Magnolol suppresses NF-κB activation and NF-κB regulated gene expression through inhibition of IkappaB kinase activation

Molecular Immunology 44 (2007) 2647–2658

Magnolol suppresses NF-␬B activation and NF-␬B regulated gene expression through inhibition of IkappaB kinase activation Anfernee Kai-Wing Tse a,b , Chi-Keung Wan a , Guo-Yuan Zhu a , Xiao-Ling Shen a , Hon-Yeung Cheung a , Mengsu Yang a , Wang-Fun Fong b,∗ a

Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, China b School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China Received 14 November 2006; accepted 10 December 2006 Available online 22 January 2007

Abstract The mis-regulation of nuclear factor-kappa B (NF-␬B) signal pathway is involved in a variety of inflammatory diseases that leds to the production of inflammatory mediators. Our studies using human U937 promonocytes cells suggested that magnolol, a low molecular weight lignan isolated from the medicinal plant Magnolia officinalis, differentially down-regulated the pharmacologically induced expression of NF-␬B-regulated inflammatory gene products MMP-9, IL-8, MCP-1, MIP-1␣, TNF-␣. Pre-treatment of magnolol blocked TNF-␣-induced NF-␬B activation in different cell types as evidenced by EMSA. Magnolol did not directly affect the binding of p65/p50 heterodimer to DNA. Immunoblot analysis demonstrated that magnolol inhibited the TNF-␣-stimulated phosphorylation and degradation of the cytosolic NF-␬B inhibitor I␬B␣ and the effects were dosedependent. Mechanistically, a non-radioactive I␬B kinases (IKK) assay using immunoprecipitated IKKs protein demonstrated that magnolol inhibited both intrinsic and TNF-␣-stimulated IKK activity, thus suggesting a critical role of magnolol in abrogating the phosphorylation and degradation of I␬B␣. The involvement of IKK was further verified in a HeLa cell NF-␬B-dependent luciferase reporter system. In this system magnolol suppressed luciferase expression stimulated by TNF-␣ and by the transient transfection and expression of NIK (NF-␬B-inducing kinase), wild type IKK␤, constitutively active IKK␣ and IKK␤, or the p65 subunit. Magnolol was also found to inhibit the nuclear translocation and phosphorylation of p65 subunit of NF-␬B. In line with the observation that NF-␬B activation may up-regulate anti-apoptotic genes, it was shown in U937 cells that magnolol enhanced TNF-␣-induced apoptotic cell death. Our results suggest that magnolol or its derivatives may have potential anti-inflammatory actions through IKK inactivation. © 2006 Elsevier Ltd. All rights reserved. Keywords: Magnolol; Nuclear factor kappa B; IKK; Anti-inflammation

1. Introduction The bark of Magnolia officinalis (Cortex Magnoliae Officinalis) is widely used as a folk remedy for gastrointestinal disorders, cough, anxiety and allergic diseases. Magnolol, a low molecular weight lignan originally isolated from the Chinese medicinal plant (Wang et al., 2004), shows a number of diverse pharmacological effects including inducing apoptosis (Lin et al., 2001; Ikeda and Nagase, 2002; Yang et al., 2003; Zhong et al., 2003), differentiation (Fong et al., 2005), calcium

∗ Corresponding author at: Division of Research & Development, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China. Tel.: +852 3411 2928; fax: +852 3411 2902. E-mail address: [email protected] (W.-F. Fong).

0161-5890/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2006.12.004

mobilization (Teng et al., 1990; Wang and Chen, 1998; Zhai et al., 2003), and anti-platelet aggregation (Teng et al., 1988; Pyo et al., 2002). It has also been suggested to have anxiolytic (Maruyama et al., 1998), anti-oxidative (Lo et al., 1994; Shen et al., 1998), anti-fungal and anti-bacterial (Chang et al., 1998; Bang et al., 2000; Ho et al., 2001; Park et al., 2004), anti-viral and anti-carcinogenic (Konoshima et al., 1991) and anti-metastatic activities (Nagase et al., 2001; Ikeda et al., 2003). Magnolol has a board spectrum anti-inflammatory effect. It suppresses the expression of the inducible nitric oxide synthase (iNOS) in macrophages (Son et al., 2000; Matsuda et al., 2001), the production of inflammatory cytokines interleukin-8 and tumor necrosis factor ␣ (TNF-␣) in THP-1 cells (Park et al., 2004; Lee et al., 2005), the formation of prostaglandin E2 (Wang et al., 1995; Lee et al., 2000), and the artherosclerosis mediators monocyte chemotactic protein-1 (MCP-1) and vascular cell

