17 at doses of 1 and 5 mg/kg.During treatment, the tumor sizes in these groups weremeasured 2 times per week (Fig. 4A). At the end of theexperiment, tumor tissues were removed from the mice,weighed, and photographed (Fig. 4B and C). The inhibi-tion of tumor growth byAC17 at 1 and 5mg/kg compared

with the vehicle-administered control group were 34.5%and 45.6%, respectively. These results imply that AC17possesses effective antitumor activity in vivo.

To determine whether the observed antitumor effect ofAC17 in vivo is associated with its proteasome-inhibitory,p53-reactivation, and NF-kB-blocking activities, samplesof the control or AC17-treated tumors were subjected tomultiple assays. Figure 4D shows that the accumulation ofubiquitinated proteins was obvious in AC17-treated

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Figure 3. AC17 reactivatesproapoptotic protein p53 byinhibiting p53 degradation. A,extracts from A549 cells treatedwith different concentrations ofAC17 for 12 hourswas analyzedbyWestern blot for p53, p21, andMDM2 expression. B, A549 cellswere treated with AC17 for 24hours, stained with PI, andanalyzed by flow cytometry. Cell-cycle distributions were analyzedusing FlowJo software. C, A549cells were treated for 24 hours withAC17, followed by photographingof cellular morphologic changes(�200), and apoptotic nuclearchanges (�400) after staining withHoechst 33342. D, total RNA wasisolated from A549 cells aftertreatment of AC17 for 12 hours andsubjected to RT-PCR analysis forp53, p21, and MDM2 mRNAlevels. Values obtained from3 separate experiments andexpressed as mean � SD,�, P < 0.05. E and F, A549 cellswere treated for 12 hours withAC17 or MG-132, and theextracted proteins wasimmunoprecipitated with p53 orMDM2 antibody. The p53 andMDM2 complexes (E) or levels ofubiquitinated p53 (F) weredetermined by Western blottingusing p53, MDM2, and ubiquitinantibodies. Nutlin-3a and IgGwere used for comparison,and GAPDH was a control forimmunoprecipitation.






tumors, indicating that AC17 inhibited proteasome activ-ity in vivo. Accompanying proteasome inhibition, p53wasreactivated in the A549 xenografts, as shown by increasedp53, MDM2, and p21 expression in tissue extracts (Fig.4D). Increased p53 expression compared with vehicle-treated mice was further verified by immunostaining intumors fromAC17-treated mice (Fig. 5A). The interactionbetween IkBa and p65 in tumor tissues was analyzed todeterminewhetherAC17-inducedproteasomal inhibitioninduces a consequent inhibition ofNF-kBpathway,whichis constitutively activated in human lung cancer cell lines(33), and plays a key role in tumor proliferation, invasion,and metastasis (34, 35). Indeed, the activated NF-kB p65subunit in the corresponding vehicle-treated controltumorswasmostly dissociated from IkBa (Fig. 4E), whichwas in sharp contrast to the cultured A549 cells (Fig. 1G).At 24 hours after the last treatment, tumors from AC17-treated mice showed dose-dependent increase of theinteraction between IkBa and p65, suggesting that NF-kB pathway was inhibited by AC17 in vivo (Fig. 4E).

Immunofluorescence staining of tumor tissues furthershowed that p65 was localized in the nucleus in thevehicle-treated control group, whereasmostly in the cyto-plasm in the AC17-treated groups (Fig. 5B). Taken togeth-er, these observations indicate that AC17 has the ability toinhibit proteasomal activity, resulting in reactivation oftumor suppressor p53 and blockade of NF-kB pathwaywithin the tumor, most likely accounting in part for theobserved antitumor activity of AC17 in vivo.

In addition, histologic examination of H&E-stainedlung, liver, and kidney tissues from the treatment groupswas conducted. The lungs of AC17-treated mice seemednormalwith less consolidation comparedwith the controlgroup (Fig. 5C, top). Livers from control mice showedhepatomegaly and necrosis, whereas AC17-treated miceshowed normally structured hepatocytes (Fig. 5C, bot-tom). There were no detectable changes in kidneys aftertreatment with AC17 compared with the vehicle control(data not shown). These results indicate that AC17 isconsiderably less toxic to normal cells than tumor cells,

