Berberine, an isoquinoline alkaloid derived from a plant used traditionally in Chinese and Ayurvedic medicine, has been reported to exhibit chemopreventive and anti-inflammatory activities through unknown mechanism. Because of the critical role of the transcription factor nuclear factor-κB (NF-κB) in these processes, we investigated the effect of berberine on this pathway. We found that berberine suppressed NF-κB activation induced by various inflammatory agents and carcinogens. This alkaloid also suppressed constitutive NF-κB activation found in certain tumor cells. Suppression of NF-κB activation occurred through the inhibition of phosphorylation and degradation of IκBα by the inhibition of IκB kinase (IKK) activation, leading to suppression of phosphorylation and nuclear translocation of p65, and finally to inhibition of NF-κB reporter activity. Inhibition of IKK by berbeine was direct and could be reversed by reducing agents. Site-specific mutagenesis suggested the involvement of cysteine residue 179 in IKK. Berberine also suppressed the expression of NF-κB–regulated gene products involved in antiapoptosis (Bcl-xL, Survivin, IAP1, IAP2, and cFLIP), proliferation (cyclin D1), inflammation (cyclooxygenase-2), and invasion (matrix metalloproteinase-9). Suppression of antiapoptotic gene products correlated with enhancement of apoptosis induced by tumor necrosis factor (TNF)-α and chemotherapeutic agents and with inhibition of TNF-induced cellular invasion. Overall, our results indicate that chemopreventive, apoptotic, and anti-inflammatory activities displayed by berberine may be mediated in part through the suppression of the NF-κB activation pathway. This may provide the molecular basis for the ability of berberine to act as an anticancer and anti-inflammatory agent. [Cancer Res 2008;68(13):5370–9]
Almost 80% of the world population cannot afford modern medicine. Traditional medicine is inexpensive but generally neither active principles nor their molecular targets are well-defined. For instance, extracts of golden seal (Hydrastis canadensis), oregon grape (Berberis aquifolium), barberry (Berberis vulgaris), coptis or golden 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). In Chinese pharmacopoeia, Huangbai and Huanglian are described as “heat removing” agents for their fever-reducing therapeutic applications. Almost four decades ago, berberine, an isoquinoline alkaloid, 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), epidermoid carcinoma ( 4), hepatoma ( 5), oral carcinoma ( 6), glioblastoma ( 7), prostate carcinoma ( 8), and gastric carcinoma ( 9). Animal studies have shown that berberine can suppress chemical-induced carcinogenesis ( 10), tumor promotion ( 11), and tumor invasion ( 12). It is a radiosensitzer of tumor cells but not of normal cells ( 13). How berberine mediates these effects is not fully understood, but its ability to modulate Mcl-1 ( 6), Bcl-xL ( 5), cyclooxygenase (COX)-2 ( 6), MDR ( 14), tumor necrosis factor (TNF)-α and IL-6 ( 15), iNOS ( 16), IL-12 ( 17), intercellular adhesion molecule-1 and ELAM-1 expression ( 12), MCP-1 and CINC-1 ( 18), cyclin D1 ( 19), activator protein (AP-1; ref. 20), HIF-1α ( 21), PPAR-γ ( 22), and topoisomerase II ( 23) has been shown. By using yeast mutants, berberine was found to bind and inhibit stress-induced mitogen-activated protein kinase kinase activation ( 17). Because apoptotic, carcinogenic, and inflammatory effects and various gene products (such as TNF-α, IL-6, COX-2, adhesion molecules, cyclin D1, and MDR) modulated by berberine are regulated by the transcription factor nuclear factor-κB (NF-κB), we postulated that this pathway plays a major role in the action of berberine.
