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Original Papers

Xanthohumol and Related Prenylated Flavonoids Inhibit Inflammatory Cytokine Production in LPS-Activated THP-1 Monocytes: Structure-Activity Relationships and In Silico Binding to Myeloid Differentiation Protein-2 (MD-2)
Authors Affiliations Michael R. Peluso 1, 2, Cristobal L. Miranda 1, 2, Deborah J. Hobbs 1, Rosita R. Proteau 2, Jan Frederik Stevens 1, 2
1 2

Linus Pauling Institute, Oregon State University, Corvallis, Oregon, USA Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon, USA

Key words " l xanthohumol " l THP1 " l antiinflammatory " l MCP1 " l IL6 " l Tolllike receptor 4

Abstract
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received revised accepted

Dec. 20, 2009 February 14, 2010 February 24, 2010

Bibliography DOI http://dx.doi.org/ 10.1055/s-0029-1241013 Planta Med 2010; 76: 19 Georg Thieme Verlag KG Stuttgart New York ISSN 00320943 Correspondence Prof. Jan Frederik Stevens, Ph.D. Oregon State University Pharmaceutical Sciences 203 Pharmacy Building 571 Weniger Hall or: 1601 SW Jefferson? Corvallis, OR 97331 United States Phone: + 1 54 17 37 95 34 or + 19 73 31 35 07? Fax: + 1 54 17 37 39 99 fred.stevens@oregonstate.edu

Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops and beer. Our objective of this study was to determine the anti-inflammatory activities of XN, isoxanthohumol (IX), and 15 related prenylated chalcones and flavanones, as well as their structure-activity relationships. The anti-inflammatory activities of the flavonoids were measured by their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production in human monocytic THP-1 cells. The position, number, and length of the prenyl groups had a marked influence on the inhibitory activity of the prenylfavonoids towards MCP-1 and IL-6 production. The ,-unsaturated carbonyl moiety present in chalcones such as XN was not an absolute requirement for inhibitory activity, as the saturated XN derivative, tetrahydroxanthohumol (TX), showed inhibitory activity comparable to XN. With the aim to determine the mechanism of the observed anti-inflammatory effects, cellular protein levels of Toll-like receptor 4 (TLR4) were measured by Western blot 24 h following coexposure of THP-1 cells to LPS and either XN, TX, or IX. Only XN reduced the cellular TLR4 protein content. Therefore, an additional hypothesis was developed for an anti-inflammatory mechanism that involves the TLR4 coreceptor myeloid differentiation protein-2 (MD-2), which provides the actual binding site for LPS. Molecular docking studies showed that the complementarity of prenylated flavonoids with the hydrophobic MD-2 pocket (indicating goodness of fit) directly predicted their relative ability to inhibit MCP-1 and IL-6 production. In conclusion, prenylated flavonoids may suppress LPS-induced TLR4 activation at least partly by interfering with LPS binding to

the TLR4 coreceptor MD-2, and XN (but not other prenylflavonoids) exerts an additional anti-inflammatory effect by downregulating the cellular TLR4 protein content.

Abbreviations
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CH: CN: COX: DPN: 6GN: 8GN: IL-6: IX: MCN: MCP-1: MD-2: MTT: MXN: NG: NFB: 6PN: 8PN: PX: TG: TLR4: TP: TX: XG: XN: XNB:

chalcone chalconaringenin cyclooxygenase 6,8-diprenylnaringenin 6-geranylnaringenin 8-geranylnaringenin interleukin-6 isoxanthohumol 4,6-dimethoxy-2,4-dihydroxychalcone monocyte chemo-attractant protein-1 myeloid differentiation protein-2 or lymphocyte antigen 96 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide 4-O-methylxanthohumol naringenin nuclear factor kappa B 6-prenylnaringenin 8-prenylnaringenin 5-prenylxanthohumol 2,4,6,4-tetrahydroxy-3-geranylchalcone Toll-like receptor 4 2,4,6,4-tetrahydroxy-3-C-prenylchalcone tetrahydroxanthohumol xanthogalenol xanthohumol xanthohumol B

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Introduction
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Materials and Methods

