Berberine Modifies Cysteine 179 of I B Kinase, Suppresses Nuclear Factor- B-Regulated Antiapoptotic Gene Products, and Potentiates Apoptosis

2008;68:5370-5379. Cancer Res Manoj K. Pandey, Bokyung Sung, Ajaikumar B. Kunnumakkara, et al. and Potentiates Apoptosis

Regulated Antiapoptotic Gene Products,−BκNuclear Factor- Kinase, SuppressesαBκBerberine Modifies Cysteine 179 of I

Updated version

http://cancerres.aacrjournals.org/content/68/13/5370

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2008/06/30/68.13.5370.DC1.html

Access the most recent supplemental material at:

Cited Articles

http://cancerres.aacrjournals.org/content/68/13/5370.full.html#ref-list-1

This article cites by 46 articles, 18 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/68/13/5370.full.html#related-urls

This article has been cited by 7 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications

Research. on June 1, 2013. © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/content/68/13/5370

http://cancerres.aacrjournals.org/content/suppl/2008/06/30/68.13.5370.DC1.html

http://cancerres.aacrjournals.org/content/68/13/5370.full.html#ref-list-1

http://cancerres.aacrjournals.org/content/68/13/5370.full.html#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]

http://cancerres.aacrjournals.org/

Berberine Modifies Cysteine 179 of IKBA Kinase, Suppresses Nuclear

Factor-KB–Regulated Antiapoptotic Gene Products,

and Potentiates Apoptosis

Manoj K. Pandey, Bokyung Sung, Ajaikumar B. Kunnumakkara, Gautam Sethi,Madan M. Chaturvedi, and Bharat B. Aggarwal

Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center,Houston, Texas

Abstract

Berberine, an isoquinoline alkaloid derived from a plantused traditionally in Chinese and Ayurvedic medicine, hasbeen reported to exhibit chemopreventive and anti-inflam-matory activities through unknown mechanism. Because ofthe critical role of the transcription factor nuclear factor-KB(NF-KB) in these processes, we investigated the effect ofberberine on this pathway. We found that berberine sup-pressed NF-KB activation induced by various inflammatoryagents and carcinogens. This alkaloid also suppressedconstitutive NF-KB activation found in certain tumor cells.Suppression of NF-KB activation occurred through theinhibition of phosphorylation and degradation of IKBA bythe inhibition of IKB kinase (IKK) activation, leading tosuppression of phosphorylation and nuclear translocation ofp65, and finally to inhibition of NF-KB reporter activity.Inhibition of IKK by berbeine was direct and could bereversed by reducing agents. Site-specific mutagenesis sug-gested the involvement of cysteine residue 179 in IKK.Berberine also suppressed the expression of NF-KB–regulatedgene products involved in antiapoptosis (Bcl-xL, Survivin,IAP1, IAP2, and cFLIP), proliferation (cyclin D1), inflamma-tion (cyclooxygenase-2), and invasion (matrix metallopro-teinase-9). Suppression of antiapoptotic gene productscorrelated with enhancement of apoptosis induced by tumornecrosis factor (TNF)-A and chemotherapeutic agents andwith inhibition of TNF-induced cellular invasion. Overall, ourresults indicate that chemopreventive, apoptotic, and anti-inflammatory activities displayed by berberine may bemediated in part through the suppression of the NF-KBactivation pathway. This may provide the molecular basis forthe ability of berberine to act as an anticancer and anti-inflammatory agent. [Cancer Res 2008;68(13):5370–9]

Introduction

Almost 80% of the world population cannot afford modernmedicine. Traditional medicine is inexpensive but generally neither

active principles nor their molecular targets are well-defined.For instance, extracts of golden seal (Hydrastis canadensis), oregongrape (Berberis aquifolium), barberry (Berberis vulgaris), coptis orgolden thread (Coptis chinensis), tumeric tree (Berberis arisata),Huangbai (Cortex phellodendri ), and Huanglian Letasiova S(Rhizoma coptidis) have been traditionally used for bacterial diarrhea,intestinal parasite infections, and ocular trachoma infections (1). InChinese pharmacopoeia, Huangbai and Huanglian are described as‘‘heat removing’’ agents for their fever-reducing therapeutic appli-cations. Almost four decades ago, berberine, an isoquinolinealkaloid, was identified as an active component in all these plants.Berberine has been shown to suppress the growth of a wide

variety of tumor cells including leukemia (2), melanoma (3), epi-dermoid carcinoma (4), hepatoma (5), oral carcinoma (6), glio-blastoma (7), prostate carcinoma (8), and gastric carcinoma (9).Animal studies have shown that berberine can suppress chemical-induced carcinogenesis (10), tumor promotion (11), and tumorinvasion (12). It is a radiosensitzer of tumor cells but not of normalcells (13). How berberine mediates these effects is not fullyunderstood, but its ability to modulate Mcl-1 (6), Bcl-xL (5),cyclooxygenase (COX)-2 (6), MDR (14), tumor necrosis factor(TNF)-a and IL-6 (15), iNOS (16), IL-12 (17), intercellular adhesionmolecule-1 and ELAM-1 expression (12), MCP-1 and CINC-1 (18),cyclin D1 (19), activator protein (AP-1; ref. 20), HIF-1a (21), PPAR-g(22), and topoisomerase II (23) has been shown. By using yeastmutants, berberine was found to bind and inhibit stress-inducedmitogen-activated protein kinase kinase activation (17). Becauseapoptotic, carcinogenic, and inflammatory effects and variousgene products (such as TNF-a, IL-6, COX-2, adhesion molecules,cyclin D1, and MDR) modulated by berberine are regulated bythe transcription factor nuclear factor-nB (NF-nB), we postulatedthat this pathway plays a major role in the action of berberine.NF-nB represents a group of five proteins, c-Rel, RelA (p65), RelB,

NF-nB1 (p50 and p105), and NF-nB2 (p52; ref. 24). In an inactivestate, NF-nB is sequestered in the cytoplasm as a heterotrimerconsisting of p50, p65, and InB subunits. On activation, InBaundergoes phosphorylation and ubiquitination-dependent degra-dation leading to p65 nuclear translocation and binding to aspecific consensus sequence in the DNA, which results in genetranscription. Most carcinogens, inflammatory agents, and tumorpromoters, including cigarette smoke, phorbol ester, okadaic acid(OA), H2O2, and TNF-a, have been shown to activate NF-nB. NF-nBhas been shown to regulate the expression of several genes whoseproducts are involved in tumorigenesis. These include antiapop-totic genes (e.g., Bcl-xL, cIAP, survivin, and cFLIP), COX-2, matrixmetalloproteinase 9 (MMP-9), genes encoding adhesion molecules,chemokines, and inflammatory cytokines and cell cycle regulatorygenes (e.g., cyclin D1 and c-myc; ref. 25).

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org/).