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adhesion molecule-1 (VCAM-1) (Chen et al., 2001, 2002, 2006). Moreover, magnolol was also reported as a cyclooxygenase (COX) inhibitor (Hsu et al., 2004; Lee et al., 2005). Nuclear factor-␬B (NF-␬B) is an ubiquitous nuclear transcription factor regulating dozens of genes involved in inflammation, and also in growth regulation, apoptosis, cancer invasion/metastasis, tumor promotion, carcinogenesis (reviewed in Aggarwal, 2004). NF-␬B consists of a family of transcription factors including p65 (RelA), p105/p50, p100/p52, RelB and c-Rel. The classic form of NF-␬B is the p65/p50 heterodimer that contains the transcriptional activation domain and is sequestered in the cytoplasm as an inactive complex by I␬B (Baldwin, 1996). Acute stimuli such as TNF-␣, LPS or PMA led to the activation of I␬B kinases (IKK) which in turn phosphorylate Ser32 and Ser36 within the N-terminal response domain of I␬B (Karin and Ben-Neriah, 2000). Phosphorylated I␬B would undergo ubiquitination-dependent proteolysis and the release of I␬B unmasks the nuclear localization signal and results in the translocation of NF-␬B to the nucleus, followed by the activation of specific target genes (Karin and Ben-Neriah, 2000). We investigated whether the attenuation of NF-␬B activity may account for the anti-inflammatory and pharmacological effects of magnolol. In a previous study magnolol was shown to reduce the nuclear NF-␬B content in TNF-␣-stimulated endothelial cells (Chen et al., 2002). However, the action mechanisms are poorly understood. We first established the effects of magnolol on NF-␬B-regulated gene expression induced by a number of inflammatory agents and carcinogens. Then, we demonstrated that magnolol inhibited TNF-␣ stimulated activation of NF-␬B in different cells. We further showed that magnolol suppressed IKK activity, stabilized cytoplasmic I␬B␣ and subsequently reduced the nuclear translocation and phosphorylation of the p65 subunit of NF-␬B. Magnolol also inhibited NF-␬B-dependent reporter gene expression induced by TNF-␣, over-expression of NIK, IKK and p65 subunit and enhanced TNF-␣-mediated apoptosis. 2. Materials and methods 2.1. Materials Magnolol (Fig. 1A) was obtained from Wako Pure Chemical Industries Ltd., Japan, dissolved in DMSO to make a 100 mM stock solution and stored at −20 ◦ C. LPS (E. coli 0127:B8) and PMA were obtained from Sigma (St. Louis, MO, USA). TNF-␣ was from Wako Pure Chemical Industries Ltd., Japan, dissolved in 0.1% (w/v) BSA and stored at −80 o C. [␥-32 P] ATP was from Perkin-Elmer Life Sciences (Hong Kong) Ltd. Phosphop65 (Ser536 ) and phosphor-I␬B␣ (Ser32 ) antibodies were from Cell Signaling Technology. NF-␬B and Sp1 consensus gel shift oligonucleotides, Protein A/G Plus-agarose, glutathione S-transferase (GST)-I␬B␣, anti-p65, anti-I␬B␣, anti-IKK␣/␤, anti-caspase-3, anti-lamin B, anti-␤-tubulin and anti-actin were purchased from Santa Cruz Biotechnology Inc. Bright-Glo luciferase assay system and Beta-Glo assay system were purchased from Promega.

Fig. 1. (A) Structure of magnolol. (B) Magnolol suppresses NF-␬Bregulated gene expression induced by TNF-␣, LPS and PMA. U937 cells (1.0 × 106 cells/ml) were treated with 60 ␮M of magnolol for 12 h and stimulated with PMA (25 ng/ml), LPS (1 ␮g/ml) or TNF-␣ (5 ng/ml) for 12 h. The mRNA levels of matrix metalloproteinase-9 (MMP-9), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1␣ (MIP1␣) and tumor necrosis factor-␣ (TNF-␣) were examined using RT-PCR assay. Densitometry estimations of the levels of gene expressed are normalized with GAPDH and the medium control and shown under each lane.

2.2. Cell culture The cell lines used in this study were obtained from American Type Culture Collection (Manassas, VA). U937 (human histiocytic lymphoma) and HL-60 (human promyelocytic leukemia) cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (Gibco, NY, USA) at 37 ◦ C in humidified 5% CO2 atmosphere. MCF-7 (human breast epithelial) and HeLa (human cervix epithelial) cells were cultured in Eagles’ minimum essential medium containing 10% fetal bovine serum under the same conditions. 2.3. Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR was performed to evaluate NF-␬B-regulated gene expression induced by TNF-␣, LPS and PMA in U937 cells. One ␮g of total RNA was subjected to a RT reaction using random oligonucleotide primers and M-MLV reverse transcriptase (Promega). One microliter of the RT reaction

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product was then amplified by PCR using HotStar Taq DNA polymerase (Qiagen) under the following conditions: 95 ◦ C for 1 min; 55 ◦ C for 1 min; 72 ◦ C for 1 min. The PCR regimen was: 5 -ACCGGAAGGAACCATCTCACTG-3 , 5 -GCATCTGGCAACCCTACAACA-3 for IL-8 (444 bp, 25 cycles); 5 -GCTCATAGCAGCCACCTTCATTC-3 , 5 -TGCAGATTCTTGGGTTGTGGAG-3 for MCP-1 (297 bp, 25 cycles); 5 -TCACCTGCTCAGAATCATGC-3 , 5 -TCCATAGAAGAGGTAGCTGTGG-3 for MIP-1␣ (404 bp, 28 cycles); 5 GAGCACTGAAAGCATGATCCGGGAC-3 , 5 -TTGGTCTGGTAGGAGACGGCGATGC-3 for TNF-␣ (495 bp, 28 cycles); 5 -GGAGACCTGAGAACCAATCTC-3 , 5 -TCCAATAGGTGATGTTGTCGT-3 for MMP-9 (276 bp, 31 cycles); 5 -ACAACATCGCCCTGTGGATGAC-3 , 5 -ATAGCTGATTCGACGTTTTGCC-3 for bcl-2 (408 bp, 31 cycles); 5 -TCCTAGCTGCAGATTCGTTC-3 , 5 -GGTAACTGGC TTGAACTTGAC-3 for c-IAP2 (745 bp, 31 cycles); 5 TGAAGGTCGGAGTCAACGGATTTGGT-3 , 5 -CATGTGGGCCATGAGGTCCACCAC-3 for GAPDH (983 bp, 28 cycles). Samples containing 5 ␮l of the PCR product were run on a 1% agarose gel and visualized by ethidium bromide staining.