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Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops, the inflorescences of the female hop plant (Humulus lupulus). In hops, XN is excreted by glandular trichomes together with a dozen other prenylated flavonoids, as well as bitter acids (humulones and lupulones) and essential oils. The content of XN in hops varies from 0.1% to about 1.0 %, depending on hop variety and age. The other prenylated flavonoids occur in fresh hops at 10100 times lower levels compared to XN [1]. Because hops are an ingredient of beer, humans are primarily exposed to XN and its flavanone isomer, isoxanthohumol (IX), which spontaneously forms during the brewing process [2]. Major brand hopped beers contain about 0.5 mg of prenylflavonoids per liter, while more bitter microbrew beers may contain up to 4 mg/L [3]. Previous work conducted in our research group was focused on the isolation and structure elucidation of XN and related hop prenylflavonoids [2, 4], their detection and quantification in beer [3], as well as their cancer-related activities and antioxidant activities [5, 6]. Monocytes/macrophages play a key role in the initiation and progression of many chronic inflammatory diseases. Activated macrophages produce a variety of inflammatory mediators, including the pro-inflammatory cytokines monocyte chemo-attractant protein-1 (MCP-1) and interleukin-6 (IL-6) [7]. The Toll-like receptors (TLRs) are a family of transmembrane glycoproteins that sense microbial constituents such as lipopolysaccharide (LPS) as part of the innate immune response and host defense against invading microorganisms [8]. Activation of TLR4 stimulates nuclear factor kappa B (NFB) downstream signaling pathways that generate inflammatory cytokines, including MCP-1 and IL-6 [9]. The activation of TLR4 by LPS requires the heterodimerization of TLR4 with myeloid differentiation protein-2 (MD-2), also known as lymphocyte antigen 96 [10]. MD-2 is an obligate partner for TLR4, and it is required for TLR4 cell surface localization and responsiveness to LPS [11]. LPS binds to a hydrophobic cavity in MD-2, which induces TLR4-MD-2 recruitment to and clustering in plasma membrane lipid rafts for signal propagation [12]. Dietary phytochemicals and components of dietary supplements have received much attention in recent years as inhibitors of inflammatory processes to control or reverse chronic diseases [13]. For example, XN has previously been shown to suppress the production of nitric oxide through inhibition of inducible nitric oxide synthase and to inhibit cyclooxygenases in LPS-activated RAW 264.7 mouse macrophages [14, 15]. XN was also shown to inhibit the LPS-induced production of TNF and IL-1 in RAW 264.7 cells in part through downregulation of TLR4 gene expression and inhibition of NFB activation [14]. Furthermore, XN was shown to inhibit LPS-induced production of MCP-1 and TNF in LPS-activated RAW 264.7 cells and U937 human monocytes [16]. In the present study, we determined the anti-inflammatory activity of XN and 16 structurally related flavonoids by measuring the inhibitory effects of these flavonoids on the production of MCP-1 and IL-6 in human monocytic THP-1 cells stimulated with LPS. Our data suggests that flavonoids with a prenyl or geranyl moiety are capable of suppressing LPS-stimulated inflammatory signaling, possibly through direct competitive binding to the hydrophobic MD-2 cavity. The chemical diversity of our flavonoid library offers a unique opportunity to relate structure to anti-inflammatory potency and to potential anti-inflammatory mechanisms.

Reagents
LPS, RPMI 1640, and 2-mercaptoethanol were purchased from Sigma Chemical Co. LPS (Sigma cat. no. L6529) was -irradiated lyophilized powder derived from E. coli, with 3 % protein impurity. Fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Invitrogen. ELISA kits for determination of MCP-1 and IL-6 were purchased from R & D Systems. XN, IX, and other prenylflavonoids (see list of abbreviations for compound names " and l Fig. 1 for compound structures) were either isolated from hops or obtained by semisynthesis as described earlier, with > 98%? purity as determined by HPLC [2, 4, 17]. Stock solutions (50 mM) of flavonoids were prepared in absolute ethanol before adding them to the culture media.

Cell culture
THP-1 cells, obtained from the American Type Culture Collection, were propagated in complete medium (RPMI 1640 supplemented with 2 mM glutamine, 100 U/mL penicillin, 100 g/mL streptomycin, 10 % heat-inactivated FBS, and 50 M 2-mercaptoethanol). The cells were grown in 75 cm2 culture flasks kept in a 37 C humidified incubator with 5% CO2 prior to seeding of 6well, 48-well, or 96-well plates for the designated experiments.