Dr. Aggarwal is a Ransom Horne, Jr., Professor of Cancer Research.Current address for M.M. Chaturvedi: The Department of Zoology, University of

Delhi, Delhi 110007, India.Requests for reprints: Bharat B. Aggarwal, Cytokine Research Laboratory,

Department of Experimental Therapeutics, The University of Texas M. D. AndersonCancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-1817;Fax: 713-745-6339; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-0511

Cancer Res 2008; 68: (13). July 1, 2008 5370 www.aacrjournals.org

Research Article



Whether berberine exerts its apoptotic, chemopreventive, anti-inflammatory, and anti-invasive effects through suppression of theNF-nB pathway was investigated in detail. We found that berberineinhibited the activation of NF-nB through the direct inhibition ofInBa kinase, and subsequently of InBa phosphorylation anddegradation, p65 nuclear translocation, and DNA binding. Thesuppression of NF-nB by this agent led to the down-regulation ofgene products that prevent apoptosis (Bcl-xL, survivin, IAP1, IAP2,and cFLIP) and promote inflammation (COX-2), proliferation(cyclin D1), and tumor metastasis (MMP-9).

Materials and Methods

Reagents. Berberine, with chemical structure as shown in Fig. 1A , wasobtained from Sigma-Aldrich. A 50 mmol/L solution of berberine was

prepared in DMSO, stored as small aliquots at �20jC, and then diluted as

needed in cell culture medium. Bacteria-derived human recombinanthuman TNF-a, purified to homogeneity with a specific activity of 5 �107 U/mg, was kindly provided by Genentech. Cigarette smoke condensate

(CSC) was kindly supplied by Dr. C. Gary Gairola (University of Kentucky,

Lexington, KY). Penicillin, streptomycin, RPMI 1640, and fetal bovine serumwere purchased from Invitrogen. Phorbol myristate acetate (PMA), OA, and

anti–h-actin antibody were obtained from Aldrich-Sigma. Antibodies

against p65, p50, InBa, cyclin D1, MMP-9, IAP1, IAP2, Bcl-xL, FLIP, poly

(ADP-ribose) polymerase (PARP), c-Jun-NH2-kinase (JNK)1, and COX-2 andAnnexin V staining kit were purchased from Santa Cruz Biotechnology.

Phospho-specific anti-InBa (Ser32/36) and phospho-specific anti-p65 (Ser536)

were purchased from Cell Signaling. Anti–InB kinase (IKK)-a and anti–IKK-

h antibodies were kindly provided by Imgenex. IKK plasmids, wild-type, andthat with mutated cysteine residue 179 (Cys179A) were kindly provided by

Dr. T.D. Gilmore from Boston University (Boston, MA).

Cell lines. The cell lines H1299 (lung adenocarcinoma), Jurkat (humanleukemia), HEK293 (human embryonic kidney), and U266 (human multiple

myeloma) were obtained from American Type Culture Collection. Normal

human foreskin diploid fibroblast cells were supplied by Dr. Olivia Smith of

the University of Texas, San Antonio, Texas. The H1299, Jurkat, and U266cells were cultured in RPMI 1640, and the HEK293 cells were cultured in

DMEM supplemented with 10% FBS. All culture medium were also

supplemented with 100 U/mL penicillin and 100 Ag/mL streptomycin.

Electrophoretic mobility shift assay. To determine NF-nB activation,we prepared nuclear extracts and performed electrophoretic mobility shift

assays (EMSA) as described previously (26). For supershift assays, nuclear

extracts prepared from TNF-a–treated cells were incubated with antibodiesagainst either p50 or p65 of NF-nB for 30 min at 37jC before the complex

was analyzed by EMSA. PIS was included as the negative control. The dried

gels were visualized, and the radioactive bands were quantitated with a

Storm 820 and Image quant software (Amersham).AP-1 activation assay. To assay AP-1 activation by EMSA, 10 Ag of

nuclear extract protein were incubated with 16 fmol of the 32P-end–labeled

AP-1 consensus oligonucleotide 5¶-CGCTTGATGACTCAGCCGGAA-3¶ (boldindicates the AP-1 binding site) for 30 min at 37jC, and then the DNA-protein complexes formed were resolved from free oligonucleotide on 6%

native polyacrylamide gels. The radioactive bands were visualized and

quantified as indicated above.Western blot analysis. To determine the effect of berberine on TNF-a–

dependent InBa phosphorylation, InBa degradation, p65 translocation, and

p65 phosphorylation, cytoplasmic or nuclear extracts were prepared. For

detection of cleavage products of PARP, antiapoptotic and angiogenesismarkers whole-cell extracts were prepared by subjecting berberine-treated

cells to lysis in lysis buffer [20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl,

2 mmol/L EDTA (pH 8.0), 0.1% Triton X-100, 0.01 Ag/mL aprotinin,

0.005 Ag/mL leupeptin, 0.4 mol/L phenylmethyl-sulfonyl fluoride, and4 mmol/L NaVO4]. Lysates were spun at 14,000 rpm for 10 min to remove

insoluble material. Supernatant were collected and kept at �80jC. Eithercytosolic or nuclear extract or whole-cell lysates were resolved by SDS-

PAGE. After electrophoresis, the proteins were electro-transferred to

nitrocellulose membranes, blotted with the relevant antibody, and detectedby enhanced chemiluminescence reagent (Amersham).

IKK assay. To determine the effect of berberine on TNF-a–induced IKK

activation, an IKK assay was performed. Briefly, the IKK complex from

whole-cell extracts was precipitated with antibody against IKK-h and thentreated with protein A/G-agarose beads (Pierce). After 2 h, the beads were

washed with whole-cell extract buffer and then resuspended in a kinase

assay mixture containing 50 mmol/L HEPES (pH 7.4), 20 mmol/L MgCl2,

2 mmol/L DTT, 20 ACi [32P] ATP, 10 Amol/L unlabeled ATP, and 2 Ag ofsubstrate glutathione S-transferase-InBa [amino acid (aa) 1–54]. After

Figure 1. A, the chemical structure of berberine. B, berberine blocks NF-nBactivation induced by TNF-a, PMA, OA, and CSC. Human leukemic cells (Jurkat)were preincubated with 50 Amol/L berberine for 18 h and then treated with0.1 nmol/L TNF-a for 30 min, 25 ng/mL PMA for 2 h, 500 nmol/L OA for 4 h,and 40 Ag/mL CSC for 1 h each. Nuclear extracts were analyzed for NF-nBactivation. C, dose-dependent effect of berberine on NF-nB activation inducedby TNF-a. Jurkat cells were incubated with the indicated concentrations ofberberine for 18 h and treated with 0.1 nmol/L TNF-a for 30 min. The nuclearextracts were assayed for NF-nB activation by EMSA. D, Jurkat cells werepreincubated with 50 Amol/L berberine for the indicated times and then treatedwith 0.1 nmol/L TNF-a for 30 min. The nuclear extracts were prepared andassayed for NF-nB activation by EMSA.

