0022-3565/06/3161-271–278$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics JPET 316:271–278, 2006 Vol 316, No 92486/3067111 Printed in U.S.A Blockade of Nuclear Factor-B Signaling Pathway and Anti-Inflammatory Activity of Cardamomin, a Chalcone Analog from Alpinia conchigera Jeong-Hyung Lee, Haeng Sun Jung, Phan Minh Giang, Xuejun Jin, Sangku Lee, Phan Tong Son, Dongho Lee, Young-Soo Hong, Kyeong Lee, and Jung Joon Lee Anticancer Research Laboratory (J.-H.L., H.S.J., X.J., D.L., Y.-S.H., K.L., J.J.L.) and Laboratory of Immune Modulator (S.L.), Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea; and Faculty of Chemistry, Vietnam National University, Hanoi, Vietnam (P.M.G., P.T.S.) ABSTRACT Nuclear factor-B (NF-B) and the signaling pathways that regulate its activity have become a focal point for intense drug discovery and development efforts NF-B regulates the transcription of a large number of genes, particularly those involved in immune, inflammatory, and antiapoptotic responses In our search for NF-B inhibitors from natural resources, we identified cardamomin, 2Ј,4Ј-dihydroxy-6Ј-methoxychalcone, as an inhibitor of NF-B activation from Alpinia conchigera Griff (Zingiberaceae) In present study, we demonstrated the effect of cardamomin on NF-B activation in lipopolysaccharide (LPS)stimulated RAW264.7 cells and LPS-induced mortality This compound significantly inhibited the induced expression of NF-B reporter gene by LPS or tumor necrosis factor (TNF)-␣ in a dose-dependent manner LPS-induced production of TNF-␣ and NO as well as expression of inducible nitric-oxide synthase and cyclooxygenase-2 was significantly suppressed by the treatment of cardamomin in RAW264.7 cells Also, cardamomin NF-B 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 (Ghosh et al., 1998) In most cell types, the pleiotropic-inducible form of NF-B is a heterodimer composed of p50 and RelA/p65 (Baeuerle and Baltimore, 1989) RelA/p65 contains a C-terminal transactivation domain in This study was partially supported by a grant from Plant Diversity Research Center of 21st Century Frontier Research Program and Korea Research Institute of Bioscience and Biotechnology Research Initiative Program funded by Ministry of Science and Technology of Korean government Article, publication date, and citation information can be found at http://jpet.aspetjournals.org doi:10.1124/jpet.105.092486 inhibited not only LPS-induced degradation and phosphorylation of inhibitor B␣ (IB␣) but also activation of inhibitor B (IB) kinases and nuclear translocation of NF-B Further analyses revealed that cardamomin did not directly inhibit IB kinases, but it significantly suppressed LPS-induced activation of Akt Moreover, cardamomin suppressed transcriptional activity and phosphorylation of Ser536 of RelA/p65 subunit of NF-B However, this compound did not inhibit LPS-induced activation of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun NH2-terminal kinase, but significantly impaired activation of p38 mitogen-activated protein kinase We also demonstrated that pretreatment of cardamomin rescued C57BL/6 mice from LPS-induced mortality in conjunction with decreased serum level of TNF-␣ Together, cardamomin could be valuable candidate for the intervention of NF-B-dependent pathological condition such as inflammation addition to the N-terminal Rel-homology domain, thus serving as a critical transactivation subunit of NF-B p50 lacks a transactivation domain, and it is believed to serve as a regulatory subunit modulating the DNA binding affinity of RelA/p65 (Schmitz and Baeuerle, 1991; Ballard et al., 1992) The p50-RelA/p65 NF-B heterodimer is normally sequestered in the cytoplasmic compartment by physical association with inhibitory proteins, including IB␣ and related proteins (Henkel et al., 1992; Baldwin, 1996) Activation of NF-B is a multistep process, including IB kinase (IKK) complex activation, that not only facilitates movement of NF-B heterodimer from the cytoplasm to the nucleus but also can phosphorylate the NF-B RelA/p65 subunit (Viatour et al., 2005) This activation requires phosphor- ABBREVIATIONS: NF-B, nuclear factor B; IB␣, inhibitor of B␣; IB, inhibitor of B; IKK, IB