Piperlongumine Chemosensitizes Tumor Cells Through ... · 31/07/2014 · 2General Surgery, Beijing...

Han et al 1 8/16/13

Piperlongumine Chemosensitizes Tumor Cells Through Interaction with

Cysteine 179 of IκBα Kinase, Leading to Suppression of NF-κB –regulated

Gene Products

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

1Cytokine Research Laboratory, Department of Experimental Therapeutics,

The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2General Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China

3 University of Mississippi Medical Center, Arthur C. Guyton Research Complex, 2500 North

State Street Jackson, MS 39216, USA

Running title: Regulation of NF-κB Activation by Piperlongumine

Keywords: Cancer; Inflammation; NF-κB; Piperlongumine

Financial Support: B.B. Aggarwal was supported in part by a grant from Malaysian Palm Oil

Board, and a grant from the Center for Targeted Therapy of The University of Texas MD

Anderson Cancer Center (chartstring 404400-80-111139-21). B.B. Aggarwal is also Ransom

Horne Jr. Professor of Cancer Research. J.G. Han was supported by a training scheme for

excellent talents from China (2011D003034000003), Program for Outstanding Medical

Academic Leader of Beijing (2009-1-03), the Basic and Clinical Cooperation Project of Capital

Medical University (10JL04), and a Youth Training Abroad Grant of Beijing Chaoyang Hospital.

Conflict of interest: The authors disclose no potential conflicts of interest.

Corresponding Author: Bharat B. Aggarwal, Cytokine Research Laboratory, Department of

Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston,

Texas, 77030. Phone: 713-794-1817; fax: 713-745-6339; E-mail: [email protected]

on March 16, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 31, 2014; DOI: 10.1158/1535-7163.MCT-14-0171

http://mct.aacrjournals.org/

Han et al 2 8/16/13

ABSTRACT

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

piperlonguimine, a pyridine alkaloid, mediates anticancer effects. In the current report, we

describe another novel mechanism by which this alkaloid mediates its anticellular effects. We

found that piperlonguimine blocked both constitutive and inducible NF-κB activated by TNF-α

and various other cancer promoters. This downregulation was accompanied with inhibition of

phosphorylation and degradation of IκBα. Further investigation revealed that this pyridine

alkaloid directly interacts with IκBα 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-κB 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, IL-6) 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 proinflammatory pathway.




Han et al 3 8/16/13

INTRODUCTION

Cancer is a major public health problem in the United States and many other parts of the

world. Currently, one in three women and one in two men in the United States will develop

cancer in his or her lifetime (1). Although chemotherapy is the standard treatment for most kinds

of cancers, resistance to chemotherapeutic drugs has become a major obstacle in treating cancer.

In fact, multidrug resistance is now considered a main reason for the failure of chemotherapy (2).

Alternatives that are inexpensive, efficacious, and safe compared with synthetic agents are sorely

needed.

Epidemiological, clinical, and experimental evidence suggests that medicines derived from

plants play a pivotal role in treating most diseases, including cancer. As much as 80% of the

cancer therapeutic agents currently used have their roots in natural products. One such product is

the compound 5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)-

pyridinone (Fig. 1A). Called piperlongumine (PL) or piplartine, it is the pyridine alkaloid found

in members of the Piper species, in particular in the fruit of the long pepper (Piper longum

Linn.). Piperlongumine has been used widely in traditional medicine, including the Indian

Ayurvedic system of medicine, traditional Chinese medicine, Tibetan medicine, and the folk

medicine of Latin America. Piperlongumine has multiple pharmacological activities: it has been

described as a platelet aggregation inhibitor (3), an anxiolytic agent (4), an antidepressant (5), an

antinociceptive agent (6), an anti-atherosclerotic agent (7), an antidiabetic agent (8), an anti-

inflammatory agent (9), and an infectious pathogen inhibitor (10). Furthermore, piperlongumine

has been reported to kill multiple types of cancer cells, inhibit metastasis, and have antitumor

activities in a variety of animal models (11-22).

Although this compound was first isolated in 1967, major interest in it did not emerge until




Han et al 4 8/16/13

the 2001 publication in Nature by Raj et al. (18). This group reported that piperlongumine

selectively induces cell death in a wide variety of tumor cell types and does not affect

noncancerous cell types even at high doses. They also reported on its antitumor effects in murine

models of melanoma, bladder cancer, breast cancer, and lung cancer while having only minimal

toxic effects in these models. How piperlongumine acts as an anticancer agent is not yet clear.

Several studies have demonstrated that it induces cytotoxicity through a variety of mechanisms,

such as activation of caspases (14,20,21), activation of the ERK pathway (11) inhibition of cdc-2,

cdk2, and cyclin D1 (19,20), and down-regulation of anti-apoptotic genes (Bcl-2, Raf-1, and

Survivin) and metastatic genes (VEGF, CD31, HIF-2, and Twist) (17-20). The compound has

also been shown to inhibit the enzymatic activity of glutathione S-transferase (GST) pi 1 and

carbonyl reductases and to decrease glutathione levels, indicating that piperlongumine-mediated

apoptosis of cancer cells is induced by a reactive oxygen species (ROS)-dependent mechanism

(18). In addition, piperlongumine-dependent cytotoxicity may involve suppression of nuclear

factor κB (NF-κB), MYC, and LMP-1 in Burkitt lymphoma (14). PL was shown to inhibit NF-

κB activity in prostate cancer cells recently (23), however, the underlying mechanism is unclear.

NF-kB activation has been linked to the survival, proliferation, invasion, angiogenesis and

metastasis of various types of cancers. Activation of NF-kB requires the activation of IκB kinase

(IKK). IKKβ, a subunit of IKK complex is essential for the activation of NF-κB in response to

various proinflammatory stimuli. Cys-179 in the activation loop of this IKKβ plays a major role

in IKK activation as mutation of this residue to alanine decreased its activity (24). Mutation of

Cys179 to alanine in IKKβ also caused reduced phosphorylation of serine residues at position

177 and 181, required for IKKβ activation. In addition, phosphorylation of tyrosine residues at




Han et al 5 8/16/13

positions 188 and 199 are also crucial for IKKβ activation as mutation of these residues have

been shown to abolish the NF-κB activity (25).

