Allelochemicals of the phenoxazinone class act at physiologically relevant concentrations

<p>Plants compete with their neighbors via the release of chemical compounds into the rhizosphere. These phytotoxins originate from a series of secondary metabolites and can be processed further by soil-living microorganisms before exerting their activity on the target plant. To determine the molecular mode of action and the physiological relevance of potential phytotoxins, it is important to simulate true-to-life conditions in laboratory experiments, for example by applying physiologically relevant concentrations. Here, we report on an improved experimental setting to study the function of allelochemicals of the benzoxazolinone class. By adjusting the solvent and the application of the chemicals, we reduced by more than 2fold the concentration that is necessary to induce growth defects in the model plant Arabidopsis thaliana.</p

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

<div><p>The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by <i>in silico</i> docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. <i>In vitro</i> analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol <i>ex vivo</i>. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas <i>in vivo</i> chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.</p></div

<i>In silico</i> docking analysis with resveratrol illustrates an inhibitory potential for human HDAC enzymes of classes I and II.

<p>(A–D) Results of the <i>in silico</i> docking analysis of resveratrol with crystal structures of HDAC2 (A), HDAC4 (B), HDAC7 (C) and HDAC8 (D). The analysis demonstrates the predicted binding mode of resveratrol in the different HDAC binding pockets. The predicted interactions of resveratrol with the zinc ion (turquoise sphere) and other residues of the catalytic center are highlighted in the left row. 2D depiction of resveratrol along with interacting amino acids is shown in the right row. Green circles represent hydrophobic, purple circles polar, red circles acidic and blue circles basic residues. Blue halolike discs around amino acids are calculated based on the reduction of solvent exposure by the ligand. Blue arrows represent backbone H-bond interactions, green ones depict sidechain H-bond interactions. Green benzol rings with a “+” describe arene-cation interaction, 2 benzol rings an arene-arene interaction. Areas with a blue background are solvent exposed parts of the ligand. The purple dotted lines represent metal contact.</p

HDAC inhibition by resveratrol in human blood.

<p>Anticoagulated human blood was incubated with 100 µM of resveratrol for 4 h, 6 h and 8 h. HDAC activity of lysates of peripheral blood cells was measured <i>ex vivo</i>. Inhibition values of HDAC activity were yielded by three independent experiments, each performed in duplicates. Shown are mean ± SEM.</p

HDAC inhibition mediated by resveratrol.

<p>(A) Overall inhibition of human HDAC enzymes in HeLa nuclear extracts by increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM). As a reference inhibitor suberoylanilide hydroxamic acid (SAHA; 100 µM) was used. Every concentration was tested three times in triplicates; One-way ANOVA Dunnetts multiple comparison test, *<i>P</i><0.01. (B) Specific fluorometric profiling assay using recombinant human HDACs of classes I, II and IV. Specific inhibition values were generated for the treatment with 50 µM and 100 µM resveratrol. Inhibition values for every HDAC were yielded by four independent experiments, each performed in duplicates; Students t-test *<i>P</i><0.01. Shown are mean ± SD (A and B).</p

Resveratrol induces overall hyperacetylation in HepG2 but not Hep3B or HuH7 cells.

<p>(A) Detection of intracellular acetylated protein levels in HepG2, Hep3B and HuH7 hepatoma cells after incubation of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control for 6 h. (B) Overall HDAC inhibition in nuclear extracts of HepG2 cells by increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control. As a reference inhibitor suberoylanilide hydroxamic acid (SAHA; 100 µM) was used. (C) Western blot analysis of acetylated histone complex H3 in HepG2 tumor cells treated with 50 µM and 100 µM of resveratrol or solvent as control. Acetylation of H3 was examined using cellular lysates. Equal protein loading was verified by vinculin staining (upper row). As a reference and positive control for hyperacetylation the cells were treated with 2 µM SAHA. Acetylation levels were calculated performing a densitometric analysis. Shown are mean ± SD of three independent experiments, each performed in triplicates (A and B); One-way ANOVA Dunnetts multiple comparison test, *<i>P</i><0.01, n.s. indicates not significant (A and B).</p

Reduced proliferation and viability of hepatoma cells by resveratrol.

<p>(A–C) HepG2 (A), Hep3B (B) and HuH7 (C) hepatoma tumor cells were treated with different concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control and monitored for an additional time period of 96 h. Cellular impedance was measured over the entire observation time using the xCELLigence™ SP system and calculated by the RTCA Software 1.2.1.1002. All cell index (CI) values were normalized when treatment started. Displayed are normalized CI values every 5 h. As a positive control for cell death, Triton X-100 0.1% was used. (D–F) Sulforhodamine B (SRB) assay of HepG2 (D), Hep3B (E) and HuH7 (F) cells treated with increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control for 96 h. Shown are mean ± SD of three independent experiments, each performed in triplicates (A–F); One-way ANOVA Dunnetts multiple comparison test, *<i>P</i><0.01 (D–F).</p

Toxicity profile of resveratrol in non-malignant cells.

<p>(A–C) Non-malignant primary human hepatocytes (PHH) from different donors were treated with increasing concentrations of resveratrol (5 µM, 10 µM, 20 µM, 50 µM and 100 µM) or solvent as control. Lactate dehydrogenase (LDH) release (A) and aspartate aminotransferase (AST) release (B) into the supernatant fluid were determined after 48 h and 96 h. Sulforhodamine B (SRB) assay (C) of PHH treated for 96 h. The incubation with Triton X-100 1% was performed as a positive control for cell death in all experiments. Bars represent mean ± SD of three independent experiments with PHH from three different human donors, each performed in triplicates (A–C). (D) Chicken embryotoxicity assay with different concentrations of resveratrol or solvent as control. Chicken embryos stage 13 (corresponding to 6 human gestational weeks) were exposed to rising concentrations of resveratrol to determine embryotoxic effects. Survival rates after 24 h, 48 h and 72 h are depicted in a Kaplan-Meier plot.</p

Fructose-loading induces <i>transient</i> ATP depletion in primary human hepatocytes.

<p>(<b>A–C</b>) PHH of human donors were treated with increasing concentrations of fructose (<b>A</b>) or treated with a fixed concentration of 50 mM fructose (<b>B</b>); ATP content was determined after 30 min (<b>A</b>) or at varying time points as indicated (<b>B</b>); LDH release of PHH after incubation with 50 mM fructose was determined after indicated time points (<b>C</b>). Data are given as mean ± SEM.</p

Neither ATP level nor cytotoxicity induction is depleted in hepatic tumor cell lines in presence of fructose.

<p>The hepatic tumor cell lines HepG2, Hep3B, HuH7 and PLC/PRF/5 were treated with increasing concentrations of fructose (<b>A</b>). ATP content was determined after 30 min (<b>A</b>) or at varying time points as indicated. Data are given as mean ± SEM (n = 15). The hepatic tumor cell lines were treated with 1 µg/ml ActD in combination with 100 ng/ml TNF and 50 mM fructose as indicated. Cytotoxicity was determined by LDH release assay after 24 hours (n = 15) (<b>B</b>). Data are depicted as mean fold changes to untreated control, being set to 1.</p