Cell viability
The effect of flavonoids and LPS on the viability of THP-1 cells was evaluated by the MTT assay. THP-1 cells were plated in 48-well plates at a density of 1 106 cells/mL and treated with test compounds (20 M) and LPS (1 g/mL) for 4 or 24 h at 37 C in 5% CO2. MTT (0.05 mg/mL) was then added to each well and after 3 h of incubation, the cells were centrifuged at 1000 g for 5 min. The medium was removed and isopropanol was added to the cell pellet to solubilize the formazan blue that formed inside the cells. The cells were transferred to a 96-well plate before reading the absorbance at 595 nm.

Determination of MCP-1 and IL-6 in culture media by ELISA

THP-1 cells were plated in 96-well culture plates at a density of 5 105 cells/mL. The cells were cotreated with LPS (1 g/mL) and various concentrations of test compounds in complete medium. Negative controls contained media alone whereas positive controls contained LPS alone. Other cells were treated with flavonoids alone or ethanol alone (as a vehicle control, not to exceed 0.1 % in the culture media). After 4 h of incubation at 37 C, the media were transferred to microcentrifuge tubes and centrifuged at 1000 g for 5 min. The cell-free media were then analyzed for the levels of MCP-1 and IL-6 using ELISA kits according to the manufacturers instructions (R & D Systems).

Effect of flavonoids on TLR4 and MD-2 expression by Western blot analysis

THP-1 cells were plated in 6-well plates at a density of 1.5 106 " cells/mL, 2 mL/well. In the first experiment (l Fig. 5 A), the cells were exposed to LPS (1 g/mL) alone or LPS plus 5, 10, or 20 M XN or IX in complete medium for 24 h at 37 C. In the second ex" periment (l Fig. 5 B), THP-1 cells were exposed to LPS alone or LPS plus 20 M XN or IX in an incomplete medium (RPMI 1640 with 10% FBS and no antibiotics or 2-mercaptoethanol) for 24 h " at 37 C. In the third experiment (l Fig. 5 C), the cells were exposed to 0, 5, 10, and 20 M XN in the presence and absence of

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Fig. 1 Structures of chalcones and flavanones. For compound acronyms, see Abbreviations list.

LPS (1 g/mL) in the incomplete medium for 24 h. In a fourth ex" periment (l Fig. 5 D and 5 E), THP-1 cells were exposed to LPS (1 g/mL) plus 20 M chalcones (TX and XN) and flavanones (NG, 6PN, 8PN and DPN) for 24 h. After the 24 h incubations, cells were harvested and pelleted by centrifugation at 500 g for 5 min. Cell pellets were washed once with Dulbeccos phosphate buffered saline (DPBS; Invitrogen) and then resuspended in DPBS prior to cell lysis by sonication. The protein content of cell lysates was determined with a modified Bradford assay using Coomassie Plus staining. The cell lysates were stored at 80 C prior to Western blot analysis. Cell lysates were fractionated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with rabbit polyclonal anti-TLR antibody, rabbit polyclonal anti-MD2 antibody, or mouse monoclonal anti--actin antibody (Santa Cruz Biotechnology, Inc.). The nitrocellulose membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody and the immunoreactive bands were detected on X-ray film using the enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminescent Substrate; Pierce Biotechnology, Inc.).

Molecular docking studies

" Chemical drawings of the flavonoid compounds shown in l Fig. 1 were submitted to the PRODRG server [18], and coordinates of energy-minimized 3-dimensional models were obtained for docking experiments. The X-ray crystallographic structural coordinates of human lymphocyte antigen 96 (MD-2 protein; PDB 2Z65) [10] were obtained from the Protein Data Bank (PDB). The X-ray crystallographic structural coordinates of human MD-2 (PDB 3FXI) [19] were also obtained from the PDB and aligned to " PDB 2Z65 for the purpose of generating l Fig. 7 B. Molecular docking of each flavonoid in the pocket of human MD2 (PDB 2Z65) was carried out using AutoDock 4 and a Lamarckian genetic algorithm [20]. Torsional freedom was allowed for rotatable bonds in each flavonoid molecule, but the receptor (MD-2) was held rigid. Ligand-protein atomic contacts (LPC) were derived with LPC software [21], and the docking conformer with highest normalized complementarity to the MD-2 pocket was determined for each flavonoid. The predictive value of flavonoid complementarity with the MD-2 pocket on LPS-induced MCP-1 and IL-6 production was determined with a simple regression " analysis (l Fig. 8).

