Yangonin Blocks Tumor Necrosis Factor-Α–Induced Nuclear Factor-Κb–Dependent Transcription by Inhibiting the Transactivation Potential of the Rela/P65 Subunit

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J Pharmacol Sci 118, 447 – 454 (2012) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Yangonin Blocks Tumor Necrosis Factor-α–Induced Nuclear Factor-κB–Dependent Transcription by Inhibiting the Transactivation Potential of the RelA/p65 Subunit

Juan Ma1,†, He Liang1,†, Hong Ri Jin2, Nguyen Tien Dat3, Shan Yu Zhang1, Ying Zi Jiang1, Ji Xing Nan1, Donghao Li1, Xue Wu1, Jung Joon Lee1,2,*a, and Xuejun Jin1,*b 1Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules, Yanbian University, Ministry of Education, Yanji Jilin 133002, China 2Center for Molecular Cancer Research, Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk 363-883, Republic of Korea 3Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Vietnam

Received November 13, 2011; Accepted January 23, 2012

Abstract. The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, and apoptosis. In our search for NF-κB inhibitors from natural resources, we identified yangonin from Piper methysticum as an inhibitor of NF-κB activation. In the present study, we demonstrate that yangonin potently inhibits NF-κB activation through suppression of the transcriptional activity of the RelA/p65 subunit of NF-κB. This compound significantly inhibited the induced expression of the NF-κB-reporter gene. However, this compound did not interfere with tumor necrosis factor-α (TNF-α)-induced inhibitor of κBα (IκBα) degradation, p65 nuclear translocation, and DNA-binding activity of NF-κB. Further analysis revealed that yangonin inhibited not only the induced NF-κB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Moreover, yangonin did not inhibit TNF-α-induced activation of p38, but it significantly impaired activation of extracellular signal-regulated kinase 1/2 and

stress-activated protein kinase/c-Jun NH2-terminal kinase. We also demonstrated that pretreatment of cells with this compound prevented TNF-α-induced expression of NF-κB target genes, such as interleukin 6, interleukin 8, monocyte chemotactic protein 1, cyclooxygenase-2 and inducible nitric oxide. Taken together, yangonin could be a valuable candidate for the intervention of NF-κB- dependent pathological conditions such as inflammation.

Keywords: yangonin, nuclear factor-κB (NF-κB), RelA/p65, transactivation, inflammation

Introduction including p65 (RelA), p105/p50, p100/p52, RelB, and c-Rel. The classic form of NF-κB is the p65/p50 het- Nuclear factor-κB (NF-κB) represents a family of eu- erodimer that contains the transcriptional activation do- karyotic transcription factors participating in the regula- main and is sequestered in the cytoplasm as an inactive tion of various cellular genes involved in the immediate complex by inhibitor of κB (IκB) (2). In response to early processes of immune, acute phase, and inflamma- stimulation, such as lipopolysaccharide (LPS) or tumor tory responses as well as genes involved in cell survival necrosis factor-α (TNF-α), IκBs are rapidly ubiquitinated (1). NF-κB consists of a family of transcription factors and degraded by 26S proteasome complex and the release of IκB unmasks the nuclear localization signal and results in the translocation of NF-κB to the nucleus, followed by † These two authors equally contributed to this work. the activation of specific target genes (3). Furthermore, Corresponding authors. *[email protected], *[email protected] NF-κB activation can be positively regulated by p65 Published online in J-STAGE post-translational modification, in which phosphoryla- doi: 10.1254/jphs.11215FP tion at various amino acid residues in p65 were found to