kinase; COX, cyclooxygenase; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase; SAPK/JNK, stressactivated protein kinase/c-Jun NH2-terminal kinase; EMSA, electrophoretic mobility shift assay; DTT, dithiothreitol; AP-1, activator protein-1; MAP, mitogen-activated protein; PI3K, phosphatidylinositol 3-kinase 271 Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 Received July 11, 2005; accepted September 21, 2005 272 Lee et al Materials and Methods Cell Culture and Chemicals RAW264.7 cells and human embryonic kidney 293 cells were maintained in Dulbecco’s modified essential medium (Invitrogen, Carlsbad, CA) supplemented with 100 units/ml penicillin/100 g/ml streptomycin (Invitrogen) and 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT) Cardamomin was isolated from dried roots of the fresh rhizomes of A conchigera and confirmed its structure in comparison with a previous report (Itokawa et al., 1981) The structure of cardamomin is shown in Fig The purity of cardamomin is more than 98% in high-performance liquid chromatography analysis Measurement of NO, TNF-␣, and Cell Viability Assay RAW264.7 cells grown on 100-mm culture dish were harvested and Fig Chemical structure of cardamomin seeded in 96-well plates at ϫ 105 cells/well for NO or at ϫ 104 cells/well for TNF-␣ The plates were pretreated with various concentrations of cardamomin for 30 and then incubated for another 24 h with or without g/ml LPS Nitrite concentration in the culture supernatant was measured by the Griess reaction The amount of TNF-␣ in the culture supernatant was measured using a TNF-␣ enzyme-linked immunosorbent assay kit (Genzyme, Cambridge, MA) Cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based colorimetric assay Plasmids, Transfections, and Luciferase Assay A pNFBLuc and a pAP-1-Luc were obtained from Stratagene (La Jolla, CA) A vector encoding a fusion protein between the DNA binding domain of Gal4 and activation domains of RelA/p65, Gal4-RelA268 –551 and a 5XGal4-luciferase reporter gene were described previously (Lee et al., 2004) Expression vectors for IKK␣ and IKK were kindly provided from Dr M Karin (University of California, San Diego, San Diego, CA) Transfections were performed using Lipofectamine Plus reagent (Invitrogen) Luciferase assay was performed using DualLuciferase reporter assay system according to the instructions of the manufacturer (Promega, Madison, WI) Luciferase activity was determined in Microlumat Plus luminometer (Berthold Technologies, Bad Wildbad, Germany) by measuring light emission for 10 s The results were normalized to the activity of Renilla expressed by cotransfected Rluc gene under the control of a constitutive promoter Western Blot Analysis Proteins were extracted from cells in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, mM EDTA, mM phenylmethylsulfonyl fluoride, g/ml leupeptin, mM sodium vanadate, and 150 mM NaCl) In certain experiments, the cytoplasmic extracts were prepared using NE-PER reagent (Pierce Chemical, Rockford, IL) An aliquot of protein extracts was used to determine protein concentration by the Bradford method Fifty micrograms of protein per lane was separated by SDS-polyacrylamide gels and followed by transferring to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA) The membrane was blocked with 5% skim milk and then incubated with the corresponding antibody Antibodies for IB␣, phospho-specific IB␣, were obtained from New England Biolabs (Beverly, MA) Antibodies for Akt, phospho-(Ser473)-specific Akt, ERK1/2, phosphospecific ERK1/2, p38, phospho-specific p38, SAPK/JNK, phosphospecific SAPK/JNK, phospho-(Ser536)-specific RelA, RelA, phospho (Ser180 for IKK␣, Ser181 for IKK)-specific IKK␣/, and IKK␣/ were purchased from Cell Signaling Technology Inc (Beverly, MA) Antibody for COX-2 was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) Antibody for iNOS was from Upstate Biotechnology (Lake Placid, NY) and antibody for ␣-tubulin was from SigmaAldrich (St Louis, MO) After binding of an appropriate secondary antibody coupled to horseradish peroxidase, proteins were visualized by enhanced chemiluminescence according to the instructions of the manufacturer (GE Healthcare, Little Chalfont, Buckinghamshire, UK) Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay (EMSA) was performed