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Figure 4. AC17 suppresses tumorgrowth in the A549 xenograftmouse model, associated withproteasomal inhibition, NF-kBblockage, and p53 reactivation.Female nude mice werexenografted by subcutaneousinjection of A549 cells (1 � 107) in1 flank. When tumor size reachedapproximately 120 mm3, micewere divided into 3 groups andtreated with vehicle solvent orAC17 (1 and 5 mg/kg). Tumorswere collected and weighted after23-day treatment (B, C), and theprepared tissue extracts wereanalyzed for Western blotting (D)and immunoprecipitation analysis(E). A, inhibition of A549 tumorgrowth by AC17. Points, meantumor volume in eachexperimentalgroup containing 5mice; bars, SD.�, P < 0.05. B, the effect of AC17-induced growth inhibition on tumorweight at the endpoint. Columns,mean (n¼ 5); bars, SD. �, P < 0.05.C, example images of tumorsexcised frommice of each group atthe endpoint. D, Western blotanalysis of tumor tissue extractswith antibodies of ubiquitin, p53,p21, MDM2, and GAPDH. E, theassociation of p65 and IkBa intumor tissue extracts was verifiedby immunoprecipitation analysis.GAPDH was used as a loadingcontrol.

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Figure 5. Immunofluorescencep65, immunohistochemistry p53,and H&E staining assays usingtissue samples. Tumors, lungs,and livers were collected after 23-day treatment and the preparedtissue slides (4 mm) were used forimmunohistochemistry with p53antibody (A), immunofluorescencestaining with p65 antibody (B), andH&E staining assays (A, C). A,slides of tumor tissues were usedfor immunostaining with p53antibody andH&E staining assays.Magnifications,�200 and�400 asindicated. B, NF-kB subcellularlocalization in tumor tissues wasstained with p65 antibody,followed DyLight 549-labeled anti-rabbit IgG (HþL), and nuclearstaining was used by Hoechst33342. Magnifications, �630. C,microscopic pictures of lungsand livers from different treatedgroups were obtained by H&Estaining. Magnifications, �100.






in agreementwith several previous studies of proteasomeinhibitors (36, 37).

DiscussionCurcumin has attracted much attention because of its

surprisingly wide range of beneficial properties, includ-ing antiinflammatory, antioxidative, and anticancer activ-ities, as well as its pharmacologic safety and potency as achemopreventive agent by regulating a variety of molec-ular targets (38, 39). Unfortunately, therapeutic potentialof curcumin is limited by its relatively low potency andpoor cellular bioavailability (40). Therefore, there is a needto develop highly active and clinically promising curcu-min analogs. In the present study, we showed that oursynthetic 4-arylidene curcumin analog (AC17) was apotent 19S deubiquitinase inhibitor that was much moreeffective in suppressing the proliferation of severalhuman cancers than its parent compound curcumin (Sup-plementary Fig. S1) and with improved cellular bioavail-ability and metabolic stability (22, 23).

Due to the emerging role in tumor cell proliferation andresistance, UPS seems to be an ideal target for the devel-opment of novel cancer therapies (2, 41–43). The success ofthe 20S proteolytic inhibitor bortezomib for the treatmentof multiple myeloma and mantle cell lymphoma hasverified this and stimulated interest in the developmentof UPS inhibitors. As a natural medicinal component,curcumin has been confirmed to inhibit proteolytic activ-ities of purified rabbit 20S with low IC50 values (44).Surprisingly, our current results showed that AC17, a4-arylidene curcumin analog, had little effect on protea-somal CT-like, T-like, and PGPH-like activities in vitro(Fig. 2D). Interestingly, AC17 caused a rapid and markedaccumulation of polyubiquitinated proteins in both A549and NCI-H1299 cells (Fig. 2A and B). Moreover, it wasenvisaged thatAC17was adeubiquitinase inhibitor basedonmolecular structure analysis (28). Again unexpectedly,the total cellular deubiquitinase activity was not dramat-ically inhibited in AC17-treated cells (Fig. 2E). Therefore,the exact mechanism by which AC17 induces the accu-mulation of ubiquitinated proteins is drastically differentfrom its parent compound curcumin.

Recent advances have revealed that the 19S RP isconsidered a novel anticancer drug target (29, 41). Func-tions of 19S RP include binding ubiquitinated proteins,recycling ubiquitin, unfolding proteins, and threadingthem into the 20S CP (45). Therefore, compounds thatinhibit one or more functions of 19S RP are potential 19Sinhibitors. The first reported 19S inhibitors were ubista-tins, which block crucial interaction of ubiquitinated pro-teins and the 19S RP (46). As a dual reversible inhibitorof USP14 and UCHL5 of the 19S RP, b-AP15 has beenshown to induce accumulation of protein-ubiquitin con-jugates similar to the traditional proteasome inhibitors,and yet there are some differences between b-AP15 andbortezomib in sensitivity to Bcl-2 overexpression and p53status as well as their anticancer activity against solidtumors (29, 41). Herein, we reported that AC17 irrevers-

ibly inhibited 19S deubiquitinase activity selectively,without inhibiting total deubiquitinase activity in cells(Fig. 2E and F, Supplementary Fig. S4–S6). AC17 treat-ment increased the levels of p53 and p21 expression,reactivated p53 (which is associated with the inductionof apoptosis in A549 cells; Fig. 3A–D), all of which areconsistentwith previous reports onproteasome inhibitors(47, 48). Although p53 partially contributed to the cyto-toxic effects of AC17, p53 depletion did not prevent theAC17-induced cytotoxicity completely (SupplementaryFig. S3 and S8). In contrast, bortezomib-induced cytotoxicactivity was much more sensitive to p53 depletion (Sup-plementary Fig. S8; refs. 21, 29). These findings haveimportant implications because p53 is involved in medi-ating bortezomib resistance (49); a therapeutic approachusing 19S deubiquitinase inhibitors would potentiallyovercome bortezomib resistance.