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Fig. 2 Effect of flavonoids on MCP-1 production in LPS-activated THP-1 monocytes. A Cells were exposed to LPS (1 g/mL) and flavonoids (20 M) for 4 h before analysis of culture media for MCP-1 protein levels by ELISA. The resulting concentration (pg/mL) of MCP-1 for the vehicle control (+LPS) was set to 100%, and the other values were referenced to it. MCP-1 values in nonstimulated and LPS-stimulated THP-1 cells were 134 and 551 pg/mL of culture media, respectively. Data are means SE of three experiments and asterisk (*) denotes significant difference (p 0.05) from the vehicle control. B Effect of increasing concentrations of XN and IX on MCP-1 production in LPS-activated THP-1 cells.

Fig. 3 Effect of flavonoids on IL-6 production in LPS-activated THP-1 monocytes. A Cells were exposed to LPS (1 g/mL) and flavonoids (20 M) for 4 h before analysis of culture media for IL-6 protein levels by ELISA. The resulting concentration (pg/mL) of IL-6 for the vehicle control (+LPS) was set to 100 %, and the other values were referenced to it. IL-6 values in nonstimulated and LPS-stimulated THP-1 cells were 0 and 113 2 pg/mL of culture media, respectively. Data are means SE of three experiments and asterisk (*) denotes significant difference (p 0.05) from the vehicle control. B Effect of increasing concentrations of XN and IX on IL-6 production in LPS-activated THP-1 cells.

Statistical analysis and graphics

Statistical analysis of experimental data was performed with Graphpad Instat 3 software (Graphpad Software, Inc.), using a 1way ANOVA. Statistically significant differences (p 0.05) between vehicle control and treated groups were obtained with the Tukey-Kramer multiple comparisons test. The correlation be" tween IL-6 production and MCP-1 production (l Fig. 4), as well as the regression analysis of MCP-1 and IL-6 production on flavo" noid-MD2 complementarity (l Fig. 8), were performed with StatView5 (SAS Institute, Inc.). Graphical presentation of data was performed with Graphpad Prism 4 software, and molecular renderings were developed with PyMOL [22]. in the absence of LPS (data not shown). In LPS-treated cells, the concentration of MCP-1 in the medium was increased 4-fold (551 4 pg/mL). MCP-1 production in LPS-activated THP-1 cells was inhibited by the flavonoids in the order: 8GN > 6GN > IX = MCN > XN = XG = PX = TX = XNB > MXN = DPN > TG = TP = CN > " NG (l Fig. 2 A). 6PN and 8PN did not inhibit LPS-induced MCP-1 production in THP-1 cells. XN and IX dose dependently inhibited LPS-induced MCP-1 production with IC50 values of 14.4 and " 12.6 M, respectively (l Fig. 2 B). In nonstimulated and LPS-activated THP-1 cells, the amounts of IL-6 secreted into the culture medium were 0 and 113 2 pg/mL, respectively. The flavonoids inhibited IL-6 production in LPS-activated THP-1 cells in the following order: 8GN > 6GN = IX = TX > XG > XN > PX = MCN = XNB > DPN = TP = MXN = 8PN = TG > 6PN " (l Fig. 3 A). CN and NG did not inhibit IL-6 production. XN and IX inhibited IL-6 production in a dose-dependent manner " (l Fig. 3 B), with IC50 values of 14.5 and 10.9 M, respectively. The flavonoid-mediated reduction (% of control) of IL-6 produc" tion was plotted against MCP-1 production (l Fig. 4). A linear relationship was observed for all flavonoids (n = 17, r = 0.89,

Results
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We examined the anti-inflammatory activities of the hop flavonoids in LPS-stimulated THP-1 monocytes. We chose an LPS concentration of 1 g/mL to stimulate the cells, as this concentration has been found to cause sufficient induction of MCP-1 production in THP-1 cells [23]. In nonstimulated cells, small amounts of MCP-1 (134 pg/mL) were secreted into the medium. This basal MCP-1 secretion was not affected by treatment with flavonoids