Therefore, we postulated that piperlongumine modulates this NF-κB signaling pathway. In this

study, we investigated in detail the effect of piperlongumine on NF-κB pathway regulation. We

found that the compound suppressed NF-κB activation pathways induced by inflammatory

stimuli, growth factors, carcinogens, and tumor promoters through the direct inhibition of

cysteine 179 (Cys179) of IKKβ, which led to the inhibition of IκBα, the suppression of NF-κB–

regulated gene products, and the enhancement of apoptosis in tumor cells.

Materials and Methods

Materials

Piperlongumine was obtained from Indofine Chemical Company (Hillsborough, NJ). Bacteria-

derived human recombinant tumor necrosis factor α (TNF-α) was provided by Genentech (South

San Francisco, CA). Penicillin, streptomycin, RPMI 1640 medium, Dulbecco’s modified Eagle’s

medium (DMEM), fetal bovine serum (FBS), and the kit for the LIVE/DEAD assay were

obtained from Invitrogen (Eugene, OR). PMA, LPS, OA, H2O2, MTT, Hoechst 33342, and

antibodies against FLAG and β-actin were obtained from Sigma (St. Louis, MO). 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 were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA), as was the Annexin V staining kit. Phospho-specific anti-IκBα

(Ser32/36), and anti-p65 (Ser536) antibodies were obtained from Cell Signaling (Danvers, MA).

Phospho-IKK β (Tyr188) and IKKα/β (Ser180/181) antibody was obtained from Abcam (Cambridge,

MA). Antibody against Survivin and an ELISA system kit for human IL-6 were purchased from




Han et al 6 8/16/13

R&D Systems (Minneapolis MN). Anti-VEGF antibody was purchased from NeoMarkers

(Fremont, CA). Antibodies against IκBα, IKK-α, and IKK-β were obtained from Imgenex (San

Diego, CA). Velcade (PS-341) was obtained from Millennium Pharmaceuticals (Cambridge,

MA).

Cell Lines

Human multiple myeloma U266 cells, human embryonic kidney A293 cells and human breast

MCF-7 cells were obtained in 2000 and human T-cell leukemia Jurkat cells were obtained in

1997 from American Type Culture Collection (Manassas, VA). Human lung adenocarcinoma

H1299 cells and human squamous cells carcinoma SCC4 cells were obtained in 2005 from Dr.

Reuben Lotan and human chronic myeloid leukemia KBM-5 cells were obtained from Dr.

Michael Andreeff of The University of Texas MD Anderson Cancer Center. KBM-5 cells were

cultured in Iscove’s modified Dulbecco’s medium with 15% FBS; Jurkat, H1299, MCF-7, and

U266 cells were cultured in RPMI 1640 medium with 10% FBS; and A293 cells were cultured in

DMEM supplemented with 10% FBS. SCC-4 cells were cultured in DMEM containing 10%

FBS, nonessential amino acids, pyruvate, glutamine, and vitamins. All culture media were

supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin. The above-mentioned

cell lines have not been recently tested for authentication in our laboratory.

Cytotoxicity Assay

The effect of piperlongumine on the cytotoxic potential of TNF-α was determined using the

MTT dye uptake method as described previously (26).




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LIVE/DEAD Assay

To measure the effect of piperlongumine on apoptosis induced by TNF-α, we used the

LIVE/DEAD assay to determine plasma membrane integrity (red fluorescent ethidium

homodimer-1) and intracellular esterase activity (green fluorescent calcein-AM). This assay was

performed as described previously (27).

Annexin V/PI Assay

The Annexin V assay is used to detect early apoptosis. This assay takes advantage of the rapid

translocation and accumulation of the membrane phospholipid phosphatidylserine from the

cytoplasmic interface of the cell to the extracellular surface. This loss of membrane asymmetry

can be detected using the binding properties of Annexin V. Briefly, 1 × 106 cells were pretreated

with 10 μM piperlongumine for 4 hours, treated with 1 nM TNF-α for 24 hours, and subjected to

Annexin V staining. Cells were washed in phosphate-buffered saline and resuspended in 100 μL

of binding buffer containing fluorescein isothiocyanate-conjugated Annexin V and then analyzed

by flow cytometry (FACS Calibur; BD Biosciences) after the addition of propidium iodide.

Invasion Assay

The BD BioCoat tumor invasion system (BD Biosciences), which includes a light-tight

polyethylene terephthalate membrane with 8-μm-diameter pores and a thin layer of reconstituted

Matrigel basement membrane matrix, was used to assess cell invasion. H1299 cells (2.5 × 104)

were suspended in serum-free medium and seeded into the upper wells. After incubation

overnight, cells were treated with different concentrations of piperlongumine for 4 hours and

then stimulated with 1 nM TNF-α for 24 hours in the presence of 1% FBS. The invasive cells




Han et al 8 8/16/13

were fixed and stained with Diff-Quik stain (Siemens Healthcare Diagnostics, Newark, DE), and

counted in 5 random microscopic fields (Nikon, Tokyo, Japan).

ELISA

An ELISA kit (R&D Systems) was used to detect human IL-6. U266 cells were treated with

different concentrations of piperlongumine for 24 hours, and cell-free supernatants were

collected for detection of IL-6 by following the manufacturer’s protocol.

EMSA

To assess NF-κB activation, we performed EMSA as described previously (28). Briefly, nuclear

extracts prepared from TNF-α–treated cells (2 × 106/mL) were incubated with 32P-end-labeled

45-mer double-stranded NF-κB oligonucleotide (15 μg of protein with 16 fmol DNA) from the

human immunodeficiency virus long terminal repeat (5′-TTGTTACAA GGGACTTTC CGCTG

GGGACTTTC CAGGGAGGCGTGG-3′) for 30 minutes at 37°C, and the DNA–protein

complex that formed was separated from free oligonucleotide on 6.6% native polyacrylamide

gels. A double-stranded mutated oligonucleotide (5′-TTGTTACAA CTCACTTTC CGCTG

CTCACTTTC CAGGGAGGCGTGG-3′) was used to examine the specificity of binding of NF-

κB to the DNA. The dried gels were visualized with a Storm 820 phosphorimager (Molecular

Dynamics, Sunnyvale, CA) and radioactive bands were quantitated using ImageQuant software

(Molecular Dynamics, Sunnyvale, CA).

