piperlongumine chemosensitizes tumor cells through interaction … · cancer biology and signal...

Cancer Biology and Signal Transduction

Piperlongumine Chemosensitizes Tumor Cells throughInteraction with Cysteine 179 of IkBa Kinase, Leading toSuppression of NF-kB–Regulated Gene Products

Jia Gang Han1,2, Subash C. Gupta1,3, Sahdeo Prasad1, and Bharat B. Aggarwal1

AbstractRecently, two different reports appeared in prominent journals suggesting a mechanism by which

piperlongumine, a pyridine alkaloid, mediates anticancer effects. In the current report, we describe another

novel mechanism by which this alkaloid mediates its anticancer effects. We found that piperlongumine

blocked NF-kB activated by TNFa and various other cancer promoters. This downregulation was

accompanied by inhibition of phosphorylation and degradation of IkBa. Further investigation revealed

that this pyridine alkaloid directly interacts with IkBa kinase (IKK) and inhibits its activity. Inhibition of

IKK occurred through interaction with its cysteine 179 as the mutation of this residue to alanine abolished

the activity of piperlongumine. Inhibition in NF-kB activity downregulated the expression of proteins

involved in cell survival (Bcl-2, Bcl-xL, c-IAP-1, c-IAP-2, survivin), proliferation (c-Myc, cyclin D1),

inflammation (COX-2, IL6), and invasion (ICAM-1, -9, CXCR-4, VEGF). Overall, our results reveal a novel

mechanism by which piperlongumine can exhibit antitumor activity through downmodulation of proin-

flammatory pathway. Mol Cancer Ther; 13(10); 1–14. �2014 AACR.

IntroductionCancer is a major public health problem in the United

States andmany other parts of the world. Currently, 1 in 3women and 1 in 2 men in the United States will developcancer in his or her lifetime (1). Although chemotherapy isthe standard treatment for most kinds of cancers, resis-tance to chemotherapeutic drugs has become a majorobstacle in treating cancer. In fact, multidrug resistanceis now considered a main reason for the failure of che-motherapy (2). Alternatives that are inexpensive, effica-cious, and safe compared with synthetic agents are sorelyneeded.Epidemiologic, clinical, and experimental evidence

suggests that medicines derived from plants play apivotal role in treating most diseases, including cancer.As much as 80% of the cancer therapeutic agents cur-rently used have their roots in natural products. Onesuch product is the compound 5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)-pyr-

idinone (Fig. 1A), called piperlongumine or piplartine,it is the pyridine alkaloid found in members of the Piperspecies, in particular in the fruit of the long pepper(Piper longum Linn.). Piperlongumine has been usedwidely in traditional medicine, including the IndianAyurvedic system of medicine, traditional Chinesemedicine, Tibetan medicine, and the folk medicine ofLatin America. Piperlongumine has multiple pharma-cologic activities: it has been described as a plateletaggregation inhibitor (3), an anxiolytic agent (4), anantidepressant (5), an antinociceptive agent (6), ananti-atherosclerotic agent (7), an antidiabetic agent(8), an anti-inflammatory agent (9), and an infectiouspathogen inhibitor (10). Furthermore, piperlonguminehas been reported to kill multiple types of cancer cells,inhibit metastasis, and have antitumor activities in avariety of animal models (11–22).

Although this compound was first isolated in 1967,major interest in it did not emerge until the 2011 publi-cation in Nature by Raj and colleagues (18). This groupreported that piperlongumine selectively induces celldeath in a wide variety of tumor cell types and does notaffect noncancerous cell types even at high doses. Theyalso reported on its antitumor effects in murine models ofmelanoma, bladder cancer, breast cancer, and lung cancerwhile having only minimal toxic effects in these models.Howpiperlongumine acts as an anticancer agent is not yetclear. Several studies have demonstrated that it inducescytotoxicity through a variety of mechanisms, such asactivation of caspases (14, 20, 21), activation of the ERKpathway (11), inhibition of cdc-2, cdk2, and cyclin D1 (19,

1Cytokine Research Laboratory, Department of Experimental Therapeu-tics, TheUniversity of TexasMDAndersonCancerCenter, Houston, Texas.2General Surgery, Beijing Chaoyang Hospital, Capital Medical University,Beijing, China. 3University of Mississippi Medical Center, Jackson,Mississippi.

Corresponding Author: Bharat B. Aggarwal, Cytokine Research Labora-tory, Department of Experimental Therapeutics, The University of TexasMDAnderson Cancer Center, Houston, Texas. Phone: 713-794-1817; Fax:713-745-6339; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0171

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on April 20, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst July 31, 2014; DOI: 10.1158/1535-7163.MCT-14-0171

http://mct.aacrjournals.org/

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Figure 1. Piperlongumine (PL) suppresses constitutive and TNFa-induced NF-kB activation in cancer cells. A, chemical structure of piperlongumine.B, dose-dependent effect of piperlongumine on TNFa-induced NF-kB activation. KBM-5 cells were treated with the indicated concentrations ofpiperlongumine for 4 hours and then exposed to 0.1 nmol/L TNFa for 30 minutes. C, time-dependent effect of piperlongumine on TNFa-induced NF-kBactivation. KBM-5 cells were treated with 10 mmol/L piperlongumine for the indicated times and then exposed to 0.1 nmol/L TNFa for 30 minutes. D,TNFa-induced NF-kB is composed of p50 and p65 subunits. Nuclear extracts from untreated KBM-5 cells or KBM-5 cells treated with 0.1 nmol/LTNFa were incubated with the indicated antibodies, pre-immune serum (PIS), an unlabeled competitor NF-kB oligonucleotide probe, or a mutantoligonucleotide probe. E, piperlongumine suppresses NF-kB activation induced by LPS, OA, PMA, H2O2, or CSC. KBM-5 cells were preincubatedwith the indicated concentrations of piperlongumine for 4 hours and then treated with 100 ng/mL LPS for 2 hours, 500 nmol/L OA for 4 hours, 25 ng/mLPMA for 1 hour, 500 mmol/L H2O2 for 2 hours, or 10 mg/mL CSC for 1 hour. F, effect of piperlongumine on TNFa-induced NF-kB activation in humanU266, Jurkat, SCC4, MCF-7, H1299, and A293 cells. Cells were incubated with the indicated concentrations of piperlongumine for 4 hours andthen treated with 0.1 nmol/L TNFa for 30 minutes. B, C, E, and F, nuclear extracts were assayed for NF-kB activation by EMSA. Results arerepresentative of 3 independent experiments. Results are expressed as fold activity over the group incubated without piperlongumine and not treatedwith TNFa, which was set at 1.0.