high-speed centrifugation. Each sample containing 500 ␮g (at 1 ␮g/␮l) of protein was incubated with 0.25 ␮g of the appropriate control IgG together with 20 ␮l of Protein A/G Plus agarose conjugate (25%, v/v) for 30 min at 4 ◦ C. The soluble fraction was incubated with 1 ␮g of anti-IKK␣/␤ for 2 h at 4 ◦ C and 20 ␮l of Protein A/G Plus agarose was added and the mixtures were incubated at 4 ◦ C on a rocker platform overnight. After several washes with IP buffer and PBS, beads coated with IKK␣/␤ were incubated with 0.5 ␮g GST-I␬B␣ substrate, 200 ␮M ATP in 20 ␮l kinase buffer (50 mM Tris-Cl pH 7.4, 20 mM MgCl2 , 20 mM ␤-glycerophosphate, 1 mM NaF, 1 mM Na3 VO4 , 1 mM PMSF, 0.5 mM DTT, 1 mM benzamidine, 10 ␮g/ml aprotinin and 1 ␮g/ml leupeptin) at 30 ◦ C for 30 min. The kinase reaction was stopped by the addition of 5 ␮l of a 5× Laemmli’s loading buffer and heated at 100 ◦ C for 5 min. Proteins were resolved by 8% SDS-PAGE, electro-transferred to nitrocellulose membrane and probed with anti-phosphor-I␬B␣ (Ser32 ) antibody (1:1000). Membranes were re-probed with anti-IKK to ensure equal loading and the presence of total IKK protein.

2.4. Electrophoretic mobility shift assay (EMSA)

The NF-␬B-dependent luciferase reporter plasmid (p3EnhConA-Luc) and its carrier control (pControl-Luc) were a gift from Dr. Ronald T Hay (School of Biology, University of St Andrews, UK). The plasmid is driven by three synthetic copies of the NF-␬B-consensus sequence from the immunoglobulin ␬ chain promoter. The pControl-Luc is identical to p3EnhConA-Luc except for the absence of the NF-␬B-consensus sequence. ␤-galactosidase control vector (pTracer-EF/Bsd/lacZ) was purchased from Invitrogen Life Technologies Inc. pCMV4 -HA-mI␬B␣ encoding both the wide type and the phosphorylation mutant I␬B␣ and pCMV4 -p65 encoding NF-␬B subunit p65 (Sun et al., 1996) were kindly provided by Dr. Warner C. Greene (Gladstone Institute of Virology and Immunology, University of California, USA). The expression plasmids, pCMV2 -Flag-IKK␤ (WT), pCMV2 -FlagIKK␣ (SS/EE) and pCMV2 -Flag-IKK␤ (SS/EE), encoding wide type or constitutively active IKKs (Mercurio et al., 1997) were generously supplied by Dr. Richard B. Gaynor (Department of Medicine, University of Texas Southwestern Medical Center, USA). The pCS3MT-NIK expression plasmid encoding wide type NF-␬B-inducing kinase (NIK) was provided by Dr. M. Kracht (Institute of Pharmacology, Medical School at Hannover, Germany). HeLa cells were seeded into 24-well plates at a density of 1.6 × 105 cells/well and grown for 24 h. Cells were transiently transfected with p3EnhConA-Luc or pControl-Luc (0.75 ␮g) using LipofectAMINE 2000 (Invitrogen), co-transfected with 0.25 ␮g of ␤-galactosidase control vector as an internal transfection efficiency standard and incubated overnight. Transfected cells were treated with magnolol for 12 h followed by the addition of 5 ng/ml of TNF-␣ and incubated for an additional 15 h. Cells were harvested in 1× reporter lysis buffer (Promega, Madison, WI). Relative luciferase activity was measured with a Bright-GLO luciferase assay system using

EMSA was performed as described (Chaturvedi et al., 2000). Briefly, 5 ␮g of nuclear protein was incubated with radio-labeled gel shift oligonucleotides for 15 min at 37 ◦ C and then separated on a non-denaturing 5% (w/v) polyacrylamide gel. The gel was dried onto a piece of 3 MM blotting paper that was used to expose X-ray film overnight at −70 ◦ C. For supershift assays, 1 ␮l of antiserum recognizing each of the NF-␬B subunits was added to EMSA reaction mixtures 30 min before electrophoresis. 2.5. Western blot analysis Cells (1 × 107 ) were washed twice with ice-cold PBS and lysed in 150 ␮l of modified RIPA buffer (50 mM Tris-Cl, 1% v/v NP-40, 0.35% w/v sodium-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF, 1 mM Na3 VO4 , 10 ␮g/ml each of aprotinin, leupeptin and pepstatin A for 20 min at 4 ◦ C. After centrifugation at 14,000 × g for 15 min at 4 ◦ C, the supernatant was collected. Alternatively, cytoplasmic extracts were prepared as described (Chaturvedi et al., 2000). Samples containing 30–50 ␮g of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes (0.45 ␮m, Bio-Rad) that were subsequently immunoblotted with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (1:5000). Labeled protein spots were visualized by ECL (Amersham Biosciences) according to manufacturer’s instructions. 2.6. IKK kinase assay Cells were lysed as above in modified RIPA buffer without sodium-deoxycholate and cellular debris was removed by