447 448 J Ma et al contribute to its transcriptional activities by affecting the Measurement of cell viability by MTT assay transcription regulators recruitment (4). HeLa cells were seeded at 1 × 105 cells/ml in 96-well It is reported that NF-κB regulates more than 150 plates containing 100 μl of DMEM medium with 10% genes, including those involved in immunity and inflam- FBS and incubated overnight. Yangonin was dissolved mation, anti-apoptosis, cell proliferation, tumorigenesis, in DMSO and DMSO was added to all plates to compen- and the negative feedback of the NF-κB signal (5). NF- sate the same volume of DMSO. After 24 h, the cells κB regulates major inflammatory cytokines, including were pretreated with different concentrations of yangonin interleukin 6 (IL-6), interleukin 8 (IL-8), and monocyte (0.1 – 3 μM) for 1 h, followed by stimulation with or chemotactic protein 1 (MCP1), many of which are potent without TNF-α for 24 h. Subsequently, cells were cul- activators for NF-κB (6). Therefore, NF-κB is primarily tured with MTT solution (5 mg/ml) [3-(4,5-dimethyl- an inducer of inflammatory cytokines. Its inhibitors could thiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma) be useful as anti-inflammatory agents. In addition to for 3 h. The viable cells converted MTT to formazan, regulating the expression of genes important for immune which generated a blue-purple color after dissolving in and inflammatory responses, NF-κB also controls the 150 μl of DMSO. The absorbance at 570 nm was mea- transcription of genes that are critical in the early and late sured by an ELISA plate reader. stages of aggressive cancers, including cyclooxygenase-2 (COX-2) and inducible nitric oxide (iNOS). Therefore, Plasmids, transfection, and luciferase reporter assay the NF-κB function inhibitors might also be useful as The pNF-κB-Luc plasmid for the NF-κB luciferase anticancer agents. reporter assay was obtained from Stratagene (La Jolla, Yangonin is one of the six major kavalactones found CA, USA). A vector encoding a fusion protein between in Piper methysticum. This compound was shown to have the DNA binding domain of Gal4 and activation domains not only potent antioxidant or free radical-scavenging of RelA/p65, Gal4-RelA268-551, was constructed by activities, but also the ability to inhibit cyclooxygenase inserting cDNA for Gal4-RelA268-551 into pFA-CMV enzyme cyclooxygenase-1 (COX-1) and COX-2 (7). ( Stratagene). The 5 × Gal4-luciferase reporter gene was Yangonin also inhibited LPS- and TNF-α-induced NF- obtained from Stratagene. Flag-Akt was kindly provided κB activation (8, 9); however, the molecular mechanism by Dr. Jeong-Hyung Lee (Kangwon National University, has not been sufficiently explained. In present study, we Republic of Korea). Transfections were performed using investigated the effects of yangonin on the NF-κB activa- Lipofectamine 2000 (Invitrogen) according to the manu- tion pathway induced by TNF-α. The results described facturer’s instructions. The luciferase reporter assay was below showed that yangonin inhibited not only NF-κB performed as described previously (10). Briefly, HeLa by the suppression of transactivation activity of RelA/ cells (1 × 105 cells/well) were seeded in a 96-well plate p65 subunit without affecting the induced degradation of for 24 h. The cells were then transfected with plasmids IκBα, p65 nuclear translocation, and DNA-binding activ- for each well and then incubated for a transfection period ity of NF-κB, but also the induced expression of NF-κB of 24 h. After that, the cell culture medium was removed target genes such as IL-6, IL-8, MCP1, COX-2, and and replaced with fresh medium containing various iNOS. concentrations of yangonin for 30 min, followed by treatment with 20 ng/ml of TNF-α for 18 h. Luciferase activ- Materials and Methods ity was determined in the Microlumat plus luminometer (EG&G Berthold, Bad Wildbad, Germany) by injecting Cell culture and reagents 100 μl of assay buffer containing luciferin and measuring RAW264.7 murine macrophages (American Type light emission for 10 s. Co-transfection with pRL-CMV Culture Collection, Manassas, VA, USA) and HeLa hu- (Promega, Madison, WI, USA), which expresses Renilla man cervix carcinoma (American Type Culture Collec- luciferase, was performed to enable normalization of tion) cells were grown in DMEM (Invitrogen, Carlsbad, data for transfection efficiency. CA, USA) with penicillin (100 units/ml) – streptomycin (100 units/ml) (Invitrogen) and 10% heat-inactivated Preparation of nuclear extracts and electrophoretic fetal bovine serum (Hyclone, Logan, UT, USA). TNF-α mobility shift assay (EMSA) was obtained from R&D Systems (Minneapolis, MN, Electrophoretic mobility shift assay was performed as USA). Yangonin was obtained from the compound bank described previously (11). HeLa cells were cultured in of Korea Research Institute of Bioscience and Biotech- 6-cm dishes and allowed to adhere for 24 h and then nology (Ochang, Republic of Korea). The purity of treatment with 3 μM yangonin for 12 h following stimu- yangonin reached 99% in high-performance liquid chro- lation with TNF-α (20 ng/ml) for the indicated times. matography analysis. The cells were then harvested and washed twice with Inhibition of NF-κB by Yangonin 449 ice-cold phosphate-buffered saline (PBS), and then nu- Real-time PCR clear extracts were prepared using NE-PER reagent Real-time PCR was performed as previously described (Pierce, Rockford, IL, USA), according to the instruc- (2). In brief, HeLa cells were preincubated with the indi- tions of the manufacturer. Electrophoretic mobility shift cated concentrations of yangonin at 37°C for 12 h. Fol- assay was performed using a gel-shift assay system lowing the preincubation, the HeLa cells were stimulated (Promega), according to the instructions of the manufac- with TNF-α (20 ng/ml) for 1 h, harvested, and washed turer. A double-stranded oligonucleotide for NF-κB