NF-κB represents a group of five proteins, c-Rel, RelA (p65), RelB, NF-κB1 (p50 and p105), and NF-κB2 (p52; ref. 24). In an inactive state, NF-κB is sequestered in the cytoplasm as a heterotrimer consisting of p50, p65, and IκB subunits. On activation, IκBα undergoes phosphorylation and ubiquitination-dependent degradation leading to p65 nuclear translocation and binding to a specific consensus sequence in the DNA, which results in gene transcription. Most carcinogens, inflammatory agents, and tumor promoters, including cigarette smoke, phorbol ester, okadaic acid (OA), H2O2, and TNF-α, have been shown to activate NF-κB. NF-κB has been shown to regulate the expression of several genes whose products are involved in tumorigenesis. These include antiapoptotic genes (e.g., Bcl-xL, cIAP, survivin, and cFLIP), COX-2, matrix metalloproteinase 9 (MMP-9), genes encoding adhesion molecules, chemokines, and inflammatory cytokines and cell cycle regulatory genes (e.g., cyclin D1 and c-myc; ref. 25).
Whether berberine exerts its apoptotic, chemopreventive, anti-inflammatory, and anti-invasive effects through suppression of the NF-κB pathway was investigated in detail. We found that berberine inhibited the activation of NF-κB through the direct inhibition of IκBα kinase, and subsequently of IκBα phosphorylation and degradation, p65 nuclear translocation, and DNA binding. The suppression of NF-κB by this agent led to the down-regulation of gene 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 , was obtained from Sigma-Aldrich. A 50 mmol/L solution of berberine was prepared in DMSO, stored as small aliquots at −20°C, and then diluted as needed in cell culture medium. Bacteria-derived human recombinant human TNF-α, 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 serum were purchased from Invitrogen. Phorbol myristate acetate (PMA), OA, and anti–β-actin antibody were obtained from Aldrich-Sigma. Antibodies against p65, p50, IκBα, cyclin D1, MMP-9, IAP1, IAP2, Bcl-xL, FLIP, poly (ADP-ribose) polymerase (PARP), c-Jun-NH2-kinase (JNK)1, and COX-2 and Annexin V staining kit were purchased from Santa Cruz Biotechnology. Phospho-specific anti-IκBα (Ser32/36) and phospho-specific anti-p65 (Ser536) were purchased from Cell Signaling. Anti–IκB kinase (IKK)-α and anti–IKK-β antibodies were kindly provided by Imgenex. IKK plasmids, wild-type, and that 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 (human leukemia), 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 U266 cells 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 μg/mL streptomycin.
Electrophoretic mobility shift assay. To determine NF-κB activation, we prepared nuclear extracts and performed electrophoretic mobility shift assays (EMSA) as described previously ( 26). For supershift assays, nuclear extracts prepared from TNF-α–treated cells were incubated with antibodies against either p50 or p65 of NF-κB for 30 min at 37°C 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 μg of nuclear extract protein were incubated with 16 fmol of the 32P-end–labeled AP-1 consensus oligonucleotide 5′-CGCTTGATGACTCAGCCGGAA-3′ (bold indicates the AP-1 binding site) for 30 min at 37°C, 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-α–dependent IκBα phosphorylation, IκBα degradation, p65 translocation, and p65 phosphorylation, cytoplasmic or nuclear extracts were prepared. For detection of cleavage products of PARP, antiapoptotic and angiogenesis markers 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 μg/mL aprotinin, 0.005 μg/mL leupeptin, 0.4 mol/L phenylmethyl-sulfonyl fluoride, and 4 mmol/L NaVO4]. Lysates were spun at 14,000 rpm for 10 min to remove insoluble material. Supernatant were collected and kept at −80°C. Either cytosolic 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 detected by enhanced chemiluminescence reagent (Amersham).