as described previously (Lee et al., 2002) In brief, 30 before stimulation, RAW264.7 cells were preincubated with the indicated concentrations of cardamomin at 37°C Then, cells were stimulated with g/ml LPS for the indicated times, washed twice with ice-cold phosphate-buffered saline, and then nuclear extracts were prepared Nuclear extracts were incubated for 20 at room temperature with a gel shift binding buffer [5% glycerol, mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, and 50 g/ml poly(dI-dC)] and 32 P-labeled oligonucleotide The DNA-protein complex formed was separated on 4% native polyacrylamide gels, and the gel was transferred to Whatman MM paper (Clifton, NJ), dried, and exposed to X-ray film In Vitro IKK Assay Human 293 cells grown in 100-mm plates were transfected with expression vectors for IKK␣ or IKK and incubated for 48 h The cells transfected with IKK␣ or IKK were stimulated with 20 ng/ml TNF-␣ for 20 and then washed three Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 ylation by IKK of IB proteins at specific serine residues, which target these proteins for ubiquitin conjugation and degradation by the 26S proteasome (Ghosh and Karin, 2002) The IKK complex contains two catalytic subunits, IKK␣ and IKK, and a regulatory subunit, IKK␥ (DiDonato et al., 1997; Zandi et al., 1997; Zandi and Karin, 1999) Activation of IKK is mediated by phosphorylation through various upstream kinases such as NF-B-inducing kinase, NF-B-activating kinase, Akt, and protein kinase C, which are involved in cellular signaling in response to proinflammatory stimuli (Ozes et al., 1999; Romashkova and Makarov, 1999; Viatour et al., 2005) Following activation, the NF-B heterodimer is rapidly translocated to the nucleus, where it activates the transcription of target genes These include various inflammatory cytokines, genes encoding cyclooxygenase-2 (COX-2), inducible nitric-oxide synthase (iNOS), immunoreceptors, cell adhesion molecules, and antiapoptotic genes (Wu and Kral, 2005) As part of our continuing search for NF-B inhibitors from natural products, a chalcone derivative, cardamomin (2Ј,4Јdihydroxy-6Ј-methoxychalcone), was identified as an inhibitor of NF-B activation from the spicy and herbaceous plant Alpinia conchigera Griff (Zingiberaceae) This plant growing in northeastern Vietnam has been used in Vietnamese traditional medicine for the treatment of inflammatory diseases (Vo, 1997) We here describe the anti-inflammatory effect of cardamomin This compound inhibited the induced activation of NF-B and expression of NF-B target genes such as COX-2 and iNOS in LPS-stimulated RAW264.7 cells Cardamomin inhibited not only the degradation of IB␣ but also the transcriptional activity and phosphorylation of RelA/p65 Furthermore, we also described that pretreatment of cardamomin protected the mice from death caused by LPS administration A Chalcone, Cardamomin, Inhibits NF-B Signaling Results Cardamomin Inhibits NF-B Activation by TNF-␣ and LPS In an effort to identify NF-B inhibitors from herbal medicines, we have identified a chalcone derivative, cardamomin, from a traditional Vietnamese medicinal plant, A conchigera (Fig 1) To investigate the effect of this compound on the induced NF-B activation by various stimuli, we performed a NF-B reporter gene assay (Fig 2A) Cardamomin dose dependently inhibited the LPS-induced expression of NF-B reporter gene construct with IC50 value of 1.2 M This compound also inhibited TNF␣-induced expression of NF-B reporter gene construct to a similar extent In contrast, cardamomin weakly inhibited the LPS-induced expression of AP-1 reporter gene construct with IC50 values more than 30 M (Fig 2B) Cardamomin Inhibits the Expression of NF-B Target Genes Next, we investigated the effect of cardamomin on the LPS-induced TNF␣ and NO production in RAW264.7 cells After RAW264.7 cells were stimulated with g/ml LPS for 24 h in the presence or absence of various concentrations of cardamomin, the amount of TNF-␣ and NO in the culture supernatant was measured (Fig 3) Cardamomin suppressed the LPS-induced TNF-␣ and NO production in a dose-dependent manner with IC50 values of 1.0 and 1.5 M, Fig Effect of cardamomin on NF-B- and AP-1-dependent reporter gene expression in