twice with ice-cold PBS; then total RNA was isolated (Promega) was end-labeled with [γ-32P]ATP and purified from cells using RNeasy Mini kits according to the manu- with a G-25 spin column (Boehringer Mannheim, facturer’s instructions (Qiagen, Valencia, CA, USA). Mannheim, Germany). Approximately 20,000 cpm of Complementary DNA was synthesized from 1 μg of total probe was used per assay. Nuclear extracts (10 μg) were RNA in a 20-μl reverse transcription reaction mixture incubated for 20 min at room temperature with a gel according to the manufacturer’s protocol (Takara Bio, shift–binding buffer [5% glycerol, 1 mM MgCl2, 0.5 mM Kyoto). The following primer pairs were used for real- EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl, time PCR amplification: human IL-6, 5′-GAACTCCT pH 7.5, 50 μg/ml poly(dI-dC) poly(dI-dC)] and 32P- TCTCCACAAGCGCCTT-3′ and 5′-CAAAAGACCA labeled oligonucleotide. The DNA–protein complex GTGATGATTTTCACCAGG-3′; human IL-8, 5′-TCT formed was separated on 4% native polyacrylamide gels, GCAGCTCTGTGTGAAGG-3′ and 5′-ACTTCTCCAC and the gel was transferred to Whatman 3 MM paper, AACCCTCTG-3′; human MCP1, 5′-CCCCAGTCACC dried, and exposed to X-ray film. TGCTGTTAT-3′ and 5′-AGATCTCCTTGGCCACAA TG-3′; GAPDH, 5′-ACCAGGTGGTCTCCTCT-3′ and Western blot analysis 5′-TGCTGTAGCCAAATTCGTTG-3′. GAPDH was HeLa cells were cultured in 10-cm dishes and allowed used as the housekeeping gene control. The quantitative to adhere for 24 h. The culture medium was removed and real-time PCR was carried out using power SYBR green replaced with fresh medium containing yangonin at 3 μM (Bio-Rad, Hercules, CA, USA). The reactions were per- for 12 h and then treatment with 20 ng/ml of TNF-α for formed in 25-μl (total volume) mixtures containing the indicated times. The cells were washed twice with primers at a concentration of 400 nM. The reaction con- ice-cold PBS and lysed in ice-cold lysis buffer (50 mM ditions consisted of 10 min at 95°C and then 40 cycles of Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM EDTA, 1 mM 15 s at 95°C 15 s at 58°C and 30 s at 72°C. Melting curve phenylmethyl sulfonylfluoride) supplemented with the analysis was used to determine PCR specificity. All reac- protease inhibitor cocktail (BD Biosciences, San Diego, tions were carried out in triplicate for each sample. The CA, USA). In certain experiments, the nuclear extracts standard curve method was used to determine the amount were prepared using NE-PER reagent. An aliquot of of each transcript. Relative induction was determined by protein extracts was used to determine protein concentra- normalizing the relative expression to the untreated tion by the Bradford method. A 50-μg sample of whole- control samples. cell extracts or 30 μg of nuclear extract protein per lane was separated by SDS-polyacrylamide gels and followed Statistical analyses by transferring to a polyvinylidene difluoride membrane All values are expressed as the mean ± S.D. A com- (Millipore, Bedford, MA, USA). The membrane was parison of the results was performed with one-way blocked with 5% skim milk and then incubated with the ANOVA and Tukey’s multiple comparison tests (Graph- corresponding antibody. Antibodies for IκBα, p65, pad Software, Inc., San Diego, CA, USA). Statistically phospho-p65 (Ser536), phospho-Akt (Ser473), Akt, significant differences between groups were defined as ERK, phospho-ERK, JNK, phospho-JNK, p38, and P-values less than 0.05. phospho-p38 were purchased from Cell Signaling Tech- nology (Beverly, MA, USA). Antibodies for Topo-I, Results COX-2, and iNOS were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody for Yangonin inhibits TNF-α- and LPS-induced NF-κB α-tubulin was from Sigma. After binding of an appropri- activation ate secondary antibody coupled to horseradish peroxi- In an effort to identify NF-κB inhibitors from herbal dase, proteins were visualized by enhanced chemilumi- medicines, we have identified yangonin from a traditional nescence according to the instructions of the manufacturer medicinal plant, Piper methysticum (Fig. 1). To investi- (Amersham Pharmacia Biotec, Buckinghamshire, UK). gate the effect of yangonin on the induced NF-κB activation by various stimuli, we performed the NF-κB-reporter assay. Yangonin dose-dependently inhibited the TNF-α- 450 J Ma et al induced NF-κB-reporter gene expression (Fig. 2A). This analyzed TNF-α-induced expression of COX-2, iNOS, compound also inhibited LPS-induced expression of IL-6, IL-8, and MCP1. After HeLa cells were stimulated NF-κB-reporter gene expression to a similar extent (Fig. with 20 ng/ml TNF-α for different periods in the pres- 2B). Yangonin did not display any cellular toxicity on ence or absence of yangonin, the induced expression of HeLa cells up to 3 μM with or without TNF-α (20 ng/ml) COX-2 and iNOS were analyzed by western blotting. (Fig. 2: C and D). TNF-α significantly induced expression of COX-2 and iNOS in a time-dependent manner (Fig. 3A, left five Yangonin inhibits TNF-α-induced NF-κB target gene lanes), whereas yangonin markedly suppressed TNF-α- expression induced expression of all these proteins (Fig. 3A, right To investigate whether yangonin inhibits the induced four lanes). Because NF-κB regulates major inflamma- expressions of NF-κB targets genes involved in early and tory cytokines and chemokines, including IL-6, IL-8, and late stages of aggressive cancers and inflammation, we MCP1, many of which are potent activators for NF-κB (1, 6, 13), we examined whether yangonin can modulate the expression of these gene products induced by TNF-α. TNF-α significantly induced expression of IL-6, IL-8, and MCP1 mRNA levels, but the induced-expression was blocked by yangonin in a dose-dependent manner (Fig. 3B). Yangonin at 3 μM almost completely inhibited the TNF-α-induced mRNA expression of IL-6, IL-8, and MCP1. Concentrations to inhibit IL-6, IL-8, and MCP1 expression are comparable with those of NF-κB inhibition.