Western Blot Analysis




Han et al 9 8/16/13

Cytoplasmic, nuclear, or whole-cell extracts were prepared, and Western blot analysis was

performed. Briefly, 30 μg of protein was resolved with SDS–polyacrylamide gel electrophoresis

(PAGE). After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes,

probed with specific antibodies, and detected by enhanced chemiluminescence reagent (GE

Healthcare Life Sciences, Piscataway, NJ). The bands were quantitated using Densitometer Scan

version 1.30 using ImageQuant software version 3.3 (GE Healthcare Life Sciences, Piscataway,

NJ).

Kinase Assay

To determine the effect of piperlongumine on TNF-α–induced IKK activation, we performed a

kinase assay as described previously (29).

NF-κB–Dependent Reporter Gene Expression Assay

The effect of piperlongumine on the induction of NF-κB–dependent reporter gene transcription

by TNF-α, TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1-β, IKK-β, and p65 was analyzed using

a SEAP assay, as described previously (30).

RESULTS

The aim of this study was to investigate the effect of piperlongumine on the NF-κB activation

induced by various carcinogens and inflammatory stimuli in cancer cells. Most of the studies

were performed using the human myeloid cell line KBM-5 because these cells express tumor

necrosis factor (TNF) receptors and the inflammatory pathway in these cells is well understood.

Other cell types were employed to test the specificity of the effect of piperlongumine.




Han et al 10 8/16/13

Piperlongumine Inhibits TNF-α–Induced NF-κB Activation in a Dose- and Time-

Dependent Manner

We first investigated the dose and duration of piperlongumine exposure required to suppress

TNF-α–induced NF-κB activation in KBM-5 cells. Electrophoretic mobility shift assay (EMSA)

results showed that pretreatment of cells with piperlongumine inhibited TNF-α–induced NF-κB

activation in a dose- (Fig. 1B) and time- (Fig. 1C) dependent manner.

To confirm that the band visualized in TNF-α–treated cells were indeed NF-κB, we

incubated the nuclear extracts from TNF-α–pretreated KBM-5 cells with anti-p50 or -p65

antibodies. The bands shifted to higher molecular masses when incubated with the antibodies,

suggesting that the TNF-α–activated NF-κB activation complex consisted of both p50 and p65.

Addition of pre-immune serum and mutated oligonucleotide had no effect on DNA binding,

whereas the addition of excess unlabeled NF-κB (cold oligonucleotide; 100-fold excess) caused a

decrease in the intensity of the band (Fig. 1D).

Piperlongumine Inhibits NF-κB Activation Induced by Carcinogens and Inflammatory

Stimuli

The endotoxin lipopolysaccharide (LPS), okadaic acid (OA), phorbol 12-myristate 13-acetate

(PMA), hydrogen peroxide (H2O2), and cigarette smoke condensate (CSC) are known to activate

NF-κB by different mechanisms. We found that all these agents activated NF-κB in KBM-5 cells

and that piperlongumine suppressed this activation (Fig. 1E). These results suggested that

piperlongumine acts at a step in the NF-κB activation pathway that is common to all 5 of these

agents.




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Inhibition of NF-κB Activation by piperlongumine Is Not Cell Type Specific

We next determined whether piperlongumine-mediated inbibition in TNF-α–induced NF-κB

activation is cell-type specific. We observed an inhibitory effect is not only in KBM-5 cells but

also multiple myeloma (U266), T-cell leukemia (Jurkat), head and neck squamous cell

carcinoma (SCC4), breast carcinoma (MCF-7), lung adenocarcinoma (H1299), and kidney

(A293) cells (Fig 1F). Thus, piperlongumine appears to be able to suppress NF-κB activation in a

variety of human tumor cells.

Piperlongumine Does Not Directly Affect the Binding of NF-κB to DNA

Some NF-κB inhibitors, such as plumbagin (31) and bharangin (32), directly modify NF-κB to

suppress its binding to DNA. To determine whether piperlongumine suppresses NF-κB

activation through a similar mechanism, we incubated the nuclear extract from TNF-α–treated

KBM-5 cells with piperlongumine and found that the compound did not modify the DNA-

binding ability of NF-κB protein (Fig. 2A). These results suggested that inhibition of NF-κB

activation by piperlongumine does not suppress DNA binding directly. This compound may,

however, use the inhibitory mechanism of nimbolide (33) and γ-tocotrienol (34), which inhibit

NF-κB activation indirectly.

Piperlongumine Inhibits TNF-α–Induced IκBα Degradation and Phosphorylation

NF-κB activation requires the phosphorylation, polyubiquitination, and subsequent degradation

of its inhibitory subunit, IκBα. To determine whether the inhibition of TNF-α–induced NF-κB

activation we observed in KBM-5 cells was due to the inhibition of IκBα degradation, KBM-5




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cells were pretreated with piperlongumine and then exposed to TNF-α for various time periods.

We then analyzed the cells for nuclear NF-κB by EMSA and for IκBα degradation in the

cytoplasm fraction by Western blotting. TNF-α activated NF-κB in the control cells in a time-

dependent manner but not in piperlongumine-pretreated cells (Fig. 2B). Moreover, TNF-α

induced IκBα degradation in control cells as early as 5 minutes (Fig. 2C). IκBα was completely

degraded after 10–15 minutes and was resynthesized after 30 minutes. In piperlongumine-

pretreated cells, however, this degradation was completely reversed.