2.7. Plasmids, transfection and NF-κB-dependentluciferase reporter assay

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POLARStar OPTIMA luminometer (BMG Labtechnologies). Luciferase activity was normalized with ␤-galactosidase activity as measured by Beta-GLO luciferase assay system according to the manufacturer’s instructions. To study the effects of magnolol on the expression of NF␬B-dependent luciferase reporter, HeLa cells transfected with the p3EnhConA-Luc and ␤-galactosidase control vectors were transiently transfected with 0.2 ␮g of each of the expression vectors of various kinases mentioned above. After 5 h incubation, cells were treated with magnolol for 24 h, harvested and assayed as described above. 2.8. TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay Cleavage of genomic DNA during apoptosis was detected by TUNEL assay using in situ Cell Death Detection Kit (Boehringer Mannheim) and performed according to the manufacturer’s instruction. 2.9. Morphology assay of live and dead cells Treated cells were washed twice, resuspended in PBS containing 10 ␮g/ml acridine orange (AO) and 10 ␮g/ml ethidium bromide (EB) and observed immediately under a confocal microscope. The status of cells was identified and recorded as previous described (McGahon et al., 1995). 2.10. Statistical analysis and data treatment

nolol for 18 h, stimulated with 5 ng/ml of TNF-␣ for 30 min, and the nuclear extracts isolated. NF-␬B-DNA binding was investigated by EMSA (Fig. 2A–D). Magnolol at 30–80 ␮M inhibited the nuclear presence of NF-␬B in all four cell lines. TNF-␣stimulated U-937 cells were used in the following experiments due to the present of both TNF receptors on its cell surface (Higuchi and Aggarwal, 1992) and its well-established methodology in the study of NF-␬B pathways. 3.3. Effects of magnolol on NF-κB p65 and p50 subunits NF-␬B is a homo- or hetero-dimer composed of different combinations of subunits (Hayden and Ghosh, 2004). We studied the effects of magnolol on the subunit-DNA binding in the nuclear extracts isolated from TNF-␣ treated U937 cells. The identities of the DNA-retarded protein bands were detected with p65, p50 or c-Rel antibodies and evaluated by EMSA. Both the p65 and the p50 subunits, but not the c-Rel subunit, were shifted to higher molecular weight positions, showing that the activated NF-␬B complex consisted of the p65 and p50 subunits (Fig. 3A, lanes 3–6). The identity of the NF-␬B subunits was further confirmed by the disappearance of the band upon competition with 40-fold unlabeled NF-␬B consensus probes (Fig. 3A, lane 7). Furthermore, competition with non-specific Sp-1 probes did not alter the NF-␬B binding (Fig. 3A, lane 8). 3.4. Magnolol did not directly alter NF-κB-DNA binding

Activation of NF-␬B by various stimuli, such as TNF-␣, LPS or PMA, induces the expression of diverse groups of target genes that control inflammation and the immune system (Shishodia and Aggarwal, 2004). We induced NF-␬B-regulated gene expression by various stimuli and examined the effects of magnolol by RT-PCR. Under different situations treatment of cells with magnolol suppressed either the intrinsic or induced expression of NF-␬B-regulated gene products (Fig. 1B).

It has been suggested that many plant-derived compounds, such as caffeic acid phenethyl ester (Natarajan et al., 1996), avicins (Haridas et al., 2001), kamebakaurin (Lee et al., 2002) and andrographolide (Xia et al., 2004) may inhibit NF-␬B activity through the directly chemical modification of NF-␬B. Nuclear extracts from TNF-␣-stimulated cells were incubated with increasing concentrations of magnolol at 37 ◦ C for 1 h. Our results show that magnolol did not directly alter the NF-␬B-DNA binding (Fig. 3B). We next determined the effect of the length of magnolol pretreatment time on inhibiting NF-␬B activity. U937 cells were incubated with magnolol for various time intervals and subjected to TNF-␣ treatment. Prolonging magnolol pre-treatment before TNF-␣ treatment increased the inhibitory effect on NF-␬B activation (Fig. 3C). It has been reported that the activation of NF-␬B in leukemic cells by TNF-␣ is a rapid process and the level of activation depends on the TNF-␣ doses (Hohmann et al., 1990; Chaturvedi et al., 1994). We investigated the effect of magnolol on NF-␬B activation induced by various concentrations of TNF␣. Magnolol at 60 ␮M completely inhibited the NF-␬B activation by various doses of TNF-␣ from 1 to 50 ng/ml (Fig. 3D).

