29-Hydroxychalcone Inhibits Nuclear Factor-kB and Blocks Tumor Necrosis Factor-a- and Lipopolysaccharide-Induced Adhesion of Neutrophils to Human Umbilical Vein Endothelial Cells

Inhibition of expression of cell adhesion molecules (CAM), including intercellular CAM-1 (ICAM-1), vascular CAM-1 (VCAM1), and E-selectin, has been shown to be important in controlling various inflammatory diseases. The cell adhesion proteins are induced by various inflammatory cytokines, such as tumor necrosis factor-a, interleukin-1, and bacterial lipopolysaccharide. The induction process primarily takes place at the level of transcription, where nuclear factor-kB (NF-kB) plays a major role. We demonstrate here that 29-hydroxychalcone inhibits the adhesion of peripheral neutrophils to the endothelial cell monolayers by inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in a concentration-dependent manner. The inhibition by 29-hydroxychalcone is reversible. 29-Hydroxychalcone inhibits the induction of steady-state transcript levels of ICAM-1, VCAM-1, and E-selectin by tumor necrosis factor-a as determined by reverse transcription-polymerase chain reaction, and therefore it may interfere with the transcription of their genes. Because NF-kB is a major transcription factor involved in CAM expression, we studied its status in the 29-hydroxychalcone treated cells. We demonstrate that 29-hydroxychalcone inhibits the activation of NF-kB. These results have implications for using NF-kB inhibitors for the treatment of various inflammatory diseases. The recruitment and subsequent migration of the leukocytes to the site of inflammation is in part regulated by the expression of various cell adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (Osborn, 1990; Butcher, 1991; Springer, 1994). These cell adhesion molecules are induced on endothelial cells by various proinflammatory cytokines such as interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) and also by bacterial lipopolysaccharide (LPS; reviewed by Mantovani et al., 1997). These proteins are up-regulated on endothelial cells during various inflammatory diseases (Bochner et al., 1991; Calderon and Lockey, 1992; Gorski, 1994). Therefore, strategies to downregulate the expression of these molecules might have therapeutic implications. Inhibition of these molecules using specific monoclonal antibodies (mAbs) has been found to be beneficial for controlling various diseases (Gorski, 1994; Weiser et al., 1997). However, because of endotoxin contamination, unpredictable clinical manifestations, such as secondary antibody formation, cellular activation, and other complications (e.g., sensitization leading to serum sickness and anaphylaxis), the practical use of mAbs is limited (Weiser et al., 1997). Also, various small molecules from natural and synthetic sources, such as curcumin, glucocorticoids, pentoxifylline, etc., have been shown to down-regulate the expression of cell adhesion molecules and are effective in controlling various inflammatory diseases (Brojstan et al., 1997; Neuner et al., 1997; Gupta and Ghosh, 1999). The promoters of ICAM-1, VCAM-1, and E-selectin contain recognition sequences for inducible nuclear transcription factor-kB (NF-kB). NF-kB has been shown to be essential for the expression of cell adhesion molecules as demonstrated by deletion mutagenesis, gel-retardation assays, Western blots, and DNA transfection experiments (Iademarco et al., 1992; Hou et al., 1994; Schindler and Baichwal, 1994; Collins et al., 1995). TNF-a and IL-1b up-regulate ICAM-1, VCAM-1, and This work was supported by Council of Scientific and Industrial Research (CSIR), India, and Department of Biotechnology, India. B.M. is a recipient of CSIR fellowships. 1 Both authors contributed equally to this work. ABBREVIATIONS: ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; IL-1, interleukin-1; TNF-a, tumor necrosis factor-a; LPS, lipopolysaccharide; mAb, monoclonal antibody; NF-kB, nuclear factor kB; ELISA, enzyme-linked immunosorbent assay; HUVECs, human umbilical vein endothelial cells; RT-PCR, reverse transcription-polymerase chain reaction; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay. 0026-895X/00/030526-00$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 3 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 11/848361 Mol Pharmacol 58:526–534, 2000 Printed in U.S.A. 526 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from E-selectin expression on endothelial cells at the transcriptional level through