IKK assay. To determine the effect of berberine on TNF-α–induced IKK activation, an IKK assay was performed. Briefly, the IKK complex from whole-cell extracts was precipitated with antibody against IKK-β and then treated 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 μCi [32P] ATP, 10 μmol/L unlabeled ATP, and 2 μg of substrate glutathione S-transferase-IκBα [amino acid (aa) 1–54]. After incubation at 30°C 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 a Storm 820. To determine the total amounts of IKK-α and IKK-β in each sample, 30 μg of whole-cell proteins was resolved on 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti–IKK-α or anti–IKK-β 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 μCi of [32P] ATP, 10 μmol/L unlabeled ATP, and 2 μg of substrate glutathione S-transferase (GST)-c-Jun (aa 1–79). The immunocomplex was incubated at 30°C 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 each sample, whole-cell extracts were subjected to Western blot analysis using anti-JNK1 antibody.
NF-κB–dependent reporter gene expression assay. NF-κB–dependent reporter gene expression was performed. The effect of berberine on TNF-α, TNF receptor (TNFR), TNFR-associated death domain (TRADD-), TNFR-associated factor 2 (TRAF2-), NF-κB–inducing kinase (NIK-), and IKK-NF-κB–dependent reporter gene transcription was analyzed by the secretory alkaline phosphatase (SEAP) assay.
Immunocytochemistry for NF-κB p65 localization. Immunocytochemistry 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 slides were 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 4°C, the slides were washed, incubated with goat anti-rabbit IgG-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 activity and 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 nucleic acids, thereby producing a bright red fluorescence in dead cells. Briefly, 2 × 105 cells were incubated with 25 μmol/L berberine and treated with 1 nmol/L TNF-α for up to 24 h at 37°C. Cells were stained with the Live/Dead reagent (5 μmol/L ethidium homodimer and 5 μmol/L calcein-AM) and incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2; Nikon).
Cytotoxicity assay. The effects of berberine on the cytotoxic effects of TNF-α 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 a 96-well plate and then treated with the 1 nmol/L TNF-α, 0.1 μmol/L 5-5-fluorouracil, and 0.1 μmol/L doxorubicin for 24 h at 37°C. An MTT solution was added to each well and incubated for 2 h at 37°C. An extraction buffer (20% SDS and 50% dimethylformamide) was added, and the cells were incubated overnight at 37°C. Then, the absorbance was measured at 570 nm using a 96-well multiscanner (Dynex Technologies; MRX Revelation).
Annexin V assay. An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface of membrane to the extracellular surface. 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-α for 24 h at 37°C, and subjected to Annexin V staining. The cells were washed in PBS, resuspended in 100 μL of binding buffer containing a FITC-conjugated anti-Annexin V antibody, and then analyzed with a flow cytometer (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 lightproof polyethylene terephthalate membrane coated with a reconstituted basement membrane gel with 8-μm-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 incubation overnight, the cells were treated with 25 μmol/L berberine and stimulated with 1 nmol/L TNF-α for 24 h in the presence of 1% FBS. The cells that invaded the lower chamber by migrating through the Matrigel during incubation were stained with 4 μg/mL calcein-AM in PBS for 30 min at 37°C 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 test and Student's t test with Microsoft excel software.
We investigated the effect of berberine on constitutively active NF-κB and on that activated by various carcinogens and inflammatory stimuli, on NF-κB–regulated gene expression, on apoptosis induced by cytokines and chemotherapeutic agents, and on invasion. We focused on TNF-α–induced NF-κB activation because the role of TNF-α role in the NF-κB activation pathway has been relatively well-established.
Berberine inhibits NF-κB activation induced by carcinogens and inflammatory stimuli. TNF-α, PMA, OA, and CSC are well known potent activators of NF-κB but by different mechanisms ( 26). We examined the effect of berberine on the activation of NF-κB by these agents using DNA binding assays. TNF-α, PMA, OA, and CSC induced NF-κB, and berberine suppressed this activation in Jurkat cells ( Fig. 1B). These results suggest that berberine acts at a step in the NF-κB activation pathway that is common to all four agents.