3.2. Magnolol inhibited TNF-α-stimulated NF-κB activation in different cell types

3.5. Magnolol inhibited TNF-α-stimulated IκBα phosphorylation and degradation

To study the effects of magnolol on TNF-␣-stimulated NF␬B activation, Hela (human cervix epithelial), MCF-7 (human breast epithelial), HL-60 (human promyelocytic leukemia) and U937 (human promonocytic) cells were pretreated with mag-

TNF-␣ induces phosphorylation of I␬B␣ which marks it for proteolytic degradation, thereby releasing the active NF-␬B dimmers for translocation to the nucleus to activate specific target genes (Henkel et al., 1993). We examined the phosphorylation

Statistical analyses were preformed using an unpaired twotailed Student’s t-test. Two compounds (A and B) were considered enhancing each other’s actions if the effect of combined treatment (AB) was larger then the sum of their individual effects (AB > A + B) after subtraction of the respective background control valves. 3. Results 3.1. Magnolol suppressed NF-κB-regulated gene expression stimulated by TNF-α, LPS and PMA

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Fig. 2. Magnolol suppresses TNF-␣-induced NF-␬B activation in different cell lines. U937, HL-60, MCF-7 and HeLa cells were pre-incubated with different doses of magnolol at 37 ◦ C for 18 h and stimulated by TNF-␣ for 30 min. Nuclear extracts were analyzed by EMSA described in Section 2. Densitometry values for NF-␬B binding (normalized to the medium control) are shown under each lane. NS represents non-specific binding.

and protein levels of I␬B␣ by immunoblot analysis. As shown in Fig. 4A and as previously reported (Sun et al., 1993), TNF␣ caused a rapid degradation of I␬B␣ after a 5 min treatment, which was followed by a slow but dramatic restoration of the I␬B␣ level at 120 min. Magnolol at 60 ␮M completely blocked the degradation of I␬B␣ (Fig. 4A) and the inhibitory effects were dose dependent (Fig. 4B). To evaluate the level of I␬B␣ phosphorylation we used proteasome inhibitor MG132 to block degradation of phosphorylated I␬B␣ (Palombella et al., 1994), and the phosphorylation status was assessed by immunoblot with an I␬B␣ phospoho-Ser32 specific antibody. At 30 min of treatment, magnolol inhibited the TNF-␣-stimulated phosphorylation of I␬B␣ (Fig. 4C). 3.6. Magnolol inhibited TNF-α-stimulated IKK activity TNF-␣ stimulates I␬B␣ phosphorylation and degradation via activation of the IKK complex (Zandi et al.,

1997). We performed a non-radioactive in vitro kinase assay using anti-IKK-antibody-precipitated protein from cell lysates as an enzyme source and GST-I␬B␣ as the substrate. TNF-␣ treatment stimulated IKK kinase activity and the maximum phosphorylation activity was achieved in about 5 min of treatment (Fig. 5A). Magnolol inhibited both the intrinsic and TNF-␣-stimulated IKK activity in U937 cells (Fig. 5B) but did not affect the immuno-precipitated IKK protein levels. Direct cell-free IKK kinase assay with lysates obtained from TNF-␣-treated cells showed that at 60 ␮M magnolol had only a minor direct effect on IKK activity (Fig. 5C). The role of IKK in magnolol’s action was further elucidated with studies with NF-␬B-dependent reporter gene expression. HeLa cells were transfected with NF-␬B-dependent luciferase reporter. Genes encoding the two catalytic subunits of IKK, including wide-type IKK␤, constitutively active IKK␣ and IKK␤, and also NF-␬B-inducing kinase (NIK), which is a com-

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Fig. 3. (A) NF-␬B-DNA binding involves the p65 and p50 subunits. U937 cells (1.5 × 106 ml−1 ) were treated with 5 ng/ml of TNF-␣ for 30 min and the nuclear extracts were isolated, followed up by incubation with indicated antibodies or unlabeled NF-␬B probe (cold probe) at 37 ◦ C for 30 min. The mixtures were assayed by EMSA. (B) Magnolol does not directly modify NF-␬B-DNA binding. Nuclear extracts from TNF-␣ treated U937 cells were incubated with indicated concentrations of magnolol for 45 min at 37 ◦ C, and then assayed for NF-␬B binding by EMSA. (C) Effects of magnolol pre-treatement on TNF-␣-induced NF-␬B activities. U937 cells (1.5 × 106 ml−1 ) were pre-incubated with 60 ␮M magnolol for the indicated time intervals and treated with 5 ng/ml of TNF-␣ for 30 min at 37 ◦ C. NF-␬B activities were assayed by EMSA. (D) Magnolol inhibits NF-␬B activities induced by different concentrations of TNF-␣. U937 cells (1.5 × 106 ml−1 ) were pre-incubated with 60 ␮M magnolol for 18 h, treated with different concentrations of TNF-␣ for 30 min at 37 ◦ C and assayed for NF-␬B activities by EMSA. Densitometry values for DNA binding (normalized to the medium control) are shown under each lane.

ponent of the TNF-receptor-induced IKK activation pathway (Ling et al., 1998), and NF-␬B p65 subunit were transiently transfected and overexpressed in the HeLa cells with the luciferase reporter gene (Fig. 5D). The luciferase reporter was

activated in all cases and magnolol significantly inhibited the NF-␬B-dependent gene expression (Fig. 5D). These results indicate that magnolol inhibit IKK-mediated NF-␬B activation pathway.

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Fig. 4. Magnolol inhibits TNF-␣-induced degradation and phosphorylation of I␬B␣. (A) Magnolol inhibits TNF-␣-induced degradation of I␬B␣. U937 cells (1.5 × 106 ml−1 ) were treated with 60 ␮M magnolol for 18 h and stimulated with 5 ng/ml of TNF-␣ at indicated time intervals. The cytosolic extracts were obtained and analyzed by Western blot using anti-I␬B␣ antibody. (B) Dose-dependent inhibition of I␬B␣ degradation by magnolol. U937 cells (1.5 × 106 cells/ml) were pre-incubated with indicated concentrations of magnolol for 18 h and treated with different concentrations of TNF-␣ for 30 min at 37 ◦ C. Whole cell lysates were analyzed by Western blot. (C) Magnolol blocked the phosphorylation of I␬B␣ by TNF-␣. U937 cells (1.5 × 106 ml−1 ) were treated with 60 ␮M magnolol for 18 h, 20 ␮M protease inhibitor MG132 for 1 h, and stimulated with 5 ng/ml of TNF-␣ for 30 min. Whole cell extracts were fractionated and analyzed by Western blot using an anti-p-I␬B␣ antibody.