the activation of NF-kB. Identification of inhibitors of NF-kB can thus serve to prevent the up-regulation of adhesion molecules on the surface of endothelial cells. Chalcones are obtained from natural plant sources and can also be synthesized in the laboratory. Chalcones have been reported to possess a variety of biological properties, including anti-inflammatory, analgesic, antioxidant, antibacterial, antifungal, and antiprotozoal activities (Haraguchi et al., 1998; Hsieh et al., 1998). They are also reported to be gastric protectant, antimutagenic, and antitumorogenic (Makita et al., 1996). Various 29-substituted chalcones have been shown to possess anti-inflammatory and antioxidant properties (Yu et al., 1995; Wegener et al., 1997). For example, 29,59-dihydroxychalcone prevents platelet aggregation (Lin et al., 1997), and 29,3-dihydroxychalcone and 29,59-dihydroxychalcone inhibit polymixin B-induced hind-paw edema (Hsieh et al., 1998). Butein (3,4,29,49-tetrahydroxychalcone) prevents antiglomerular basement membrane antibody-associated glomerulonephritis in rats (Hayashi et al., 1996). 29-Substituted chalcones have also been shown to inhibit production of IL-1b from monocytes stimulated with LPS and also prevent LPS-induced septic shock in mice (Batt et al., 1993). LPSinduced septic shock involves excessive infiltration of neutrophils into the liver because of uncontrolled up-regulation of ICAM-1 expression in the liver (Xu et al., 1994; B. Gupta and B. Ghosh, unpublished observations). As a result, ICAM-1 deficient mice are protected against septic shock (Xu et al., 1994) and inhibitors of ICAM-1 prevent lethality induced by septic shock in mice (B. Gupta and B. Ghosh, unpublished observations). Inhibition of LPS-induced septic shock by 29substituted chalcones, therefore, might be caused by the inhibition of infiltration of neutrophils into the liver. Recently, 29-hydroxychalcone has been shown to be a potent antioxidant, it inhibits lipid peroxidation and is antitumorogenic (Anto et al., 1995). Having the hydroxyl group at the orthoposition on the benzene ring of chalcone increases its antioxidant property compared with other substituted chalcones (Anto et al., 1995). Very little is known in regard to its mechanism of action. The effect of 29-hydroxychalcone on the expression of cell adhesion molecules has also not been studied so far. Because 29-hydroxychalcone has been found to be pharmacologically important, we were interested to study its mechanism of action on cell trafficking. In this study, we show that 29-hydroxychalcone blocks the adhesion of neutrophils to endothelial monolayers by preventing TNF-aand LPS-induced up-regulation of cell adhesion molecule expression on endothelial cells. We also show that 29-hydroxychalcone inhibits TNF-a-induced cell adhesion molecule expression by blocking the activation of NF-kB in endothelial cells. Experimental Procedures Materials. TNF-a, anti-ICAM-1 (BBA3), anti-VCAM-1 (BBA6), and anti-E-selectin (BBA1) antibodies were purchased from R & D Systems (Minneapolis, MN). M199, L-glutamine, penicillin, streptomycin, amphotericin, endothelial cell growth factor, trypsin, Pucks saline, HEPES, o-phenylenediamine dihydrochloride, FicollHypaque, tetramethyl benzidine, cetitrimethyl ammonium bromide, 3-amino-1,2,4 triazole and anti-mouse IgG-horseradish peroxidase was purchased from Sigma Chemical (St. Louis, MO). NF-kB oligonucleotide was purchased from Promega (Madison, WI). The structure of 29-hydroxychalcone has been shown in Fig. 1. It has been prepared by Clasien-Schmidt condensation between 2-hydroxyacetophenone and benzaldehyde done in ethanol, using partially dehydrated barium oxide as a catalyst and characterized by H NMR (Sathyanarayana and Krishnamurthy, 1988). The ICAM-1, VCAM-1, E-selectin, and b-actin primer sets were synthesized by Genset Corp. (Tokyo, Japan). Fetal calf serum was purchased from Biological Industries (Kibbutz Beit Haemek, Israel). Anti-mouse-IgG-fluorescein isothiocyanate was purchased from Becton Dickinson (Mountain View, CA). Anti-NF-kB antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cells and Cell Culture. Primary endothelial cells were isolated from umbilical cord by mild trypsinization. The endothelial cells obtained were grown on gelatin-coated tissue culture flasks in M 199 medium supplemented with 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml amphotericin, endothelial cell growth factor (50 mg/ml), and heparin (5 U/ml). For subculturing, the cells