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Originally published In Press as doi:10.1074/jbc.M201368200 on March 4, 2002

J. Biol. Chem., Vol. 277, Issue 21, 18411-18420, May 24, 2002
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Kaurane Diterpene, Kamebakaurin, Inhibits NF-kappa B by Directly Targeting the DNA-binding Activity of p50 and Blocks the Expression of Antiapoptotic NF-kappa B Target Genes*

Jeong-Hyung Lee, Tae Hyeon Koo, Bang Yeon Hwang, and Jung Joon LeeDagger

From the Anticancer Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yuseong, Daejeon 305-600, Korea

Received for publication, February 11, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Kaurane diterpenes have been identified from numerous medicinal plants, which have been used for treatment of inflammation and cancer, however, their molecular mechanism of action remains unclear. We have previously shown that kamebakaurin and other three kaurane diterpenes selectively inhibit activation of transcription factor NF-kappa B, a central mediator of apoptosis and immune responses. We here demonstrate that kamebakaurin is a potent inhibitor of NF-kappa B activation by directly targeting DNA-binding activity of p50. Kamebakaurin prevented the activation of NF-kappa B by different stimuli in various cell types. Kamebakaurin did not prevent either stimuli-induced degradation of Ikappa B-alpha or nuclear translocation of NF-kappa B, however, it significantly interfered DNA binding activity of activated NF-kappa B in cell and in vitro and preferentially prevented p50-mediated DNA-binding activity of NF-kappa B rather than that of RelA as measured using in vitro translated p50 and RelA proteins. Moreover, a p50 mutant with a Cys-62 right-arrow Ser mutation was not inhibited with kamebakaurin, indicating that the effect of kamebakaurin was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by kamebakaurin was further demonstrated by mass spectrometry analysis that showed an increase in the molecular mass of kamebakaurin-treated p50, and this modification was not reverted by addition of dithiothreitol. These results suggested that kamebakaurin exhibited its inhibitory activity by a direct covalent modification of cysteine 62 in the p50. Also, treatment of cells with kamebakaurin prevented the tumor necrosis factor-alpha (TNF-alpha )-induced expression of antiapoptotic NF-kappa B target genes encoding c-IAP1 (hiap-2) and c-IAP2 (hiap-1), members of the inhibitor of apoptosis family, and Bfl-1/A1, a prosurvival Bcl-2 homologue, and augmented the TNF-alpha -induced caspase 8 activity, thereby resulting in sensitizing MCF-7 cells to TNF-alpha -induced apoptosis. Taken together, kamebakaurin is a valuable candidate for the intervention of NF-kappa B-dependent pathological conditions such as inflammation and cancer.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear factor kappa B (NF-kappa B)1 represents a family of eukaryotic transcription factors participating in the regulation of various cellular genes involved in the immediate early processes of immune, acute phase, and inflammatory responses as well as genes involved in cell survival (1). In most cell types, the pleiotropic-inducible form of NF-kappa B is a heterodimer composed of p50 and RelA (previously termed p65) (2). RelA contains a C-terminal transactivation domain in addition to the N-terminal Rel homology domain, thus serving as a critical transactivation subunit of NF-kappa B (3, 4). p50 lacks a transactivation domain and is believed to serve as a regulatory subunit modulating the DNA binding affinity of RelA (3, 4). The p50·RelA NF-kappa B heterodimer is normally sequestered in the cytoplasmic compartment by physical association with inhibitory proteins, including Ikappa B-alpha and related proteins (5). Ikappa B-alpha specifically binds to and masks the nuclear localization signals of RelA and p50, thereby preventing the nuclear translocation of the NF-kappa B heterodimer (6). The latent cytoplasmic NF-kappa B RelA·p50 complex can be post-translationally activated by a variety of cellular stimuli, which trigger site-specific phosphorylation of Ikappa B-alpha by a multisubunit Ikappa B kinase (7-9). The phosphorylated Ikappa B-alpha becomes rapidly ubiquitinated and degraded by the proteasome complex (10, 11). Following Ikappa B-alpha degradation, the NF-kappa B heterodimer is rapidly translocated to the nucleus, where it activates the transcription of target genes.

NF-kappa B regulates the transcription of various inflammatory cytokines, such as interleukin-1, -2, -6, and -8 and TNF-alpha , as well as genes encoding cyclooxygenase II, inducible nitric oxide synthase, immunoreceptors, cell adhesion molecules, hematopoietic growth factors, and growth factor receptors (12). In addition to regulating the expression of genes important for immune and inflammatory responses, NF-kappa B also controls the transcription of genes that confer resistance to death-inducing signals. Candidate target genes include those encoding the caspase inhibitors c-IAP1, c-IAP2, and X-IAP, the TNF receptor-associated factors TRAF1 and TRAF2, the zinc finger protein A20, the immediate-early response gene IEX-1L, and the prosurvival Bcl-2 homologue Bfl-1/A1 (13-16). Therefore, pharmacological inhibition of NF-kappa B could be a valuable strategy to modulate the inflammatory processes as well as cell death.