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Fig. 4 Direct linear relationship between flavonoid-mediated inhibition of IL-6 and MCP-1 production in LPS-activated THP-1 monocytes (r = 0.89, p < 0.0001).

p < 0.0001), for the subgroup of the chalcones (n = 10, r = 0.88, p = 0.0008), and for the flavanones (n = 7, r = 0.93, p = 0.0021). XN (5 g/mL or 14.1 M) has been shown to inhibit TLR4 and MD2 mRNA expression in LPS-activated RAW 264.7 cells [14]. The present study determined the inhibitory effect of XN on the levels of TLR4 and MD-2 protein in THP-1 cells, as determined by West" ern blot analysis (l Fig. 5). After a 24-h incubation, TLR4 protein levels were significantly reduced by XN, and to a lower extent by IX, in LPS-activated THP-1 cells maintained in complete medium " containing 50 M 2-mercaptoethanol (l Fig. 5 A). However, the inhibitory effect of XN on TLR4 protein level was not dose-dependent. When 2-mercaptoethanol was omitted from the culture medium, TLR4 protein was completely eliminated in LPS-activated THP-1 cells after a 24-h treatment with XN (20 M), but " not after IX treatment (l Fig. 5 B). Furthermore, XN dose-dependently lowered TLR4 protein levels in both unstimulated and LPSactivated THP-1 cells cultured without 2-mercaptoethanol " (l Fig. 5 C). Other flavonoids (TX, IX, NG, 6PN, 8PN, and DPN) failed to reduce TLR4 protein levels in LPS-activated THP-1 cells " cultured without 2-mercaptoethanol (l Fig. 5 D). MD-2 protein levels were not suppressed by XN or by any of the other flavonoids tested in LPS-activated THP-1 cells cultured without 2-

Fig. 5 Representative Western blots depicting the effect of XN and other flavonoids on TLR4 and MD-2 expression in LPS-activated THP-1 monocytes. A Cell lysates were prepared from control and LPStreated cells, with or without cotreatment with 5, 10, or 20 M XN or IX for 24 h in complete medium (with 50 M 2-mercaptoethanol), and then analyzed for TLR4 protein by immunoblotting using an antiTLR4 antibody probe. B Cell lysates were prepared from LPS-treated cells, with or without cotreatment with 20 M XN or 20 M IX in an incomplete medium (RPMI 1640 with 10% FBS) for 24 h at 37 C, and then analyzed for TLR4 protein with the antiTLR4 probe. C THP-1 cells were treated either with XN alone or with XN plus LPS in an incomplete medium for 24 h prior to immunoblotting with antiTLR4 antibody. D Cell lysates were prepared from control and LPS-treated cells, with or without cotreatment with flavonoids (20 M in an incomplete medium) for 24 h prior to immunoblotting with anti-TLR4 antibody. E Cell lysates were prepared from control and LPS-treated cells, with or without co-treatment with flavonoids (20 M in an incomplete medium) for 24 h, and then analyzed for MD-2 by immunoblotting using an anti-MD-2 antibody probe.

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Fig. 6 Effect of flavonoids on viability of LPS-activated THP-1 cells. Cells were exposed to LPS (1 g/mL) and flavonoids (20 M) for 4 h before analysis of cell viability by the MTT assay. Data are means SE of three experiments and asterisk (*) denotes significant difference (p 0.05) from vehicle controls.

" mercaptoethanol (l Fig. 5 E). The inhibitory activity of the flavonoids on LPS-induced MCP-1 or IL-6 production in THP-1 cells could have been due to cytotoxicity of the compounds themselves. To rule out this possibility, we determined the cytotoxicity of the test compounds at 20 M concentrations using the MTT assay. Among all the flavonoids tested, only 8GN showed evidence " of cytotoxicity based on the MTT assay (l Fig. 6). At 20 M, 8GN decreased cell viability by 32 % after a 4-h exposure period. This slight reduction in cell viability does not fully explain the almost complete inhibition of MCP-1 or IL-6 production by 8GN in LPSactivated THP-1 cells. Cell viability was not affected by exposing the cells to XN and IX at concentrations of 1 to 20 M for 24 h (data not shown).