were dislodged using 0.125% trypsin/0.01 M EDTA solution in Pucks saline and HEPES buffer. The cells were used between passages three to four. The viability of cells was determined by trypan blue staining and purity of endothelial cells was determined by E-selectin expression. Neutrophil Isolation. Neutrophils were isolated from peripheral blood of healthy individuals as described previously (Clark and Naseef, 1996). Briefly, the peripheral blood was collected in heparin solution (final concentration, 20 U/ml) and erythrocytes were removed by sedimentation with 6% dextran solution. The white-bloodcell-rich plasma layer was collected and layered over Ficoll-Hypaque solution followed by centrifugation at 300g for 20 min at 20°C. The top saline layer and the Ficoll-Hypaque layer were aspirated, leaving the neutrophil/red blood cell pellet. The residual red blood cells were removed by hypotonic lysis. The isolated cells were washed with PBS and resuspended in PBS containing 5 mM glucose, 1 mM CaCl2, and 1 mM MgCl2 at a final concentration of 6 3 10 5 cells/ml. This procedure usually resulted in approximately 95% neutrophils and the cell viability was more than 95% as detected by trypan blue exclusion test. Cell Adherence Assay. Adhesion of neutrophils to endothelial monolayers was assayed as described previously (Dobrina et al., 1991). Briefly, the endothelial cells were plated in 96-well culture plates at a density of 2 3 10 cells/well and allowed to adhere for 24 h. The cells were incubated without or with 29-hydroxychalcone for 1 h followed by induction with LPS (1 mg/ml) for 6 h. The endothelial monolayers were washed twice with PBS, and neutrophils (6 3 10/well) were added and allowed to adhere for 1 h at 37°C. Nonadherent neutrophils were removed by washing the wells with PBS thrice. Adherent neutrophils were assayed colorimetrically by adding a substrate solution (100 ml/well) consisting of o-phenylenediamine dihydrochloride (40 mg/100 ml in citrate phosphate buffer, pH 4.5) containing 0.1% cetitrimethyl ammonium bromide as peroxFig. 1. Structure of 29-hydroxychalcone 2*-Hydroxychalcone Inhibits NF-kB 527 at A PE T Jornals on A uust 7, 2017 m oharm .aspeurnals.org D ow nladed from idase solubilizing agent. The interference by the few contaminating eosinophils was abolished by adding a selective eosinophil peroxidase inhibitor, 3-amino-1,2,4 triazole (1 mM) to the substrate solution. After 2 min of incubation, 2N H2SO4 (50 ml/well) was added to stop the reaction. The absorbance was determined at 490 nm using an automated microplate reader (Spectramax 190; Molecular Devices, Menlo Park, CA). Modified Enzyme-Linked Immunosorbent Assay (ELISA) for Measurement of ICAM-1. Expression of ICAM-1 on surface of endothelial cells was quantified using cell-ELISA as described before (Gupta and Ghosh, 1999). Human umbilical vein endothelial cells (HUVECs) were plated to confluency in gelatin-coated, 96-well plates. The cells were then incubated without or with 29-hydroxychalcone at desired concentrations for desired time periods followed by induction with LPS (1 mg/ml) for 16 h. After incubation, the cells were fixed with 1.0% glutaraldehyde and nonspecific binding was blocked using nonfat dry milk (3.0% in PBS). The cells were incubated overnight at 4°C with ICAM-1 mAb or control IgG antibody (0.25 mg/ml, diluted in blocking buffer), followed by washing with PBS and incubation with peroxidase-conjugated goat antimouse secondary antibody (1 mg/ml, diluted in PBS). After this, the cells were again washed with PBS and exposed to the peroxidase substrate (o-phenylenediamine dihydrochloride, 40 mg/100 ml in citrate phosphate buffer, pH 4.5). Color development reaction was stopped by the addition of 2 N sulfuric acid. Absorbance was determined at 490 nm by an automated microplate reader (Spectramax 190). Flow Cytometry. The expression of ICAM-1, VCAM-1, and Eselectin expression on endothelial cells was measured by flow cytometry as described previously (Gupta and Ghosh, 1999). Briefly, the endothelial cells were incubated without or with 29-hydroxychalcone for 1 h followed by induction with TNF-a (10 ng/ml) for 16 h (for ICAM-1 and VCAM-1) or for 4 h (for E-selectin). After incubation, the cells were dislodged and incubated with anti-ICAM-1, anti-VCAM-1, anti-E-selectin, or control IgG antibody (1.0 mg/10 cells) for 30 min at 4°C. The cells were washed twice with PBS for removing the unbound antibody and then incubated with goat anti-mouse IgGfluorescein