Whole plant extracts of Isodon japonicus have been used in folk medicine in China, Japan, and Korea for a remedy for gastrointestinal disorder, tumor, and inflammatory diseases (17, 18). The genus Isodon (also called Rabdosia) is a rich source of diterpenes, especially the highly oxidized kaurane diterpenes. Previously, we have shown that four diterpenes, including kamebakaurin (KA) inhibit the LPS-induced NO and prostaglandin E2 production in RAW264.7 cells (19). We here show that KA inhibits NF-kappa B by directly targeting DNA-binding activity of p50, possibly through a covalent modification of cysteine 62 within the DNA-binding domain, without affecting the induced degradation of Ikappa B-alpha and nuclear translocation of NF-kappa B. Also, KA not only prevented the TNF-alpha -induced expression of antiapoptotic NF-kappa B target such as c-IAP1 and Bfl-1/A1 genes but also augmented TNF-alpha -induced caspase-8 activity, resulting in sensitizing MCF-7 cells to TNF-alpha -induced apoptosis. This study shows that KA is a potential candidate for modulation of NF-kappa B-dependent pathological conditions.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture and Chemicals-- Jurkat T leukemia cells, THP-1 cells, and MCF-7 cells were maintained in RPMI 1640 medium. HeLa cells, RAW264.7 cells, and HT-1080 cells were maintained in Dulbecco's modified Eagle's medium. Both media were supplemented with penicillin (100 units/ml)-streptomycin (100 µg/ml) (Invitrogen, Gaithersburg, MD) and 10% heat-inactivated fetal bovine serum (Invitrogen). All cells were grown in an incubator at 37 °C and 5% CO2. TNF-alpha was obtained from Invitrogen and phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) from Sigma Chemical Co. KA (compound 1) and three other kaurane diterpenes (compounds 2-4) were isolated from dried whole plants of I. japonicus as described previously (19), and their structures are shown in Fig. 1 (see below). The purity of KA, recrystallized with MeOH as colorless plates, was over 98% in an HPLC analysis. KA's physicochemical and spectral data were comparable to previously reported values (20).

Plasmids and Transfections-- A pNFkB-Luc plasmid for NF-kappa B luciferase reporter assay was obtained from Stratagene (La Jolla, CA). Expression vectors for RelA and p50 were kindly provided from Dr. M. Jung (Georgetown University, Washington, D.C.) and. Dr. J. Lee (Pohang Institute of Science and Technology, Pohang, Korea), respectively. Full-length cDNA for Bfl-1/A1 was kindly provided by Dr. S. Hong (Korea Cancer Research Center, Seoul, Korea). Transfections were performed using LipofectAMINE plus reagent (Invitrogen), according to the instructions of the manufacturer. The recombinant wild type and mutant cDNA in the DNA-binding domains of human p50 (amino acids 36-385, GenBankTM accession number M55643) were a generous gift from Dr. D. Perez-Sala (Departamento de Estructuray Función de Proteínas, Madrid, Spain) and were expressed in Escherichia coli as hexahistidine fusion proteins and purified as described previously (21).

Electrophoretic Mobility Shift Assay-- Thirty minutes prior to stimulation with TNF-alpha , LPS, or PMA, cells were preincubated with the indicated concentrations of KA at 37 °C. In the following, cells were stimulated with TNF-alpha (20 ng/ml), PMA (50 ng/ml), or LPS (10 µg/ml), harvested by centrifugation, washed twice with cold phosphate-buffered saline, and nuclear extracts were then prepared using a protocol described previously (22). In certain experiments, nuclear extracts were prepared from p50- or RelA-overexpressed MCF-7 cells without stimulation, and p50 and RelA proteins were prepared by in vitro translation using a TnT quick-coupled transcription/translation system (Promega, Madison, WI). The recombinant wild- and mutant-type p50 proteins were purified using the QIA expressionist system (Qiagen, Valencia, CA) according to the instructions of the manufacturer. The electrophoretic mobility shift assay was performed using a gel-shift assay system (Promega, Madison, WI), according to the instructions of the manufacturer. A double-stranded oligonucleotide for NF-kappa B (Promega) or AP-1 (Promega) was end-labeled with [gamma -32P]ATP and purified with a G-25 spin column (Roche Molecular Biochemicals, Mannheim, Germany). Nuclear extracts (10 µg), in vitro translated p50, RelA, or recombinant wild type and mutant proteins in the binding domains of human p50 (amino acids 36-385) were incubated for 20 min at room temperature with a gel-shift binding buffer (5% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 50 µg/ml poly(dI-dC)/poly(dI-dC)) and 32P-labeled oligonucleotide. The DNA· protein complex formed was separated on 4% native polyacrylamide gels. The gel was transferred to Whatman 3 MM paper, dried, and exposed to x-ray film at -70 °C with an intensifying screen. The specificity of binding was examined by competition with an excess of unlabeled oligonucleotide. Supershift studies were performed by incubation with antibodies against either RelA and c-Rel (Oncogene Research Products, Boston, MA) or p50 subunits (Santa Cruz Biochemical, Santa Cruz, CA) of NF-kappa B for 20 min at room temperature.

HPLC Purification and MALDI-TOF Analysis of p50-- The purified p50 (amino acid 36-385) incubated in the absence or presence of KA was injected into a reverse-phase HPLC column (Beckman Ultrasphere, 5-µm particle size, 4.6 mm × 25 cm), equilibrated with solvent A (0.1% trifluoroacetic acid), and eluted with a gradient of 0-100% eluant B (100% acetonitrile in solvent A). Fractions containing p50 were pooled and concentrated by evaporation. 0.5 µl of the fractions to be analyzed were applied onto target and dried out along with 0.5 µl of sinapinic acid (10 mg/ml) matrix in water:acetonitrile (1:1) containing 0.1% trifluoroacetic acid. Mass spectrometry analysis by MALDI-TOF was performed using a Voyager-DE STR instrument (Applied Biosystems, Foster City, CA), operating in a linear mode. Calibration was performed externally using bovine serum albumin and control p50 as standards.