Han et al.

Mol Cancer Ther; 13(10) October 2014 Molecular Cancer TherapeuticsOF2




20), and downregulation of anti-apoptotic genes (Bcl-2,Raf-1, and survivin) and metastatic genes (VEGF,CD31, HIF-2, and Twist; refs. 17–20). The compound hasalso been shown to inhibit the enzymatic activity of GSTp 1 and carbonyl reductases and to decrease glutathionelevels, indicating that piperlongumine-mediated apo-ptosis of cancer cells is induced by a reactive oxygenspecies (ROS)–dependent mechanism (18). In addition,piperlongumine-dependent cytotoxicity may involvesuppression of NF-kB, MYC, and LMP-1 in Burkittlymphoma (14). Piperlongumine was shown to inhibitNF-kB activity in prostate cancer cells recently (23);however, the underlying mechanism is unclear.NF-kB activation has been linked to the survival, pro-

liferation, invasion, angiogenesis, and metastasis of var-ious types of cancers. Activation of NF-kB requires theactivation of IkB kinase (IKK). IKKb, a subunit of the IKKcomplex, is essential for the activation of NF-kB inresponse to various proinflammatory stimuli. Cys-179 inthe activation loop of this IKKb plays a major role in IKKactivation asmutation of this residue to alanine decreasedits activity (24).Mutation ofCys179 to alanine in IKKb alsocaused reduced phosphorylation of serine residues atpositions 177 and 181, required for IKKb activation. Inaddition, phosphorylation of tyrosine residues at posi-tions 188 and 199 are also crucial for IKKb activation asmutations of these residues have been shown to abolishthe NF-kB activity (25).Therefore, we postulated that piperlongumine mod-

ulates this NF-kB signaling pathway. In this study, weinvestigated in detail the effect of piperlongumineon NF-kB pathway regulation. We found that the com-pound suppressed NF-kB activation pathways inducedby inflammatory stimuli, growth factors, carcinogens,and tumor promoters through the direct inhibitionof cysteine 179 (Cys179) of IKKb, which led to theinhibition of IkBa, the suppression of NF-kB–regulatedgene products, and the enhancement of apoptosis intumor cells.

Materials and MethodsMaterialsPiperlongumine was obtained from Indofine Chemical

Company. Bacteria-derived human recombinant TNFawas provided by Genentech. Penicillin, streptomycin,RPMI-1640 medium, DMEM, FBS, and the kit for theLIVE/DEAD assay were obtained from Invitrogen. Phor-bol 12-myristate 13-acetate (PMA), lipopolysaccharide(LPS), okadaic acid (OA), H2O2, MTT, Hoechst 33342, andantibodies against FLAG and b-actin were obtained fromSigma. Antibodies against p65, p50, cyclin D1, MMP-9,COX-2, PARP, IAP-1, IAP-2, Bcl-2, Bcl-xL, survivin,c-Myc, ICAM-1, caspase-3, caspase-8, and caspase-9 werepurchased from Santa Cruz Biotechnology, as wasthe Annexin V staining kit. Phospho-specific anti-IkBa(Ser32/36) and anti-p65 (Ser536) antibodies were obtainedfromCell Signaling.Antibodies against CXCR4, phospho-

IKKb (Tyr188) and IKKa/b (Ser180/181) antibody wasobtained from Abcam. Antibody against survivin and anELISA system kit for human IL6 were purchased fromR&D Systems. Anti-VEGF antibody was purchased fromNeoMarkers. Antibodies against IkBa, IKKa, and IKKbwere obtained from Imgenex. Velcade (PS-341) wasobtained from Millennium Pharmaceuticals.

Cell linesHuman multiple myeloma U266 cells, human embry-

onic kidney A293 cells, and human breast MCF-7 cellswere obtained in 2000, and human T-cell leukemia Jurkatcells were obtained in 1997 from ATCC. Human lungadenocarcinoma H1299 cells and human squamous cellscarcinoma SCC4 cells were obtained in 2005 from Dr.Reuben Lotan, and human chronic myeloid leukemiaKBM-5 cells were obtained from Dr. Michael Andreeff ofThe University of Texas MD Anderson Cancer Center(Houston, TX). KBM-5 cells were cultured in Iscove’smodified Dulbecco’s medium with 15% FBS; Jurkat,H1299, MCF-7, and U266 cells were cultured in RPMI-1640mediumwith 10% FBS; andA293 cells were culturedin DMEM supplemented with 10% FBS. SCC-4 cells werecultured in DMEM containing 10% FBS, nonessentialamino acids, pyruvate, glutamine, and vitamins. All cul-ture media were supplemented with 100 mg/mL strepto-mycin and 100 units/mLpenicillin. The above-mentionedcell lines have not been recently tested for authenticationin our laboratory.

Cytotoxicity assayThe effect of piperlongumine on the cytotoxic potential

of TNFa was determined using the MTT dye uptakemethod as described previously (26).

LIVE/DEAD assayTo measure the effect of piperlongumine on apoptosis

induced by TNFa, we used the LIVE/DEAD assay todetermine plasma membrane integrity (red fluorescentethidiumhomodimer-1) and intracellular esterase activity(greenfluorescent calcein-AM). This assaywasperformedas described previously (27).

Annexin V/propidium iodide assayThe Annexin V assay is used to detect early apoptosis.