Berberine Inhibits NF-kB Activation

www.aacrjournals.org 5371 Cancer Res 2008; 68: (13). July 1, 2008



incubation at 30jC for 30 min, the reaction was terminated by boiling with

SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-

PAGE, the gel was dried, and the radioactive bands were visualized with aStorm 820. To determine the total amounts of IKK-a and IKK-h in each

sample, 30 Ag of whole-cell proteins was resolved on 10% SDS-PAGE,

electrotransferred to a nitrocellulose membrane, and then blotted witheither anti–IKK-a or anti–IKK-h antibody.

JNK assay. To determine the effect of berberine on the kinase activity of

JNK, JNK complex from whole-cell extracts was precipitated with antibody

against JNK1, followed by treatment with protein A/G-agarose beads(Pierce). After 2 h of incubation, the beads were washed with lysis buffer

and then assayed in kinase assay mixture containing 50 mmol/L HEPES

(pH 7.4), 20 mmol/L MgCl2, 2 mmol/L DTT, 20 ACi of [32P] ATP, 10 Amol/L

unlabeled ATP, and 2 Ag of substrate glutathione S-transferase (GST)-c-Jun(aa 1–79). The immunocomplex was incubated at 30jC for 30 min and then

boiled with SDS sample buffer for 5 min. Finally, the protein was resolved on

10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized

using the PhosphorImager. To determine the total amount of JNK1 in eachsample, whole-cell extracts were subjected to Western blot analysis using

anti-JNK1 antibody.

NF-KB–dependent reporter gene expression assay. NF-nB–dependentreporter gene expression was performed. The effect of berberine on TNF-a,TNF receptor (TNFR), TNFR-associated death domain (TRADD-), TNFR-

associated factor 2 (TRAF2-), NF-nB–inducing kinase (NIK-), and IKK-

NF-nB–dependent reporter gene transcription was analyzed by thesecretory alkaline phosphatase (SEAP) assay.

Immunocytochemistry for NF-KB p65 localization. Immunocyto-

chemistry was used to examine the effect of berberine on the nuclear

translocation of p65. Briefly, treated cells were plated on a poly-L-lysine–

coated glass slide by centrifugation (Cytospin 4; Thermoshendon), air dried,

and fixed with 4% paraformaldehyde. After being washed in PBS, the slideswere blocked with 5% normal goat serum for 1 h and then incubated with

rabbit polyclonal anti-human p65 at a 1/200 dilution. After overnight

incubation at 4jC, the slides were washed, incubated with goat anti-rabbitIgG-Alexa Fluor 594 (Molecular Probes) at a 1/200 dilution for 1 h, and

counterstained for nuclei with Hoechst 33342 (50 ng/mL) for 5 min. Stained

slides were mounted with mounting medium purchased from Sigma-

Aldrich and analyzed under a fluorescence microscope (Labophot-2; Nikon).Pictures were captured using a Photometrics Coolsnap CF color camera

(Nikon) and MetaMorph version 4.6.5 software (Universal Imaging).

Live/dead assay. To measure apoptosis, we also used the Live/Dead

assay (Molecular Probes), which determines intracellular esterase activityand plasma membrane integrity. Calcein-AM, a nonfluorescent polyanionic

dye, is retained by live cells, in which it produces intense green fluorescence

through enzymatic (esterase) conversion. In addition, the ethidium

homodimer enters cells with damaged membranes and binds to nucleicacids, thereby producing a bright red fluorescence in dead cells. Briefly,

2 � 105 cells were incubated with 25 Amol/L berberine and treated with

1 nmol/L TNF-a for up to 24 h at 37jC. Cells were stained with the Live/Dead reagent (5 Amol/L ethidium homodimer and 5 Amol/L calcein-AM)

and incubated at 37jC for 30 min. Cells were analyzed under a fluorescence

microscope (Labophot-2; Nikon).

Cytotoxicity assay. The effects of berberine on the cytotoxic effects ofTNF-a and other chemotherapeutic agents were determined by the 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake

method. Briefly, 5, 000 cells were incubated with berberine in triplicate in

Figure 2. A, effect of berberine on constitutive NF-nBin human multiple myeloma U266 cells. Cells wereincubated with the indicated concentrations ofberberine for 18 h. Nuclear extracts were preparedand analyzed for NF-nB activation by EMSA. B,NF-nB induced by TNF-a is composed of p65 andp50 subunits. Nuclear extracts from untreated cellsor cells treated with 0.1 nmol/L TNF-a were incubatedwith the indicated antibodies, an unlabeled NF-nBoligo-probe, PIS, or a mutant oligo-probe. They werethen assayed for NF-nB activation by EMSA. C,berberine inhibits TNF-a–induced activation of NF-nB.Jurkat cells were incubated with 50 Amol/L berberinefor 18 h, treated with 0.1 nmol/L TNF-a for the indicatedtimes, prepared nuclear extract, and then analyzedfor NF-nB activation by EMSA. D, effect of berberineon TNF-a–induced degradation of InBa. Cytoplasmicextracts were prepared and analyzed by Westernblotting using antibody against anti-InBa. Equal proteinloading was evaluated by h-actin.

Cancer Research




a 96-well plate and then treated with the 1 nmol/L TNF-a, 0.1 Amol/L 5-5-fluorouracil, and 0.1 Amol/L doxorubicin for 24 h at 37jC. An MTT solution

was added to each well and incubated for 2 h at 37jC. An extraction buffer

(20% SDS and 50% dimethylformamide) was added, and the cells were incu-

bated overnight at 37jC. Then, the absorbance was measured at 570 nmusing a 96-well multiscanner (Dynex Technologies; MRX Revelation).

Annexin V assay. An early indicator of apoptosis is the rapid trans-

location and accumulation of the membrane phospholipid phosphatidyl-

serine from the cytoplasmic interface of membrane to the extracellularsurface. This loss of membrane asymmetry can be detected by using

the binding properties of Annexin V. To identify apoptosis, we used an

Annexin V antibody, which was conjugated with a FITC fluorescence dye.

Briefly, 1 � 106 cells were pretreated with berberine, treated with TNF-afor 24 h at 37jC, and subjected to Annexin V staining. The cells were

washed in PBS, resuspended in 100 AL of binding buffer containing a FITC-

conjugated anti-Annexin V antibody, and then analyzed with a flowcytometer (FACS Calibur).

Invasion assay. Because invasion is a crucial step in tumor metastasis, a

membrane invasion culture system was used to assess cell invasion. The BD

BioCoat tumor invasion system consists of chambers with a lightproofpolyethylene terephthalate membrane coated with a reconstituted base-

ment membrane gel with 8-Am-diameter pores (BD Biosciences). We

suspended 2.5 � 104 non–small cell adenocarcinoma H1299 cells in serum-

free medium and seeded the upper wells with them. After incubationovernight, the cells were treated with 25 Amol/L berberine and stimulated

with 1 nmol/L TNF-a for 24 h in the presence of 1% FBS. The cells that

invaded the lower chamber by migrating through the Matrigel duringincubation were stained with 4 Ag/mL calcein-AM in PBS for 30 min at

37jC and scanned for fluorescence with a Victor 3 multiplate reader

(Perkin-Elmer); fluorescent cells were counted.