Western Blotting-- Proteins were extracted from cells in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin). 50 µg of protein per lane was separated by SDS-polyacrylamide gels before blotting a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was blocked with 5% skim milk and then incubated with various primary antibodies (alpha -Ikappa B-alpha , New England BioLabs Inc., Beverly, MA; alpha -RelA, alpha -c-IAP1, alpha -c-IAP2, alpha -bcl-2, and alpha -bax, Santa Cruz Biochemicals, Santa Cruz, CA). The respective proteins were detected by incubation with primary antibodies. After binding of an appropriate secondary antibody coupled to horseradish peroxidase, proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (Amersham Biosciences, Inc., Buckinghamshire, UK).

Northern Blot Analysis-- RNA was isolated from cells using RNeasy Mini kits according to the manufacturer's instructions (Qiagen, Valencia, CA). 10 µg of total RNA were resolved on 1% agarose-formaldehyde gel and transferred to a nylon membrane by capillary action. Membranes were probed and washed according to the instructions of the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). 32P-Labeled probes were generated by the random priming method using Rediprime II (Amersham Biosciences, Inc., Buckinghamshire, UK) and 50 µCi of [alpha -32P]dCTP (3000 Ci/mmol, PerkinElmer Life Sciences). Unincorporated nucleotides were removed by purification through a G-25 spin column. The results were visualized by autoradiography. Quantitation was determined by densitometry.

Luciferase Assay-- Luciferase assay was performed using a luciferase assay system according to the manufacturer's instructions (Promega, Madison, WI). Luciferase activity was determined in Microlumat Plus luminometer (EG&G Berthold, Bad Wildbad, Germany) by injecting 100 µl of assay buffer containing luciferin and measuring light emission for 10 s. The results were normalized to the activity of beta -galactosidase expressed by co-transfected lacZ gene under the control of a constitutive promoter.

Cell Viability and Caspase-8 Assays-- Cell viability was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, untreated cells or treated cells with KA and/or TNF-alpha in a 96-well plate were incubated for 48 h followed by the addition of MTT to the cells. Caspase-8 activity was determined using caspase-8 colorimetric assay kit according to the manufacturer's instructions (CLONTECH, Palo Alto, CA). Optical densities were determined on a microplate reader (Molecular Devices, Sunnyvale, CA) at 405 nm.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

KA Inhibits NF-kappa B Activation by LPS, TNF-alpha , and PMA-- In an effort to identify NF-kappa B inhibitors from anti-inflammatory herbal medicine, we have identified KA (compound 1) together with three other kaurane diterpenes, kamebanin, kamebacetal A, and excisanin A (compounds 2-4, respectively) from a traditional medical plant, I. japonicus (Fig. 1), which has been used in the treatment of inflammatory diseases and cancer (17, 18). All compounds inhibited the LPS-induced NF-kappa B activation as well as the LPS-induced productions of NO and prostaglandin E2 in RAW264.7 cells without affecting cell viability, and KA was more abundant and more potent than the others (19). The effect of KA on the NF-kappa B activation by various stimuli was investigated in a NF-kappa B reporter assay. KA inhibited TNF-alpha -, PMA-, and LPS-induced expression of NF-kappa B reporter gene construct in a dose-dependent manner (Fig. 2). Basal NF-kappa B activity was also suppressed by KA. To confirm that KA inhibits NF-kappa B activation, we performed electrophoretic mobility shift assays (Fig. 3). Three cell lines, human breast cancer MCF-7, human lymphoma Jurkat, and human monocyte THP-1, were preincubated with various concentrations of KA for 30 min prior to stimulation. THP-1 cells were stimulated for 30 min with LPS, Jurkat cells for 30 min with PMA, and MCF-7 cells for 90 min with TNF-alpha . After the stimulation, nuclear extracts were prepared and DNA-binding activity of NF-kappa B in the nuclear extracts was measured. We found that these cell lines stimulated with the corresponding stimuli strongly induced DNA-binding activity of NF-kappa B. However, pretreatment of KA dose-dependently inhibited DNA-binding activity of NF-kappa B induced by above stimuli. Similar to the reporter assay, basal DNA-binding activity of NF-kappa B was significantly reduced at 10 µg/ml of KA. All of these results indicate that KA interferes with one or more common steps during NF-kappa B activation in different cell types rather than with one single event specific for an individual stimuli.


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  		Fig. 1.   Structures of kamebakaurin (1) and three other kaurane diterpenes, kamebanin (2), kamebacetal A (3), and excisanin A (4) isolated from I. japonicus.


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  		Fig. 2.   Effect of KA on NF-kappa B-dependent reporter gene expression by different stimuli. HeLa (A), Jurkat (B), and THP-1 (C) cells, which were transiently transfected with a NF-kappa B-dependent reporter gene, were grown for 1 day, pretreated for 30 min with the indicated concentrations of KA, then stimulated for 8 h with TNF-alpha (A, 20 ng/ml), PMA (B, 50 ng/ml), or LPS (C, 10 µg/ml). Luciferase activities were determined as described under "Materials and Methods." Mean values from three independent experiments performed in triplicate are shown: bars, S.D.