Yangonin does not interfere with the TNF-α-induced IκBα degradation, p65 nuclear translocation, and DNA- binding activity of NF-κB The translocation of NF-κB to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic Fig. 1. Structure of yangonin. degradation of IκBα (14). To determine whether inhibi-

A B 14 10 12 8 Fig. 2. Effect of yangonin on the TNF-α- and LPS- 10 6 induced NF-κB-dependent reporter gene expression. 8 * * * * HeLa cells (A) and RAW264.7 cells (B), transiently 6 4 transfected with a NF-κB-dependent reporter gene, 4 * * * * * * 2 were grown for 48 h and then pretreated for 8 h with * * * 2 * * * the indicated concentrations of yangonin followed by 0 Relative Luciferase (Fold) 0 Relative Luciferase (Fold) stimulation for 8 h with TNF-α (20 ng/ml) and LPS (1 TNF- -+++++ LPS -+++++ μg/ml), respectively; and the luciferase activity was YGN (μM) --0.1 0.3 1 3 YGN (μM) --0.1 0.3 1 3 determined as described in “Materials and Methods”. Data represent the mean ± S.D. of three independent experiments. **P < 0.01, ***P < 0.001, significantly C D different when compared with TNF-α- and LPS-stimu- 140 120 lated normal cells. C) HeLa cells were treated with the 120 100 indicated concentrations of yangonin. After 24-h incu- 100 80 bation, cell viability was determined by MTT assays. 80 60 Data represent the mean ± S.D. of three independent 60 experiments. D) HeLa cells were pretreated with differ- 40 40 ent concentrations of yangonin (0.1 – 3 μM) for 1 h, 20 20 followed by stimulation with TNF-α for 24 h. Cell Viability (% of control) Viability (% of control) 0 0 viability was determined by MTT assays. Reported TNF- +++++- 0310.30.1 data represent the mean ± S.D. of three independent YGN (μM) 0 0310.30.1 YGN (μM) experiments. Inhibition of NF-κB by Yangonin 451