isothiocyanate antibody (1:10 diluted) for 30 min at 4°C. The cells were fixed with 0.1% paraformaldehyde and were analyzed by using a flow cytometer (FACSVantage; Becton Dickinson). For each analysis, 20,000 events were collected and histograms were generated. Total RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RNA was isolated according to a modified guanidinium thiocyanate procedure (Chomczynski and Sacchi, 1987). The expression of the transcripts for ICAM-1, VCAM-1 and E-selectin was evaluated by RT-PCR as described previously (Gupta and Ghosh, 1999). The primers were synthesized according to the published cDNA sequences to yield products of length 555, 533, and 479 base pairs, respectively. As a control, b-actin mRNA was also amplified by RT-PCR and a product of 661 base pairs was obtained. The RT-PCR was performed following the manufacturer’s protocol (Access RT-PCR system; Promega). Briefly, 100 ng of the total RNA was reverse transcribed using AMV reverse transcriptase at 48°C for 45 min followed by amplification using Tfl polymerase for 25 cycles. The conditions for PCR were as follows: denaturation at 92°C for 1 min, primer annealing at 52°C for 90 s, extension at 72°C for 2 min, and a final extension at 72°C for 10 min. The PCR products were analyzed in 1% agarose gel electrophoresis and visualized by ethidium bromide staining. Preparation of Nuclear Extracts. Nuclear extracts were prepared using a modification of previously published methods (Dignam et al., 1983). Primary endothelial cells (2 3 10 cells/ml) were incubated without or with 50 mM 29-hydroxychalcone for 1 h followed by induction with TNF-a (10 ng/ml) for 30 min. The cells were washed with PBS, dislodged using a cell scraper, and pelleted by centrifugation at 300g. The cells were resuspended in cell lysis buffer [10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM phenylymethylsulfonyl fluoride, 1 mM dithiothreitol (DTT), 0.5% Nonidet P40, 0.1 mM EGTA, and 0.1 mM EDTA[ and allowed to swell on ice for 5 min. This was followed by centrifugation at 3300g for 15 min. The supernatant was stored as cytoplasmic extract and the nuclear pellet resuspended in nuclear extraction buffer (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM phenylymethylsulfonyl fluoride, and 1 mM DTT) and incubated for 30 min at 4°C. The extracted nuclei were pelleted at 25,000g for 15 min at 4°C and the supernatant was collected as nuclear extract. The protein concentration was estimated by Bradford’s protein estimation method. The nuclear and cytoplasmic extracts were stored at 270°C. Electrophoretic Mobility Shift Assay (EMSA). EMSA was performed with modifications of a previously published procedure (Marrugo et al., 1996). Briefly, 10 mg of nuclear extract was incubated with 40 to 80 fmol of P-end labeled double-stranded NF-kB oligonucleotide (59AGTTGAGGGGACTTTCCCAGG-39) in binding buffer [12 mM HEPES, 50 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA, 1 mM DTT, and 1.0 mg of poly(dI-dC)] for 30 min at RT. The DNA-protein complexes were analyzed by electrophoresis on a 4% native polyacrylamide gel using Tris-glycine buffer, pH 8.5, and visualized by autoradiography. Western Blot Analysis. Nuclear and cytoplasmic extracts from endothelial cells treated without or with 50 mM 29-hydroxychalcone were electrophoresed on 10% SDS polyacrylamide gels and transferred to Hybond-C membrane (Amersham, Paisley, UK) in 25 mM Tris, 192 mM glycine, 20% methanol at 15 V overnight. Nonspecific binding sites were blocked by incubating the membrane in 3.0% nonfat dry milk in HEPES-buffered saline (10 mM HEPES, pH 7.4, 100 mM NaCl) at 37°C for 1 h. After being washed twice in HEPESbuffered saline, the membranes were incubated in polyclonal antip65 antibody (0.1 mg/ml) overnight at 4°C. After this the membranes were washed with HEPES-buffered saline and incubated with antirabbit IgG antibody conjugated with horseradish peroxidase for 30 min at 37°C. After extensive washing with HEPES-buffered saline, the blots were exposed to peroxidase substrate (0.25 mg/ml tetramethylbenzidine in 12 mM HEPES, 2 mg/ml dioctyl sodium sulfosuccinate). The color development reactions were stopped by addition of HEPES buffer. Statistical Analysis. Results are given as mean 6 S.D. Independent two-tailed Student’s t test was performed. Differences were considered statistically significant for P , .050. All statistical analysis was performed using the software Microcal Origin (ver 3.0; Microcal Software Inc., Northampton, MA).