Statistical analysis. The statistical analysis was done using ANOVA testand Student’s t test with Microsoft excel software.

Results

We investigated the effect of berberine on constitutively activeNF-nB and on that activated by various carcinogens andinflammatory stimuli, on NF-nB–regulated gene expression, onapoptosis induced by cytokines and chemotherapeutic agents, andon invasion. We focused on TNF-a–induced NF-nB activationbecause the role of TNF-a role in the NF-nB activation pathway hasbeen relatively well-established.Berberine inhibits NF-KB activation induced by carcinogens

and inflammatory stimuli. TNF-a, PMA, OA, and CSC are wellknown potent activators of NF-nB but by different mechanisms(26). We examined the effect of berberine on the activation ofNF-nB by these agents using DNA binding assays. TNF-a, PMA, OA,and CSC induced NF-nB, and berberine suppressed this activationin Jurkat cells (Fig. 1B). These results suggest that berberine acts ata step in the NF-nB activation pathway that is common to all fouragents.We then determined the dose and time of exposure to berberine

required to suppress TNF-a induced NF-nB activation in Jurkatcells. EMSA showed that berberine alone had no effect on NF-nBactivation, but it inhibited TNF-a–mediated NF-nB activation in adose- (Fig. 1C) and time-dependent (Fig. 1D) manner, respectively,and that 18 h exposure to 50 Amol/L berberine was sufficient tosuppress activation.Most tumor cells have constitutively active NF-nB that arises

through mechanisms that vary with the cell type. Whetherberberine can inhibit constitutively active NF-nB in multiplemyeloma U266 cells was determined. Berberine at a concentration40 Amol/L and higher completely suppressed constitutively activeNF-nB in U266 cells (Fig. 2A).

NF-nB is a protein complex in which various combinations ofRel/NF-nB constitute active NF-nB heterodimers that bind tospecific DNA sequences. To show that the band visualized by EMSAin TNF-a–treated cells was indeed NF-nB, nuclear extracts fromTNF-a–activated Jurkat cells were incubated with antibodies tothe p50 and the p65 (RelA) subunit of NF-nB. The preincubation ofnuclear extracts with anti-p65 and mixture of anti-p65 and anti-p50 antibodies shifted the band to a higher molecular complex(Fig. 2B), suggesting that the TNF-a–activated complex consistedof p50 and p65. The band lower to NF-nB is a nonspecific band.Preimmune serum (PIS) did not cause any super shift. Addition ofexcess unlabeled oligonucleotide (competitor; 100-fold molarexcess) caused a complete disappearance of the band, whereasmutated oligonucleotide had no effect on DNA binding.Some NF-nB inhibitors, such as caffeic acid phenethyl ester and

plumbagin (27, 28), directly suppress binding of NF-nB to DNA. Wedetermined whether berberine mediates suppression of NF-nBactivation through a similar mechanism. Berberine did not modifythe DNA-binding ability of NF-nB proteins (Supplementary Fig. S1).These results suggest that berberine inhibits NF-nB activation at astep upstream to its DNA binding.Whether berberine suppresses other transcription factor such as

AP-1 under the conditions it suppresses NF-nB was examined. AP-1showed constitutive activation in Jurkat cells, and berberine had noeffect on it (Supplementary Fig. S1).Berberine inhibits TNF-A–dependent IKBA degradation and

phosphorylation. The translocation of NF-nB to the nucleus ispreceded by the phosphorylation, ubiquitination, and proteolyticdegradation of InBa. To determine whether inhibition of TNF-ainduced NF-nB activation was due to inhibition of InBa degrada-tion, we pretreated Jurkat cells with berberine and then exposedthem to TNF-a for various time periods. We then examined the cellsfor NF-nB in the nucleus by EMSA and for InBa degradation in thecytoplasm by Western blot analysis. As shown in Fig. 2C , TNF-aactivated NF-nB in the control cells. TNF-a induced NF-nB as earlyas 30 minutes and that continued to increase at 60 minutes but hadno effect on berberine-pretreated cells. Moreover, TNF-a inducedInBa degradation as early as 10 minutes, but in berberine,pretreated cells showed TNF-a had no effect on InBa degradation(Fig. 2D). These results indicate that berberine inhibited bothTNF-a induced InBa degradation and NF-nB activation.To determine whether the inhibition of TNF-a–induced InBa

degradation was due to an inhibition of InBa phosphorylation, weused the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal(ALLN) to block degradation of InBa. Western blot using anantibody that recognizes the serine-phosphorylated (Ser32/36) formof InBa showed that TNF-a induced InBa phosphorylation wasstrongly suppressed by berberine (Fig. 3A).Berberine directly inhibits TNF-A–induced IKBA kinase

activation. Because berberine inhibits the phosphorylation anddegradation of InBa, we tested the effect of berberine on TNF-a–induced IKK activation, which is required for TNF-a–inducedphosphorylation of InBa. As shown in Fig. 3B , TNF-a induced theactivation of IKK and berberine completely suppressed it (top).Neither TNF-a nor berberine had any effect on the expression ofIKK-a (middle) or IKK-h proteins (bottom).To evaluate whether berberine suppresses IKK activity directly

by binding to IKK or indirectly by suppressing its activation, weimmunoprecipitated IKK complex from whole-cell extracts fromTNF-a–stimulated cells with anti–IKK-h antibody. The immuno-complexes were treated in vitro with various concentrations of





berberine, and kinase assay for IKK was performed. Results fromthe immune complex kinase assay showed that berberine directlyinhibited the activity of IKK (Fig. 3C, left). This finding suggestedthat berberine directly modulates TNF-a induced IKK activation.IKK-h contains various cysteine residues. The modulation of IKK

activity by berberine through the modification of cysteine residueswas investigated using reducing agents, such as DTT. We foundthat addition of DTT to the kinase reactions reversed theberberine-mediated inhibition of IKK activity induced by TNF-a(Fig. 3C, right).