RAW264.7 cells A, RAW264.7 cells, transiently transfected with a NF-B-dependent reporter gene, were grown for 48 h and then pretreated for 30 with the indicated concentrations of cardamomin followed by stimulation for h with LPS (1 g/ml) or TNF-␣ (20 ng/ml) B, RAW264.7 cells, which were transiently transfected with a AP-1-dependent reporter gene, were grown for 48 h and then pretreated for 30 with the indicated concentrations of cardamomin followed by stimulation for h with LPS (1 g/ml) Luciferase activities were determined as described under Materials and Methods Bars indicate the S.D Statistical significance judged by paired Student’s t test is marked with an asterisk (ءء, p Ͻ 0.01; ء, p Ͻ 0.05) respectively To further confirm the NF-B inhibitory activity of cardamomin, we analyzed the effect of cardamomin on expression of some NF-B target genes such as iNOS and COX-2 After RAW264.7 cells were stimulated for 18 h with g/ml LPS in the presence of various concentrations of cardamomin, total cell lysates were prepared, and then immunoblot analysis was performed LPS-induced iNOS and COX-2 expression were suppressed by cardamomin in a dosedependent manner (Fig 3C) Cardamomin Interferes with the Degradation of IB␣ and DNA Binding Activity of NF-B Since degradation of IB proteins is an essential step for NF-B activation by various stimuli, we examined the effect of cardamomin on the induced degradation and phosphorylation of IB␣ protein by LPS (Fig 4A) RAW264.7 cells were pretreated with 30 M cardamomin for 30 and subsequently stimulated with LPS for the indicated times Cytoplasmic extracts were analyzed in the presence of IB␣ with Western Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 times with ice-cold PBS containing mM Na3VO4 and mM EDTA Cell lysates prepared in lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 0.25% Triton X-100, mM EDTA, mM EGTA, 10 mM -glycerophosphate, 10 mM NaF, 10 mM 4-nitrophenylphosphate, 300 M Na3VO4, mM benzamidine, M phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, g/ml leupeptin, g/ml pepstatin, and mM DTT) were incubated with IKK␣ or IKK antibody on ice for h Protein A- or protein G-conjugated agarose beads were then added and incubated for additional h at 4°C Kinase assays were performed by incubating the immune complexes in kinase reaction buffer (20 mM HEPES, pH 7.7, mM MgCl2, mM MnCl2, 10 M ATP, 10 mM -glycerophosphate, 10 mM NaF, 300 M Na3VO4, mM benzamidine, M phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, g/ml leupeptin, g/ml pepstatin, and mM DTT) with Ci of [␥-32P]ATP and bacterially expressed glutathione S-transferase-IB␣ in a reaction volume of 20 l for 30 at 30°C in the presence of various concentrations of cardamomin Samples were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis, autoradiography, and Western blotting Effect of Cardamomin on LPS-Induced Serum Level of TNF-␣ and Mortality in C57BL/6 Mice Male C57BL/6 mice (20 – 22g) were housed in plastic cages and maintained at 22 Ϯ 2°C and 50 – 60% relative humidity with 12-h light-dark cycles throughout the experiment The animals were maintained in these facilities for at least week before the experiment Mice (12 mice per group) were twice injected intraperitoneally with cardamomin (50 or 20 mg/kg) or control vehicle at 12 and h before LPS (Escherichia coli 055:B5; Sigma-Aldrich) One hour after the second cardamomin treatment, mice were given an intraperitoneally injection of LPS (25 mg/kg body weight) The survival of the mice was monitored for days after injection of LPS, after which no further loss of mice occurred For the determination serum level of TNF-␣, the mice (n ϭ 10) were injected cardamomin as described above One and a half hours after injection of LPS, mice were sacrificed Serum samples from mice were determined the level of TNF-␣ using a TNF-␣ enzyme-linked immunosorbent assay kit (Genzyme, Cambridge, MA) Statistical Analysis Statistical differences were determined by paired Student’s t test Statistical differences in survival curves among the groups of mice were analyzed by log rank test 273 274 Lee et al blots IB␣ was almost completely degraded in 15 after stimulation with LPS and resynthesized in 30 However, preincubation with cardamomin significantly