Incensole Acetate, a Novel Anti-Inflammatory Compound Isolated from Boswellia Resin, Inhibits Nuclear Factor- B Activation

Boswellia resin is a major anti-inflammatory agent in herbal medical tradition, as well as a common food supplement. Its anti-inflammatory activity has been attributed to boswellic acid and its derivatives. Here, we re-examined the antiinflammatory effect of the resin, using inhibitor of nuclear factorB (I B ) degradation in tumor necrosis factor (TNF) -stimulated HeLa cells for a bioassay-guided fractionation. We thus isolated two novel nuclear factorB (NFB) inhibitors from the resin, their structures elucidated as incensole acetate (IA) and its nonacetylated form, incensole (IN). IA inhibited TAK/TAB-mediated I B kinase (IKK) activation loop phosphorylation, resulting in the inhibition of cytokine and lipopolysaccharide-mediated NFB activation. It had no effect on IKK activity in vitro, and it did not suppress I B phosphorylation in costimulated T-cells, indicating that the kinase inhibition is neither direct nor does it affect all NFB activation pathways. The inhibitory effect seems specific; IA did not interfere with TNF -induced activation of c-Jun Nterminal kinase (JNK) and p38 mitogen-activated protein kinase. IA treatment had a robust anti-inflammatory effect in a mouse inflamed paw model. Cembrenoid diterpenoids, specifically IA and its derivatives, may thus constitute a potential novel group of NFB inhibitors, originating from an ancient anti-inflammatory herbal remedy. Boswellia species are natives of Eastern Africa, where their resin, commonly known as “frankincense” or “olibanum,” is used and exported as incense. It has been extensively used for many centuries for various medical purposes, especially for the treatment of inflammatory diseases, in European, Middle Eastern, and African medical traditions. In India, Boswellia resin is widely used in the treatment of inflammatory conditions, including Crohn’s disease, arthritic diseases, and asthma; hence, a considerable amount of work has been done on the antiinflammatory properties of Boswellia (for example, see Gupta et al., 1998; Gerhardt et al., 2001; Altmann et al., 2004). Numerous previous reports attribute the anti-inflammatory and cytotoxic properties of Boswellia resin solely to boswellic acid and its derivatives (e.g., Gerhardt et al., 2001; Altmann et al., 2004; Xia et al., 2005; Khanna et al., 2007). NFB is an inducible transcription factor that plays a central role in the mammalian innate immune response and This work was supported by a EC 5th framework consortium grant number QLK3-CT-2000-00-463 (Anti-Inflammatory Natural Products consortium), and by the Miriam and Sheldon Adelson program in Neural Repair and Rehabilitation for support (to R.M.). Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.038810. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: NFB, nuclear factor B; I B, inhibitor of nuclear factorB; IKK, I B kinase; TAK, transforming growth factor -activated kinase; TAB, transforming growth factor -activated kinase-binding protein; IA, incensole acetate; IN, incensole; PE, petroleum ether; HPLC, high-performance liquid chromatography; GC-MS, gas chromatograph-mass spectrometry; LTR, long-terminal repeat; TNF , tumor necrosis factor; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; NP-40, Nonidet P-40; WB, Western blotting; GST, glutathione transferase; JNK, C-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; WB, Western blotting. 0026-895X/07/7206-1657–1664$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 6 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 38810/3281935 Mol Pharmacol 72:1657–1664, 2007 Printed in U.S.A. 1657 http://molpharm.aspetjournals.org/content/suppl/2007/09/25/mol.107.038810.