3.7. Magnolol inhibited TNF-α-stimulated p65 nuclear accumulation and phosphorylation

3.8. Magnolol inhibited TNF-α-stimulated NF-κB-dependent luciferase gene expression

EMSA results indicate that there were increases in nuclear NF-␬B level after 15–120 min of TNF-␣ treatments and the increase was diminished by the presence of magnolol (Fig. 6A). We determined p65 nuclear accumulation using Western blot analysis since p65 subunit is responsible for the transcriptional activity of NF-␬B (Schmitz and Baeuerle, 1991). As illustrated in Fig. 6B, magnolol caused a dose-dependent reduction in TNF-␣-stimulated nuclear accumulation of p65 (Fig. 6C). These results are in line with observations mentioned above on magnolol’s effect on TNF-␣-stimulated I␬B␣ degradation (Fig. 4). Phosphorylation of p65 subunit by a variety of kinases, including IKK, leds to the modification of NF-␬B activity (Vermeulen et al., 2002). Our results showed that magnolol completely blocked TNF-␣-mediated p65 phosphorylation in whole cell extracts (Fig. 6D).

HeLa cells were transfected with a NF-kB promoterdependent luciferase reporter construct and stimulated with 5 ng/ml TNF-␣. Treatment with TNF-␣ resulted in a 4.3-fold increase in reporter gene expression, which was suppressed by over 50% by magnolol pre-treatment (Fig. 7). Magnolol also inhibited PMA-induced NF-␬B-dependent luciferase gene expression (data not shown). Co-transfection of I␬B␣ plasmid abrogated the luciferase gene expression induced by TNF-␣ (Fig. 7, lane 5). Luciferase reporter activation was not detected in cells transfected with the promoter-less control reporter (Fig. 7, lane 6). 3.9. Magnolol sensitizes TNF-α-induced apoptosis TNF-␣ has dual actions on apoptosis. On the one hand it triggers the death receptor-mediated pro-apoptotic pathways, but

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Fig. 5. Effect of magnolol on the TNF-␣-induced IKK activation. (A) TNF-␣-induced, time-dependent I␬B␣ phosphorylation. Whole cell lysates of U937 cells (1.5 × 106 cells/ml) were treated with 5 ng/ml of TNF-␣ for various durations, immunoprecipitated using anti-IKK␣/␤ antibody, and assayed for IKK kinase activity. The phosphorylation of GST-I␬B␣ was detected by Western blot analysis using p-I␬B␣ antibody. The same nitrocellulose membrane was re-probed with anti-IKK␣/␤ antibody to detect the level of immunoprecipitated IKK protein. (B) Magnolol inhibits TNF-␣-induced IKK activity. U937 cells (1.5 × 106 cells/ml) were treated with 60 ␮M magnolol for 18 h, stimulated with 5 ng/ml of TNF-␣ for 5 min, and IKK protein were immunoprecipitated with anti-IKK␣/␤ antibody. The level of phosphorylation of GST-I␬B␣ was detected by IKK assay. (C) Direct effect of magnolol on TNF-␣-induced IKK activity. U937 cells (1.5 × 106 cells/ml) were treated with 5 ng/ml of TNF-␣ for 5 min and whole cell extracts were prepared and immunoprecipitated with anti-IKK␣/␤ antibody. The IKK assay was performed in the absence or presence of 60 ␮M magnolol for 30 min at 30 ◦ C. Densitometry results (normalized to IKK␣/␤ protein and the medium control) are shown under each lane. (D) Magnolol suppresses NF-␬B-dependent gene expression induced by the over-expression of NIK, IKK␣/␤ and p65 subunit. HeLa cells transfected with the NF-␬B-dependent luciferase reporter were transiently transfected with NIK, wide-type IKK␤, and constitutively active IKK␣/␤ and p65 plasmids. Cells were incubated with 60 ␮M magnolol for 24 h and the luciferase activities detected. Results are normalized with ␤-galactosidase activity and expressed as folds changed compared to the medium control. Results are mean ± standard derivation of triplicate determinations. Statistically significant differences between with or without magnolol treatments: *p < 0.001.