This assay takes advantage of the rapid translocationand accumulation of the membrane phospholipid phos-phatidylserine from the cytoplasmic interface of the cellto the extracellular surface. This loss of membraneasymmetry can be detected using the binding propertiesof Annexin V. Briefly, 1 � 106 cells were pretreatedwith 10 mmol/L piperlongumine for 4 hours, treatedwith 1 nmol/L TNFa for 24 hours, and subjected toAnnexin V staining. Cells were washed in PBS andresuspended in 100 mL of binding buffer containingFITC-conjugated Annexin V and then analyzed by flowcytometry (FACS Calibur; BD Biosciences) after theaddition of propidium iodide.

Regulation of NF-kB Activation by Piperlongumine

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Invasion assayThe BD BioCoat tumor invasion system (BD Bios-

ciences), which includes a light-tight polyethylene tere-phthalatemembranewith 8-mmdiameter pores and a thinlayer of reconstituted Matrigel basement membranematrix, was used to assess cell invasion. H1299 cells(2.5 � 104) were suspended in serum-free medium andseeded into the upper wells. After incubation overnight,cells were treated with different concentrations of piper-longumine for 4 hours and then stimulatedwith 1 nmol/LTNFa for 24 hours in the presence of 1% FBS. The invasivecells were fixed and stainedwithDiff-Quik stain (SiemensHealthcare Diagnostics) and counted in 5 random micro-scopic fields (Nikon).

ELISAAnELISA kit (R&DSystems)was used to detect human

IL6. U266 cells were treated with different concentrationsof piperlongumine for 24 hours, and cell-free superna-tants were collected for detection of IL6 by following themanufacturer’s protocol.

Electrophoretic mobility shift assayTo assess NF-kB activation, we performed electropho-

retic mobility shift assay (EMSA) as described previously(28). Briefly, nuclear extracts prepared from TNFa-treatedcells (2 � 106/mL) were incubated with 32P-end–labeled45-mer double-stranded NF-kB oligonucleotide (15 mg ofprotein with 16 fmol DNA) from the human immunodefi-ciency virus long terminal repeat (50-TTGTTACAA GGG-ACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-30)for 30 minutes at 37�C, and the DNA–protein complex thatformed was separated from free oligonucleotide on 6.6%native PAGE. A double-stranded mutated oligonucleotide(50-TTGTTACAA CTCACTTTC CGCTG CTCACTTTCCAGGGAGGCGTGG-30) was used to examine the speci-ficity of binding of NF-kB to the DNA. The dried gels werevisualized with a Storm 820 phosphorimager (MolecularDynamics), and radioactive bands were quantitated usingImageQuant software (Molecular Dynamics).

Western blot analysisCytoplasmic, nuclear, or whole-cell extracts were pre-

pared, and Western blot analysis was performed. Briefly,30 mg of protein was resolved with SDS-PAGE. Afterelectrophoresis, the proteins were electrotransferredto nitrocellulose membranes, probed with specific anti-bodies, and detected by enhanced chemiluminescencereagent (GE Healthcare Life Sciences).

Kinase assayTo determine the effect of piperlongumine on TNFa-

induced IKK activation, we performed a kinase assay asdescribed previously (29).

NF-kB–dependent reporter gene expression assayThe effect of piperlongumine on the induction of NF-

kB–dependent reporter gene transcription by TNFa,

TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1-b, IKKb, andp65 was analyzed using a SEAP assay, as describedpreviously (30).

ResultsThe aim of this study was to investigate the effect of

piperlongumine on the NF-kB activation induced byvarious carcinogens and inflammatory stimuli in cancercells.Most of the studieswereperformedusing thehumanmyeloid cell line KBM-5 because these cells express TNFreceptors, and the inflammatory pathway in these cells iswell understood. Other cell types were used to test thespecificity of the effect of piperlongumine.

Piperlongumine inhibits TNFa-induced NF-kBactivation in a dose- and time-dependent manner

We first investigated the dose and duration of piper-longumine exposure required to suppress TNFa-inducedNF-kB activation in KBM-5 cells. EMSA results showedthat pretreatment of cells with piperlongumine inhibitedTNFa-induced NF-kB activation in a dose- (Fig. 1B) andtime-dependent manner (Fig. 1C).

To confirm that the band visualized in TNFa-treatedcellswas indeedNF-kB,we incubated the nuclear extractsfrom TNFa-pretreated KBM-5 cells with anti-p50 or -p65antibodies. The bands shifted to higher molecular masseswhen incubated with the antibodies, suggesting that theTNFa-activated NF-kB activation complex consisted ofboth p50 and p65. Addition of preimmune serum andmutated oligonucleotide had no effect on DNA binding,whereas the addition of excess unlabeled NF-kB (coldoligonucleotide; 100-fold excess) caused a decrease in theintensity of the band (Fig. 1D).

Piperlongumine inhibits NF-kB activation inducedby carcinogens and inflammatory stimuli

The endotoxin LPS, OA, PMA, H2O2, and cigarettesmoke condensate (CSC) are known to activate NF-kBby different mechanisms. We found that all these agentsactivated NF-kB in KBM-5 cells and that piperlongu-mine suppressed this activation (Fig. 1E). These resultssuggested that piperlongumine acts at a step in theNF-kB activation pathway that is common to all 5 ofthese agents.

Inhibition of NF-kB activation by piperlongumine isnot cell-type–specific

We next determined whether piperlongumine-mediat-ed inhibition in TNFa-induced NF-kB activation is cell-type–specific.We observed an inhibitory effect in not onlyKBM-5 cells but also multiple myeloma (U266), T-cellleukemia (Jurkat), head and neck squamous cell carcino-ma (SCC4), breast carcinoma (MCF-7), lung adenocarci-noma (H1299), and kidney (A293) cells (Fig. 1F). Thus,piperlongumine appears to be able to suppress NF-kBactivation in a variety of human tumor cells.

Han et al.





Piperlongumine does not directly affect the bindingof NF-kB to DNASome NF-kB inhibitors, such as plumbagin (31) and

bharangin (32), directly modify NF-kB to suppress itsbinding to DNA. To determine whether piperlonguminesuppresses NF-kB activation through a similar mecha-nism,we incubated thenuclear extract fromTNFa-treatedKBM-5 cells with piperlongumine and found that thecompound did not modify the DNA-binding ability ofNF-kB protein (Fig. 2A). These results suggested thatinhibition of NF-kB activation by piperlongumine doesnot suppressDNAbinding directly. This compoundmay,however, use the inhibitory mechanism of nimbolide (33)and g-tocotrienol (34), which inhibit NF-kB activationindirectly.