In search of a potential mechanism for the observed flavonoidmediated inhibition of MCP-1 and IL-6 production as shown in " l Figs. 2 A and 3 A, respectively, molecular docking studies were conducted to determine the relative complementarity of the fla" vonoids shown in l Fig. 1 with the hydrophobic cavity of human MD-2. Normalized complementarity, as determined with LPC analysis [21], is based on the atomic contact surface area and the chemical properties of contacting atoms between the flavonoid " and the protein side-chains. l Fig. 7 A represents the X-ray crystallographic structure of human MD-2 (PDB 2Z65), devoid of any ligand, with hydrophobic residues (Ala, Gly, Val, Ile, Leu, Phe, and Met) colored orange, illustrating the generally hydrophobic character of the MD-2 pocket. Highest normalized complementarity between docked flavonoid and MD-2 protein was often found in a corner of the MD-2 pocket that tunnels under Tyr 102 as indicated. Therefore, this area was chosen as the primary site for " docking conformer analysis. l Fig. 7 B illustrates the positioning of both an agonist (LPS, colored red) and an antagonist (eritoran, colored blue) in the hydrophobic MD-2 cavity. The primary docking site (adjacent to Tyr 102) is occupied by two acyl chains of both the agonist and antagonist, deep into the MD-2 cavity. The binding of all chosen flavonoid docking conformers to MD-2 was energetically favorable. The AutoDock calculated free energy of binding ranged from 4.95 kcal/mol to 8.70 kcal/mol, corresponding to calculated inhibition constant (Ki) values of 234 M " to 421 nM. l Fig. 7 C and 7 D illustrate the final positioning of the IX and 8GN docking conformers in MD-2, adjacent to and hydrogen-bonded with Tyr 102, with a normalized complementarity of 75 % and 73 %, respectively, to the MD-2 contact surface. The polyphenol curcumin has been shown to interact with human MD-2 and was previously modeled with AutoDock in the LPS binding site of a theoretical model of MD-2 [24]. Therefore, as a positive control, curcumin was docked into PDB 2Z65 so that at least part of the curcumin structure occupied the LPS binding " area shown in l Fig. 7 A. The maximum normalized complementarity of curcumin with MD-2 was 64 %, corresponding to an Au-

Fig. 7 Molecular modeling of prenylated flavonoids in the hydrophobic cavity of human MD-2. Energy-minimized 3-dimensional models of the fla" vonoids shown in l Fig. 1 were docked into the hydrophobic cavity of human MD-2, using AutoDock4 as described in Materials and Methods. A Molecular surface rendering of human MD-2 (PDB 2Z65). The hydrophobic residues (Ala, Gly, Val, Ile, Leu, Phe, and Met) are colored orange. The flavonoid primary target docking site adjacent to Tyr 102 is indicated with arrows. B Positioning of the known TLR4 agonist, LPS (colored red), and the TLR4 antagonist, eritoran (colored blue), within the hydrophobic MD2 cavity. The overlay of LPS was generated through a structural alignment of MD-2 from PDB 3FXI with MD-2 from PDB 2Z65. Positioning of the docking solutions for IX (C) and 8GN (D) within the hydrophobic MD-2 cavity, adjacent to and hydrogenbonded with Tyr 102. Molecular renderings were generated with PyMOL [22].

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subsequent recruitment of LPSMD-2-TLR4 complexes to signaling rafts.

Discussion
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Fig. 8 Complementarity of docked prenylflavonoids with the hydrophobic cavity of human MD-2 predicts relative flavonoid efficacy to inhibit LPSstimulated inflammatory cytokine production in THP-1 cells. The flavonoids " shown in l Fig. 1 were docked into the hydrophobic cavity of human MD-2 as described in Materials and Methods and as illustrated by the IX and 8GN " " examples shown in l Fig. 7 C and 7 D, respectively. l Fig. 8 shows an inverse linear relationship between the normalized complementarity of docked flavonoids (with contacting residues of MD-2) and (A) LPS-stimulated MCP-1 production (r = 0.63, p = 0.007) and (B) LPS-stimulated IL-6 production (r = 0.81, p < 0.0001).