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  		Fig. 3.   KA inhibits NF-kappa B activation by different stimuli. A, MCF-7 cells were preincubated for 30 min with the indicated concentrations of KA and stimulated with TNF-alpha (20 ng/ml) for 90 min. Subsequently nuclear extracts were prepared and tested for DNA binding of activated NF-kappa B by EMSA. B, Jurkat cells were preincubated for 30 min with the indicated concentrations of KA followed by the stimulation of PMA (50 ng/ml) for 30 min. Subsequently, nuclear extracts were prepared and tested for DNA binding of activated NF-kappa B by EMSA. C, THP-1 cells were preincubated for 30 min with the indicated concentrations of KA and stimulated with LPS (10 µg/ml) for 30 min. Subsequently, nuclear extracts were prepared and tested for DNA binding of activated NF-kappa B by EMSA. In lane Ap-1, a 100-fold excess of unlabeled AP-1 consensus oligonucleotide was added to the reaction mixture. In lane kappa B, a 100-fold excess of unlabeled NF-kappa B oligonucleotide was added to the reaction mixture. The arrow indicates the location of the DNA·NF-kappa B complex. The amount of DNA·NF-kappa B complex formed was estimated by image scanning and is expressed in arbitrary units at the bottom of each panel.

KA Does Not Significantly Inhibit Degradation of Ikappa B-alpha and Translocation of NF-kappa B to Nucleus-- Because degradation of Ikappa B proteins is an essential step for NF-kappa B activation by various stimuli, we firstly examined the effect of KA on the induced degradation of Ikappa B-alpha protein by TNF-alpha (Fig. 4). MCF-7 cells were pretreated with 10 µg/ml KA for 30 min and subsequently stimulated with TNF-alpha for indicated times. Total cell extracts were analyzed for the presence of Ikappa B-alpha with Western blots. Ikappa B-alpha was completely degraded in 30 min after stimulation with TNF-alpha and re-synthesized in 60 min. However, preincubation with KA did not prevent the induced degradation of Ikappa B-alpha protein. Interestingly, resynthesis of Ikappa B-alpha , which is under control of NF-kappa B, was significantly suppressed by KA. Identical results were obtained for all stimuli described here with KA (data not shown). To further examine the inhibitory effect of KA on NF-kappa B activation, we measured the amount of NF-kappa B translocated into nucleus after stimulation. Nuclear extracts from stimulated cells were tested for the amount of NF-kappa B by Western blot analysis. KA did not significantly prevented nuclear translocation of NF-kappa B after stimulation (data not shown).


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  		Fig. 4.   Effect of KA on the degradation of Ikappa B-alpha induced by TNF-alpha . MCF-7 cells were pretreated for 30 min with 10 µg/ml KA prior to stimulation with TNF-alpha (20 ng/ml). Cells were harvested at the indicated time points, and total cell extracts were prepared. Ikappa B-alpha protein was detected by Western blot analysis as described under "Materials and Methods."

KA Directly Inhibits DNA-binding Activity of Active NF-kappa B Complex-- To further investigate the molecular target of KA, we examined the effect of KA on DNA-binding activity of activated NF-kappa B in vitro by EMSA. After stimulation of MCF-7 cells with TNF-alpha for 1.5 h, the nuclear extract was prepared and then incubated with KA in vitro. This compound significantly inhibited DNA-binding activity of activated NF-kappa B in a dose-dependent manner without affecting DNA-binding activity of AP-1 (Fig. 5, A and B). However, addition of 5 mM DTT in the reaction mixture completely reversed the inhibitory effect of KA (data not shown). It is important to mention that concentration to inhibit NF-kappa B activation in vitro is comparable with those of in cells. To address that KA inhibits active NF-kappa B in cells, RAW264.7 cells were pretreated with LPS for 30 min and subsequently treated with KA for indicated times. Nuclear extracts were analyzed for the DNA-binding activity of NF-kappa B by EMSA. Postincubation with KA after LPS stimulation significantly suppressed the DNA-binding activity of NF-kappa B (Fig. 5C). The inhibition was time-dependent. Identical results were obtained for Jurkat cells stimulated with TNF-alpha (data not shown). These observations suggested that KA directly interfered DNA-binding activity of NF-kappa B.


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  		Fig. 5.   Effect of KA on the DNA-binding activity of the activated NF-kappa B. The nuclear extracts were prepared from MCF-7 cells after the stimulation with TNF-alpha (20 ng/ml) for 90 min. The indicated amounts of KA were directly added to the reaction mixture to determine the effect of KA on DNA-binding activity of the activated NF-kappa B (A) or AP-1 (B) by EMSA. In lane kappa B, a 100-fold excess of unlabeled NF-kappa B oligonucleotide was added to the reaction mixture. In lane AP, a 100-fold excess of unlabeled AP-1 oligonucleotide was added to the reaction mixture. C, KA inhibits DNA-binding activity of activated NF-kappa B in cells. Lane 1 shows unstimulated RAW264.7 cells. In lane 2 cells were stimulated with 1 µg/ml LPS for 60 min without KA. In lanes 3-5, cells were treated with KA (10 µg/ml) for the last 30 (lane 3), 15 (lane 4), or 5 min (lane 5) during LPS stimulation for 60 min. The nuclear extracts were prepared and analyzed for DNA-binding activity of NF-kappa B by EMSA. An arrow indicates the position of the specific DNA·NF-kappa B complex. The amount of DNA·NF-kappa B complex formed was estimated by image scanning and is expressed in arbitrary units at the bottom of each panel.