Piperlongumine inhibits TNFa-induced IkBadegradation and phosphorylationNF-kB activation requires the phosphorylation, poly-

ubiquitination, and subsequent degradation of its inhib-itory subunit, IkBa. To determine whether the inhibitionof TNFa-induced NF-kB activation we observed in KBM-5 cells was due to the inhibition of IkBa degradation,KBM-5 cells were pretreated with piperlongumine andthen exposed to TNFa for various time periods. We thenanalyzed the cells for nuclear NF-kB by EMSA and forIkBa degradation in the cytoplasm fraction by Westernblotting. TNFa activated NF-kB in the control cells in atime-dependent manner but not in piperlongumine-pre-treated cells (Fig. 2B). Moreover, TNFa induced IkBadegradation in control cells as early as 5 minutes (Fig.2C). IkBawas completely degraded after 10 to 15minutesand was resynthesized after 30 minutes. In piperlongu-mine-pretreated cells, however, this degradation wascompletely reversed.Because IkBa degradation requires IkBa phosphoryla-

tion (35), we explored whether the inhibition of TNFa-induced IkBa degradation was due to the inhibition ofIkBa phosphorylation. We blocked IkBa degradationby using the proteasome inhibitorN-acetyl-leucyl-leucyl-norleucinal (ALLN).Western blotting results showed thatco-treatment with TNFa and ALLN induced IkBa phos-phorylation and that pretreatment with piperlonguminestrongly suppressed this phosphorylation (Fig. 2D).These results indicated that piperlongumine suppressesTNFa-induced IkBa degradation by inhibiting IkBaphosphorylation.

Piperlongumine directly inhibits TNFa-induced IKKactivationPhosphorylation of IkBa is regulated by the upstream

kinase IKK complex. Because piperlongumine inhibitsthe phosphorylation and degradation of TNFa-inducedIkBa, we determined the effect of this compound onIKK activation. Our results revealed that TNFa activat-ed IKK in a time-dependent manner and piperlongu-mine suppressed this activation (Fig. 2E). Expression of

IKKa or IKKb proteins was not notably affected byTNFa or piperlongumine.

To determine whether piperlongumine inhibits IKKactivity directly or indirectly, we treated the kinase assaymixture prepared from TNFa-pretreated KBM-5 cellswith various concentrations of piperlongumine. Resultsfrom thekinase assay showed that it inhibited IKKactivity(Fig. 2F), suggesting that the compound suppressesTNFa-induced IKK activation directly.

Because IKKb contains various cysteine residues, weinvestigated whether piperlongumine suppresses IKKactivation by modifying one or more of these cysteineresidues. The reducing agent dithiothreitol (DTT) wasused to determinewhether themodulation of IKK activityby piperlongumine was through the modification of crit-ical cysteine residues. We found that the addition of DTTto the kinase reaction mixture reversed the piperlongu-mine-mediated inhibition of TNFa-induced IKK activity(Fig. 2G). The finding suggested that a cysteine residue isinvolved in this pathway.

The cysteine residue Cys179, which is positionedwithinthe activation loop of the IKK catalytic subunits, is criticalfor IKKb activity. To determine whether Cys179 isinvolved in piperlongumine-induced IKK inhibition, wetransfected A293 cells with wild-type FLAG-IKKb ormutated FLAG-IKKb (alanine substituting for Cys179).Piperlongumine inhibited wild-type IKKb but had noapparent effect on mutated IKKb (Fig. 2H). These resultssuggested that piperlongumine inhibits IKKb activity bydirectly modifying the Cys179 residue.

Furthermore, we determined whether piperlonguminemodulates phosphorylation of other residues of IKKbsuch as serine-181 and tyrosine-188, which are alsoresponsible for its full activity. Results showed thatTNF-induced phosphorylation of IKKb at both serine-181 and tyrosine-188 residues and piperlongumine inhib-ited the phosphorylation of both the residues (Fig. 2I).

Piperlongumine inhibits TNFa-induced p65 nucleartranslocation and phosphorylation

The phosphorylation and degradation of IkBa areessential to the NF-kB activation pathway, whereas thestabilization of IkBa is important in preventing thenuclear translocation of p65. The effect of piperlongu-mine on TNF-induced nuclear translocation of p65 wasexamined byWestern blot analysis. For this experiment,KBM-5 cells were treated with piperlongumine andthen exposed to TNFa for different time periods. Thenuclear extracts were prepared and examined for p65 byWestern blot analysis. We observed that TNFa inducedthe nuclear translocation of p65 and that piperlongu-mine suppressed the TNFa-induced p65 nuclear trans-location (Fig. 3A).

Whether piperlongumine affects TNFa-induced p65phosphorylation at Ser536 was also examined. The resultsshowed that TNFa induced the phosphorylation of p65and that piperlongumine suppressed this phosphoryla-tion (Fig. 3A).