Furthermore, previous studies have shown that NF-kBactivation, regulated by UPS (50), is important for tumorproliferation, invasion, and metastasis, accounting forradiation and chemotherapy resistance in cancer cells(34, 51). Chemotherapeutic drugs activate NF-kB throughdiverse pathways, leading to resistance to and failure ofchemotherapy (52). Inhibition of NF-kB activation wouldtherefore contribute to the anticancer activity of UPSinhibitors. We have reported that 4-arylidene curcuminanalogs inhibit NF-kB activation in part by blocking IKKactivity (22). Herein we showed that AC17 induced theaccumulation of polyubiquitinated IkBa and IkBa bind-ing to NF-kB p65 subunit (Fig. 1D and E), resulting ininhibition of NF-kB activation by stabilizing the intrinsicinhibitor, IkB. The 2 mechanisms of AC17 NF-kB inhib-itory activity are cooperative and shed some light on themechanism of AC17-induced cancer cell death.

Using an A549 xenograft mouse model, treatment ofAC17 resulted in the accumulation of ubiquitinated pro-teins, reactivation of tumor suppressor protein p53 (Fig.4D), and an enhanced interaction between IkBa andp65 invivo (Fig. 4E). Immunostaining and immunofluorescenceassays of tumor tissues further confirmed that p53 wasoverexpressed and that NF-kB translocation to the nucle-us was inhibited in AC17-treated mice (Fig. 5A and B).Consistent with these, AC17 treatment effectively inhib-ited tumor growth in vivo (Fig. 4A–C). Notably, micetreated with AC17 under the present experimental con-ditions showed no renal toxicity, hepatotoxicity, or pul-monary consolidation (Fig. 5C). These results stronglysuggest that 4-arylidene curcumin analogs could be usedas new agents for cancer chemoprevention, cancer che-motherapy, or both.

In the present study, the exactmolecularmechanism bywhich AC17 inhibits the deubiquitinase activity of 19S RPwas not completely elucidated. The a,b-unsaturated die-none in AC17 may serve as a Michael acceptor thattheoretically interacts with the thiol groups of cysteineresidues in deubiquitinases (53). This may also explainhow AC17 acts as an irreversible 19S deubiquitinaseinhibitor (Supplementary Fig. S6) and entirely loses its

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19S deubiquitinase inhibition in the presence of GSH(Supplementary Fig. S7). However, the type of deubiqui-tinases specifically inhibited by AC17 remains unclear.Therefore, the full explanation of the activity of AC17requires further study.In addition, structure–activity rela-tionship studies of 4-arylidene curcumin analogswith 19Sdeubiquitinase inhibitory activity may help to improvesuch proteasome deubiquitinase inhibitors and aid newanticancer agent development in clinical settings.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Zuo, J. Du, X. BuDevelopment of methodology: B. Zhou, H. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Zhou, S. WenAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): B. ZhouWriting, review, and/or revision of the manuscript: B. Zhou, B. Li

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Zuo, H. Wang, H. Liu, X. Wang,X. Qiu, Y. Hu, S. WenStudy supervision: X. Bu

AcknowledgmentsThe authors thank Dr. Peng Huang in the State Key Laboratory of

Oncology in South China for helpful advice and assistance.

Grant SupportThis work was supported by the National Natural Science Foundation

ofChina (No. 30973619&81172931),NationalHigh-techR&D863Program(No. 2008AA02Z304), and Fundamental Research Funds for the CentralUniversities (No. 09ykpy66; to X. Bu); National Basic Research Program ofChina (973 Program, No. 2011CB9358003; to J. Du).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 9, 2012; revised May 3, 2013; acceptedMay 9, 2013;published OnlineFirst May 21, 2013.

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2013;12:1381-1392. Published OnlineFirst May 21, 2013.Mol Cancer Ther Binhua Zhou, Yinglin Zuo, Baojian Li, et al. p53 Reactivation in Human Lung Cancer Cells

B Inhibition andκ4-Arylidene Curcumin Analog AC17 Causes NF-Deubiquitinase Inhibition of 19S Regulatory Particles by

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