KA Directly Inhibits DNA-binding Activity of p50-overexpressed Cells-- Next, we investigated whether KA inhibits DNA-binding activity of p50 or RelA subunit. We prepared nuclear extracts from p50-overexpressed cells, and then analyzed the effect of KA on the DNA-binding activity in vitro (Fig. 6, A and B). In nuclear extract from the p50-overexpressed cells, DNA-binding activity of NF-kappa B was not interfered by the addition of RelA- or c-Rel-antibody but p50-antibody. Similar to p50-antibody, KA significantly inhibited DNA-binding activity of NF-kappa B (Fig. 6A). Furthermore, preincubation of the p50-overexpressed cells with KA for 30 min significantly prevented DNA-binding activity of NF-kappa B (Fig. 6A, lane 7). An identical experiment was also carried out with the nuclear extracts from RelA overexpressed MCF-7 cells. DNA-binding activity of NF-kappa B was interfered by the addition of KA, however, the major form of NF-kappa B was a heterodimer of RelA and p50, and RelA homodimer was barely detectable (data not shown). We next explored how DTT suppressed the effect of KA on the DNA-binding activity of p50. Co-treatment of various concentrations of DTT with KA reduced the potency of KA in a dose-dependent manner (Fig. 6B, upper panel), and the effect of KA (10 mg/ml) was completely abolished by 5 mM DTT. However, the effect of KA was not reverted by a post-treatment of DTT even at concentration of 25 mM DTT (Fig. 6B, lower panel), suggesting that covalent modification of p50 with KA is stable and that the effect of KA is not a redox-sensitive manner. In both cases EMSA was performed in the presence of 0.1 mM DTT, which was not sufficient to revert the inhibition. To verify the above results, we prepared p50 and RelA proteins by in vitro translation, and then analyzed the effect of KA on DNA-binding activities of p50 and RelA molecules. KA preferentially inhibited DNA-binding activity of p50 homodimer rather than that of RelA homodimer (Fig. 6, C and D). Taken together, these results suggest that KA inhibits DNA-binding activity of NF-kappa B by directly modifying DNA-binding activity of p50 subunit and that the inhibitory effect of KA may arise from its interaction with cysteine residues in p50.


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  		Fig. 6.   KA inhibits DNA-binding activity of p50 subunit. MCF-7 cells were transiently transfected with a p50 expression vector, and nuclear extracts were prepared 48 h later after transfection and then analyzed for the effect of KA (A) or DTT (B) on the DNA-binding activity of NF-kappa B by EMSA. A, lane 1 shows untreated control. In lanes 2-7, EMSA was performed in the presence of antibody for RelA (lane 2), c-Rel (lane 3), p50 (lane 4), 5 µg/ml KA (lane 5), or 1 µg/ml KA (lane 6). In lane 7, prior to cell harvest, the cells were treated for 30 min with 5 µg/ml KA, nuclear extracts were prepared, and analyzed for DNA-binding activity of NF-kappa B by EMSA. B, co-treatment of DTT abolishes inhibitory effect of KA dose-dependently, however, post-treatment of DTT does not. The nuclear extracts were incubated with 10 µg/ml KA (lane 2-5) in the presence of various concentrations of DTT (upper panel) or incubated with 10 µg/ml KA followed by addition of various concentrations of DTT for last 10 min (lower panel). An arrow indicates the position of each specific band. In another experiment p50 or RelA proteins were prepared by in vitro translation as described under "Materials and Methods" and analyzed for the effect of KA on the DNA-binding activity of p50 (C) or RelA (D). Lane 1 shows untreated control. In lanes 2-9, EMSA was performed in the presence of RelA antibody (lane 2), p50 antibody (lane 3), a 100-fold excess of unlabeled NF-kappa B oligonucleotide (lane 4), a 100-fold excess of unlabeled AP-1 oligonucleotide (lane 5), vehicle (lane 6), 1 µg/ml KA (lane 7), 5 µg/ml KA (lane 8), and 10 µg/ml KA (lane 9). The amount of p50·DNA or p65·DNA complex formed was estimated by image scanning and is expressed in arbitrary units at the bottom of each panel. Results are average values of three experiments

KA Requires Cys-62 in the Inhibition of p50 DNA Binding and Forms a Covalent Adduct with p50-- p50 DNA-binding domain contains a cysteine residue that has been proposed to be a target for redox regulation (26). We therefore explored whether this cysteine was important for the effect of KA with protein purified from a p50 mutant with a Cys-62 right-arrow Ser mutation in comparison with that of wild type p50. As it can be observed, the DNA binding ability of a p50 mutant in which Cys-62 was substituted by serine was virtually unaffected by KA treatment (Fig. 7A, lower panel). Meanwhile, the DNA-binding ability of wild type p50 was inhibited by KA dose-dependently (Fig. 7A, upper panel), and again this inhibition was abolished by co-treatment of 5 mM DTT (Fig. 7A, middle panel). To gain insight into the interaction between KA and p50, wild type p50 was incubated with vehicle control (Me2SO) or KA and p50 was then purified with reverse-phase HPLC. Fractions corresponding to the major peaks from control and KA-treated wild type p50 were subsequently analyzed by mass spectrometry. The MALDI-TOF spectrum of control p50 showed a peak of m/z = 40,564, which is close to the calculated molecular mass (40,600) of the p50 construct used (30), together with peaks of m/z = 20,276 (doubly charged) and 81,266 (dimer of p50) (Fig. 7B, upper panel). The spectrum of KA-treated p50 showed peaks of m/z = 40,894, m/z = 20,426 (doubly charged), which are compatible with the formation of a covalent adduct between one molecule of p50 and one molecule of KA (expected m/z 40,950 and 20,475, respectively), and a peak of m/z = 81,266 (Fig. 7B, lower panel). These results do not exactly map the cysteine modified by KA, however, they suggest that KA covalently modifies p50 possibly targeting cysteine 62. 