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Figure 2. Downregulation of TNFa-induced NF-kB activation by piperlongumine (PL) involves Cys179 of IKK. A, in vitro effect of piperlongumineon NF-kB–DNA binding. Nuclear extracts (NE) prepared from KBM-5 cells treated with 0.1 nmol/L TNFa were incubated with the indicatedconcentrations of piperlongumine for 30 minutes. B, KBM-5 cells were preincubated with 10 mmol/L piperlongumine for 4 hours and then treated withTNFa for the indicated times. A and B, nuclear extracts from KBM-5 cells were subjected to EMSA for NF-kB activation. Results are expressedas fold activity over the group incubated without piperlongumine and not treated with TNFa, which was set at 1.0. C, effect of piperlongumineon TNFa-induced IkBa degradation. KBM-5 cells were preincubated with 10 mmol/L piperlongumine for 4 hours and treated with TNFa for the indicatedtimes. Cytoplasmic extracts were analyzed for Western blotting with antibodies against IkBa and b-actin. D, piperlongumine inhibits TNFa-inducedIkBa phosphorylation. KBM-5 cells were preincubated with 10 mmol/L piperlongumine for 4 hours, treated with 50 mg/mL ALLN for 30 minutes,and then treated with 0.1 nmol/L TNFa for 10 minutes. Cytoplasmic extracts were analyzed for Western blotting using antibodies against phospho-specific IkBa (Ser32/36), IkBa, and b-actin. E, effect of piperlongumine on the TNFa-induced IKK activation. KBM-5 cells were preincubated with10 mmol/L piperlongumine for 4 hours and then treated with 1 nmol/L TNFa for the indicated times. Whole-cell extracts were immunoprecipitated withantibody against IKKa and analyzed with an immune complex kinase assay. The effect of piperlongumine on IKK protein expression wasdetermined by Western blotting using anti-IKKa and anti-IKKb antibodies. F, direct effect of piperlongumine on IKK activation induced by TNFa.Whole-cell extracts were prepared from KBM-5 cells treated with 1 nmol/L TNFa and immunoprecipitated with an anti-IKKa antibody. Theimmunocomplex kinase assay was performed in the presence of the indicated concentrations of piperlongumine. G, effect of the reducing agent DTT onpiperlongumine-induced inhibition of IKK activation. KBM-5 cells were treated with 1 nmol/L TNFa and immunoprecipitated with antibodies againstIKKa and IKKb. The immunocomplex kinase assay was performed in the presence of 10 mmol/L piperlongumine, with or without DTT. H, effect ofpiperlongumine on the kinase activity of IKKC179A. A293 cells were transfected with wild-type (WT) or mutated (MT) FLAG-IKKb. Whole-cell extractswere prepared, immunoprecipitated, incubated with 10 mmol/L piperlongumine, and subjected to an IKK assay. I, effect of piperlongumine onphosphorylation of IKKb. KBM-5 cells were treated with 10 mmol/L piperlongumine for 4 hours and exposed with 0.1nmol/L of TNFa for indicated timeperiod. Whole-cell extracts were prepared and subjected to Western blotting. A–H, results shown are representative of 3 independent experiments.

Han et al.





Piperlongumine represses TNFa-induced NF-kB–dependent reporter gene expressionAlthough we observed that piperlongumine inhibits

NF-kB activation, DNA binding alone does not alwaysresult in NF-kB–dependent gene transcription. For thisreason, we assessed whether piperlongumine can affectTNFa-induced reporter gene transcription. A 12-foldincrease in SEAP activity was observed after simulationwith TNFa compared with the vector control, and theinduction was nearly abolished by dominant-negativeIkBa (Fig. 3B), indicating specificity. When the A293 cellswere pretreated with piperlongumine, TNFa-regulatedNF-kB–dependent SEAP expression was inhibited in adose-dependent manner (Fig. 3B). These results sug-gested that piperlongumine inhibits the NF-kB–depen-dent reporter gene expression induced by TNFa.TNFa-induced NF-kB activation is mediated through

the sequential interaction of TNF receptor 1 (TNFR1) withTNFR-associated death domain (TRADD), TNFR-associ-ated factor 2 (TRAF2), NF-kB–inducing kinase (NIK),TGFb-activated protein kinase 1 (TAK1)/TAK1-bindingprotein-1 (TAB1), and IKKb, which results in phosphor-ylation of IkBa and leads to degradation of IkBa and p65nuclear translocation.We determined the site of action for

piperlongumine in TNFa signaling in A293 cells. Thisagent significantly suppressed NF-kB–dependent report-er gene expression induced by TNFR1, TRADD, TRAF2,NIK, TAK1/TAB1, and IKKb plasmids (Fig. 3C), indicat-ing that piperlongumine acts at a step upstream of p65.

Piperlongumine represses TNFa-induced NF-kB–dependent gene products associated with survival,proliferation, invasion, and metastasis

NF-kB regulates the expression of the antiapoptoticproteins Bcl-2, Bcl-xL, cellular inhibitor of apoptosis pro-teins 1 and 2 (c-IAP-1, c-IAP-2), and survivin, which areknown to be induced by TNFa and to play an importantrole in cell survival. We used Western blotting to deter-mine whether piperlongumine inhibits the expression ofthese gene products in KBM-5 cells. Our results showedthat the compound downregulated the expression of all 5of these TNFa-induced proteins (Fig. 4A).

We also used Western blotting to investigate whetherpiperlongumine modulates the expression of the prolif-erative proteins c-myc, cyclin D1, and COX-2. Weobserved that TNFa induced the expression of theseproliferative proteins and that piperlongumine inhibitedit (Fig. 4B).

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Figure 3. Piperlongumine inhibits TNFa-induced phosphorylation and nuclear translocation of p65 and NF-kB–dependent reporter gene expression inducedby TNFa and other molecules in the NF-kB signaling pathway. A, KBM-5 cells were pretreated with 10 mmol/L piperlongumine for 4 hours and then treatedwith 0.1 nmol/L TNFa for the indicated times. Nuclear extracts were prepared and analyzed by Western blotting using antibodies against p65 andphospho-p65 (Ser536). PARP was used as an internal control. B, piperlongumine inhibits TNFa-induced NF-kB–dependent SEAP expression. A293 cellswere transiently transfected with a plasmid containing an NF-kB–SEAP gene. Cells were treated with piperlongumine for 4 hours at the indicatedconcentrations, which was followed by treatment with 1 nmol/L TNFa for 24 hours. Cell supernatants were collected and assayed for SEAP activity.Results are expressed as fold activity over the vector control (Con), which was set at 1. Data are presented as mean � SD. DN, dominant negative. C,piperlongumine inhibits NF-kB–dependent reporter gene expression induced by TNFa, TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, IKKb, or p65. A293cells were transiently transfected with pNF-kB–SEAP plasmid, expression plasmid, and control plasmid for 24 hours and treated with 10 mmol/Lpiperlongumine for 4 hours. The cell supernatants were then assayed for SEAP activity. For TNFa-treated cells, cells were incubated with 10 mmol/Lpiperlongumine for 4 hours and then treated with 1 nmol/L TNF for an additional 12 hours of incubation. Results are expressed as fold activity over the vectorcontrol (Con), which was set at 1. Data are presented as mean � SD. Results shown are representative of 3 independent replicates.