We then determined the dose and time of exposure to berberine required to suppress TNF-α induced NF-κB activation in Jurkat cells. EMSA showed that berberine alone had no effect on NF-κB activation, but it inhibited TNF-α–mediated NF-κB activation in a dose- ( Fig. 1C) and time-dependent ( Fig. 1D) manner, respectively, and that 18 h exposure to 50 μmol/L berberine was sufficient to suppress activation.
Most tumor cells have constitutively active NF-κB that arises through mechanisms that vary with the cell type. Whether berberine can inhibit constitutively active NF-κB in multiple myeloma U266 cells was determined. Berberine at a concentration 40 μmol/L and higher completely suppressed constitutively active NF-κB in U266 cells ( Fig. 2A ).
NF-κB is a protein complex in which various combinations of Rel/NF-κB constitute active NF-κB heterodimers that bind to specific DNA sequences. To show that the band visualized by EMSA in TNF-α–treated cells was indeed NF-κB, nuclear extracts from TNF-α–activated Jurkat cells were incubated with antibodies to the p50 and the p65 (RelA) subunit of NF-κB. The preincubation of nuclear 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-α–activated complex consisted of p50 and p65. The band lower to NF-κB is a nonspecific band. Preimmune serum (PIS) did not cause any super shift. Addition of excess unlabeled oligonucleotide (competitor; 100-fold molar excess) caused a complete disappearance of the band, whereas mutated oligonucleotide had no effect on DNA binding.
Some NF-κB inhibitors, such as caffeic acid phenethyl ester and plumbagin ( 27, 28), directly suppress binding of NF-κB to DNA. We determined whether berberine mediates suppression of NF-κB activation through a similar mechanism. Berberine did not modify the DNA-binding ability of NF-κB proteins (Supplementary Fig. S1). These results suggest that berberine inhibits NF-κB activation at a step upstream to its DNA binding.
Whether berberine suppresses other transcription factor such as AP-1 under the conditions it suppresses NF-κB was examined. AP-1 showed constitutive activation in Jurkat cells, and berberine had no effect on it (Supplementary Fig. S1).
Berberine inhibits TNF-α–dependent IκBα degradation and phosphorylation. The translocation of NF-κB to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of IκBα. To determine whether inhibition of TNF-α induced NF-κB activation was due to inhibition of IκBα degradation, we pretreated Jurkat cells with berberine and then exposed them to TNF-α for various time periods. We then examined the cells for NF-κB in the nucleus by EMSA and for IκBα degradation in the cytoplasm by Western blot analysis. As shown in Fig. 2C, TNF-α activated NF-κB in the control cells. TNF-α induced NF-κB as early as 30 minutes and that continued to increase at 60 minutes but had no effect on berberine-pretreated cells. Moreover, TNF-α induced IκBα degradation as early as 10 minutes, but in berberine, pretreated cells showed TNF-α had no effect on IκBα degradation ( Fig. 2D). These results indicate that berberine inhibited both TNF-α induced IκBα degradation and NF-κB activation.
To determine whether the inhibition of TNF-α–induced IκBα degradation was due to an inhibition of IκBα phosphorylation, we used the proteasome inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN) to block degradation of IκBα. Western blot using an antibody that recognizes the serine-phosphorylated (Ser32/36) form of IκBα showed that TNF-α induced IκBα phosphorylation was strongly suppressed by berberine ( Fig. 3A ).
Berberine directly inhibits TNF-α–induced IκBα kinase activation. Because berberine inhibits the phosphorylation and degradation of IκBα, we tested the effect of berberine on TNF-α–induced IKK activation, which is required for TNF-α–induced phosphorylation of IκBα. As shown in Fig. 3B, TNF-α induced the activation of IKK and berberine completely suppressed it (top). Neither TNF-α nor berberine had any effect on the expression of IKK-α (middle) or IKK-β proteins (bottom).