Because IκBα degradation requires IκBα phosphorylation (35), we explored whether the

inhibition of TNF-α–induced IκBα degradation was due to the inhibition of IκBα

phosphorylation. We blocked IκBα degradation by using the proteasome inhibitor N-acetyl-

leucyl-leucylnorleucinal (ALLN). Western blotting results showed that co-treatment with TNF-α

and ALLN induced IκBα phosphorylation and that pretreatment with piperlongumine strongly

suppressed this phosphorylation (Fig. 2D). These results indicated that piperlongumine

suppresses TNF-α–induced IκBα degradation by inhibiting IκBα phosphorylation.

Piperlongumine Directly Inhibits TNF-α–Induced IKK Activation

Phosphorylation of IκBα is regulated by the upstream kinase IKK complex. Because

piperlongumine inhibits the phosphorylation and degradation of TNF-α–induced IκBα, we

determined the effect of this compound on IKK activation. Our results revealed that TNF-α

activated IKK in a time-dependent manner and piperlongumine suppressed this activation (Fig.

2E). Expression of IKK-α or IKK-β proteins was not notably affected by TNF-α or

piperlongumine.




Han et al 13 8/16/13

To determine whether piperlongumine inhibits IKK activity directly or indirectly, we treated

the kinase assay mixture prepared from TNF-α–pretreated KBM-5 cells with various

concentrations of piperlongumine. Results from the kinase assay showed that it inhibited IKK

activity (Fig 2F), suggesting that the compound suppresses TNF-α–induced IKK activation

directly.

Because IKK-β contains various cysteine residues, we investigated whether piperlongumine

suppresses IKK activation by modifying one or more of these cysteine residues. The reducing

agent dithiothreitol (DTT) was used to determine whether the modulation of IKK activity by

piperlongumine was through the modification of critical cysteine residues. We found that the

addition of DTT to the kinase reaction mixture reversed the piperlongumine-mediated inhibition

of TNF-α–induced IKK activity (Fig. 2G). The finding suggested that a cysteine residue is

involved in this pathway.

The cysteine residue Cys179, which is positioned within the activation loop of the IKK

catalytic subunits, is critical for IKK-β activity. To determine whether Cys179 is involved in

piperlongumine-induced IKK inhibition, we transfected A293 cells with wild-type FLAG-IKK-β

or mutated FLAG-IKK-β (alanine substituting for Cys179A). Piperlongumine inhibited wild-type

IKK-β but had no apparent effect on mutated IKK-β (Fig. 2H). These results suggested that

piperlongumine inhibits IKK-β activity by directly modifying the Cys179 residue.

Further we determined whether PL modulates phosphorylation of other residues of IKK-β

such as serine-181 and tyrosine-188, which are also responsible for its full activity. Results

showed that TNF-induced phosphorylation of IKK-β at both serine-181 and tyrosine-188

residues and PL inhibited the phosphorylation of both the residues (Fig 2I).




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Piperlongumine Inhibits TNF-α–Induced p65 Nuclear Translocation and Phosphorylation

The phosphorylation and degradation of IκBα are essential to the NF-κB activation pathway,

whereas the stabilization of IκBα is important in preventing the nuclear translocation of p65. The

effect of piperlongumine on TNF-induced nuclear translocation of p65 was examined by

Western blot analysis. For this experiment, KBM-5 cells were treated with piperlongumine and

then exposed to TNF-α for different time periods. The nuclear extracts were prepared and

examined for p65 by Western blot analysis. We observed that TNF-α induced the nuclear

translocation of p65 and that piperlongumine suppressed the TNF-α–induced p65 nuclear

translocation (Fig. 3A).

Whether piperlongumine affects TNF-α–induced p65 phosphorylation at Ser536 was also

examined. The results showed that TNF-α induced the phosphorylation of p65 and that

piperlongumine suppressed this phosphorylation (Fig. 3A).

Piperlongumine Represses TNF-α–Induced NF-κB–Dependent Reporter Gene Expression

Although we observed that piperlongumine inhibits NF-κB activation, DNA binding alone does

not always result in NF-κB–dependent gene transcription. For this reason, we assessed whether

piperlongumine can affect TNF-α–induced reporter gene transcription. A 12-fold increase in

SEAP activity was observed after simulation with TNF-α compared with the vector control, and

the induction was nearly abolished by dominant-negative IκBα (Fig. 3B), indicating specificity.

When the A293 cells were pretreated with piperlongumine, TNF-α–regulated NF-κB–dependent

SEAP expression was inhibited in a dose-dependent manner (Fig. 3B). These results suggested

that piperlongumine inhibits the NF-κB–dependent reporter gene expression induced by TNF-α.




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TNF-α–induced NF-κB activation is mediated through the sequential interaction of TNF

receptor 1 (TNFR1) with TNF receptor-associated death domain (TRADD), TNF receptor-

associated factor 2 (TRAF2), NF-κB–inducing kinase (NIK), transforming growth factor-β

activated protein kinase 1 (TAK1)/TAK1-binding-protein-1 (TAB1), and IKK-β, which results in

phosphorylation of IκBα and leads to degradation of IκBα and p65 nuclear translocation. We

determined the site of action for piperlongumine in TNF-α signaling in A293 cells. This agent

significantly suppressed NF-κB–dependent reporter gene expression induced by TNFR1,

TRADD, TRAF2, NIK, TAK1/TAB1, and IKK-β plasmids (Fig. 3C), indicating that

piperlongumine acts at a step upstream of p65.

Piperlongumine Represses TNF-α–Induced NF-κB–Dependent Gene Products Associated

with Survival, Proliferation, Invasion, and Metastasis

NF-κB regulates the expression of the anti-apoptotic proteins Bcl-2, Bcl-xL, cellular inhibitor of

apoptosis proteins 1 and 2 (c-IAP-1, c-IAP-2), and Survivin, which are known to be induced by

TNF-α and to play an important role in cell survival. We used Western blotting to determine

whether piperlongumine inhibits the expression of these gene products in KBM-5 cells. Our

results showed that the compound down-regulated the expression of all 5 of these TNF-α–

induced proteins (Fig 4A).