Finally, we examined whether piperlongumine affectsNF-kB–regulated gene products involved in tumorcell invasion and metastasis. Western blotting revealedthat TNFa induced the expression of intercellular adhe-sion molecule-1 (ICAM-1), matrix metallopeptidase 9(MMP-9), CXC chemokine receptor 4 (CXCR-4), andVEGF and that piperlongumine downregulated theexpression of all these proteins (Fig. 4C).

Piperlongumine suppresses proinflammatorycytokine production

IL6 is a proinflammatory cytokine that is regulatedbyNF-kBandaugments theproliferation of awidevariety

of tumor cells. We examined whether piperlongumineaffects the IL6 level produced by human multiplemyeloma U266 cells. Our ELISA results showed thatpiperlongumine suppressed IL6 production in a dose-dependent manner (Fig. 4D).

Piperlonguminepotentiates the cell death inducedbyTNFa and chemotherapeutic agents

Using KBM-5 cells, we next examined whether thedownregulation by piperlongumine of the tumor cellsurvival proteins leads to potentiation of the apoptosisinduced by TNFa or chemotherapeutic agents. Usingthe MTT assay, we found that piperlongumine did,in fact, potentiate the cytotoxicity induced by TNFa,5-fluorouracil (5-FU), or bortezomib in a dose-depen-dent manner (Fig. 5A). The apoptotic effect of piper-longumine was also examined by phosphatidylserineexternalization, a marker of early apoptosis, usingAnnexin V staining and flow cytometry. The numberof Annexin V–positive cells was increased significantlywhen KBM-5 cells were pretreated with piperlongu-mine before TNFa (Fig. 5B).

Whether piperlongumine can enhance the TNFa-induced activation of caspases-3, -8, and -9 was alsoexamined. Our Western blotting results showed that thepiperlongumine sensitized the cells to caspase activation(Fig. 5C).

We also used Western blotting to examine whetherpiperlongumine can enhance TNFa-induced PARP cleav-age in KBM-5 cells. We found that TNFa alone had littleeffect on PARP cleavage, but when the cells were pre-treated with piperlongumine, a remarkable increase inPARP cleavage was observed (Fig. 5D). These results alsodemonstrate a synergistic interaction between the 2agents. Taken together, these results suggested that piper-longumine potentiates the apoptotic effects of TNFa andchemotherapeutic agents.

Ubiquitous intracellular esterase activity and an intactplasmamembrane are characteristics of live cells.Weusedthe LIVE/DEAD assay to discriminate between live anddead KBM-5 cells by simultaneously staining with cal-cein-AM and ethidium homodimer-1. This assay indicat-ed that piperlongumine alone induces loss of membraneintegrity (an indicator of apoptosis) and that treatmentwith TNFa enhances the piperlongumine-induced apo-ptosis (Fig. 6A).

Piperlongumine suppresses TNFa-induced tumorcell invasion

Because piperlongumine suppressed the TNFa-induced expression of proteins such as CXCR-4 orMMP-9, which are linked to tumor cell invasion, wenext examined whether piperlongumine suppressesTNFa-induced tumor cell invasion. For this experiment,we used H1299 cells. Our results showed that TNFaincreased tumor cell invasion by 1.4-fold and that pre-treatment with piperlongumine decreased the level of

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Figure 4. Piperlongumine (PL) inhibits TNFa-induced expression of NF-kB–dependent antiapoptotic, proliferative, and metastatic proteins.KBM-5 cells were incubated with 10 mmol/L piperlongumine for 4 hoursand then treated with 1 nmol/L TNFa for the indicated times. A–C, whole-cell extracts were prepared and analyzed by Western blotting usingantibodies against antiapoptotic (A), proliferative (B), and metastatic (C)proteins. Results of 1 of the 3 independent experiments are shown. D,piperlongumine downregulates IL6 production in U266 cells in aconcentration-dependent manner. Cells were treated with the indicatedconcentrations of piperlongumine, and cell-free supernatants wereharvested after 24 hours. The level of IL6wasdetected byELISA.Data arepresented as mean � SD. Results shown are representative of 3independent replications.

Han et al.





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Figure 5. Piperlongumine (PL) potentiates the apoptotic effects of TNFa and chemotherapeutic agents in KBM-5 cells. A, piperlongumine enhancesTNFa-, 5-FU–, and bortezomib (Velcade)–induced cytotoxicity. Cells (5,000) were seeded in 4 replicates, pretreated with the indicated concentrations ofpiperlongumine, and incubated with a chemotherapeutic agent [1 nmol/L TNFa (left), 0.1 mmol/L 5-FU (middle), or 20 mmol/L bortezomib (right)] for24 hours. Cytotoxicity was analyzed by the MTT assay. B, piperlongumine potentiates TNFa-induced early apoptosis. Cells were pretreated with10 mmol/L piperlongumine for 4 hours and then incubated with 1 nmol/L TNFa for 24 hours. Cells were incubated with FITC-conjugated AnnexinV antibody and then analyzed using flow cytometry (left) and displayed as histogram (right). A and B, data are presented as mean � SD. Results shownare representative of 3 independent replications. C, piperlongumine induces TNFa-induced caspase activation. Cells were incubated with 10 mmol/Lpiperlongumine for 4 hours and then treated with 1 nmol/L TNFa for 24 hours. Whole-cell extracts were prepared and analyzed by Western blotting.D, piperlongumine induces PARP cleavage. Cells were pretreated with 10 mmol/L piperlongumine for 4 hours and then incubated with 1 nmol/LTNFa for the indicated times. Whole-cell extracts were prepared and analyzed by Western blotting. C and D, figures are representative of 1 of3 independent experiments.






TNFa-induced tumor cell invasion to below baseline(Fig. 6B).