To evaluate whether berberine suppresses IKK activity directly by binding to IKK or indirectly by suppressing its activation, we immunoprecipitated IKK complex from whole-cell extracts from TNF-α–stimulated cells with anti–IKK-β antibody. The immunocomplexes were treated in vitro with various concentrations of berberine, and kinase assay for IKK was performed. Results from the immune complex kinase assay showed that berberine directly inhibited the activity of IKK ( Fig. 3C, left). This finding suggested that berberine directly modulates TNF-α induced IKK activation.
IKK-β contains various cysteine residues. The modulation of IKK activity by berberine through the modification of cysteine residues was investigated using reducing agents, such as DTT. We found that addition of DTT to the kinase reactions reversed the berberine-mediated inhibition of IKK activity induced by TNF-α ( Fig. 3C, right).
IKK-β contains a cysteine at position 179 in its activation loop that is critical for its biological activity. To determine whether this cysteine is involved in berberine-mediated inhibition, we transfected cells with plasmids for either wild-type Flag-IKK-β or Flag-IKK-β with a C179A mutation. Berberine inhibited constitutive wild-type IKK-β activity ( Fig. 3D). In contrast, berberine had no effect on IKK-β (C179A) activity ( Fig. 3D). These findings proved that berberine inhibited IKK-β activity through the modification of the Cys179 residue.
We also examined whether berberine affects other protein kinases. The results show that TNF activates JNK, and berberine slightly inhibited the TNF-α–induced activation of JNK at later times (Supplementary Fig. S2).
Berberine inhibits TNF-α–induced phosphorylation and nuclear translocation of p65. We also investigated the effect of berberine on TNF-α–induced phosphorylation of p65 because phosphorylation is also required for its transcriptional activity ( 24). In the nuclear fraction from the TNF-α–treated cells, berberine suppressed the phosphorylated form of p65 (Ser536; Fig. 4A, top ). We further showed that berberine suppressed TNF-α–induced nuclear translocation of p65, as measured by Western blotting ( Fig. 4A, middle).
An immunocytochemistry assay confirmed that berberine suppressed TNF-α–induced translocation of p65 to the nucleus ( Fig. 4B).
Berberine represses NF-κB–dependent reporter gene expression. Because DNA binding alone does not always correlate with NF-κB–dependent gene transcription, suggesting there are additional regulatory steps ( 29). We transiently transfected the cells with NF-κB–regulated SEAP reporter construct and pretreated with berberine or left untreated and then stimulated the cells with TNF-α. A 3-fold increase in SEAP activity was noted after stimulation with TNF-α ( Fig. 5A ), and that was abolished by dominant-negative IκBα, indicating the specificity. When the cells were pretreated with berberine, TNF-α–induced NF-κB–dependent SEAP expression was inhibited in a dose-dependent manner ( Fig. 5A). These results indicate that berberine, inhibits NF-κB–dependent reporter gene expression induced by TNF-α.
TNF-α–induced NF-κB activation is mediated through sequential interaction of the TNFR with TRADD, TRAF2, NIK, and IKK-β, resulting in phosphorylation of IκBα, which leads to degradation of IκBα and p65 nuclear translocation ( 30). To delineate the site of action of berberine in the TNF-α–signaling pathway leading to NF-κB activation, cells were transiently transfected with TNFR1, TRADD, TRAF2, NIK, and IKK-β, and then NF-κB–dependent SEAP expression was monitored with or without berberine treatment. As shown in Fig. 5B, berberine suppressed TNFR1, TRADD, TRAF2, NIK, and IKK plasmid–induced reporter gene expression. Because IKK activation can cause the phosphorylation of IκBα and p65 ( 31), we suggest that berberine inhibits NF-κB activation through inhibition of IKK.