We also used Western blotting to investigate whether piperlongumine modulates the

expression of the proliferative proteins c-myc, cyclin D1, and cyclooxygenase-2 (COX-2). We

observed that TNF-α induced the expression of these proliferative proteins and that

piperlongumine inhibited it (Fig. 4B).




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Finally, we examined whether piperlongumine affects NF-κB–regulated gene products

involved in tumor cell invasion and metastasis. Western blotting revealed that TNF-α induced

the expression of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9

(MMP-9), CXC chemokine receptor 4 (CXCR-4), and vascular endothelial growth factor

(VEGF) and that piperlongumine down-regulated the expression of all these proteins (Fig. 4C).

Piperlongumine Suppresses Pro-Inflammatory Cytokine Production

Interleukin 6 (IL-6) is a proinflammatory cytokine that is regulated by NF-κB and augments the

proliferation of a wide variety of tumor cells. We examined whether piperlongumine affects the

IL-6 level produced by human multiple myeloma U266 cells. Our enzyme-linked

immunosorbent assay (ELISA) results showed that piperlongumine suppressed IL-6 production

in a dose-dependent manner (Fig. 4D).

Piperlongumine Potentiates the Cell Death Induced by TNF-α and Chemotherapeutic

Agents

Using KBM-5 cells, we next examined whether the down-regulation by piperlongumine of the

tumor cell survival proteins leads to potentiation of the apoptosis induced by TNF-α or

chemotherapeutic agents. Using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay, we found that piperlongumine did, in fact, potentiate the cytotoxicity

induced by TNF-α, 5-fluorouracil (5-FU), or bortezomib in a dose-dependent manner (Fig. 5A).

The apoptotic effect of piperlongumine was also examined by phosphatidylserine

externalization, a marker of early apoptosis, using Annexin V staining and flow cytometry. The




Han et al 17 8/16/13

number of Annexin V–positive cells induced by TNF-α increased significantly when KBM-5

cells were pretreated with piperlongumine (Fig. 5B).

Whether piperlongumine can enhance the TNF-α–induced activation of caspases-3, -8, and -9

was also examined. Our Western blotting results showed that the piperlongumine sensitized the

cells to caspase activation (Fig. 5C).

We also used Western blotting to examine whether piperlongumine can enhance TNF-α–

induced poly(ADP-ribose) polymerase (PARP) cleavage in KBM-5 cells. We found that TNF-α

alone had little effect on PARP cleavage, but when the cells were pretreated with

piperlongumine, a remarkable increase in PARP cleavage was observed (Fig. 5D). These results

also demonstrate a synergistic interaction between the 2 agents. Taken together, these results

suggested that piperlongumine potentiates the apoptotic effects of TNF-α and chemotherapeutic

agents.

Ubiquitous intracellular esterase activity and an intact plasma membrane are characteristics

of live cells. We used the LIVE/DEAD assay to discriminate between live and dead KBM-5 cells

by simultaneously staining with calcein-AM and ethidium homodimer-1. This assay indicated

that piperlongumine alone induces loss of membrane integrity (an indicator of apoptosis) and

that treatment with TNF-α enhances the piperlongumine-induced apoptosis (Fig. 6A).

Piperlongumine Suppresses TNF-α–Induced Tumor Cell Invasion

Because piperlongumine suppressed the TNF-α induced expression of proteins such as

CXCR-4 or MMP-9, which are linked to tumor cell invasion, we next examined whether

piperlongumine suppresses TNF-α–induced tumor cell invasion. For this experiment, we used

H1299 cells. Our results showed that TNF-α increased tumor cell invasion by 1.4-fold and that




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pretreatment with piperlongumine decreased the level of TNF-α-induced tumor cell invasion to

below baseline (Fig. 6B).

We also determined whether PL inhibits the expression of MMP-9 and CXCR4 involved in

invasion and metastasis, respectively. We found that TNF-α induced the expression of MMP-9

and CXCR4, and the pretreatment with PL significantly suppressed the expression of these

proteins in H1299 cells (Fig 6C). These results suggest that inhibition in MMP-9 and CXCR4

protein expression may contribute to the inhibitory affects of PL on invasion activity of H1299

cells.

DISCUSSION

Although radiotherapy and chemotherapy are used to treat cancer, both activate the NF-κB

pathway, which leads to radioresistance and chemoresistance, respectively. Thus, agents that are

safe and efficacious in down-modulating the NF-κB pathway are urgently needed for radio- and

chemosensitization. One such agent is piperlongumine, which is derived from the “long pepper”

plant and is consumed traditionally to address a range of inflammatory diseases. However, the

mechanism of action of this compound is not fully understood. Two recently studies from

Harvard researchers published in Nature (18) and PNAS (15) described the activity of

piperlongumine against a wide variety of human cancers in cell culture and animal models, but

how this compound mediates its anticancer and other activities is poorly understood. Our current

study provides insight into piperlongumine’s mechanism of action.

The specific objective of our study was to determine the effect of piperlongumine on the NF-

κB activation pathway and on NF-κB–regulated gene products that are associated with

inflammation, tumor cell proliferation, invasion, and angiogenesis. We demonstrated that




Han et al 19 8/16/13

piperlongumine suppressed NF-κB activation that was induced by various carcinogens and

inflammatory agents through the inhibition of IKK activation, which led to the suppression of

IκBα phosphorylation and degradation and the suppression of p65 nuclear translocation and

phosphorylation. Piperlongumine also promoted the apoptosis induced by TNF-α and various

chemotherapeutic agents, and it suppressed cancer cell invasion.

We found that piperlongumine inhibited NF-κB activation that was induced by a wide variety

of agents (TNF-α, LPS, OA, PMA, H2O2, and CSC2) and that this suppression was not cell type

specific. These results are in agreement with recent reports that piperlongumine can suppress

LPS-induced NF-kB activation in endothelial cells (9) and PMA-induced NF-κB activation in

murine macrophages (36). Our results are also consistent with another recent report that

piperlongumine inhibits constitutive NF-κB expression in Burkitt lymphoma cells (14).