We also determined whether piperlongumine inhibitsthe expression ofMMP-9 andCXCR4 involved in invasionand metastasis, respectively. We found that TNFainduced the expression of MMP-9 and CXCR4, and thepretreatment with piperlongumine significantly sup-pressed the expression of these proteins in H1299 cells

(Fig. 6C). These results suggest that inhibition in MMP-9and CXCR4 protein expression may contribute to theinhibitory effects of piperlongumine on invasion activityof H1299 cells.

DiscussionAlthough radiotherapy and chemotherapy are used

to treat cancer, both activate the NF-kB pathway, which

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Figure 6. Piperlongumine suppresses proliferation and potentiates TNFa-induced apoptosis in tumor cells. A, piperlongumine enhances TNFa-inducedapoptosis. KBM-5 cells were pretreated with the indicated concentrations of piperlongumine for 4 hours and then incubated with 1 nmol/L TNFa for24 hours. Cells were stained with LIVE/DEAD assay reagent for 30 minutes and analyzed under a fluorescence microscope. Magnification, 100�.Percentages represent mean � SD number of apoptotic cells. Green, intracellular esterase activity; red, loss of plasma membrane integrity. B,piperlongumine suppresses TNFa-induced cell invasion. H1299 cells (2.5 � 104 cells) were seeded into the top chamber of a Matrigel invasionchamber system overnight in the absence of serum and then treated with the indicated concentrations of piperlongumine. After incubation for 4 hours,the cells were treated with TNFa in the presence of 1% serum for 24 hours, stained with Diff-Quick stain, and assayed for invasion. Controlvalue was set at 1.0. Values represent mean � SD of 3 independent replicates. C, piperlongumine suppresses TNFa-induced expression of MMP-9and CXCR4 proteins. H1299 (1 � 106 cells) were treated with indicated concentration of piperlongumine for 4 hours and then exposed with TNFafor 24 hours. Whole-cell extracts were prepared and subjected to Western blotting.

Han et al.





leads to radioresistance and chemoresistance, respec-tively. Thus, agents that are safe and efficacious indownmodulating the NF-kB pathway are urgentlyneeded for radio- and chemosensitization. One suchagent is piperlongumine, which is derived from thefruit of the "long pepper" plant and is consumed tradi-tionally to address a range of inflammatory diseases.However, the mechanism of action of this compound isnot fully understood. Two recent studies from Harvardresearchers published in Nature (18) and PNAS (15)described the activity of piperlongumine against a widevariety of human cancers in cell culture and animalmodels, but how this compound mediates its anticancerand other activities is poorly understood. Our currentstudy provides insight into the mechanism of action ofpiperlongumine.The specific objective of our study was to determine

the effect of piperlongumine on the NF-kB activationpathway and on NF-kB–regulated gene products thatare associated with inflammation, tumor cell prolifera-tion, invasion, and angiogenesis. We demonstrated thatpiperlongumine suppressed NF-kB activation that wasinduced by various carcinogens and inflammatoryagents through the inhibition of IKK activation, whichled to the suppression of IkBa phosphorylation anddegradation and the suppression of p65 nuclear trans-location and phosphorylation. Piperlongumine alsopromoted the apoptosis induced by TNFa and variouschemotherapeutic agents, and it suppressed cancer cellinvasion.We found that piperlongumine inhibited NF-kB acti-

vation that was induced by a wide variety of agents(TNFa, LPS, OA, PMA, H2O2, and CSC2) and that thissuppression was not cell-type–specific. These results arein agreement with recent reports that piperlonguminecan suppress LPS-induced NF-kB activation in endo-thelial cells (9) and PMA-induced NF-kB activation inmurine macrophages (36). Our results are also consis-tent with another recent report that piperlongumineinhibits constitutive NF-kB expression in Burkitt lym-phoma cells (14).Howpiperlongumine inhibits NF-kB activation has not

been reported and thus was investigated in detail. Wefound, for the first time, that piperlongumine suppressesTNFa-induced IKK activation, which leads to suppres-sion of the phosphorylation and degradation of IkBa. Ourstudy also showed that piperlongumine directly inhibitsTNFa-induced IKK activity. This inhibition could bereversed with the reducing agent DTT, which suggeststhat inhibition of IKKactivation involves the interaction ofpiperlongumine with -SH groups within IKK. Cys179 ofIKK, a redox-sensitive cysteine residue, is positionedwithin the activation loop of the IKK catalytic subunits(37). We found that substitution of Cys179 with alanineprevents the inhibitionof IKKactivity bypiperlongumine,thus suggesting the role of this residue in the action of thisalkaloid. This is first report to suggest the role of piper-longumine with -SH groups of any protein, although

irreversible protein glutathionylation as a process associ-ated with cellular toxicity of this alkaloid has been dem-onstrated (15).

We also found, for the first time, that TNFa induces thephosphorylation of the p65 subunit of NF-kB at Ser536 andthat piperlongumine completely suppresses the phos-phorylation. IKK has been shown to cause the phosphor-ylation of p65 at Ser536 (38). Unlike IkBa, multiple kinaseshave been implicated in the phosphorylation of p65 atSer536 (39), and it is very likely that suppression of IKKactivity is involved in inhibiting the phosphorylation ofboth IkBa and p65 by piperlongumine. Interestingly,phosphorylation of RelA/p65 on Ser536 has been shownto be independent of IkBa (40), indicating that IKK hasnumerous substrates.NF-kBp65phosphorylated at Ser536

was also shown to be an independent prognostic factor inSwedish patients with colorectal cancer (41), suggestingthe potential of using piperlongumine to treat suchpatients.

Our results indicate that despite the involvement of atleast 50 different proteins in the NF-kB activation path-way, each containing multiple cysteines (42), piperlon-gumine inhibits NF-kB by modifying Cys179 in the IKKbactivation loop. Nimbolide (33), butein (43), 3-formyl-chromone (44), and other compounds have beenreported to exhibit similar effects on Cys179. In addition,we found that piperlongumine also blocked the phos-phorylation of serine 181 and tyrosine 188 in IKKbinduced by TNFa. Serine kinases, such as TAK-1, havebeen shown to phosphorylate IKKb at residues 181(see 25for reference). In addition, tyrosine residue 188is within the activation loop of IKKb and is in closeproximity to serine181. The fact that the tyrosine kinasec-Src binds to IKKb suggests that tyrosine phosphory-lation of IKKb is potentially important for regulationof its activity (25). Thus, these results suggest thatpiperlongumine may inhibit IKKb through multiplemechanisms.