toDock calculated free energy of binding of 6.51 kcal/mol. " l Fig. 8 represents the simple regression analyses used to illustrate the relationship of flavonoid-mediated inhibition of LPS-in" duced MCP-1 and IL-6 production (as shown in l Figs. 2 A and " 3 A, respectively) to flavonoid-MD-2 complementarity. l Fig. 8 A shows that flavonoid complementarity with the MD-2 pocket was inversely-related to LPS-induced MCP-1 production (r = " 0.63, p = 0.007). l Fig. 8 B shows a similar inverse relationship between flavonoid-MD-2 complementarity and IL-6 production (r = 0.81, p < 0.0001). However, the AutoDock calculated free energy of binding was not a significant predictor of MCP-1 (p = 0.22) or IL-6 (p = 0.43) production. These data show that flavonoids with increasing complementarity to the MD-2 chosen docking site were increasingly more effective at reducing LPS-induced MCP-1 and IL-6 production. Thus, one possible mechanism for flavonoid-mediated attenuation of inflammatory cytokine production is through flavonoid occupation of part of the MD-2 cavity, inhibition of LPS binding to MD-2, and suppression of any

The anti-inflammatory activities of flavonoids have received considerable interest because they may provide an alternative or complementary therapy for a number of inflammation-associated disorders [25, 26]. Activated immune cells produce NO and cytokines such as MCP-1, IL-6, and TNF. In the present study, we examined the anti-inflammatory activities of XN, IX, and other prenylflavonoids as shown by their ability to inhibit the production of MCP-1 and IL-6 by LPS-activated human monocytic THP-1 cells. We report for the first time that XN, IX, and other prenylated chalcones and flavanones, notably 8GN, are potent inhibitors of MCP-1 and IL-6 secretion in LPS-activated THP-1 cells. Our results are consistent with the inhibition of MCP-1 release by XN that has been previously shown in LPS-stimulated RAW 264.7 cells and in U937 human monocytes [16]. Several studies have focused on structure-activity relationships that may ultimately predict anti-inflammatory activities of flavonoids [2527]. For example, flavones (with a C2=C3 double bond) were found to be more potent than flavanones (with a C2C3 single bond), and flavonoids with a 4-OH group in the B-ring were more potent than those with a 3-OH4-methoxy substitution [28]. However, little is known about the impact of the number and position of prenyl or geranyl groups on the anti-inflammatory properties of flavonoids. In the present study, methylation and prenylation of the core chalcone structure (CN) markedly in" creased the inhibitory activity towards MCP-1 (l Fig. 2 A) and IL" Fig. 3 A) production. Furthermore, replacement of prenyl 6 (l groups with geranyl groups (6PN vs. 6GN and 8PN vs. 8GN) profoundly increased the inhibitory effects on inflammatory cytokine production. The ,-unsaturated bond present in chalcones appeared to have no influence on anti-inflammatory activity. Indeed, TX, without an ,-unsaturated functionality, was as effective or better than XN at inhibiting MCP-1 and IL-6 production " " (TX vs. XN in l Figs. 2 A and 3 A). Finally, l Fig. 4 shows that flavonoid-mediated inhibition of MCP-1 and IL-6 production in LPSactivated THP-1 cells were directly correlated (r = 0.89, p < 0.0001), and this positive linear relationship was significant for both the chalcone (r = 0.88, p = 0.0008) and flavanone (r = 0.93, p = 0.0021) subgroups. This suggests that the production of MCP-1 and IL-6 is regulated by prenylated flavonoids through a common but unidentified pathway, and it also suggests that the closure of the C-ring and saturation of the ,-double bond in chalcones to form flavanones does not influence the mechanism of anti-inflammatory activity through this pathway. The anti-inflammatory action of XN has been postulated to occur through several mechanisms. XN was shown to reduce the expression of TLR4 and inhibit NFB activity in LPS-activated RAW 264.7 cells [14]. Albini et al. [29] have shown that XN inhibits NFB nuclear translocation and activation and represses the Akt pathway in TNF-treated HUVEC cells. XN also inhibited IL-2, IFN, and TNF production in activated lymphocytes through inhibition of NFB via suppression of IB phosphorylation [30]. Other studies revealed that XN can bind covalently to Cys residues of IKK, the kinase for the inhibitory subunit of NFB, as well as to Cys residues of the p65 subunit of NFB, resulting in the inhibition of NFB activation and suppression of NFB-regulated gene product formation in leukemia cells [31]. Our structure-ac-

