17 research outputs found

    The computed binding energy values (Δ<i>E</i><sub>binding</sub>) for the molecular docking study for the binding of myricetin, quercetin, chrysin or flavone with human COX I or COX II.

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    <p>The ligand-enzyme interaction energy value (Δ<i>E</i><sub>binding</sub>) was calculated using the following equation: Δ<i>E</i><sub>binding</sub> = <i>E</i><sub>complex</sub>−(<i>E</i><sub>COX</sub>+<i>E</i><sub>ligand</sub>), where <i>E</i><sub>complex</sub> was the potential energy for the complex of COX bound with the ligand, <i>E</i><sub>COX</sub> was the potential energy of the enzyme alone, and <i>E</i><sub>ligand</sub> was the potential energy for the ligand alone.</p

    Identification of the binding sites for COX I and II by molecular docking approach.

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    <p><b>A</b>. The superimposed structures of COX I and II in complex with hematin and AA. The white labels indicate the two potential binding sites for quercetin as identified by the <i>Active-Site-Search</i> function in <i>InsightII</i>. <b>B</b>. Docking results for quercetin in Site-2 of COX I. <b>C</b>. Docking results for quercetin in Site-2 of COX II. <b>D</b>. Enlarged view of the interaction of quercetin with hematin and key amino acid residues in the peroxidase active sites of COX I. <b>E</b>. Enlarged view of the interaction of quercetin with hematin and key amino acid residues in the peroxidase active site of COX II. The protein structure was shown in the ribbon format in <b>A</b>, <b>B</b> and <b>C</b>. COX I was colored in pink and COX II in purple. AA was colored in light green for COX I and dark green for COX II. Quercetin was colored in light red for COX I and magenta for COX II. Carbon atoms in hematin were colored in yellow for COX I and orange for COX II whereas nitrogen atoms were colored in blue, oxygen atoms in red, and magnesium in silver. The green dashes represent the hydrogen bonds. Hematin, AA, key amino acid residues, and quercetin are shown in the ball and stick format. For amino acid residues, oxygen atoms are shown in red, carbon atoms in gray, and nitrogen atoms in blue. Hydrogens are omitted in these molecules.</p

    Primers used in the site-directed mutagenesis study.

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    <p>The sequences that are changed for each of the mutant COX proteins developed in this study are marked with underlines.</p

    Chemical structures of the bioflavonoids used in this study.

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    <p>The structure of flavone is enlarged to show the numbering of different carbon positions.</p

    The 3-D QSAR/CoMFA analysis showing the correlation between the experimentally-determined COX-stimulating activity values and the predicted values.

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    <p>Nine representative bioflavonoids plus flavone were used in the analysis. The experimental values were based on measuring PGE<sub>2</sub> production, which was determined in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a>. Statistical parameters (<i>q</i><sup>2</sup>, <i>r</i><sup>2</sup>, PC, SEE, and F) for the CoMFA models of COX I and II are also listed in the figure.</p

    Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [<sup>14</sup>C]AA or PGG<sub>2</sub> is used as substrate.

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    <p>The incubation mixtures consisted of 20 µM [<sup>14</sup>C]AA (0.2 µCi) or 10 µM PGG<sub>2</sub> as substrate, COX I or COX II as enzyme (0.5 or 0.97 µg/mL, respectively), 10 mM EDTA, 1 mM reduced glutathione, 1 µM hematin, and myricetin in 200 µL Tris-HCl buffer (100 mM, pH 7.4). The reaction was incubated at 37°C for 5 min and terminated by adding 15 µL of 0.5 N HCl to each test tube. Ethyl acetate (600 µL) was added immediately for extraction. The dried extracts were re-dissolved in acetonitrile or EIA buffer (Cayman Co. Michigan, USA), and the metabolites were analyzed using HPLC (with radioactivity detection) when [<sup>14</sup>C]AA was used as substrate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a> or using an EIA kit when PGG<sub>2</sub> was used as substrate. Note that in this experiment, the COX I and II enzymes with or without aspirin pretreatment were both tested. For aspirin pretreatment, enzymes were pre-incubated with aspirin at 0.5 mM for COX I or 5 mM for COX II for 30 min at room temperature and then were immediately used as the enzyme source in the assay.</p

    Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.