The suppression of NF-kB activation induced byTNFa, LPS, OA, PMA, H2O2, and CSC suggests thatpiperlongumine acts at a step common to all theseactivators. NF-kB activation by TNFa is mediatedthrough sequential recruitment of TNFR1, TRADD,TRAF2, NIK, TAK1/TAB1, and IKK. Our transfectionexperiments showed that piperlongumine inhibited theNF-kB reporter gene expression induced by these mole-cules. Similar results have been reported with g-toco-trienol (34), flavopiridol (27), and celastrol (45).

Most approaches used in cancer treatment, such aschemotherapy and radiotherapy, kill cancer cells byinducing apoptosis; however, cancer cells often developresistance to such treatment. Furthermore, many cancertherapies indirectly activate apoptosis by chemically orphysically damaging DNA. This damage may havethe unintended effect of generating a pool of heavilymutated cancer cells and increasing the chances oftheir developing resistance (46). We showed that piper-longumine potentiated cell death induced by TNFa or the






chemotherapeutic agents 5-FU and bortezomib in KBM-5cells. Our results suggest that chemotherapy or radiother-apy followed by treatment with piperlongumine couldreduce the tumor burden.

The loss of caspase activation appears to be centralto the prevention of most cell death events in cancer.Finding strategies to overcome caspase inhibition will bevaluable for the development of novel cancer treatments(47). We investigated in detail how piperlonguminepotentiates apoptosis and found that piperlongumineenhanced the cleavage of precursors of caspases-3, -8,and -9, which suggested that the compound activated celldeath signaling through 2 pathways: the extrinsic, deathreceptor pathway (caspase-8) and the intrinsic, mitochon-drial pathway (caspase-9). NF-kB–regulated cell survivalproteins, such as the Bcl-2 family of proteins and IAPs, areselectively overexpressed in various types of tumors andare involved in the apoptotic pathway defect in manytumor cells (48). Overexpression of these proteins isinvolved in tumor cell survival (49) and might be used

to reverse the apoptotic pathway defect in cancer. Bcl-2was found to be downregulated by piperlongumine inprevious studies (18, 20). Furthermore, we found thatpiperlongumine suppressed the expression of cell surviv-al proteins, including Bcl-2, Bcl-xl, c-IAP-1, c-IAP-2, andsurvivin, suggesting that these gene products could betherapeutic targets for potentiation of apoptosis bypiperlongumine.

We also found that treatment with piperlonguminedownregulated the expression of NF-kB–regulated geneproducts involved in cell proliferation (c-Myc, cyclin D1,and COX-2). The previously reported antiproliferativeeffects of piperlongumine against various tumor cells(7, 14, 16) could be due to downregulation of these geneproducts. In agreementwith our results, compounds suchas bharangin (32), nimbolide (33) flavopiridol (27), andcelastrol (45) have also shown antiproliferative effects indifferent tumor cells.

Furthermore, our results demonstrated that piperlon-gumine suppressed TNFa-induced expression of ICAM-

Okadaic acid LPS TNFα

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Figure 7. Molecular mechanism bywhich piperlongumine inhibits theNF-kB pathway and regulates NF-kB–associated biomarkers that areinvolved in cell proliferation,angiogenesis, invasion, andmetastasis.

Han et al.





1,MMP-9,CXCR-4, andVEGF,which aremajormediatorsinvolved in tumor invasion and metastasis (50–52). Thedownregulation of these invasion-related proteins sug-gests that piperlongumine has a role in preventing tumorcell invasion and metastasis. That this compound sup-pressed TNFa-induced invasion in cancer cells furthersuggests its anti-invasive activity. The inflammatory cyto-kine IL6, which is known to promote tumorigenesis, wasalso downregulated by piperlongumine.Overall, our results suggest that piperlongumine has

significant potential for tumor treatment: it inhibits theNF-kB pathway by targeting IKK as well as NF-kB–asso-ciated biomarkers that are involved in cell proliferation,angiogenesis, invasion, and metastasis (Fig. 7). On thebasis of these results, further studies are required inanimals and in patients to explore the potential of piper-longumine as an anticancer agent.

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

Authors' ContributionsConception and design: J.G. Han, S.C. Gupta, B.B. AggarwalDevelopment of methodology: J.G. HanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.G. Han

Analysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): J.G. Han, S.C. Gupta, B.B. AggarwalWriting, review, and/or revision of the manuscript: J.G. Han, S.C. Gupta,S. Prasad, B.B. AggarwalAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.G. HanStudy supervision: B.B. Aggarwal

AcknowledgmentsThe authors thank Elizabeth L. Hess from the Department of Scientific

Publications for carefully proofreading the article.

Grant SupportB.B. Aggarwal was supported in part by a grant from Malaysian

Palm Oil Board and a grant from the Center for Targeted Therapy ofThe University of Texas MD Anderson Cancer Center (chartstring404400-80-111139-21). B.B. Aggarwal is also Ransom Horne Jr. Profes-sor of Cancer Research. J.G. Han was supported by a training schemefor excellent talents from China (2011D003034000003), Program forOutstanding Medical Academic Leader of Beijing (2009-1-03), the Basicand Clinical Cooperation Project of Capital Medical University(10JL04), and a Youth Training Abroad Grant of Beijing ChaoyangHospital.

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 February 26, 2014; revised July 8, 2014; accepted July 25, 2014;published OnlineFirst July 31, 2014.

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Han et al.




Published OnlineFirst July 31, 2014.Mol Cancer Ther Jia Gang Han, Subash C. Gupta, Sahdeo Prasad, et al.

Regulated Gene Products−BκSuppression of NF- Kinase, Leading toαBκInteraction with Cysteine 179 of I

Piperlongumine Chemosensitizes Tumor Cells through

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