78 research outputs found

    UV-Vis spectra of H<sub>2</sub>O<sub>2</sub> and its quenching by apigenin.

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    <p>(<b>A</b>) UV-Vis spectra of calf thymus (CT) DNA incubated with varying concentration of H<sub>2</sub>O<sub>2</sub> ranging from 1.95 mM to 125 mM. (<b>B</b>) Quenching of UV-Vis spectra by apigenin after H<sub>2</sub>O<sub>2</sub> treatment. CT-DNA was incubated with varying concentration of apigenin ranging from 0.2 mM to 0.8 mM followed by H<sub>2</sub>O<sub>2</sub> treatment. The experiment was repeated three times with similar results. Details are described in materials and methods section.</p

    Effect of apigenin on reactive oxygen species (ROS) generation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels with H<sub>2</sub>O<sub>2</sub> in transformed human prostate epithelial RWPE-1 cells.

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    <p>(<b>A</b>) ROS assay with DCF-DA on RWPE-1 cells treated with 10 µM and 20 µM apigenin for 16 h followed by 200 µM H<sub>2</sub>O<sub>2</sub> incubation for 6 h. (<b>B</b>) 8-OHdG levels in RWPE-1 cells treated with 10 µM and 20 µM apigenin for 16 h followed by 200 µM H<sub>2</sub>O<sub>2</sub> incubation for 6 h. Bars±SD of experiments performed three times. **P<0.001, compared to H<sub>2</sub>O<sub>2</sub> treated group. Details are described in materials and methods section.</p

    Effect of apigenin on H<sub>2</sub>O<sub>2</sub> -mediated RWPE-1 cell death.

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    <p>(<b>A</b>) Effect of H<sub>2</sub>O<sub>2</sub> on cell death in RWPE-1 cells. The cells were treated with 200 µM H<sub>2</sub>O<sub>2</sub> for 6 h. Negative, PI and annexin V only treatments are included as controls. (<b>B</b>) Cells were treated with 10 µM and 20 µM concentration of apigenin for 16 h followed by 200 µM H<sub>2</sub>O<sub>2</sub> incubation for 6 h, stained with PI and annexin V for 15 min and analyzed using fluorescence activated cell sorter (FACS). Data shown is representation of FACS graphs analyzed two times in duplicate. Details are described in materials and methods section.</p

    Ligands used for protein-ligand interaction analysis.

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    <p>The IUPAC name, structure, molecular weight and PubChem CID is provided for the ligands.</p

    Proposed model of the dual action of dietary flavones in inhibiting DNA methyltransferase and histone methyltransferase.

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    <p>(A) The two major epigenetic mechanisms, DNA methylation and histone methylation, acted in concert to regulate gene transcription. The DNA double helix backbone is shown in blue and red. In DNA methylation, methyl groups are added to a cytosine that is immediately 5′ to a guanine. In general, an increase in DNA methylation leads to a decrease in gene transcription. Histone methylation is accomplished by trimethylation of histone H3 lysine 27 (H3K27me3), which is catalyzed by the enhancer of zeste homolog 2 enzyme (EZH2) resulting in gene transcriptional repression; occurring to the tails of histones (histones shown in brown, with green H3K27 marks on tails), leading to a condensed chromatin state. (B) Dietary flavones viz. Apigenin, Chrysin and Luteolin can bind to the DNA bases, dock on the catalytic pocket of DNMT and HMT to inhibit methylation resulting in relaxed chromatin, increasing the likelihood of gene transcription.</p

    Effect of dietary flavones on DNA methyltransferase activity and <i>in vitro</i> methylation.

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    <p>(A) Dose-dependent inhibition of DNMT activity by 10- μM and 20-μM concentrations of 5-Aza-dC, Apigenin, Chrysin and Luteolin. The results are the mean of 3–4 determinations and were analyzed with a one way ANOVA, bars ± SD. **P<0.001 (B) Inhibition of purified recombinant DNA methyltransferase activity by dietary flavones. A 728 bp fragment (-428/+243 relative to the initiation codon) isolated from RWPE-1 cells within the promoter region of the human GSTP1 gene was used as substrate DNA. The methylation reactions were carried out in 1X M.SssI buffer with 160 mM SAM S-adinosylmethionine. The addition of 20 μg of flavones resulted in a detectable decrease in DNA methyltransferase activity (Lanes 4–6), compared to controls (Lane 2). No inhibitory affect was observed with an equal concentration of 5-Aza-dC. (C) MS-PCR for GSTP1 promoter on genomic DNA isolated from RWPE1 cells after treatment with 20 μM of 5-Aza-dC and dietary flavones for 48 h and treatment with methylation using M.SssI. The products generated with primers specific for unmethylated GSTP1 CpG island alleles (U) and for hypermethylated GSTP1 CpG island alleles (M) are displayed. L, DNA ladder. Details are described in the Materials and Methods section.</p

    Sub-cellular distribution of apigenin in human prostate cancer LNCaP cells.

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    <p>The cells were incubated with 20 µM apigenin for 48 h with approximately 5×10<sup>6</sup> cells and processed for different fractions. Bars±SD of experiments performed three times. Distribution is represented as 100% apigenin in all the fractions. Details are described in materials and methods section.</p

    Molecular modeling of the interaction between dietary flavones and EZH2.

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    <p>(A) 3-Deazaneplanocin A (in sticks) docked into the active pocket of EZH2 (shown as surface), (B) Apigenin, (C) Chrysin, and (D) Luteolin (Left panel). Schematic representation of different non-bonded interactions between ligand and amino acid residues of EZH2 is shown in the right panel. The pink arrow represents a hydrogen bond and amino acids are colored according to their chemical characteristics. The Glide scores for docking of DZNep, Apigenin, Luteolin, and Chrysin were -7.62 Kcal/mol, -10.07 Kcal/mol, -9.73 Kcal/mol, and -11.23 Kcal/mol, respectively. Details are described in the Materials and Methods section.</p

    Chemical structure and stability of apigenin.

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    <p>(<b>A</b>) 4′, 5, 7-trihydroxyflavone (<b>B</b>) Apigenin stability was determined at 37°C by incubating 20 µM apigenin with or without human prostate cancer PC-3 cells for up to 96 h. The concentration of apigenin at each time point was measured using UV-HPLC. Points±SD, percentage of remaining apigenin performed three times. Details are described in materials and methods section.</p

    Uptake of apigenin by various human prostate cell lines.

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    <p>(<b>A</b>) Time course apigenin uptake by transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells incubated with 20 µM apigenin for up to 16 h. Cellular uptake of apigenin (apigenin/million cells) was determined using UV-HPLC. (<b>B</b>) Bar graph of apignin uptake by various cell lines at 16 h. Bars±SD of experiments performed three times. **P<0.001. Details are described in materials and methods section.</p
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