on the other it may up-regulate NF-␬B-regulated anti-apoptotic genes, which results in the blockage of cell death (Yamamoto and Gaynor, 2001). Thus, the sensitivity of cells to apoptotic signals can be increased by the inhibition of NF-␬B-mediated survival and anti-apoptotic pathways. We therefore hypothesized that magnolol pre-treatment might potentiate the TNF-␣’s apoptotic action. Apoptosis was detected by DNA fragmentation (TUNEL assay) coupled with flow cytometry analysis (Fig. 8A), caspase-3 cleavage (Fig. 8B) and the live/dead morphological assay (Fig. 8C). All the experiments confirmed that magnolol enhanced the apoptotic effect of TNF-␣. 4. Discussion In this paper we have established that magnolol may exert some of its anti-inflammatory and pharmacological effects by affecting the activity of NF-␬B. In U937 promonocytes cells magnolol suppresses chemically induced, NF-␬B-regulated inflammatory gene products and the suppression is mediated by interfering the binding of NF-␬B p65/50 heterdimer to DNA. Mechanistically, magnolol abrogates the IKK activation, results in blockage of I␬B␣ phosphorylation/degradation and p65 subunit translocation. Since the activation of NF-␬B induces anti-apoptotic signaling, the anti-NF-␬B effect of magnolol has been verified by its enhancement of TNF-␣-induced apoptosis. Our data suggest that the signal pathways and kinases upstream of IKK activation may be involved in the action of

magnolol. Magnolol has an effect on the intrinsically expressed IKK and in addition it also inhibits NIK-induced NF-␬B activation. The IKK complex, consisting of two catalytic subunits IKK␣ and IKK␤ and a regulatory subunit IKK␥, targets at two distinct serine residues on I␬B␣. The phosphorylation of the serine residues leds to a rapid ubiquitin-dependent proteolysis of I␬B␣ and finally NF-␬B activation (Zandi et al., 1997; Hayden and Ghosh, 2004). In a HeLa cell NF-␬B-regulated luciferase reporter system, we have demonstrated that magnolol suppresses the reporter expression stimulated by the transfection and expression of the wide-type IKK␤, and constitutively active IKK␣ and IKK␤. Phosphorylation/activation of IKK involves multiple upstream kinases including NIK, MEKK3, Tpl2, PKC, Akt (Hayden and Ghosh, 2004) and calcium-binding protein calmodulin (Hughes et al., 2001). Magnolol have been reported to alter the MAPK (Yang et al., 2003), Ca2+ (Teng et al., 1990; Wang and Chen, 1998; Zhai et al., 2003) and PKC (Wang et al., 1998) signaling events. At the present time, it is only possible to assume that some of these molecular signals may be involved in IKK inactivation and the exact molecular targets affected by magnolol remain to be identified. Our study reveals that magnolol inhibited extrinsic p65induced NF-␬B-dependent luciferase activity (Fig. 6D). This finding leds to the possible hypotheses that (i) a fraction of extrinsic p65 was sequestered and inactivated by intrinsic I␬B␣, which in turn re-activated by intrinsic IKK. Magnolol was shown to inhibit intrinsic IKK activity (Fig. 5B), resulting in the blockage

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Fig. 7. Magnolol inhibits TNF-␣-induced NF-␬B-dependent luciferase reporter gene expression. HeLa cells were transfected with the NF-␬B-dependent luciferase reporter p3EnhConA-Luc, treated with magnolol for 12 h, and then stimulated with 5 ng/ml of TNF-␣ for 15 h. The luciferase expression was measured and normalized with ␤-galactosidase activity. Results are expressed as folds changed compared to the medium control and expressed as mean ± standard derivation of triplicate determinations. Statistically significant differences between TNF-␣ stimulation alone and other treatments: *p < 0.02 and **p < 0.005.

Fig. 6. Magnolol suppresses TNF-␣-induced p65 nuclear translocation and phosphorylation. (A) Magnolol blocks the time-dependent TNF-␣-induced NF␬B activation. U937 cells (1.5 × 106 cells/ml) were treated with 60 ␮M magnolol for 18 h and stimulated with 5 ng/ml of TNF-␣ for various durations at 37 ◦ C. The NF-␬B activities were detected by EMSA. Densitometry measurements of NF-␬B are normalized with the medium control and shown under each lane. (B) Magnolol inhibits TNF-␣-induced p65 nuclear translocation. U937 cells (1.5 × 106 cells/ml) were treated as described in (A). Nuclear and cytoplasmic extracts were obtained and the p65 protein levels were measured by Western blot analysis as described under Section 2. Densitometry measurements of the p65 protein are normalized to lamin B, tubulin or the medium control and shown under each lane. (C) Dose-dependent inhibition of p65 nuclear translocation by magnolol. U937 cells (1.5 × 106 cells/ml) were treated with indicated concentrations of magnolol for 18 h and stimulated with 5 ng/ml of TNF-␣ for 30 min at 37 ◦ C. Nuclear extracts were obtained and the p65 protein levels were measured by Western blot analysis. Densitometry measurements of the p65 protein are normalized with lamin B and the medium control and shown under each lane. (D) Magnolol inhibits TNF-␣-induced p65 phosphorylation. U937 cells (1.5 × 106 cells/ml) were treated with 60 ␮M of magnolol for 18 h and stimulated with 5 ng/ml of TNF-␣ for various durations. Whole cell extracts were prepared, fractionated and analyzed by Western blot using anti-phosphorylatedp65 antibody. Densitometry measurements of the phosphorylated p65 protein are normalized with total p65 and the medium control and shown under each lane.