prevented the induced degradation of IB␣ protein at and 15 min, and resynthesis of IB␣, which is under control of NF-B, was also significantly suppressed by cardamomin Furthermore, determination of phosphorylation of IB␣ by Western blot using a phospho-specific IB␣ antibody revealed that cardamomin significantly affected the LPS-induced phosphorylation of IB␣ at Next, we measured the effect of cardamomin on LPS-induced DNA-binding activity of NF-B RAW264.7 cells were pretreated with 30 M of cardamomin for 30 min, and nuclear extracts were prepared from the cells stimulated with LPS for the indicated times DNA binding activity of NF-B in each nuclear extract was measured by electrophoretic mobility shift assays (Fig 4B) Stimulation of Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 Fig Effect of cardamomin on the inflammatory NF-B target genes A and B, RAW264.7 cells were pretreated with the indicated concentrations of cardamomin (CD) for 30 and treated with LPS (1 g/ml) After 24-h incubation, the amounts of TNF-␣ (A) and NO (B) in culture supernatants were measured as described under Materials and Methods Mean values from two independent experiments performed in triplicate are shown; bars indicate the S.D Statistical significance (p Ͻ 0.01) judged by paired Student’s t test is marked with an asterisk (ءء, p Ͻ 0.01; ء, p Ͻ 0.05) C, RAW264.7 cells were pretreated for 30 with the indicated concentrations of cardamomin followed by stimulation with g/ml LPS for 18 h Subsequently, total lysates were isolated, and Western blot analysis was performed as described under Materials and Methods The bottom gel represents ␣-tubulin to show the equal loading of cell lysates Fig Effect of cardamomin on the LPS-induced degradation and phosphorylation of IB␣ and DNA-binding activity of NF-B A, RAW264.7 cells were pretreated for 30 with cardamomin (CD) before stimulation with g/ml LPS Cells were harvested at the indicated time points, and the cytoplasmic extracts were prepared IB␣ protein (top) and phospho-IB␣ protein (bottom) were detected by Western blot analysis as described under Materials and Methods The bottom gel represents ␣-tubulin to show the equal loading of cell lysates B, RAW264.7 cells were preincubated for 30 with CD and then stimulated with g/ml LPS for the indicated times Subsequently nuclear extracts were prepared and tested for DNA binding of activated NF-B by EMSA as described under Materials and Methods A representative autoradiographic exposure is shown, and the arrow indicates the location of the DNA-NF-B complex The bottom graph represents the amount of NF-B complex estimated by image scanning and is expressed in arbitrary units Results are average values of three independent experiments C, nuclear extract was prepared from RAW264.7 cells stimulated with g/ml LPS for h The indicated amounts of kamebakaurin (KA) or CD were directly added to the reaction mixture to determine the effect of the compound on DNAbinding activity of the activated NF-B by EMSA The representative autoradiographic exposure is shown, and the arrow indicates the location of the DNA-NF-B complex The bottom graph represents the amount of NF-B complex estimated by image scanning and is expressed in arbitrary units Results are average values of three independent experiments A Chalcone, Cardamomin, Inhibits NF-B Signaling RAW264.7 cells were pretreated with cardamomin for 30 and then stimulated with g/ml LPS for the indicated times (Fig 6A) Stimulation of RAW264.7 cells with LPS significantly induced the phosphorylation of Akt with the maximum at 15 In contrast, pretreatment of cardamomin suppressed the LPS-induced phosphorylation of Akt Since it has been also reported that Akt regulates transcriptional activity of NF-B through a mechanism dependent on phosphorylation of the RelA/p65 subunit of NF-B (Madrid et al., 2000; Mayo et al., 2002), we also examined the effect of cardamomin on the transcriptional activity of RelA/p65 A plasmid encoding a fusion protein of the transactivating domains of RelA, Gal4-RelA268 –551, with the DNA binding domain of the yeast transcription factor Gal4, was transfected into 293 cells along with a luciferase reporter containing upstream Gal4 binding sites Cardamomin suppressed