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  		Fig. 7.   Effect of KA on the DNA-binding activity of wild type or C62S mutant p50 and mass spectrometry analysis of control and KA-treated p50. A, the recombinant wild type and mutant type p50 proteins purified as described under "Materials and Methods" were analyzed for the effect of KA on the DNA-binding activity in the presence or absence of DTT. Wild type (WT) or mutant p50 (Cys-62 right-arrow Ser) was incubated with the indicated concentrations of KA for 30 min at 37 °C before being analyzed by EMSA. The wild type p50 was also incubated with KA in the presence of 5 mM DTT. The amount of p50·DNA complex formed was estimated by image scanning and is expressed in arbitrary units. Mean values from three independent experiments performed in triplicate are shown: S.D. is indicated by bars. B, the recombinant wild type p50 proteins purified were treated with vehicle (control) or KA (10 mg/ml) for 30 min at 37 °C and subsequently purified by reverse-phase HPLC. The fractions of the p50 peak were collected and analyzed by MALDI-TOF mass spectrometry. Upper panel, control wild type p50; lower panel, KA-treated wild type p50.

KA Prevents the Induced Expression of the NF-kappa B Target Genes-- Recent studies demonstrate that a number of gene involved in inflammation and apoptosis is under control of NF-kappa B. It is well known that several antiapoptotic proteins such as Bfl-1/A1, c-IAP1, and c-IAP2 are regulated by NF-kappa B and block the induced apoptosis by TNF-alpha as well as chemotherapy agents such as etoposide (16, 23). Therefore, we examined the effect of KA on the TNF-alpha -induced expression of these antiapoptotic proteins (Fig. 8, A and B). After preincubation of HT-1080 and MCF-7 cells with KA with indicated concentrations for 30 min and subsequently stimulation with TNF-alpha for 3h, the induced expression of Bfl-1/A1 was analyzed by Northern blot. TNF-alpha induced a 15- and 7-fold increase of Bfl-1/A1 mRNA in HT-1080 and MCF-7 cells, respectively, however, the induced expression was blocked by KA in a dose-dependent manner. The suppression of TNF-alpha -induced expression of c-IAP1 (hiap-2) and c-IAP2 (hiap-1) by KA was investigated by Western blot analysis in MCF-7 cells (Fig. 8B). TNF-alpha induced a 4-fold increase of c-IAP1 proteins in MCF-7 cells. This induction was completely blocked by KA. Interestingly, KA suppressed the basal level of c-IAP1 protein expression at over 5 µg/ml. Also, KA suppressed induced expression of c-IAP2 protein. The same lysates were analyzed for Bcl-2 and Bax expression as control. Neither TNF-alpha nor KA modulated the expression of Bcl-2 and Bax in this cell line.


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  		Fig. 8.   Effect of KA on the expression of some antiapoptotic NF-kappa B target genes. A, expression of Bfl-1/A1. MCF-7 (1) and HT-1080 (2) cells were pretreated for 30 min with the indicated concentrations of KA and stimulated with TNF-alpha (20 ng/ml) for 3 h. Subsequently, total RNAs were isolated and Northern blot analysis was performed as described under "Materials and Methods." Ethidium bromide staining of the 18 S ribosomal RNA band on the gel was shown to demonstrate equal loading of RNA. B, expression of cIAP-1 and cIAP-2. MCF-7 cells were pretreated for 30 min with the indicated concentrations of KA followed by the stimulation with TNF-alpha (20 ng/ml) for 6 h. Subsequently, total cell lysates were prepared and Western blot analysis was performed as described under "Materials and Methods."

KA Sensitizes TNF-alpha -induced Apoptosis-- Because KA suppressed TNF-alpha -induced expression of antiapoptotic proteins, we next investigated whether this compound sensitizes TNF-alpha -induced cell death in MCF-7 cells (Fig. 9A). Cells were incubated with 20 ng of TNF-alpha for 48 h either in the presence or absence of KA and then examined for cell viability by the MTT method. TNF-alpha -induced cell death in MCF-7 was potentiated by KA in a dose-dependent manner. TNF-alpha alone induced cell death in ~17% of cells and KA (1 µg/ml) alone in ~26% of cells. However, the combination of TNF-alpha and KA induced cell death in over 80% of cells. We investigated to see if KA affects TNF-alpha -induced caspase-8 activity. Treatment of MCF-7 cells with KA or TNF-alpha alone (20 ng/ml) showed similar degree of caspase-8 activity, however, KA significantly induced caspase-8 activity by co-incubation with TNF-alpha in a dose-dependent manner (Fig. 9B).