IKK-h contains a cysteine at position 179 in its activation loopthat is critical for its biological activity. To determine whether thiscysteine is involved in berberine-mediated inhibition, we trans-fected cells with plasmids for either wild-type Flag-IKK-h or Flag-IKK-h with a C179A mutation. Berberine inhibited constitutivewild-type IKK-h activity (Fig. 3D). In contrast, berberine had noeffect on IKK-h (C179A) activity (Fig. 3D). These findings provedthat berberine inhibited IKK-h activity through the modification ofthe Cys179 residue.We also examined whether berberine affects other protein

kinases. The results show that TNF activates JNK, and berberineslightly inhibited the TNF-a–induced activation of JNK at latertimes (Supplementary Fig. S2).Berberine inhibits TNF-A–induced phosphorylation and

nuclear translocation of p65. We also investigated the effect ofberberine on TNF-a–induced phosphorylation of p65 becausephosphorylation is also required for its transcriptional activity (24).In the nuclear fraction from the TNF-a–treated cells, berberinesuppressed the phosphorylated form of p65 (Ser536; Fig. 4A, top).We further showed that berberine suppressed TNF-a–inducednuclear translocation of p65, as measured by Western blotting(Fig. 4A, middle).An immunocytochemistry assay confirmed that berberine

suppressed TNF-a–induced translocation of p65 to the nucleus(Fig. 4B).Berberine represses NF-KB–dependent reporter gene

expression. Because DNA binding alone does not always correlatewith NF-nB–dependent gene transcription, suggesting there areadditional regulatory steps (29). We transiently transfected the cellswith NF-nB–regulated SEAP reporter construct and pretreated withberberine or left untreated and then stimulated the cells withTNF-a. A 3-fold increase in SEAP activity was noted afterstimulation with TNF-a (Fig. 5A), and that was abolished bydominant-negative InBa, indicating the specificity. When the cellswere pretreated with berberine, TNF-a–induced NF-nB–dependentSEAP expression was inhibited in a dose-dependent manner(Fig. 5A). These results indicate that berberine, inhibits NF-nB–dependent reporter gene expression induced by TNF-a.TNF-a–induced NF-nB activation is mediated through sequen-

tial interaction of the TNFR with TRADD, TRAF2, NIK, and IKK-h,resulting in phosphorylation of InBa, which leads to degradation ofInBa and p65 nuclear translocation (30). To delineate the site ofaction of berberine in the TNF-a–signaling pathway leading toNF-nB activation, cells were transiently transfected with TNFR1,TRADD, TRAF2, NIK, and IKK-h, and then NF-nB–dependent SEAPexpression was monitored with or without berberine treatment. Asshown in Fig. 5B , berberine suppressed TNFR1, TRADD, TRAF2,NIK, and IKK plasmid–induced reporter gene expression. BecauseIKK activation can cause the phosphorylation of InBa and p65 (31),we suggest that berberine inhibits NF-nB activation throughinhibition of IKK.Berberine represses the expression of TNF-A–induced NF-

KB–dependent antiapoptotic, proliferation, and metastaticgene products. Because NF-nB regulates the expression of theantiapoptotic proteins IAP1/2, Bcl-xL, survivin, and cFLIP, weinvestigated whether berberine could modulate TNF-a–inducedexpression of these antiapoptotic genes. We found that berberineabolished TNF-a–induced expression of Bcl-xL, survivin, IAP1/2,and cFLIP antiapoptotic proteins (Fig. 5C, top).We also investigated whether berberine can modulate NF-nB–

regulated gene products involved in the proliferation of tumor cells.

Figure 3. A, berberine inhibits TNF-a–induced phosphorylation of InBa. Cellswere preincubated with 50 Amol/L berberine for 18 h, incubated with 50 Ag/mLof ALLN for 30 min, and then treated with 0.1 nmol/L TNF-a for 10 min.Cytoplasmic extracts were fractionated and then subjected to Western blottingusing phospho-specific InBa antibody. The same membrane was reblottedwith h-actin. B, effect of berberine on the TNF-a–induced activation of IKK.Jurkat cells were preincubated with 50 Amol/L berberine for 18 h and thentreated with 1 nmol/L TNF-a for the indicated times. Whole-cell extracts wereimmunoprecipitated with antibody against IKK-h and analyzed by an immunecomplex kinase assay. To examine the effect of berberine on the level ofexpression of IKK proteins, whole-cell extracts were fractionated on SDS-PAGEand examined by Western blot analysis using anti–IKK-a and anti–IKK-hantibodies. C, effect of berberine on IKK activity (left). Whole-cell extracts wereprepared from Jurkat cells treated with 1 nmol/L TNF-a and immunoprecipitatedwith anti–IKK-h antibody. The immunocomplex kinase assay was performedin the absence or presence of the indicated concentration of berberine. Right,effect of reducing agent on the inhibition of IKK by berberine. IKK activity wasthen assayed in kinase buffer without and with 100 Amol/L DTT along withabsence or presence of berberine (50 Amol/L). D, effect of berberine onwild-type and C179A-mutated IKK. HEK293cells were transfected withflag-IKK-h or flag-IKK-h (C179A) plasmids. Whole cell extracts were preparedand the indicated concentration of berberine was added in vitro . Immunecomplex was analyzed by SDS-PAGE and autoradiography. IB, immunoblot.

Cancer Research




TNF-a has been shown to induce cyclin D1 and COX-2, both ofwhich have NF-nB binding sites in their promoters. Thus, weinvestigated whether berberine inhibits the TNF-a–inducedexpression of these proteins by Western blot analysis using specificantibodies. We found that berberine abolished TNF-a–inducedexpression of COX-2 and cyclin D1 (Fig. 5C, middle).We also investigated whether berberine modulates TNF-a

induced NF-nB–dependent MMP-9 expression, a gene involved inthe invasion of tumor cells. It has been established already thatMMP-9 is regulated by NF-nB. We found that berberine abolishedTNF-a–induced expression of MMP-9 (Fig. 5C, bottom).Berberine potentiates apoptosis induced by TNF-A and

chemotherapeutic agents. Because the activation of NF-nB hasbeen shown to inhibit apoptosis induced by TNF-a andchemotherapeutic agents (32), we investigated whether berberineaffects TNF-a and chemotherapeutic agent-induced apoptosis. Theesterase-staining method (also called Live/Dead assay) showed thatberberine up-regulated TNF-a–induced apoptosis from 5% to 65%(Fig. 6A). MTT assay showed that berberine enhanced cytotoxicityinduced by TNF-a, 5-fluorouracil (5-FU), and doxorubicin (Fig. 6B).Caspase-mediated PARP cleavage likewise showed that berberineenhanced the apoptotic effect of TNF-a substantially (Fig. 6C, left).Annexin V-FITC (Fig. 6C, middle) and terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling (TUNEL; Fig. 6C,right) staining also confirmed that berberine up-regulated TNF-ainduced early events in apoptosis. These results together indicatethat berberine potentiates the apoptotic effects of TNF-a andchemotherapeutic agents.Whether berberine has any effect on normal cells was examined

using normal human diploid fibroblasts. MTT assay showed that

under conditions as indicated above, berberine had no significanteffect on the viability of these cells (data not shown).Berberine suppresses TNF-A–induced invasion activity. The

expression of both COX-2 and MMP-9 has been linked with tumorcell invasion (33). Therefore, we investigated whether berberinecan modulate the tumor cell invasion activity induced by TNF-ain vitro in a Matrigel invasion assay. We found that berberinesuppressed TNF-a–induced tumor cell invasion (Fig. 6D).