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    <p>PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.</p

    The 3-D QSAR/CoMFA color contour maps for COX I (A) and COX II (B).

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    <p>Note that quercetin was shown in the ball and stick format inside the field for demonstration. Oxygen, carbon, and hydrogen atoms are colored in red, gray, and blue, respectively. The contours of the steric maps are shown in yellow and green, and those of the electrostatic maps are shown in red and blue. Green contours indicate regions where a relatively bulkier substitution would increase the COX activity, whereas the yellow contours indicate areas where a bulkier substituent would decrease the COX activity. The red contours are regions where a negative-charged substitution likely would increase the COX activity whereas the blue contours showed areas where a negative-charged substitution would decrease the COX activity. Bioflavonoids with a higher ability to activate the COX enzymes are correlated with: (<b><i>i</i></b>) more bulkier substitute near green; (<b><i>ii</i></b>) less bulkier substitute near yellow; (<b><i>iii</i></b>) less negative charge near blue; and/or (<b><i>iv</i></b>) more negative charge near red. The figure in the lower panel shows the 90° rotation around the <i>x</i>-axis of the figure shown in the upper panel.</p

    Dex-IR inhibits the invasiveness of H1650 cells.

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    <p>(A) Invasion assay of H1650 cells treated with the drugs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194341#pone.0194341.g001" target="_blank">Fig 1</a>. The Transwell invasion assay showed that Dex-IR suppressed the invasion of H1650 cells. Images were captured at a magnification of 400× (A, left panel). Scale bars, 100 μm. Graphical representation of the number of invasive H1650 cells per microscopic field. Each column and bar shows the SEM from three independent experiments (*<i>P</i> < 0.05 <i>vs</i>. vehicle) (A, right panel). (B) Inhibition of MMP9 activity in conditioned medium from H1650 cells treated with Dex-IR at the indicated concentration and incubated for 18 h was evaluated using gelatin zymography. Representative data from a single experiment are shown. The left lanes are standard markers. (C) qRT-PCR analysis of the MMP2, MMP9, integrin α2, and integrin α5 gene expression in cells 6 h after treatment with drugs. Each experiment was repeated three times and the results shown are representative of the three independent experiments. The bar graph shows the mean ± SEM of three independent experiments (*P < 0.05 vs. MMP2 expression in vehicle; <sup>#</sup>P < 0.05 vs. MMP9 expression in vehicle; <sup>§</sup>P < 0.05 vs. integrin α2 expression in vehicle).</p

    Dex-IR induced H1650 cell cycle arrest.

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    <p>(A) Cell cycle analysis following drug treatment for 72 h. The bar graph of the results of the cell cycle analysis of drug-treated H1650 cells shows the percentages of cells in different phases of the cell cycle (G<sub>0</sub>/G<sub>1</sub>, S, and G<sub>2</sub>/M). The bar graph shows the mean ± SEM of three independent experiments (*<i>P</i> < 0.05 <i>vs</i>. G<sub>0</sub>/G<sub>1</sub> phase in the medium; <sup>#</sup><i>P</i> < 0.05 <i>vs</i>. S phase in the medium; and <sup>§</sup><i>P</i> < 0.05 <i>vs</i>. G2/M phase in the medium). (B) Cell lysates were analyzed by Immunoblot analysis using specific antibodies against the cyclins A2, D1, B1 and Phospho-Rb. Protein loading was normalized based on GAPDH. (C) Cells were treated with 400 nM nocodazole for 24 h, and then further treated with Dex or Dex-IR for 24 h. They were then harvested, and subjected to cell cycle analysis. The bar graph shows the mean ± SEM of three independent experiments (*P < 0.05 vs. G<sub>0</sub>/G<sub>1</sub> phase in the Nocodazole).</p
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