How piperlongumine inhibits NF-κB activation has not been reported and thus was

investigated in detail. We found, for the first time, that piperlongumine suppresses TNF-α–

induced IKK activation, which leads to suppression of the phosphorylation and degradation of

IκBα. Our study also showed that piperlongumine directly inhibits TNF-α–induced IKK activity.

This inhibition could be reversed with the reducing agent DTT, which suggests that inhibition of

IKK activation involves the interaction of piperlongumine with -SH groups within IKK. Cys179

of IKK, a redox-sensitive cysteine residue, is positioned within the activation loop of the IKK

catalytic subunits (37). We found that substitution of Cys179 with alanine prevents the inhibition

of IKK activity by piperlongumine, thus suggesting the role of this residue in the action of this

alkaloid. This is first report to suggest the role of piperlongumine with -SH groups of any

protein, although irreversible protein glutathionylation as a process associated with cellular

toxicity of this alkaloid has been demonstrated (15).




Han et al 20 8/16/13

We also found, for the first time, that TNF-α induces the phosphorylation of the p65 subunit

of NF-κB at Ser536 and that piperlongumine completely suppresses the phosphorylation. IKK has

been shown to cause the phosphorylation of p65 at Ser536 (38). Unlike IκBα, multiple kinases

have been implicated in the phosphorylation of p65 at Ser536 (39), and it is very likely that

suppression of IKK activity is involved in inhibiting the phosphorylation of both IκBα and p65

by piperlongumine. Interestingly, phosphorylation of RelA/p65 on Ser536 has been shown to be

independent of IκBα (40), indicating that IKK has numerous substrates. NF-κB p65

phosphorylated at Ser536 was also shown to be an independent prognostic factor in Swedish

colorectal cancer patients (41), suggesting the potential of using piperlongumine to treat such

patients.

Our results indicate that despite the involvement of at least 50 different proteins in the NF-κB

activation pathway, each containing multiple cysteines (42), piperlongumine inhibits NF-κB by

modifying Cys179 in the IKK-β activation loop. Nimbolide (33), butein (43), 3-formylchromone

(44), and other compounds have been reported to exhibit similar effects on Cys179. In addition,

we found that PL also blocked the phosphorylation of serine 181 and tyrosine 188 in IKKβ

induced by TNF-α. Serine kinases, such as TAK-1, has been shown to phosphorylate IKKβ at

residues 181 (for references 25). In addition, tyrosine residue 188 is within the activation loop of

IKKβ and is in close proximity to serine181. The fact that the tyrosine kinase c-Src binds to

IKKβ suggests that tyrosine phosphorylation of IKKβ is potentially important for regulation of

its activity (25). Thus these results suggest that PL may inhibit IKKβ through multiple

mechanisms.

The suppression of NF-κB activation induced by TNF-α, LPS, OA, PMA, H2O2, and CSC

suggests that piperlongumine acts at a step common to all these activators. NF-κB activation by




Han et al 21 8/16/13

TNF-α is mediated through sequential recruitment of TNFR1, TRADD, TRAF2, NIK,

TAK1/TAB1, and IKK. Our transfection experiments showed that piperlongumine inhibited the

NF-κB reporter gene expression induced by these molecules. Similar results have been reported

with γ-tocotrienol (34), flavopiridol (27), and celastrol (45).

Most approaches used in cancer treatment, such as chemotherapy and radiotherapy, kill

cancer cells by inducing apoptosis; however, cancer cells often develop resistance to such

treatment. Furthermore, many cancer therapies indirectly activate apoptosis by chemically or

physically damaging DNA. This damage may have the unintended effect of generating a pool of

heavily mutated cancer cells and increasing the chances of their developing resistance (46). We

showed that piperlongumine potentiated cell death induced by TNF-α or the chemotherapeutic

agents 5-FU and bortezomib in KBM-5 cells. Our results suggest that chemotherapy or

radiotherapy followed by treatment with piperlongumine could reduce the tumor burden.

The loss of caspase activation appears to be central to the prevention of most cell death

events in cancer. Finding strategies to overcome caspase inhibition will be valuable for the

development of novel cancer treatments (47). We investigated in detail how piperlongumine

potentiates apoptosis and found that piperlongumine enhanced the cleavage of precursors of

caspases-3, -8, and -9, which suggested that the compound activated cell death signaling through

2 pathways: the extrinsic, death receptor pathway (caspase-8) and the intrinsic, mitochondrial

pathway (caspase-9). NF-κB–regulated cell survival proteins, such as the Bcl-2 family of

proteins and IAPs, are selectively overexpressed in various types of tumors and are involved in

the apoptotic pathway defect in many tumor cells (48). Overexpression of these proteins is

involved in tumor cell survival (49) and might be used to reverse the apoptotic pathway defect in




Han et al 22 8/16/13

cancer. Bcl-2 was found to be downregulated by piperlongumine in previous studies (18,20).

Furthermore, we found that piperlongumine suppressed the expression of cell survival proteins,

including Bcl-2, Bcl-xl, c-IAP-1, c-IAP-2, and survivin, suggesting that these gene products

could be therapeutic targets for potentiation of apoptosis by piperlongumine.

We also found that treatment with piperlongumine down-regulated the expression of NF-κB–

regulated gene products involved in cell proliferation (c-Myc, Cyclin D1, and COX-2). The

previously reported anti-proliferative effects of piperlongumine against various tumor cells

(7,14,16) could be due to down-regulation of these gene products. In agreement with our results,

compounds such as bharangin (32), nimbolide (33) flavopiridol (27), and celastrol (45) have also

shown antiproliferative effects in different tumor cells.

Furthermore, our results demonstrated that piperlongumine suppressed TNF-α–induced

expression of ICAM-1, MMP-9, CXCR-4, and VEGF, which are major mediators involved in

tumor invasion and metastasis (50-52). The down-regulation of these invasion-related proteins

suggest that piperlongumine has a role in preventing tumor cell invasion and metastasis. That

this compound suppressed TNF-α–induced invasion in cancer cells further suggests its anti-

invasive activity. The inflammatory cytokine IL-6, which is known to promote tumorigenesis,

was also downregulated by piperlongumine.