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from chronic inflammation (Karin, 2005; Perkins, 2007). Ubiquitously expressed and involved in the activation of a multitude of genes in response to various stress stimuli, NFB plays a pivotal role in immune and inflammatory responses (Karin and Ben-Neriah, 2000). This effect is exerted through the regulation of target genes that encode proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes (Lawrence et al., 2001). Inappropriate regulation of NFB is thus directly involved in a wide range of human disorders, including arthritis, asthma, inflammatory bowel disease, a variety of cancers, ataxia telangiectasia, and neurodegenerative diseases (Karin and Ben-Neriah, 2000; Ben-Neriah and Schmitz, 2004). Hence, identification of drugs allowing modulation of the NFB transduction pathway is of considerable interest (Bremner and Heinrich, 2002; Calzado et al., 2007). In nonstimulated cells, NFB is normally sequestered in the cytoplasm and must be translocated into the nucleus for the exertion of its function. This subcellular localization is controlled by I B proteins, a family of inhibitory proteins that bind NFB, inhibit its DNA binding, and prevent its nuclear accumulation. Specific extracellular stimuli lead to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of I B, which frees NFB to translocate to the nucleus, where it regulates gene transcription (Karin and Ben-Neriah, 2000; Perkins, 2007). Cytokines act through distinct signaling pathways that converge on the activation of IKK. I B degradation, after its phosphorylation by the IKK complex at Ser-32 and Ser-36, is considered to be the major step in NFB regulation (Karin and Ben-Neriah, 2000). Thus, activation of IKK is a key event in canonical NFB activation (Häcker and Karin, 2006). The core IKK complex consists of the kinases IKK and IKK and the regulatory IKK /NEMO protein. The activation of both IKKs depends on phosphorylation of serines at their activation loop. This process probably involves transautophosphorylation of IKKs and phosphorylation by upstream kinases such as transforming growth factor -activated kinase (TAK) 1. TAK1 is recruited to the IKK complex via the ubiquitin-binding adaptor proteins TAK-binding protein (TAB) 2 and TAB3 (Häcker and Karin, 2006). We revisited the anti-inflammatory properties of Boswellia resin and examined the mechanism by which the active ingredients of the resin inhibit NFB activation. A bioassayguided fractionation, testing the inhibition of I B phosphorylation/degradation, led to the identification and isolation of incensole acetate (IA) and its nonacetylated form, incensole (IN) as inhibitors of NFB activation. Although IA and IN have previously been identified in Boswellia species (Corsano and Nicoletti, 1967) and are considered to be biomarkers of these species (Hamm et al., 2005), their biological activities have not yet been studied. Materials and Methods Extraction and Isolation of IA. Boswellia carterii resin (20 g; Pamir, Tel-Aviv, Israel) was extracted with petroleum ether (PE) (three times with 150 ml). PE extract was washed with NaOH 5% solution (three times with 200 ml). The non–acid-containing PE fraction was acidified with HCl (1 M) and then washed with a saturated NaCl solution and dried over MgSO4. After evaporation, the residue was chomatographed on a silica column. Fractions were assayed for their activity on I B degradation as described under I B Phosphorylation and Degradation. A fraction eluted with 3% diethyl-ether in PE, which contained IA, showed activity. Pure IA was obtained by HPLC separation, using an HPLC system with a UV absorbance detector (Spectra-Physics 783; Applied Biosystems, Foster City, CA) and a C18 column (10 250 mm; Vydac, Hesperia, CA). Acetonitrile and water were used as mobile phase for HPLC, and the gradient consisted of 90 to 99% acetonitrile for 30 min. Structure Elucidation. To analyze the purification process, the HPLC system consisted of a pump (660; Waters, Milford, MA) and a Photo-Diode Array detector (996; Waters) with an analytical C18 symmetry column (4 250 mm). Electrospray ionization and high resolution mass spectral analyses (Bruker APE 3 ICRMS) as well as several NMR methods (H-NMR, C-NMR, distortionless enhancement by polarization transfer, correlation spectroscopy, heteronuclear single quantum correlation, heteronuclear multiple-bond correlation spectroscopy, total correlation spectroscopy, and nuclear Overhauser effect spectroscopy) were used for the structure elucidation of the isolated active compounds. NMR spectra were recorded both in CDCl3 and in C6D6 solutions using an Avance spectrometer (Bruker, Newark, DE) at 400 MHz and repeated using a Unity Inova spectrometer (Varian, Inc., Palo Alto, CA) at 500 MHz. GC-MS analysis was performed using a gas chromatograph detector system (G1800A; Hewlett Packard, Palo Alto, CA) with a gas chomatograph with an electron ionization detector (HP5971; Hewlett Packard). An SPB-5 (30 m 0.25 mm 0.25 m film thickness) column was used. The following method was used for analysis: The column was held at 70°C for 4 min, after which a temperature gradient was applied from 70 to 280°C, at a rate of 50°/min (inlet temperature, 280°C; detector temperature, 280°C; splitless injection; gas, helium, 1 ml/min). Cell Lines. HeLa cells and 293T cells were grown in Dulbecco s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% (v/v) penicillin/streptomycin (all from Biological Industries, Kibbutz Beit Haemek, Israel) in a humidified incubator at 37°C. The RAW 264.7 macrophage cell line derived from BALB/c mice was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 1% (v/v) penicillin/streptomycin (Biological Industries, Beit Haemek, Israel), nonessential amino acid (Sigma, St. Louis, MO), 1% glutamine (Beit Haemek, Israel), and 1% pyruvate (Beit Haemek, Israel). Cells were grown in a humidified incubator at 37°C. Jurkat T leukemia cells were grown at 37°C in RPMI 1640 medium containing 10% (v/v) heat-inactivated fetal calf serum, 10 mM HEPES, 1% (v/v) penicillin/streptomycin (all from Invitrogen, Eggenstein, Germany) and 2 mM glutamine. The 5.1 Jurkat and HeLa-Tat-Luc cell lines have been described previously (Sancho et al., 2004). 5.1 cells is a Jurkat derived clone stably transfected with a plasmid containing the luciferase gene driven by the HIV-1 LTR promoter, responsive to the NFB activator cytokine TNF . The HeLa-Tat-Luc contains the luciferase gene driven by the HIV-1 LTR promoter and the Tat gene regulated by the CMV promoter. Therefore, the HIV-1 LTR is highly activated in this cell line because of high levels of intracellular Tat protein, and the luciferase activity is on the order of 10 relative light units/10 cells (considered 100% activation). A549 cells (10/ml) were transiently cotransfected with the KBFLuc reporter (0.2 g/ml) together with empty vectors or overexpressing vectors for IKK /IKK (0.5 g/ml each), TRAF-2 (1 g/ml), and TAK1/TAB2 (0.5 g/ml each). The transfections were performed using Lipofectamine Plus reagent (Invitrogen) for 24 h, according to the manufacturer’s recommendations. Isolation of Human Monocytes. Human peripheral monocytes from healthy human donors were prepared following a standardized protocol (Ficoll gradient preparation; GE Healthcare, Freiburg, Germany) using a completely endotoxin-free cultivation as described previously (Noble et al., 1968; English and Andersen, 1974). By using 50-ml tubes, 25 ml of Ficoll was loaded with 25 ml of the buffy coats 1658 Moussaieff et al. at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from from the blood of healthy donors. The gradient was established by centrifugation at 1800 rpm, 20°C for 40 min by using slow acceleration and brakes. Peripheral blood mononuclear cells in the interphase were carefully removed and resuspended in 50 ml of prewarmed phosphate-buffered saline (PBS; Invitrogen, Karlsruhe, Germany) followed by centrifugation for 10 min at 1600 rpm and 20°C. The supernatant was discarded, and the pellet was washed in 50 ml of PBS and centrifuged as described above. The pellet was then re-suspended in 50 ml of RPMI-1640 low endotoxin-medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% human serum (PAA, Coelbe, Germany). Animals. Female Sabra mice (Harlan, Jerusalem, Israel; 15–20 weeks old) were used for in vivo anti-inflammatory assessments. Ten mice were housed in each cage. The animal care and protocols met the guidelines of the U.S. National Institutes of Health, detailed in the Guide for the Care and Use of Laboratory Animals, and were applied in conformity with the Institutional Ethics Committees. Temperature in the animal room was maintained between 20 and 22°C, and there was a 12-h light/dark cycle (light from 8:00 AM–8:00 PM). I B Phosphorylation and Degradation. HeLa cells were preincubated with tested compounds (dissolved in ethanol) for 2 h, and then stimulated for 20 mins with 20 ng/ml TNF (Cetus, Emeryville, CA) or costimulated with 20 ng/ml phorbol 12-myristate 13-acetate (PMA) and 100 ng/ml ionomycin for 15 min. After removing the slides from plates for immunostaining (see p65 Subunit Immunostaining), proteins were extracted in NP-40 lysis buffer [50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, 0.5 mM sodium vanadate, 10 g/ml leupeptin, 1% (v/v) NP-40, and 