A A 04816 24 4 8 2416 Time (h) 0 5 10 60305 10 6030 Time (min) -++++++++TNF-α (20 ng/ml) -++++++++TNF-α (20 ng/ml) ----- ++++YGN (3 μM) ----- ++++YGN (3 μM) COX2 IκBα (Cyto) iNOS Tubulin Tubulin

B B 0 5 10 60305 1030 60 Time (min) 10 -++++++++TNF-α (20 ng/ml) ----- ++++YGN (3 μM)

8 p65 (Nu)

Topo-I 6 *

* 4 * *

* * C * * 01030 60 0 10 6030 Time (min) 2 * * * * * * -+++-+++TNF-α (20 ng/ml) mRNA Expression (Relative) ---- ++++YGN (3 μM) 0 - +++++ - +++++ - +++++ TNF- --0.1 0.3 1 3 --0.1 0.3 1 3 --0.1 0.3 1 3 YGN (μM) NF-κB IL-6 IL-8 MCP1

Fig. 3. Effect of yangonin on the inflammatory NF-κB target genes. A) HeLa cells were incubated with 3 μM yangonin for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Whole cell extracts were analyzed by western blotting using antibodies for COX- 2, iNOS, and tubulin. B) HeLa cells were preincubated with the indicated concentrations of yangonin for 12 h and then treated with TNF-α (20 ng/ml) for an additional 1 h. RNAs were isolated from cells, re- Fig. 4. Effect of yangonin on the TNF-α-induced IκBα degradation, verse-transcribed, and analyzed by real-time PCR for IL-6, IL-8, and p65 nuclear translocation and DNA-binding activity of NF-κB. HeLa MCP1. Reported data represent the mean ± S.D. of three independent cells were incubated with 3 μM yangonin for 12 h and then incubated experiments. *P < 0.05, **P < 0.01, ***P < 0.001, significantly dif- with 20 ng/ml TNF-α for the indicated times. Cells were harvested at ferent when compared with TNF-α-stimulated normal cells. the indicated time points and then cytosol extracts and nuclear extracts were prepared. Cytosol IκBα (A) and nuclear p65 (B) were detected by western blot analysis. C) HeLa cells were incubated with 3 μM tion of TNF-α-induced NF-κB activation by yangonin is yangonin for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Cells were harvested at the indicated time points and caused by inhibition of IκBα degradation, we pretreated subsequently nuclear extracts were prepared and tested for DNA- cells with yangonin and then exposed them to TNF-α for binding of activated NF-κB by EMSA as described in “Materials and various time periods. Cytoplasmic extracts were analyzed Methods”. for the presence of IκBα with western blot analysis. IκBα was almost completely degraded in 30 min after stimulation with TNF-α and resynthesized in 1 h (Fig. 4A, left Yangonin inhibits transactivation of RelA/p65 subunit five lanes). Preincubation with yangonin, however, could In order to further investigate how yangonin prevents not significantly prevent TNF-α-induced degradation and NF-κB activation, we examined the effect of yangonin resynthesis of IκBα (Fig. 4A, right four lanes). To con- on the induced NF-κB activity by overexpression of firm that yangonin inhibits NF-κB activation, we mea- RelA/p65 subunit, which is the critical transactivation sured the effect of yangonin on TNF-α-induced nuclear subunit of NF-κB (15,16). HeLa cells were either tran- translocation and DNA-binding activity of NF-κB. HeLa siently transfected with 3 × κB-Luc reporter or cotrans- cells stimulated with TNF-α showed strong nuclear fected with plasmids bearing genes encoding the RelA/ translocation and DNA-binding activity of NF-κB. How- p65. The results showed that yangonin significantly in- ever, pretreatment of yangonin did not inhibit nuclear hibited NF-κB-dependent transcription induced by translocation (Fig. 4B) and DNA-binding activity of NF- overexpression of RelA/p65 in dose-dependent manner κB induced by TNF-α (Fig. 4C). (Fig. 5A), suggesting that yangonin could influence the 452 J Ma et al