Overall, our results suggest that piperlongumine has significant potential for tumor treatment:

it inhibits the NF-κB pathway by targeting IKK as well as NF-κB–associated biomarkers that are

involved in cell proliferation, angiogenesis, invasion, and metastasis (Fig 7). On the basis of

these results, further studies are required in animals and in patients to explore the potential of

piperlongumine as an anticancer agent.




Han et al 23 8/16/13

Acknowledgements:

We thank Elizabeth L. Hess from the Department of Scientific Publications for carefully

proofreading the manuscript. Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer

Research.




Han et al 24 8/16/13

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Legend of Figures:

FIGURE 1. Piperlongumine suppresses constitutive and TNF-α–induced NF-κB activation in

cancer cells. A, Chemical structure of piperlongumine. B, Dose-dependent effect of

piperlongumine on TNF-α–induced NF-κB activation. KBM-5 cells were treated with the

indicated concentrations of piperlongumine for 4 hours and then exposed to 0.1 nM TNF-α for

30 minutes. C, Time-dependent effect of piperlongumine on TNF-α–induced NF-κB activation.

KBM-5 cells were treated with 10 μM piperlongumine for the indicated times and then exposed

to 0.1 nM TNF-α for 30 minutes. D, TNF-α–induced NF-κB is composed of p50 and p65

subunits. Nuclear extracts from untreated KBM-5 cells or KBM-5 cells treated with 0.1 nM

TNF-α were incubated with the indicated antibodies, pre-immune serum (PIS), an unlabeled

competitor NF-κB oligonucleotide probe, or a mutant oligonucleotide probe. E, piperlongumine

suppresses NF-κB activation induced by LPS, OA, PMA, H2O2, or CSC. KBM-5 cells were pre-

incubated with the indicated concentrations of piperlongumine for 4 hours and then treated with

100 ng/ml LPS for 2 hours, 500 nM OA for 4 hours, 25 ng/ml PMA for 1 hour, 500 μM H2O2 for

2 hours, or 10 μg/ml CSC for 1 hour. F, Effect of piperlongumine on TNF-α–induced NF-κB

activation in human U266, Jurkat, SCC4, MCF-7, H1299, and A293 cells. Cells were incubated

with the indicated concentrations of piperlongumine for 4 hours and then treated with 0.1 nM

TNF-α for 30 minutes. B, C, E and F, Nuclear extracts were assayed for NF-κB activation by

EMSA. Results are representative of 3 independent experiments. Results are expressed as fold

activity over the group incubated without piperlongumine and not treated with TNF-α, which

was set at 1.0. PL, piperlongumine.




Han et al 29 8/16/13

FIGURE 2. Down-regulation of TNF-α–induced NF-κB activation by piperlongumine involves

Cys179 of IKK. A, In vitro effect of piperlongumine on NF-κB–DNA binding. Nuclear extracts

(NE) prepared from KBM-5 cells treated with 0.1 nM TNF-α were incubated with the indicated

concentrations of piperlongumine for 30 minutes. B, KBM-5 cells were pre-incubated with 10

μM piperlongumine for 4 hours and then treated with TNF-α for the indicated times. A and B,

Nuclear extracts from KBM-5 cells were subjected to EMSA for NF-κB activation. Results are

expressed as fold activity over the group incubated without piperlongumine and not treated with

TNF-α, which was set at 1.0. C, Effect of piperlongumine on TNF-α–induced IκBα degradation.

KBM-5 cells were pre-incubated with 10 μM piperlongumine for 4 hours and treated with TNF-α

for the indicated times. Cytoplasmic extracts were analyzed for Western blotting with antibodies

against IκBα and β-actin. D, Piperlongumine inhibits TNF-α–induced IκBα phosphorylation.

KBM-5 cells were pre-incubated with 10 μM piperlongumine for 4 hours, treated with 50 μg/mL

ALLN for 30 minutes, and then treated with 0.1 nM TNF-α for 10 minutes. Cytoplasmic extracts

were analyzed for Western blotting using antibodies against phospho-specific IκBα (Ser32/36),

IκBα, and β-actin. E, Effect of piperlongumine on the TNF-α–induced IKK activation. KBM-5

cells were pre-incubated with 10 μM piperlongumine for 4 hours and then treated with 1 nM

TNF-α for the indicated times. Whole-cell extracts were immunoprecipitated with antibody

against IKK-α and analyzed with an immune complex kinase assay. The effect of

piperlongumine on IKK protein expression was determined by Western blotting using anti-IKK-

α and anti-IKK-β antibodies. F, Direct effect of piperlongumine on IKK activation induced by

TNF-α. Whole-cell extracts were prepared from KBM-5 cells treated with 1 nM TNF-α and

immunoprecipitated with an anti-IKK-α antibody. The immunocomplex kinase assay was

performed in the presence of the indicated concentrations of piperlongumine. G, Effect of the




Han et al 30 8/16/13

reducing agent DTT on piperlongumine-induced inhibition of IKK activation. KBM-5 cells were

treated with 1 nM TNF-α and immunoprecipitated with antibodies against IKK-α and IKK-β.

The immunocomplex kinase assay was performed in the presence of 10 μM piperlongumine,

with or without DTT. H, Effect of piperlongumine on the kinase activity of IKKC179A. A293 cells

were transfected with wild-type (WT) or mutated (MT) FLAG-IKK-β. Whole-cell extracts were

prepared, immunoprecipitated, incubated with 10 μM piperlongumine, and subjected to an IKK

assay. I. Effect of piperlongumine on phosphorylation of IKK-β. KBM-5 cells were treated with

10 μM piperlongumine for 4 hours and exposed with 0.1nM of TNF-α for indicated time period.

Whole-cell extracts were prepared and subjected to western blot. A–H, Results shown are

representative of 3 independent experiments. PL, piperlongumine.