Discussion

The goal of this study was to determine whether the chemo-preventive, anti-inflammatory, and apoptotic effects of berberinewere mediated through the modulation of the NF-nB–signalingpathway and NF-nB–regulated gene products. We found thatberberine suppressed NF-nB activation induced by variouscarcinogens and inflammatory agents. NF-nB inhibition was dueto inhibition of IKK activation, leading to suppression of InBaphosphorylation, InBa degradation, p65 phosphorylation, andNF-nB–dependent reporter gene expression. Berberine also down-regulated gene products involved in cell proliferation, antiapop-tosis, and invasion. This down-regulation led to the potentiationof apoptosis and inhibition of invasion.This is the first report to suggest that berberine can inhibit the

NF-nB activation induced by TNF-a, PMA, OA, and CSC. In supportof our results, this alkaloid has been shown to inhibit acetaldehyde-induced NF-nB activation in human hepatoma cells (34). Unlikeour results, however, Enk and colleagues (35) recently reported thatberberine has no effect on TNF-a–induced NF-nB activation inhuman keratinocytes. The differences in cell types, methodology

Figure 4. A, berberine inhibitsTNF-a–induced phosphorylation andnuclear translocation of p65. Jurkat cellswere either untreated or pretreated with50 Amol/L berberine for 18 h and thentreated with 0.1 nmol/L TNF-a for theindicated times. Nuclear extracts wereprepared and analyzed by Western blottingusing antibodies against phospho-specificp65 (Ser536) and p65. For loading controlof nuclear protein, the membrane wasreprobed with anti-PARP antibody. B,immunocytochemical analysis of p65localization. Jurkat cells were firsttreated with 50 Amol/L berberine for18 h and then stimulated with 1 nmol/LTNF-a for 15 min. After cytospin,immunocytochemical analysis was done.





used to measure NF-nB, and conditions used may account for thedifference in results.How berberine suppresses NF-nB activation was investigated in

detail. We found that berberine suppresses the phosphorylationand degradation of InBa, consistent with that reported earlier withacetaldehyde-induced activation (34). We further found that thisalkaloid inhibits InBa degradation through inhibition of activationof IKK induced by TNF-a. We found that berberine inhibits IKKactivity directly by modification of cysteine residue 179. Thisresidue is present in the activation loop of IKK-h and is a target fora variety of compounds such as arsenic and auranofin (36).Interactions of these compounds with IKK involving cysteineresidue 179 is easily reversed by addition of thiol-reducing agents(e.g., DTT and reduced glutathione; ref. 37). The inhibitory effects ofN-ethylmaleimid on IKK activity were abolished by the expressionof mutant IKK-h, which contains alanine at residue 179 rather thancysteine (38, 39). Like berberine, we showed recently that buteinalso inhibits the NF-nB activation by direct modification of cysteineresidue 179 in IKK-h (26). Different cysteine residues have beenidentified in IKK that interfere with kinase activity. For instance,

herbimycin A binds specifically to cysteine residue 59 (40);Withaferin A interacts with cysteine residues (41); and aspirininterferes with ATP binding site (42). Thus, the mechanism bywhich berberine and other agents inhibit IKK may involve cysteineresidue but not always the same cysteine residue.The concentration of berberine used in our study is comparable

with several commercially available inhibitors of IKK. For example,IKK inhibitor PS-1145 acts in a range of 10 to 50 Amol/L, which iscomparable with the dose of berberine used in our study (43).Another compound, BMS-345541, was found to bind to anallosteric site of IKK-h and acts as an ATP noncompetitiveinhibitor with IC50 of 0.3 Amol/L (44). Although these compoundsdo not possess positively charged imine moiety as found inberberine, they are alkaloid in nature similar to berberine.TNF-a activates NF-nB through sequential recruitment of

TNFR1, TNFR2, NIK, and IKK, and berberine suppressed NF-nBactivation by all of these signaling intermediates. IKK has beenimplicated in the phosphorylation of p65 (31), which is needed forits transcriptional activity. We found that berberine suppressed thephosphorylation of p65. Furthermore, berberine had no direct

Figure 5. A, berberine inhibited TNF-a–induced NF-nB–dependent reporter gene(SEAP) expression. HEK293 cells treatedwith the indicated concentrations ofberberine were transiently transfectedwith a NF-nB–containing plasmid linkedto the SEAP gene. After 24 h in culture with1 nmol/L TNF-a, cell supernatants werecollected and assayed for SEAP activity.Results are expressed as fold activityover the activity of the vector control (con ).**, P < 0.0001; *, P < 0.001, significantwith respect to control. B, berberineinhibited NF-nB–dependent reporter geneexpression induced by TNF-a, TNFR-1,TRADD, TRAF2, NIK, and IKK. HEK293cells were pretreated with 25 Amol/Lberberine and transiently transfectedwith the indicated plasmids along with aNF-nB–containing plasmid linked to theSEAP gene, and where indicated cellswere exposed to 1 nmol/L TNF-a for 24 h.**, P < 0.0001, significant with respectto control. C, berberine inhibits theexpression of TNF-a–induced antiapoptoticproteins (top ). Jurkat cells were incubatedwith 25 Amol/L berberine for 18 h andthen treated with 1 nmol/L TNF-a for theindicated times. Whole-cell extracts wereprepared and analyzed by Western blottingusing the indicated antibodies. Middle andbottom, berberine inhibits TNF-a–inducedCOX-2, cyclin D1, and MMP-9 expression.Jurkat cells were incubated with 25 Amol/Lberberine for 18 h and then treated with1 nmol/L TNF-a for the indicated times.Whole-cell extracts were prepared andanalyzed by Western blotting using therelevant antibodies. DN, dominantnegative.

Cancer Research




effect on the binding of p50-p65 to the DNA. Therefore, it ispossible that inhibition of IKK-mediated InBa degradation and p65phosphorylation contribute to the suppressive effects of berberineon NF-nB activation.

We found that numerous gene products that are regulated byNF-nB were down-regulated by berberine. These included proteinswith proinflammatory (COX-2), cell proliferative (cyclin D1),antiapoptotic (Bcl-xL, survivin, IAP1, IAP2, and cFLIP), and invasive

Figure 6. A, berberine enhances TNF-a–induced cytotoxicity. Jurkat cells were pretreated with 25 Amol/L berberine and then incubated with 1 nmol/L TNF-a for 24 h.The cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. The results shown are representative ofthree independent experiments. B, berberine enhances TNF-a, 5-FU, and doxorubicin-induced cytotoxicity. Five thousand cells were seeded in triplicate in 96-wellplates. The cells were pretreated with 25 Amol/L berberine and then incubated with chemotherapeutic agents. Cell viability was then analyzed by the MTT method.The results shown are representative of three independent experiments. C, effect of berberine on PARP cleavage (left). Cells were pretreated with 25 Amol/L berberinefor 18 h and then incubated with 1 nmol/L TNF-a for the indicated times. Whole-cell extracts were prepared, and analyzed by Western blotting using an anti-PARPantibody. Middle and right, cells were pretreated with 25 Amol/L berberine and then incubated with 1 nmol/L TNF-a for 24 h. The cells were incubated with eitherFITC-conjugated Annexin V antibody or TUNEL reagents and then analyzed by flow cytometry. The results shown are representative of three independentexperiments. D, berberine suppresses TNF-a induced invasion activity. H1299 cells (2.5 � 104 cells) were seeded to the top chamber of a Matrigel invasionchamber overnight in the absence of serum and then treated with 25 Amol/L berberine. After incubation, the cells were treated with TNF-a in the presence of1% serum and then assayed for invasion. The results shown are representative of three independent experiments.