transcriptional activity of the transactivation domain of RelA/p65 in a dose-dependent manner (Fig 6B) Consistent with this result, cardamomin significantly suppressed the LPS-induced phosphorylation of RelA/p65 at Ser536, which is required for efficient transcriptional activation of NF-B (Fig 6C) Effect of Cardamomin on the LPS-Induced Phosphorylation of Mitogen-Activated Protein Kinases The MAP kinases such as ERK1/2, p38, and JNK/SAPK are known to be activated by LPS and are important for the activation of NF-B (Nakano et al., 1998) To investigate whether cardamomin inhibits the LPS-induced activation of MAP kinases, we examined the effect of cardamomin on the LPS-induced phosphorylation of ERK1/2, JNK/SAPK, and p38 in RAW264.7 cells using immunoblot analysis (Fig 7) Cardamomin did not suppress the LPS-induced activation of ERK1/2 and SAPK/JNK The LPS-induced phosphorylation of p38, however, was significantly affected by cardamomin Effect of Cardamomin on LPS-Induced TNF-␣ Production and Mortality in C57BL/6 Mice Since activation of NF-B has been implicated in sepsis, we investigated Fig Effect of cardamomin on the LPS-induced activation of IKK and the activity of IKK A, RAW264.7 cells were pretreated for 30 with cardamomin (CD) before stimulation with g/ml LPS Cells were harvested at the indicated time points, and total cell extracts were prepared phospho-IKK␣/ was detected by Western blot analysis as described under Materials and Methods The bottom gel represents IKK␣ to show the equal loading of cell lysates B, IKK immunocomplex was prepared from 293 cells transfected with IKK␣ or IKK 15 after TNF-␣ stimulation In vitro kinase assays were performed with GST-IB␣ and [␥-32P]ATP in the presence or absence of various doses of cardamomin To show the equal amount of immunocomplex in each reaction, the bottom gels represent IKK␣ or IKK detected with Western blot Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 RAW264.7 cells with LPS strongly induced DNA binding activity of NF-B In contrast, treatment of cardamomin significantly suppressed the induced DNA-binding activity of NF-B by LPS at early time points, especially at and 15 After 30 min, however, the inhibitory effect of cardamomin on DNA binding activity of NF-B was not as effective as that of the early time point (Fig 4B) To examine whether cardamomin directly affects DNA binding activity of NF-B, we examined the effect of cardamomin on DNA binding activity of activated NF-B in vitro by EMSA (Fig 4C) As a positive compound, we used kamebakaurin, which is known to inhibit DNA binding activity of p50 subunit of NF-B (Lee et al., 2002) In contrast to kamebakaurin, cardamomin did not significantly inhibited DNA binding activity of activated NF-B Cardamomin Suppresses LPS-Induced Activation of IKK but Does Not Directly Inhibit IKK Since IKK complex acts as a convergence point for a variety of upstream activating kinases and plays a critical role in degradation of IB proteins, we determined whether cardamomin inhibits LPS-induced activation of IKK using phospho-specific antibody (Fig 5A) Treatment of cardamomin significantly inhibited the LPS-induced phosphorylation of IKK␣ and IKK Therefore, we examined whether cardamomin directly inhibited IKK activity with in vitro kinase assay IKK immunoprecipitates were prepared from 293 cells transfected with IKK␣ or IKK after TNF-␣ stimulation, and then in vitro kinase assays were conducted in the presence of various concentrations of cardamomin (Fig 5B) Cardamomin did not inhibit IKK␣ and IKK activity up to 30 M Together, these results suggest that cardamomin could suppress LPS-induced activation of IKK through inhibiting an upstreamactivating kinase of IKK Cardamomin Inhibits Akt Activation in LPS-Stimulated RAW264.7 Cells To further investigate how cardamomin prevents NF-B activation, we examined the effect of cardamomin on the LPS-induced activation of Akt 275 276 Lee et al whether cardamomin protected C57BL/6 mice from LPS-induced lethality Mice were injected with either cardamomin or vehicle, and h later, they were challenged with intraperitoneally with 25 mg/kg LPS (Fig 8A) Intraperitoneal injection of LPS into control mice resulted in death of all the mice (n ϭ 12) within days In contrast, all