FIGURE 3. Piperlongumine inhibits TNF-α–induced phosphorylation and nuclear translocation

of p65 and NF-κB–dependent reporter gene expression induced by TNF-α and other molecules in

the NF-κB signaling pathway. A, KBM-5 cells were pretreated with 10 μM piperlongumine for 4

hours and then treated with 0.1 nM TNF-α for the indicated times. Nuclear extracts were

prepared and analyzed by Western blotting using antibodies against p65 and phospho-p65

(Ser536). PARP was used as an internal control. B, Piperlongumine inhibits TNF-α–induced NF-

κB–dependent SEAP expression. A293 cells were transiently transfected with a plasmid

containing an NF-κB–SEAP gene. Cells were treated with piperlongumine for 4 hours at the

indicated concentrations, which was followed by treatment with 1 nM TNF-α 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 presented as mean ± S.D. DN, dominant

negative. C, Piperlongumine inhibits NF-κB–dependent reporter gene expression induced by




Han et al 31 8/16/13

TNF-α, TNFR1, TRADD, TRAF2, NIK, TAK1/TAB1, IKK-β, or p65. A293 cells were

transiently transfected with pNF-κB–SEAP plasmid, expression plasmid, and control plasmid for

24 hours and treated with 10 μM piperlongumine for 4 hours. The cell supernatants were then

assayed for SEAP activity. For TNF-α–treated cells, cells were incubated with 10 μM

piperlongumine for 4 hours and then treated with 1 nM TNF for an additional 12 hours of

incubation. Results are expressed as fold activity over the vector control (Con), which was set at

1. Data presented as mean ± S.D. B and C, Data presented as mean ± S.D. Results shown are

representative of 3 independent replicates. PL, piperlongumine.

FIGURE 4. Piperlongumine inhibits TNF-α–induced expression of NF-κB–dependent anti-

apoptotic, proliferative, and metastatic proteins. KBM-5 cells were incubated with 10 μM

piperlongumine for 4 hours and then treated with 1 nM TNF-α for the indicated times. A–C,

Whole-cell extracts were prepared and analyzed by Western blotting using antibodies against

anti-apoptotic A, proliferative B, and metastatic proteins C. Results of 1 of the 3 independent

experiments are shown. D, Piperlongumine down-regulates IL-6 production in U266 cells in a

concentration-dependent manner. Cells were treated with the indicated concentrations of

piperlongumine, and cell-free supernatants were harvested after 24 hours. The level of IL-6 was

detected by ELISA. Data presented as mean ± S.D. Results shown are representative of 3

independent replications. A–D, PL, piperlongumine.

Figure 5. Piperlongumine potentiates the apoptotic effects of TNF-α and chemotherapeutic

agents in KBM-5 cells. A, piperlongumine enhances TNF-α–, 5-FU–, and bortezomib

(Velcade)–induced cytotoxicity. Cells (5,000) were seeded in 4 replicates, pretreated with the




Han et al 32 8/16/13

indicated concentrations of piperlongumine, and incubated with a chemotherapeutic agent (1 nM

TNF-α [left], 0.1 μM 5-FU [center], or 20 μM bortezomib [right]) for 24 hours. Cytotoxicity was

analyzed by the MTT assay. B, Piperlongumine potentiates TNF-α–induced early apoptosis.

Cells were pretreated with 10 μM piperlongumine for 4 hours and then incubated with 1 nM

TNF-α for 24 hours. Cells were incubated with fluorescein isothiocyanate-conjugated Annexin V

antibody and then analyzed using flow cytometry (left), and displayed as histogram (right). A

and B, Data presented as mean ± S.D. Results shown are representative of 3 independent

replications. C, Piperlongumine induces TNF-α–induced caspase activation. Cells were

incubated with 10 μM piperlongumine for 4 hours and then treated with 1 nM TNF-α for 24

hours. Whole-cell extracts were prepared and analyzed by Western blotting. D, Piperlongumine

induces PARP cleavage. Cells were pretreated with 10 μM piperlongumine for 4 hours and then

incubated with 1 nM TNF-α for the indicated times. Whole-cell extracts were prepared and

analyzed by Western blotting. C and D, Figures are representative of 1 of 3 independent

experiments. A–D, PL, piperlongumine.

FIGURE 6. Piperlongumine suppresses proliferation and potentiates TNF-α–induced apoptosis

in tumor cells. A, Piperlongumine enhances TNF-α–induced apoptosis. KBM-5 cells were

pretreated with the indicated concentrations of piperlongumine for 4 hours and then incubated

with 1 nM TNF-α for 24 hours. Cells were stained with LIVE/DEAD assay reagent for 30

minutes and analyzed under a fluorescence microscope. Magnification, 100×. Percentages

represent mean ± S.D. number of apoptotic cells. Green, intracellular esterase activity; red, loss

of plasma membrane integrity. B, Piperlongumine suppresses TNF-α–induced cell invasion.

H1299 cells (2.5 × 104 cells) were seeded into the top chamber of a Matrigel invasion chamber




Han et al 33 8/16/13

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 TNF-α in the presence

of 1% serum for 24h, stained with Diff-Quick stain, and assayed for invasion. Control value was

set at 1.0. Values represent mean ± S.D. of 3 independent replicates. PL, piperlongumine. C.

Piperlongumine suppresses TNF-α–induced expression of MMP-9 and CXCR4 proteins. H1299

(1 × 106 cells) were treated with indicated concentration of piperlongumine for 4 hours and the

exposed with TNF-α for 24 hours. Whole cell extract were prepared and subjected to western

blotting.

Figure 7. Molecular mechanism by which piperlongumine inhibits the NF-κB pathway and

regulates NF-κB–associated biomarkers that are involved in cell proliferation, angiogenesis,

invasion, and metastasis.




Published OnlineFirst July 31, 2014.Mol Cancer Ther Jia Gang Han, Subash C. Gupta, Sahdeo Prasad, et al. to Suppression of NF-kappaB -regulated Gene ProductsInteraction with Cysteine 179 of IkappaBalpha Kinase, Leading Piperlongumine Chemosensitizes Tumor Cells Through

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