(MMP-9) activities. The down-regulation of COX-2 by berberine isin agreement with previous report (20). The decrease in TNF-a andiNOS (45); urokinase-plasminogen activator and plasminogenactivator inhibitor (12); Mcl-1 (6); MCP-1 and CINC-1 (18); andmdr1 (14) expression reported previously could also be due todown-regulation of NF-nB activation by berberine. Down-regula-tion of cyclin D1 reported here is also in agreement with previousresults (19). It is possible that suppression of proliferation ofvarious tumor cell lines by berberine is through down-regulation ofcyclin D1.Our results also show that berberine potentiated the apoptotic

effects of TNF-a and chemotherapeutic agents. This is likely linkedto the down-regulation of antiapoptotic (Bcl-xL, survivin, IAP1,IAP2, and cFLIP) gene products. The down-regulation of Bcl-xL isin agreement with previous reports (5). It is possible thatsensitization of cells to chemotherapeutic agents by berberine ismediated through the down-regulation of mdr1 as reportedpreviously (14). Consistent with our results, Lin and colleagues(14) reported that berberine potentiated the anti-tumor effects ofpaclitaxel on digestive tract cancer cells.In animal studies, berberine has been shown to suppress

chemical-induced carcinogenesis (10), phorbol-ester–induced tu-mor promotion (11), and tumor invasion (12). Various carcinogensand tumor promoters have been shown to activate NF-nB. Thus,suppression of NF-nB by berberine as shown here may contributeto its ability to suppress carcinogenesis. Tumor invasion requires

the expression of MMP-9, which was also suppressed by berberine.Whether doses of berberine used in the current studies areachievable in vivo is unclear. The doses used in vitro studies,however, are irrelevant to that in vivo as the exposure to the drugin vivo may occur over long-periods. Thus, overall, our resultssuggest that anticarcinogenic, anti-inflammatory, and proapoptoticeffects of berberine may be mediated through inhibition of NF-nBinduced by carcinogens and inflammatory agents.Limited clinical trials suggest that berberine is quite safe in

human (46). Further studies in animals and in patients are requiredto recognize the full potential of this important constituent ofancient medicine.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 2/14/2008; revised 3/27/2008; accepted 4/16/2008.Grant support: Clayton Foundation for Research (B.B. Aggarwal), and cancer

center support grant 5P30 CA016672-32 from NIH.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Dr. Aggarwal is a Ransom Horne, Jr., Professor of Cancer Research.We thank Walter Pagel for carefully proofreading the manuscript and providing

valuable comments.

Cancer Research


References

1. Sack RB, Froehlich JL. Berberine inhibits intestinalsecretory response of Vibrio cholerae and Escherichiacoli enterotoxins. Infect Immun 1982;35:471–5.2. Lin CC, Lin SY, Chung JG, et al. Down-regulation ofcyclin B1 and up-regulation of Wee1 by berberinepromotes entry of leukemia cells into the G2/M-phaseof the cell cycle. Anticancer Res 2006;26:1097–104.3. Letasiova S, Jantova S, Cipak L, et al. Berberine-antiproliferative activity in vitro and induction ofapoptosis/necrosis of the U937 and B16 cells. CancerLett 2006;239:254–62.4. Kettmann V, Kosfalova D, Jantova S, et al. In vitrocytotoxicity of berberine against HeLa and L1210 cancercell lines. Pharmazie 2004;59:548–51.5. Hwang JM, Kuo HC, Tseng TH, et al. Berberine inducesapoptosis through a mitochondria/caspases pathway inhuman hepatoma cells. Arch Toxicol 2006;80:62–73.6. Kuo CL, Chi CW, Liu TY. Modulation of apoptosis byberberine through inhibition of cyclooxygenase-2 andMcl-1 expression in oral cancer cells. In Vivo 2005;19:247–52.7. Sanders MM, Liu AA, Li TK, et al. Selective cytotoxicityof topoisomerase-directed protoberberines against glio-blastoma cells. Biochem Pharmacol 1998;56:1157–66.8. Mantena SK, Sharma SD, Katiyar SK. Berberine, anatural product, induces G1-phase cell cycle arrest andcaspase-3-dependent apoptosis in human prostatecarcinoma cells. Mol Cancer Ther 2006;5:296–308.9. Lin JP, Yang JS, Lee JH, et al. Berberine induces cellcycle arrest and apoptosis in human gastric carcinomaSNU-5 cell line. World J Gastroenterol 2006;12:21–8.10. Anis KV, Rajeshkumar NV, Kuttan R. Inhibition ofchemical carcinogenesis by berberine in rats and mice.J Pharm Pharmacol 2001;53:763–8.11. Nishino H, Kitagawa K, Fujiki H, et al. Berberinesulfate inhibits tumor-promoting activity of teleocidinin two-stage carcinogenesis on mouse skin. Oncology1986;43:131–4.12. Peng PL, Hsieh YS, Wang CJ, et al. Inhibitory effect ofberberine on the invasion of human lung cancer cells viadecreased productions of urokinase-plasminogen acti-

vator and matrix metalloproteinase-2. Toxicol ApplPharmacol 2006;214:8–15.13. Yount G, Qian Y, Moore D, et al. Berberine sensitizeshuman glioma cells, but not normal glial cells, toionizing radiation in vitro . J Exp Ther Oncol 2004;4:137–43.14. Lin HL, Liu TY, Wu CW, et al. Berberine modulatesexpression of mdr1 gene product and the responses ofdigestive track cancer cells to Paclitaxel. Br J Cancer1999;81:416–22.15. Choi BH, Ahn IS, Kim YH, et al. Berberine reduces theexpression of adipogenic enzymes and inflammatorymolecules of 3T3–1 adipocyte. Exp Mol Med 2006;38:599–605.16. Tan Y, Ming Z, Tang Q, et al. Effect of berberine onthe mRNA expression of nitric oxide synthase (NOS) inrat corpus cavernosum. J Huazhong Univ Sci TechnologMed Sci 2005;25:127–30.17. Kang BY, Chung SW, Cho D, et al. Involvement of p38mitogen-activated protein kinase in the induction ofinterleukin-12 p40 production in mouse macrophagesby berberine, a benzodioxoloquinolizine alkaloid. Bio-chem Pharmacol 2002;63:1901–10.