10% (v/v) glycerol] from remaining cells in the plates. Total protein concentration was determined using the Bradford method (Bradford, 1976). Lysates were then analyzed either by Western blotting (WB) or by in vitro kinase assays (see below). Boswellic acid mixture ( and ) was obtained from the laboratory of Dr. Gerald Culioli, Université de Toulon et du Var, La Valette-du-Var, France. Kinase Assays. The IKK complex was isolated from precleared NP-40 HeLa cell extracts (see I B Phosphorylation and Degradation) by immunoprecipitation with 2 g of IKK antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and 25 l of protein A/G Sepharose. The precipitate was washed three times in the above NP-40 lysis buffer and two times in kinase buffer (20 mM HEPES/KOH, pH 7.4, 25 mM -glycerophosphate, 2 mM dithiothreitol, and 20 mM MgCl2). The kinase assay was performed using glutathione transferase (GST) fusion proteins as substrates, in a final volume of 20 l of kinase buffer containing 2 g of bacterially expressed GST-I B(1–54), 20 M ATP, and 5 Ci of [ -P]ATP. After incubation for 20 min at 30°C, the reaction was stopped by the addition of 5 SDS sample buffer. After separation by SDS-polyacrylamide gel electrophoresis, the gel was fixed, dried, and autoradiographed. The JNK assays were performed with a similar protocol, except that the immunoprecipitation was done with JNK1 and JNK2 antibodies (Santa Cruz Biotechnology) and that GST-c-Jun (5–89) was used as a substrate protein. IKK Phosphorylation Assay. 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, Chalfont St. Giles, Buckinghamshire, UK). p65 Subunit Immunostaining. HeLa Cells were preincubated with IA and then stimulated with TNF , as described under I B Phosphorylation and Degradation above. Cells were then fixed with formaldehyde 1%, permeabilized with 0.25% Triton X-100, stained with rabbit anti-p65 (Santa Cruz Biotechnology) and visualized with anti-rabbit Rhodamine Red-labeled secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). Cells were also stained with 4,6-diamidino-2-phenylindole for nuclei location (data not shown). The cells were examined under an Axioscope Zeiss microscope with a plan-Neofluor 60 lens. Electrophoretic Mobility Shift Assay. Cells were preincubated for 1 h with IA and stimulated for 15 min as shown. The oligonucleotides were synthesized at MWG Biotech, Germany, and the singlestrand oligonucleotides were annealed according to standard procedure by heating and subsequent cooling down to 50°C in 10 mM Tris/HCl, pH 7.5, and 100 mM NaCl. Equal amounts of protein contained in TOTEX buffer [20 mM HEPES/KOH, pH 7.9, 0.35 M NaCl, 20% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, and 1 mM phenylmethylsulfonyl fluoride] were incubated with a P-labeled double-stranded oligonucleotide containing an NFB recognition site for 15 min. Bound and free oligonucleotides were separated by electrophoresis on a native 0.5 TBE 4% polyacrylamide gel. The dried gel was then exposed to X-ray film. Luciferase Assays. The various cell lines were preincubated with the compounds and stimulated as specified in the figure legends. Cells were harvested, washed with PBS and then lysed in a luciferase lysis buffer (25 mM Tris-phosphate, pH 7.8, 8 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, and 7% glycerol). Luciferase activity was measured using an Autolumat LB 953 luminometer (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, WI). Inflamed Paw Model. Vehicle (isopropanol/Emulphor/saline 1:1:18) or vehicle containing IA (50 mg/kg) was administered by i.p. injection 30 min before applying the inflammatory stimulus. Emulphor, a polyethoxylated vegetable oil, is a commercial emulsifier. Hind paws were injected with 50 l of saline (left or right alternating) or -carrageenin (4%, right or left alternating), using 26G needles. The resulting inflammatory swelling was measured by increase in foot volume in a plethysmometer (Ugo-Basile, Italy) as described previously (Calhoun et al., 1987). Paw volume as well as redness (as a measure of erythema) and licking (as a measure of pain) were assayed before carrageenin application and every 60 min until 4 h. Data Analysis. Dose response data were plotted and analyzed using GraphPad Prism 4.01 software (San Diego, CA). Differencees were considered statistically significant if the p value was 0.05.