Berberine represses the expression of TNF-α–induced NF-κB–dependent antiapoptotic, proliferation, and metastatic gene products. Because NF-κB regulates the expression of the antiapoptotic proteins IAP1/2, Bcl-xL, survivin, and cFLIP, we investigated whether berberine could modulate TNF-α–induced expression of these antiapoptotic genes. We found that berberine abolished TNF-α–induced expression of Bcl-xL, survivin, IAP1/2, and cFLIP antiapoptotic proteins ( Fig. 5C, top).
We also investigated whether berberine can modulate NF-κB–regulated gene products involved in the proliferation of tumor cells. TNF-α has been shown to induce cyclin D1 and COX-2, both of which have NF-κB binding sites in their promoters. Thus, we investigated whether berberine inhibits the TNF-α–induced expression of these proteins by Western blot analysis using specific antibodies. We found that berberine abolished TNF-α–induced expression of COX-2 and cyclin D1 ( Fig. 5C, middle).
We also investigated whether berberine modulates TNF-α induced NF-κB–dependent MMP-9 expression, a gene involved in the invasion of tumor cells. It has been established already that MMP-9 is regulated by NF-κB. We found that berberine abolished TNF-α–induced expression of MMP-9 ( Fig. 5C, bottom).
Berberine potentiates apoptosis induced by TNF-α and chemotherapeutic agents. Because the activation of NF-κB has been shown to inhibit apoptosis induced by TNF-α and chemotherapeutic agents ( 32), we investigated whether berberine affects TNF-α and chemotherapeutic agent-induced apoptosis. The esterase-staining method (also called Live/Dead assay) showed that berberine up-regulated TNF-α–induced apoptosis from 5% to 65% ( Fig. 6A ). MTT assay showed that berberine enhanced cytotoxicity induced by TNF-α, 5-fluorouracil (5-FU), and doxorubicin ( Fig. 6B). Caspase-mediated PARP cleavage likewise showed that berberine enhanced the apoptotic effect of TNF-α 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-α induced early events in apoptosis. These results together indicate that berberine potentiates the apoptotic effects of TNF-α and chemotherapeutic 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 significant effect on the viability of these cells (data not shown).
Berberine suppresses TNF-α–induced invasion activity. The expression of both COX-2 and MMP-9 has been linked with tumor cell invasion ( 33). Therefore, we investigated whether berberine can modulate the tumor cell invasion activity induced by TNF-α in vitro in a Matrigel invasion assay. We found that berberine suppressed TNF-α–induced tumor cell invasion ( Fig. 6D).
The goal of this study was to determine whether the chemopreventive, anti-inflammatory, and apoptotic effects of berberine were mediated through the modulation of the NF-κB–signaling pathway and NF-κB–regulated gene products. We found that berberine suppressed NF-κB activation induced by various carcinogens and inflammatory agents. NF-κB inhibition was due to inhibition of IKK activation, leading to suppression of IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and NF-κB–dependent reporter gene expression. Berberine also down-regulated gene products involved in cell proliferation, antiapoptosis, and invasion. This down-regulation led to the potentiation of apoptosis and inhibition of invasion.
This is the first report to suggest that berberine can inhibit the NF-κB activation induced by TNF-α, PMA, OA, and CSC. In support of our results, this alkaloid has been shown to inhibit acetaldehyde-induced NF-κB activation in human hepatoma cells ( 34). Unlike our results, however, Enk and colleagues ( 35) recently reported that berberine has no effect on TNF-α–induced NF-κB activation in human keratinocytes. The differences in cell types, methodology used to measure NF-κB, and conditions used may account for the difference in results.