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Ziegler + Mller D. Bauer 10.03.2010

tivity relationships point to multiple mechanisms of action of chalcones and flavanones. The hydrogenated derivative of XN, TX, has no reactivity towards Cys residues, but it had an anti-inflammatory activity comparable to XN. Similarly, IX has no reactivity towards Cys residues after its formation from XN, yet IX was a slightly more potent anti-inflammatory flavonoid than XN in both the MCP-1 and IL-6 assays. The TLR4-MD-2 complex acts as the LPS signaling receptor on the surface of monocytes [19]. Correspondingly, a reduction in the cellular content of either TLR4 or MD-2 may lead to impairment in the LPS inflammatory signaling pathway. We found that XN uniquely and markedly decreased the protein level of TLR4 in LPS-stimulated THP-1 cells " (l Fig. 5 D), which is consistent with a downregulation of TLR4 gene expression by XN in LPS-activated macrophages that was reported by Cho et al. [14]. In contrast, MD-2 protein levels were not affected by XN or by any of the other flavonoids tested " (l Fig. 5 E). A reduction in TLR4 protein may partially explain the anti-inflammatory activity of XN, but it cannot be used to explain the anti-inflammatory effects observed with the other flavonoids. The XN-mediated reduction in TLR4 protein may have been due to decreased protein synthesis, or it may have resulted from increased protein degradation. Interestingly, the reduction in TLR4 protein by XN was more profound in THP-1 cells grown " in culture medium without 2-mercaptoethanol (l Fig. 5 B) than " it was in cells cultured with 2-mercaptoethanol (l Fig. 5 A). It is entirely possible that the addition of 2-mercaptoethanol to the culture medium inactivated XN through covalent modification of XN to form a Michael-type adduct. Furthermore, XN reduced TLR4 protein levels in THP-1 cells with or without LPS cotreat" ment (l Fig. 5 C), suggesting that LPS stimulation was not related to the mechanism responsible for the XN-mediated reduction in TLR4 protein. Our molecular docking studies strongly suggest that the nearly universal and differential prenylflavonoid-mediated inhibition of MCP-1 and IL-6 production observed in the present studies may have been largely a result of flavonoid binding to the TLR4 coreceptor, MD-2, which attenuated LPS signaling through the TLR4-MD-2 complex. A similar anti-inflammatory mechanism involving MD-2 binding has been demonstrated for the polyphenol curcumin [24]. MD-2 is required for TLR4 cell surface localization and responsiveness to LPS [11]. LPS binds to a hydrophobic cavity in MD-2, which induces TLR4-MD-2 clustering in plasma membrane lipid rafts for signal propagation [12]. The X-ray crystallographic solutions of human MD-2 with bound " agonist LPS [19] and antagonist eritoran [10] (see l Fig. 7) show an extension of the ligand acyl chains deep into the area of the MD-2 cavity that was used for flavonoid docking in the present study. Analysis of our molecular docking data showed that flavonoids with increasing complementarity to this area of the MD-2 cavity were increasingly more effective at reducing LPS-induced " MCP-1 and IL-6 production in THP-1 cells (l Fig. 8). In conclusion, the present studies show that numerous prenylated flavonoids have anti-inflammatory effects in LPS-stimulated THP-1 monocytes through inhibition of MCP-1 and IL-6 production. The mechanism responsible for flavonoid-mediated inhibition of inflammatory cytokine production may at least partly involve competitive interference with LPS binding to the TLR4 coreceptor MD-2. Basal MCP-1 secretion was not affected by treatment with flavonoids in the absence of LPS. This suggests that the observed anti-inflammatory effects were not related to pathways not involving TLR4 and MD-2. Finally, XN (but not other prenylflavonoids) exerted a separate anti-inflammatory effect through

downregulation of the cellular TLR4 protein content via an unidentified mechanism.

Acknowledgements
!

This work was supported by the National Institutes of Health Grant P30ES000210 and the Oregon Agricultural Research Foundation, as well as by USANA Health Sciences, Inc., Salt Lake City, UT.

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Original Papers

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