transactivation activity of the RelA/p65 subunit. In order to examine this possibility, we adopted a heterologous A 10 luciferase reporter system in which gene expression from the reporter plasmid pFR-luc containing Gal4-binding 8 sites is under the control of Gal4. A construct containing * * 6 the transactivating domains of RelA, Gal4-RelA268-551, * * * which we created, was transfected into HeLa cells along 4 * * * with a luciferase reporter containing upstream Gal4 bind- * * * 2 ing sites. Yangonin blocked transcriptional activity of the

Relative Luciferase (Fold) 0 transactivation domain of RelA/p65 in a dose-dependent B vector ++++++ manner (Fig. 5B). Since the efficient transcriptional acti- RelA/p65 - +++++ vation of NF-κB depends on the phosphorylation of its YGN (μM) 000.10.313 active subunit RelA/p65, particularly at serine 536 resi- due (14), we examined whether yangonin inhibits TNF- B 12 α-induced phosphorylation of p65 at serine 536. The re- 10 sults showed that TNF-α induced phosphorylation of p65 * * as quickly as 5 min (Fig. 5C, left five lanes), and treat- 8 * * * ment of these cells with yangonin completely inhibited 6 * * * the phosphorylation of p65 (Fig. 5C, right four lanes). 4 * * * Taken together, these data suggested that yangonin regu- 2 lates the activation of NF-κB by inhibiting transactivation