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  		Fig. 9.   Effect of KA on TNF-alpha -induced apoptosis in MCF-7 cells. MCF-7 cells were pretreated with the indicated concentrations of KA for 30 min and treated with TNF-alpha (20 ng/ml). After 48-h incubation, cell viability (A) and caspase-8 activity (B) were determined as described under "Materials and Methods." Mean values from two independent experiments performed in triplicate are shown; bar indicates the S.D. Statistical significance (p < 0.001) judged by paired Student's t test is marked with an asterisk.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whole plants of I. japonicus (Labiatae) have been used in traditional oriental medicine as a remedy for gastrointestinal disorders, cancer, and inflammatory diseases and a rich source of kaurane diterpenes (17, 18). Despite various pharmacological activities, its molecular mechanism has not been sufficiently explained. In previous study, we isolated KA and three other kaurane diterpenes (Fig. 1) from the plant as inhibitors of production of inflammatory mediators and NF-kappa B activation induced by LPS (19), indicating that these activities of those compounds could explain, in part, its diverse pharmacological activities such as anticancer and anti-inflammation. However, it remained to be elucidated how the most abundant, a kaurane diterpene KA inhibits NF-kappa B activation. Here we showed that KA inhibited NF-kappa B by directly targeting on the DNA-binding activity of p50 subunit. KA prevents neither induced degradation of Ikappa B-alpha nor nuclear translocation of NF-kappa B following stimulation but inhibits NF-kappa B activation by various stimuli (Figs. 3 and 4). Also, a basal level of DNA-binding activity of NF-kappa B is significantly inhibited. These observations led us to formulate a hypothesis that KA may directly modify DNA-binding activity of NF-kappa B. To test this hypothesis, KA was incubated with activated NF-kappa B in vitro (Fig. 5A) and in cells (Fig. 5C). KA significantly inhibited DNA-binding activity of activated NF-kappa B without inhibiting that of AP-1 (Fig. 5B). Concentrations to inhibit DNA-binding activity of NF-kappa B in vitro were comparable to those in which KA inhibits NF-kappa B activation by various inducers in cells. We also proposed that KA directly targets the p50 molecule based on the finding that it selectively inhibited DNA binding activity of the p50 homodimer not the RelA homodimer (Fig. 6, A, C, and D). The p50 subunit lacks a transactivation domain but serves as a regulatory subunit modulating the DNA binding affinity of RelA, which is a critical transactivation subunit of NF-kappa B (3, 4). p50 possesses a critical cysteine residue in its DNA-binding domain. This cysteine (Cys-62 in human p50) has been proposed to be the target for inhibition of DNA-binding activity of NF-kappa B by NO, either through S-nitrosylation (24) or NO-induced S-glutathionylation (21). Indeed, this cysteine can be S-nitrosylated in vitro and in vivo to mediate the effect of redox changes on NF-kappa B activity (26).

NF-kappa B is also inhibited by a modification of the cysteine 62 in the p50 molecule with N-ethylmaleimide or other reagents such as cyclopentenone prostaglandin (27-30). Therefore, a potential role of p50 as a target for the inhibitory action of KA on the NF-kappa B pathway could be hypothesized. We showed that the inhibitory effect of KA on DNA-binding activity of active p50 was completely suppressed by the addition of more than 5 mM DTT (Fig. 6B, lane 4 and 5 of upper panel), and this suppression was a dose-dependent fashion. However, post-treatment of DTT did not suppress the inhibitory effect of KA on DNA-binding activity of active p50 (Fig. 6B, lower panel). We also found that KA reacted with the sulfhydryl group of cysteine easily to give a thioadduct but not with lysine or serine (31).2 Indeed, a p50 mutant with a C62S mutation was not inhibited by KA, indicating that the effect of KA was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by KA was further demonstrated by mass spectrometry analysis that showed an increase by mass unit 330 (calculated mass of KA, 350) in the molecular mass of KA-treated p50. Furthermore, the covalent modification was not reverted with the post-treatment of DTT (Fig 6B, lower panel), indicating that the inhibition by DTT on the co-treatment with KA is due to entrapping of KA by forming a thioadduct with excess DTT. Therefore, it is highly probable that KA would covalently modify cysteine 62 in the p50 molecule through a Michael-type reaction, although we did not map precisely the cysteine modified by KA. Recently, cyclopentenone prostaglandin 15d-PGJ2 has been demonstrated to inhibit DNA binding of NF-kappa B by direct modification of cysteine 62 of p50 (30). Sesquiterpene lactones such as parthenolide and helenarin have also exerted their potent anti-inflammatory activity by inhibiting activation of NF-kappa B (32, 33). Molecular target of parthenolide has been demonstrated to be the cysteine 179 of Ikappa B kinase beta  (34), but another report has proposed that parthenolide would modify the cysteine 38 of RelA (35). The same authors have proposed that helenalin would bind to cysteines 38 and 120 of RelA based on the EMSA and computer modeling of the RelA homodimer (33, 35). What would make anti-inflammatory compounds such as helenarin, parthenolide, 15d-PGJ2, and KA selective in the modification of cysteine residue in the different target molecules such as Ikappa B kinase beta , p50, or RelA? This difference may arise not only from chemical environment of target sulfhydryl group in the protein but also from structural environment of Michael acceptor in the NF-kappa B inhibitors (35). Helenarin and parthenolide contain a lactone ring conjugated with an exomethylene group, which can react with a biological nucleophile, especially the sulfhydryl group of cysteine residue by Michael type reaction. 15d-PGJ2 contains two possible reactive Michael acceptors, namely a cyclopentenone ring and a doubly conjugated exomethylene functional group to the carbonyl group of the pentenone ring. This would be a possible explanation for the formation of a bimolecular adduct by 15-dPGJ2 with two different cysteines in p50 (30). KA also contains an exomethylene group conjugated with a carbonyl group of cyclopentenone in a bicyclic ring system, which, however, possesses a quite different structural feature from parthenolide and helenarin, which have a fused alpha -methylene-gamma -lactone ring. These differences among NF-kappa B inhibitors could be attributed to their selective specificity toward target cysteines in Ikappa B kinase beta , RelA, or p50. Another group of kaurane diterpene compounds such as foliol and ent-kaur-19-oic acid, in which a fused five-membered ring contains only the exomethylene group without a conjugated carbonyl group, has been shown to inhibit NF-kappa B activation by interfering with NF-kappa B-inducing kinase activity (36).