How berberine suppresses NF-κB activation was investigated in detail. We found that berberine suppresses the phosphorylation and degradation of IκBα, consistent with that reported earlier with acetaldehyde-induced activation ( 34). We further found that this alkaloid inhibits IκBα degradation through inhibition of activation of IKK induced by TNF-α. We found that berberine inhibits IKK activity directly by modification of cysteine residue 179. This residue is present in the activation loop of IKK-β and is a target for a variety of compounds such as arsenic and auranofin ( 36). Interactions of these compounds with IKK involving cysteine residue 179 is easily reversed by addition of thiol-reducing agents (e.g., DTT and reduced glutathione; ref. 37). The inhibitory effects of N-ethylmaleimid on IKK activity were abolished by the expression of mutant IKK-β, which contains alanine at residue 179 rather than cysteine ( 38, 39). Like berberine, we showed recently that butein also inhibits the NF-κB activation by direct modification of cysteine residue 179 in IKK-β ( 26). Different cysteine residues have been identified 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 aspirin interferes with ATP binding site ( 42). Thus, the mechanism by which berberine and other agents inhibit IKK may involve cysteine residue 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 μmol/L, which is comparable with the dose of berberine used in our study ( 43). Another compound, BMS-345541, was found to bind to an allosteric site of IKK-β and acts as an ATP noncompetitive inhibitor with IC50 of 0.3 μmol/L ( 44). Although these compounds do not possess positively charged imine moiety as found in berberine, they are alkaloid in nature similar to berberine.
TNF-α activates NF-κB through sequential recruitment of TNFR1, TNFR2, NIK, and IKK, and berberine suppressed NF-κB activation by all of these signaling intermediates. IKK has been implicated in the phosphorylation of p65 ( 31), which is needed for its transcriptional activity. We found that berberine suppressed the phosphorylation of p65. Furthermore, berberine had no direct effect on the binding of p50-p65 to the DNA. Therefore, it is possible that inhibition of IKK-mediated IκBα degradation and p65 phosphorylation contribute to the suppressive effects of berberine on NF-κB activation.
We found that numerous gene products that are regulated by NF-κB were down-regulated by berberine. These included proteins with proinflammatory (COX-2), cell proliferative (cyclin D1), antiapoptotic (Bcl-xL, survivin, IAP1, IAP2, and cFLIP), and invasive (MMP-9) activities. The down-regulation of COX-2 by berberine is in agreement with previous report ( 20). The decrease in TNF-α and iNOS ( 45); urokinase-plasminogen activator and plasminogen activator inhibitor ( 12); Mcl-1 ( 6); MCP-1 and CINC-1 ( 18); and mdr1 ( 14) expression reported previously could also be due to down-regulation of NF-κB activation by berberine. Down-regulation of cyclin D1 reported here is also in agreement with previous results ( 19). It is possible that suppression of proliferation of various tumor cell lines by berberine is through down-regulation of cyclin D1.
Our results also show that berberine potentiated the apoptotic effects of TNF-α and chemotherapeutic agents. This is likely linked to the down-regulation of antiapoptotic (Bcl-xL, survivin, IAP1, IAP2, and cFLIP) gene products. The down-regulation of Bcl-xL is in agreement with previous reports ( 5). It is possible that sensitization of cells to chemotherapeutic agents by berberine is mediated through the down-regulation of mdr1 as reported previously ( 14). Consistent with our results, Lin and colleagues ( 14) reported that berberine potentiated the anti-tumor effects of paclitaxel on digestive tract cancer cells.
In animal studies, berberine has been shown to suppress chemical-induced carcinogenesis ( 10), phorbol-ester–induced tumor promotion ( 11), and tumor invasion ( 12). Various carcinogens and tumor promoters have been shown to activate NF-κB. Thus, suppression of NF-κB by berberine as shown here may contribute to 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 are achievable in vivo is unclear. The doses used in vitro studies, however, are irrelevant to that in vivo as the exposure to the drug in vivo may occur over long-periods. Thus, overall, our results suggest that anticarcinogenic, anti-inflammatory, and proapoptotic effects of berberine may be mediated through inhibition of NF-κB induced 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 required to recognize the full potential of this important constituent of ancient medicine.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
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 accordance with 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.
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.
- Received February 14, 2008.
- Revision received March 27, 2008.
- Accepted April 16, 2008.
- ©2008 American Association for Cancer Research.