mice rescued from death by administration of 50 mg/kg cardamomin Next, we determined the effect of cardamonin on LPSinduced serum level of TNF-␣ in C57BL/6 mice (Fig 8B) One and a half hours after LPS injection, serum level of TNF-␣ reached a mean 1420 Ϯ 297 pg/ml (n ϭ 10) Preadministration of cardamomin at a dose of 50 mg/kg decreased their serum level of TNF-␣ to 354 Ϯ 43 pg/ml (n ϭ 10; p Ͻ 0.01) However, this compound did not significantly inhibit LPSinduced mortality of C57BL/6 mice when given as a posttreatment Discussion The rhizomes of A conchigera have been used as appetizer, digestive stimulant, analgesic, and as an anti-inflammatory traditional medicine in Vietnam (Vo, 1997) Despite of its various pharmacological activities, the molecular mechanism has not been sufficiently explained In present study, we identified cardamomin as an inhibitor of NF-B activation and investigated how this compound suppressed NF-B activation Cardamomin inhibited the induced activation of NF-B and expression of NF-B target genes such as COX-2, TNF-␣, and iNOS in LPS-stimulated RAW264.7 cells Furthermore, cardamomin inhibited not only the degradation of IB␣ and DNA binding activity of NF-B but also phosphorylation of RelA/p65 and activation of Akt It is well known that the critical step in NF-B activation is IB␣ phosphorylation at Ser-32 and Ser-36 by IKK complex (Mercurio et al., 1997) IKK complex is found to be activated by a number of different protein kinases (Viatour et al., 2005) Since cardamomin did not directly inhibit the activity of IKK␣ or IKK as assessed by in vitro kinase assay, it is possible to conclude that this compound could suppress the LPS-induced NF-B activation by inhibiting upstream kinase of IKKs Although the involvement of Akt in NF-B activation has been controversial, one of the upstream kinases for IKKs is Akt, an important component of the PI3K pathway (Ozes et al., 1999; Romashkova and Makarov, 1999) Akt seemed to enhance nuclear translocation of NF-B through phosphorylation and activation of IB kinase, resulting in enhanced degradation of IB␣ (Madrid et al., 2000, 2001) Other studies have found that Akt did not affect nuclear translocation of Downloaded from jpet.aspetjournals.org at ASPET Journals on March 24, 2015 Fig Effect of cardamomin on the LPS-induced activation of Akt A, RAW264.7 cells were pretreated for 30 with cardamomin before stimulation with g/ml LPS Cells were harvested at the indicated time points, and total cell extracts were prepared phospho-Akt was detected by Western blot analysis as described under Materials and Methods The bottom represents Akt to show the equal loading of cell lysates B, 293 cells were transiently transfected with a 5XGal4-luciferase reporter gene with a plasmid encoding a fusion protein between the DNA binding domain of Gal4 and activation domains of RelA After 48 h, the cells were incubated for another 12 h in the presence of the indicated concentrations of cardamomin, and the luciferase activity was determined as described under Materials and Methods Bars indicate the S.D Statistical significance judged by paired Student’s t test is marked with an asterisk (ءء, p Ͻ 0.01; ء, p Ͻ 0.05) C, phospho-RelA/p65 or RelA/p65 was detected using total cell lysates prepared in A by Western blot analysis Fig Effect of cardamomin on the LPS-induced activation of MAP kinases RAW264.7 cells were incubated with or without LPS (1 g/ml) in the presence of the cardamomin Cells were harvested at the indicated time points, and total cell extracts were prepared Phospho-ERK1/2, -SAPK/JNK, or -p38 was detected by Western blot analysis as described under Materials and Methods Representative autoradiographic exposures performed three-independent experiments are shown The bottom represents the corresponding total MAP kinase to show the equal loading of cell lysates For p38, the amount of phospho-p38 estimated by image scanning is expressed in arbitrary units Results are average values of at least three independent experiments A Chalcone, Cardamomin, Inhibits NF-B Signaling deoxy-⌬12,14-prostaglandin J2, 5-aminoimidazole-4-carboxamide riboside, and C2-ceramide exert their antiinflammatory effects by inhibiting PI3K/Akt/NF-B signaling in LPS-stimulated astrocytes