Several studies have demonstrated an essential role for NF-kappa B in preventing apoptosis induced by TNF-alpha and chemotherapy agents. In these studies, cells were made sensitive to TNF-alpha - and chemotherapy-induced apoptosis through inhibition of NF-kappa B activity (23, 37, 38). It is now clear that several downstream effectors of NF-kappa B activation have been known to suppress TNF-alpha - and chemotherapy-induced apoptosis. These include TRAF-1, TRAF-2, c-IAP1, c-IAP2, and Bfl-1/A1. KA clearly suppressed the induced expression of c-IAP1, c-IAP2, and Bfl-1/A1 by TNF-alpha without affecting Bax and Bcl-2, whose expression is not under control of NF-kappa B (Fig. 8). Interestingly, KA completely inhibited even basal level expression of cIAP-1. Further studies are needed to show how KA regulates the expression of c-IAP1 in MCF-7 cells. We were also able to demonstrate that KA sensitizes cytotoxic potential of TNF-alpha as assessed by MTT and that this effect is likely associated with caspase-8 activity (Fig. 9). It has been demonstrated that the induction of c-IAP1 and c-IAP-2 by NF-kappa B suppressed caspase-8 activation, resulting in cell survival (13). This is consistent with our results that KA blocked the DNA-binding activity of NF-kappa B and thereby suppressed TNF-alpha -induced expression of both c-IAP1 and c-IAP2 and, in turn, enhanced TNF-alpha -induced caspase-8 activity. These results demonstrate that tumor cells are sensitized to TNF-alpha - and chemotherapy-induced apoptosis through inhibition of NF-kappa B.

The relevance of most of NF-kappa B target genes makes this transcription factor an interesting therapeutic target for the identification of inhibitors. One group of NF-kappa B inhibitors exerts its inhibitory effects by antioxidative properties (39-41). These inhibitors include N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, or curcumin. Another group of inhibitors interferes with the induced degradation of Ikappa B family members by affecting the 26S proteasome or inhibiting Ikappa B kinase complex (42, 43). Another group of inhibitors exerts their effects only in the cell nucleus by impairing the transcriptional activity of NF-kappa B already bound to DNA. Examples are PG490 (triptolide), and, at least in some cell type, glucocorticoids (25, 44). In addition, a group of inhibitors interferes with the DNA-binding activity of NF-kappa B by directly targeting the NF-kappa B subunits. This is the case of helenalin, which is a specific inhibitor of DNA-binding activity of RelA subunit (33). KA could be added to this group as a specific inhibitor of DNA-binding activity of NF-kappa B by directly targeting p50. Importantly, KA can inactivate the activated NF-kappa B complex. This property is crucial for the treatment of various diseases such as inflammation, where previously activated NF-kappa B is sustaining the pathological processes of diseases and needs to be inactivated. Taken together, we have shown that KA inhibits the NF-kappa B signal cascade by directly targeting DNA binding of the p50 subunit in the activated NF-kappa B and the induced expression of NF-kappa B target genes. Based on our results, KA could serve as an interesting lead compound for the development of new, potent anti-inflammatory or anticancer agents. Furthermore, this study extends our understanding of the molecular mechanisms underlying the anti-inflammatory and anticancer activities of traditional medicinal plants, which abundantly contain kaurane diterpenes.

    ACKNOWLEDGEMENTS

We thank Drs. M. Jung for the kind gift of RelA construct, J. Lee for p50 construct, S. Hong for Bfl-1/A1 cDNA, and Sung-Goo Park and Eun Ok Yun for help with mass spectrometry analysis. We deeply thank Dr. D. Perez-Sala for the generous gift of recombinant wild type and mutant DNA-binding domains of human p50 (amino acids 36-385).

    FOOTNOTES

* This work was supported by Grant PF002113-01 from the Plant Diversity Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Korean government.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 82-42-860-4360; Fax: 82-42-860-4595; E-mail: jjlee@mail.kribb.re.kr.

Published, JBC Papers in Press, March 4, 2002, DOI 10.1074/jbc.M201368200

2 J.-H. Lee, T. H. Koo, B. Y. Hwang, and J. J. Lee, unpublished result.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; KA, kamebakaurin; AP-1, activator protein-1; LPS, lipopolysaccharide; TNF-alpha , tumor necrosis factor-alpha ; PMA, phorbol 12-myristate 13-acetate; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 15d-PGJ2, 15-deoxy-Delta 12,14-prostaglandinJ2.

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RESULTS
DISCUSSION
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