Relative Luciferase (Fold) 0 activity of the RelA/p65 subunit. Gal4-RelA268-551 - ++++++ 5 Gal4-Luc + -+++++ Yangonin inhibits TNF-α-induced phosphorylation of YGN (μM) 0 000.10.313 Akt Since Akt has been implicated in the regulation of p65 C 0 5 10 60305 10 6030 Time (min) phosphorylation and NF-κB activation (17), we further -++++++++TNF-α (20 ng/ml) examined the effect of yangonin on the induced NF-κB ----- ++++YGN (3 μM) activity by overexpression of Akt. As shown in Fig. 6A, p-p65 (Ser536) yangonin treatment resulted in the inhibition of overexpression of Akt-induced NF-κB-dependent transcription p65 in a dose-dependent manner, suggesting that yangonin might critically influence Akt activity. To further exam- Fig. 5. Yangonin inhibits NF-κB activation induced by overexpres- ine this possibility, we exposed the HeLa cells to yan- sion of RelA/p65 and transcriptional activity of RelA/p65 subunit. A) HeLa cells were transiently transfected with a NF-κB-dependent re- gonin for 12 h and then treated them with TNF-α for porter gene together with expression vectors encoding RelA/p65. The different periods. We then prepared total extracts and cotransfected cells were subsequently grown for 24 h and for another analyzed them for phosphorylation of Akt by western 12 h in the presence of the indicated concentrations of yangonin, and blotting. The results showed that TNF-α induced phos- the luciferase activity was determined as described in “Materials and phorylation of Akt in a time-dependent manner and that Methods”. Reported data represent the mean ± S.D. of three indepen- the earliest activation occurred within 5 min after TNF-α dent experiments. **P < 0.01, ***P < 0.001, significantly different when compared with RelA/p65 and NF-κB-dependent reporter gene– addition (Fig. 6B, left five lanes). However, we did not cotransfected normal cells. B) HeLa cells were transiently transfected observe phosphorylation of Akt in cells pretreated with with a 5 × Gal4-luciferase reporter gene alone or in combination with yangonin (Fig. 6B, right four lanes). a plasmid encoding a fusion protein between the DNA binding domain 268-551 of Gal4 and transactivation domains of ReA, Gal4-RelA . After Yangonin inhibits TNF-α-induced phosphorylation of 24 h, the cells were incubated for another 12 h in the presence of the indicated concentrations of yangonin, and the luciferase activity was ERK1/2 and phosphorylation of JNK/SAPK determined as described in “Materials and Methods”. Reported data The mitogen-activated protein (MAP) kinases such as represent the mean ± S.D. of three independent experiments. **P < extracellular signal-regulated kinase 1/2 (ERK1/2), 0.01, ***P < 0.001, significantly different when compared with Gal4 stress-activated protein kinase/c-Jun NH2-terminal kinase and RelA268-551–cotransfected normal cells. C) HeLa cells were incu- (JNK/SAPK), and p38 are known to be activated by bated with 3 μM yangonin for 12 h and then incubated with 20 ng/ml TNF-α and are important signaling molecules involved TNF-α for the indicated times. Cells were harvested at the indicated time points and total extracts were prepared. Phospho-p65 and p65 in the activation of NF-κB (18). Therefore, we used protein level were detected by western blot analysis. western blotting to determine the effect of yangonin on Inhibition of NF-κB by Yangonin 453

A 0 5 10 60305 1030 60 Time (min) 8 -++++++++TNF-α (20 ng/ml) ----- ++++YGN (3 μM) 6 p-ERK * * 4 * * * ERK

2 * * * p-JNK

Relative Luciferase (Fold) 0 JNK B vector +++++ + Flag-Akt -+++++ p-p38 YGN (μM) 000.10.313 p38