of the IKK-induced extrinsic p65-NF-␬B activity; (ii) the transcription activity of extrinsic p65 was reduced by the inhibition of intrinsic IKK- or other kinases-induced p65 phosphorylation. Consistent with previous reports (Son et al., 2000; Chen et al., 2001, 2002, 2006; Matsuda et al., 2001; Park et al., 2004; Lee et al., 2005), magnolol suppresses inflammatory gene products IL-8, MCP-1, MIP-1␣, TNF-␣, and MMP-9 that are known to be regulated by NF-␬B. These genes have been implicated in inflammatory diseases and also in tumorigenesis and carcinogenesis. For example IL-8 and MCP-1 are important activators and chemo-attractants for neutrophils (Mukaida et al., 1998). MIP-1␣ is a 8kDa member of the C–C chemokine subfamily that brings about inflammatory disorders such as asthma, arthritis, or multiple sclerosis (Menten et al., 2002). TNF-␣ is a proinflammatory cytokine and has recently been implicated in the carcinogenic processes. The production of TNF-␣ by stromal cells was found to be essential for promoting inflammationassociated malignancy (Greten et al., 2004; Pikarsky et al., 2004). MMP-9 plays an important role in tissue remodeling in normal and pathological inflammatory processes (Van den Steen et al., 2002; St-Pierre et al., 2003). Some specificity has been observed in the inhibitory action of magnolol on NF-␬B-regulated inflammatory responses. For example RT-PCR gene expression experiments have shown that magnolol reduces MIP-1␣ mRNA levels in PMA- and TNF␣-stimulated cells, but not in LPS-stimulated cells (Fig. 1B). However, as suggested by Baltimore and coworkers (Hoffmann et al., 2002, 2003; Leung et al., 2004), the stimulus responsiveness of NF-␬B is not solely dependent on the DNA-NF-␬B interactions but in addition the interaction duration, or the participation of other transcriptional machinery, such as neighboring transcription factors, co-activators and chromatin components, may make various contributions to the formation of the final responses. For example the MIP-1␣ promoter can also be activated by other transcription factors such as MIP-1␣ nuclear protein and RUNX1 (Ritter et al., 1995; Bristow and Shore,

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Fig. 8. Magnolol enhances TNF-␣-induced apoptosis in U937 cells. (A) Magnolol enhances TNF-␣-induced TUNEL positive cells. U937 cells (1.0 × 106 cells/ml) were treated with 60 ␮M magnolol for 4 h and with 5 ng/ml TNF-␣ for 24 h. The TUNEL positive cells were identified by flow cytometry and results are expressed as mean ± standard derivation of triplicate determinations. Statistically significant differences from the sum of the individual effects of drugs: *p < 0.015. (B) Magnolol enhances TNF-␣-induced capases-3 activation. U937 cells (1.0 × 106 cells/ml) were treated as in (A) and the pro- and activated-caspase-3 proteins were detected by Western blot analysis using anti-caspase-3 antibody. Cycloheximide (CHX, 1 ␮g/ml) was added as a positive control for the apoptotic enhancing effect. (C) The effect of magnolol on TNF-␣-induced apoptotic nuclear condensation. A portion of U937 cells (1.0 × 106 cells/ml) treated described in (A) were stained with AO/EB and analyzed by confocal laser microscopy.

2003). We have observed that magnolol also influences other transcription factors-regulated signaling pathways (e.g. AP-1, data not shown) that may account for the individual inhibitory effects. On the cellular level we have shown that one of magnolol notable actions is the enhancement of TNF-␣-induced apoptosis. A number of mechanisms have been suggested in the enhancement of TNF-␣-induced cell death: (i) suppression of NF-␬B-regulated anti-apoptotic signaling (Beg and Baltimore, 1996; Van Antwerp et al., 1996; Wang et al., 1996); (ii) activation of caspase-8, the triggering caspase in the TNF-␣ apoptotic pathway (Hsu et al., 1996); (iii) promotion of c-Jun N-terminal kinase signaling (JNK) (Deng et al., 2003); (iv) accumulation of reactive oxygen species (Weitsman et al., 2003; Kamata et al., 2005); (v) inhibition of p38 mitogen-activated protein kinase activation (Varghese et al., 2001; Luschen et al., 2004). Magnolol activates the caspase-8 (Lin et al., 2001; Ikeda and Nagase, 2002) and the JNK pathways (Yang et al., 2003) and has been observed to suppress both the intrinsic or induced expression of NF-␬B-regulated apoptotic genes bcl-2 and cIAP2 (data not shown). The involvement of other pathways in the enhancement of TNF-␣-induced apoptosis by magnolol needs to be further clarified. Magnolol is likely a universal inhibitor for NF-␬B activation in various cell-types. Recent studies have suggested some

cell type-specificities in the inhibition of TNF-␣-induced NF-␬B activation, probably mediated by the cell type-specific NF-␬B signaling mediators such as Akt (Gustin et al., 2004) or reactive oxygen species (Panopoulos et al., 2005). Magnolol, however, blocks TNF-␣-induced NF-␬B activation in all four cell lines tested. A previous report (Chen et al., 2002) showed that magnolol, in concentration as low as 5 ␮M, inhibits NF-␬B activation in human aortic endothelial cells but in our studies higher doses (30–60 ␮M) of magnolol are needed to produce similar effects. In summary, these experiments demonstrate that magnolol suppresses TNF-␣-induced NF-␬B p65 subunit activation and the upstream IKK activation, I␬B␣ phosphorylation and degradation. And likely as a result it suppresses NF-␬B-regulated gene expression and enhances TNF-␣-induced apoptosis. Our results suggest that magnolol or its derivatives may have potential therapeutic values. Acknowledgement We thank Dr. Ronald T Hay for the NF-␬B-dependent luciferase reporter and its control plasmids, Dr. Warner C. Greene for the p65 and I␬B␣ plasmids, Dr. Richard B. Gaynor for the IKK␣/␤ plasmids, and Dr. M. Kracht for the NIK plasmids. This project is supported by special grants from CityU.

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