or RAW264.7 cells (Koide et al., 2003; Giri et al., 2004; Jhun et al., 2004) Consistent with these findings, we were able to demonstrate that cardamomin inhibited not only the activation of Akt and IKK but also the degradation of IB␣ and transcriptional activity of the RelA subunit of NF-B in LPS-stimulated RAW264.7 cells The mitogen-activated protein kinases are known to play an important role in the activation NF-B in LPS-stimulated RAW264.7 cells Cardamomin did not significantly affect activation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase However, cardamomin prevented the activation of p38 kinase A recent study has been shown that the mitogen-activated kinase p38 was activated by Akt and induces transcriptional function of NF-B by stimulating RelA transactivation in interleukin-1-stimulated NIH3T3 ells (Madrid et al., 2001) In this regard, suppression of p38 kinase activation by cardamomin could result from the inhibitory effect of cardamomin on Akt activation Chalcone, a derivative of flavonoid, have been reported to possess a variety of biological activities, including anti-inflammatory effect For example, 2Ј-hydroxychalcone and its derivatives inhibit the induced activation of NF-B by LPS or TNF-␣, although it is not investigated how these derivatives inhibits the activation of NF-B (Madan et al., 2000; Ban et al., 2004) Here, we have shown that cardamomin inhibited the NF-B signal cascade and the induced expression of inflammatory NF-B target genes such as iNOS, COX-2, and TNF-␣ in LPS-stimulated RAW264.7 cells Moreover, pretreatment of cardamomin significantly protected the C57BL/6 mice from death caused by LPS-induced mortality Together, this study extends our understanding on the molecular mechanisms underlying the diverse biological activities of A conchigera rhizomes by indigenous population as an anti-inflammatory traditional medicine Furthermore, our results imply that cardamomin is a valuable compound for modulation of NF-B-dependent pathological conditions and support pharmacological basis that the A conchigera plant has been used as a traditional herbal medicine for the treatment of inflammation Acknowledgments NF-B but did influence NF-B-dependent transcription through a mechanism dependent on phosphorylation of the NF-B RelA/p65 subunit (Koul et al., 2001; Mayo et al., 2002) It has been also proposed that expression and the ration of IKK␣ to IKK vary among cell types, and the ratio may play a role in determining the cell-type specificity of Akt in NF-B activation (Gustin et al., 2004) For example, cells containing a high proportion of IKK␣ to IKK are susceptible to inhibition of NF-B DNA binding activity by PI3K inhibitors In human monocytes containing a high proportion of IKK␣ to IKK, Akt binds to IKK␣ constitutively, and their phosphorylation occurs after LPS stimulation (Lu and Wahl, 2005) This report also describes that Akt phosphorylates IKK␣ and induces degradation of IB␣ and that inhibition of Akt blocks LPS-induced IKK␣ activation and IB␣ degradation Moreover, some anti-inflammatory agents such as 15- We thank Drs M Karin, Mira Jung, and S Hong for the kind gifts of 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phosphatidylinositol-3 kinase/Akt/IKK␣/NF-B pathway J Leukoc Biol 78:1–7 Madan B, Batra S, and Ghosh B (2000) 2Ј-Hydroxychalcone inhibits nuclear factor-B and blocks tumor necrosis factor-␣- and lipopolysaccharide-induced adhesion of neutrophils to human umbilical vein endothelial cells Mol Pharmacol 58:526 –534 Madrid LV, Mayo MW, Reuther JY, and Baldwin AS Jr (2001) Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-B through utilization of ... degradation of I B and DNA binding activity of NF- B but also phosphorylation of RelA/p65 and activation of Akt It is well known that the critical step in NF- B activation is I B phosphorylation... activation of p38 kinase A recent study has been shown that the mitogen-activated kinase p38 was activated by Akt and induces transcriptional function of NF- B by stimulating RelA transactivation... modulation of NF- B- dependent pathological conditions and support pharmacological basis that the A conchigera plant has been used as a traditional herbal medicine for the treatment of inflammation