B 0 5 10 60305 10 6030 Time (min) Tubulin -++++++++TNF-α (20 ng/ml) ----- ++++YGN (3 μM) Fig. 7. Effect of yangonin on the TNF-α-induced activation MAP p-Akt (Ser473) kinases. HeLa cells were incubated with 3 μM yangonin for 12 h and then incubated with 20 ng/ml TNF-α for the indicated times. Cells Akt were harvested at the indicated time points and total extracts were prepared. Phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, Fig. 6. Effect of yangonin on the TNF-α-induced phosphorylation and p38 were detected by western blot analysis. of Akt. A) HeLa cells were transiently transfected with the NF-κB- dependent reporter gene together with expression vectors encoding Flag-Akt. The cotransfected cells were subsequently grown for 24 h We found that yangonin did not inhibit the phosphory- and for another 12 h in the presence of the indicated concentrations of lation and degradation of IκBα and DNA-binding activity yangonin, and the luciferase activity was determined as described in of NF-κB following TNF-α stimulation, but inhibited “Materials and Methods”. Reported data represent the mean ± S.D. of NF-κB activation by RelA/p65-overexpression. These three independent experiments. **P < 0.01, ***P < 0.001, significantly different when compared with Akt and NF-κB-dependent re- observations lead us to propose the hypothesis that yan- porter gene–cotransfected normal cells. B) HeLa cells were incubated gonin may modify transactivation activity of the RelA/ with 3 μM yangonin for 12 h and then incubated with 20 ng/ml TNF-α p65 subunit of NF-κB. In order to test this hypothesis, we for the indicated times. Cells were harvested at the indicated time investigated whether yangonin suppresses the transacti- points and total extracts were prepared. Phospho-Akt and Akt protein vation activity of the RelA/p65 subunit using the Gal4 level were detected by western blot analysis. system. As expected, yangonin significantly suppressed the transactivation activity of the RelA/p65 subunit. It is well known that NF-κB activation, characterized by TNF-α-induced phosphorylation of ERK1/2, JNK/SAPK, phosphorylation of specific amino acid residues in the and p38 in HeLa cells (Fig. 7). Yangonin did not sup- p65 subunit, is one important prerequisite for transactiva- press the TNF-α-induced activation of p38. The TNF-α- tion of the target genes. We included in our analysis the induced phosphorylation of ERK1/2 and JNK/SAPK, assessment of phosphorylation of p65. Our results show however, were significantly inhibited by yangonin. that yangonin completely inhibited the phosphorylation of p65. Discussion The MAP kinases are known to play an important role in the activation NF-κB (19). Therefore, future experi- The leaves and the root of Piper methysticum have ments were performed to determine whether yangonin been used for herbal food and medicinal products and tightly regulates expression of MAP kinases to induce herbal remedies used to treat anxiety, tension, and rest- anti-inflammatory effects in TNF-α-stimulated HeLa. lessness in many Pacific Ocean islands. On the other Yangonin did not significantly affect activation of p38 hand, Piper methysticum is a rich source of kavalactones. kinase. However, yangonin prevented the activation of Extracts containing kavalactones, like yangonin, have ERK1/2 and JNK/SAPK. A recent study has been shown been used for the treatment of inflammatory disease. that MAP kinases, such as ERK1/2 and JNK/SAPK, Despite of its various pharmacological activities, the were activated by Akt, and their role in regulating NF-κB molecular mechanism has not been sufficiently explained. activation has been well-documented (20 – 23). In this In present study, we identified yangonin as an inhibitor regard, suppression of ERK1/2 and JNK/SAPK activa- of NF-κB activation and investigated how this compound tion by yangonin could result from the inhibitory effect suppressed NF-κB activation. of yangonin on Akt activation. 454 J Ma et al

Accumulating studies have shown that NF-κB regu- Pharmacol. 2006;71:1206–1218. lates a wide variety of genes whose products are involved 9 Zhou P, Gross S, Liu JH, Yu BY, Feng LL, Nolta J, et al. Flavoka- in inflammation, carcinogenesis, and evasion of apopto- wain B, the hepatotoxic constituent from kava root, induces sis (5). Constitutive activation of NF-κB may up-regulate GSH-sensitive oxidative stress through modulation of IKK/NF- kappaB and MAPK signaling pathways. FASEB J. 2010;24: the expression of inflammatory cytokines, chemokines, 4722–4732. cell adhesion molecules, and inflammatory gene products 10 Takei H, Baba Y, Hisatsune A, Katsuki H, Miyata T, Yokomizo such as IL-6, IL-8, MCP1, COX-2, and iNOS (1, 6, 24). K, et al. Glycyrrhizin inhibits interleukin-8 production and nu- Most of these genes are shown to be up-regulated in the clear factor-kappaB activity in lung epithelial cells, but not early and late of aggressive cancers. In the present study, through glucocorticoid receptors. J Pharmacol Sci. 2008;106: we have demonstrated that pretreatment with yangonin 460–468. suppressed TNF-α-induced expression IL-6, IL-8, MCP1, 11 Lee JH, Koo TH, Yoon H, Jung HS, Jin HZ, Lee K, et al. Inhibi- tion of NF-kappa B activation through targeting I kappa B kinase COX-2, and iNOS. Therefore, yangonin could serve as by celastrol, a quinone methide triterpenoid. Biochem Pharma- an interesting lead compound for the development of col. 2006;72:1311–1321. new, potent anti-inflammatory agents. 12 Jin X, Jin HR, Jung HS, Lee SJ, Lee JH, Lee JJ. An atypical E3 Taken together, we have shown that yangonin inhibits ligase zinc finger protein 91 stabilizes and activates NF-kappaB- the NF-κB signal cascade by preventing transcriptional inducing kinase via Lys63-linked ubiquitination. 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