18. Cui HS, Hayasaka S, Zheng LS, et al. Effect ofberberine on monocyte chemotactic protein-1 andcytokine-induced neutrophil chemoattractant-1 expres-sion in rat lipopolysaccharide-induced uveitis. Ophthal-mic Res 2007;39:32–9.19. Mantena SK, Sharma SD, Katiyar SK. Berberineinhibits growth, induces G1 arrest and apoptosis inhuman epidermoid carcinoma A431 cells by regulatingCdki-Cdk-cyclin cascade, disruption of mitochondrialmembrane potential and cleavage of caspase 3 andPARP. Carcinogenesis 2006;27:2018–27.20. Kuo CL, Chi CW, Liu TY. The anti-inflammatorypotential of berberine in vitro and in vivo . Cancer Lett2004;203:127–37.21. Lin S, Tsai SC, Lee CC, et al. Berberine inhibits HIF-1a expression via enhanced proteolysis. Mol Pharmacol2004;66:612–9.22. Huang C, Zhang Y, Gong Z, et al. Berberine inhibits3T3–1 adipocyte differentiation through the PPARg path-way. Biochem Biophys Res Commun 2006;348:571–8.

23. Kang MR, Chung IK. Down-regulation of DNAtopoisomerase IIa in human colorectal carcinoma cellsresistant to a protoberberine alkaloid, berberrubine. MolPharmacol 2002;61:879–84.24. Ghosh S, May MJ, Kopp EB. NF-n B and Rel proteins:evolutionarily conserved mediators of immuneresponses. Annu Rev Immunol 1998;16:225–60.25. Aggarwal BB, Takada Y, Oommen OV. From chemo-prevention to chemotherapy: common targets and com-mon goals. Expert Opin Investig Drugs 2004;13:1327–38.26. Pandey MK, Sandur SK, Sung B, et al. Butein, atetrahydroxychalcone, inhibits nuclear factor (NF)-nBand NF-nB-regulated gene expression through directinhibition of InBa kinase h on cysteine 179 residue.J Biol Chem 2007;282:17340–50.

27. Natarajan K, Singh S, Burke TR, Jr., et al. Caffeic acidphenethyl ester is a potent and specific inhibitor ofactivation of nuclear transcription factor NF-nB. ProcNatl Acad Sci U S A 1996;93:9090–5.28. Sandur SK, Ichikawa H, Sethi G, et al. Plumbagin(5-hydroxy-2-methyl-1,4-naphthoquinone) suppressesNF-nB activation and NF-nB-regulated gene productsthrough modulation of p65 and InBa kinase activation,leading to potentiation of apoptosis induced by cytokineand chemotherapeutic agents. J Biol Chem 2006;281:17023–33.

29. Campbell KJ, Rocha S, Perkins ND. Active repressionof antiapoptotic gene expression by RelA(p65) NF-nB.Mol Cell 2004;13:853–65.30. Simeonidis S, Stauber D, Chen G, et al. Mechanismsby which InB proteins control NF-nB activity. Proc NatlAcad Sci U S A 1999;96:49–54.31. Sizemore N, Lerner N, Dombrowski N, et al. Distinctroles of the In B kinase a and h subunits in liberatingnuclear factor nB (NF-nB) from InB and in phosphorylatingthe p65 subunit of NF-nB. J Biol Chem 2002;277:3863–9.32. Wang CY, Mayo MW, Korneluk RG, et al. NF-nBantiapoptosis: induction of TRAF1 and TRAF2 andc-IAP1 and c-IAP2 to suppress caspase-8 activation.Science 1998;281:1680–3.33. Liotta LA, Thorgeirsson UP, Garbisa S. Role ofcollagenases in tumor cell invasion. Cancer MetastasisRev 1982;1:277–88.





34. Hsiang CY, Wu SL, Cheng SE, et al. Acetaldehyde-induced interleukin-1h and tumor necrosis factor-aproduction is inhibited by berberine through nuclearfactor-nB signaling pathway in HepG2 cells. J BiomedSci 2005;12:791–801.35. Enk R, Ehehalt R, Graham JE, et al. Differential effectof Rhizoma coptidis and its main alkaloid compoundberberine on TNF-a induced NFnB translocation inhuman keratinocytes. J Ethnopharmacol 2007;109:170–5.36. Kapahi P, Takahashi T, Natoli G, et al. Inhibition ofNF-nB activation by arsenite through reaction with acritical cysteine in the activation loop of In B kinase.J Biol Chem 2000;275:36062–6.37. Park MH, Song HS, Kim KH, et al. Cobrotoxin inhibitsNF-nB activation and target gene expression throughreaction with NF-nB signal molecules. Biochemistry2005;44:8326–36.

38. Rossi A, Kapahi P, Natoli G, et al. Anti-inflammatorycyclopentenone prostaglandins are direct inhibitors ofInB kinase. Nature 2000;403:103–8.39. Jeon KI, Byun MS, Jue DM. Gold compound auranofininhibits InB kinase (IKK) by modifying Cys-179 of IKKhsubunit. Exp Mol Med 2003;35:61–6.40. Ogino S, Tsuruma K, Uehara T, et al. Herbimycin Aabrogates nuclear factor-nB activation by interacting prefer-entially with the InB kinase h subunit. Mol Pharmacol 2004;65:1344–51.41. Kaileh M, Vanden Berghe W, Heyerick A, et al.Withaferin a strongly elicits InB kinase h hyperphosphor-ylation concomitant with potent inhibition of its kinaseactivity. J Biol Chem 2007;282:4253–64.42. Yin MJ, Yamamoto Y, Gaynor RB. The anti-inflammatoryagents aspirin and salicylate inhibit the activity of I(n)Bkinase-h. Nature 1998;396:77–80.

43. Hideshima T, Neri P, Tassone P, et al. MLN120B, anovel InB kinase h inhibitor, blocks multiple myelomacell growth in vitro and in vivo . Clin Cancer Res 2006;12:5887–94.44. Burke JR, Pattoli MA, Gregor KR, et al. BMS-345541 is a highly selective inhibitor of I n B kinasethat binds at an allosteric site of the enzyme andblocks NF-n B-dependent transcription in mice. J BiolChem 2003;278:1450–6.45. Lee DU, Kang YJ, Park MK, et al. Effects of 13-alkyl-substituted berberine alkaloids on the expression ofCOX-II, TNF-a, iNOS, and IL-12 production in LPS-stimulated macrophages. Life Sci 2003;73:1401–12.46. Marin-Neto JA, Maciel BC, Secches AL, et al.Cardiovascular effects of berberine in patients withsevere congestive heart failure. Clin Cardiol 1988;11:253–60.



Documents

Berberine Modifies Cysteine 179 of I B Kinase, Suppresses Nuclear Factor- B-Regulated Antiapoptotic Gene Products, and Potentiates Apoptosis