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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]
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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.
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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
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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
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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
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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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Han et al 7 8/16/13
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
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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
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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.
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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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Han et al 11 8/16/13
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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Han et al 12 8/16/13
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.
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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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Han et al 14 8/16/13
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
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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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Han et al 18 8/16/13
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
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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).
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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
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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
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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.
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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.
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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.
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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
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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
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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
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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
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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.
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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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