Luteolin exhibits selective cytotoxicity towards tumorigenic cells and induces apoptosis.

<p>(A), The effects of luteolin on cell viability of tumorigenic (HeLa and U2OS) and non-tumorigenic (BEAS-2B and HEK293A) cell lines. Cells were treated with indicated concentration of luteolin for 24 hours. Afterward, cell viability was analyzed by the MTT assay. (B), Time-dependent effects of luteolin on HeLa cell viability. HeLa cells were treated with indicated luteolin 10 µM for indicated times. Cell viability was assessed by the MTT assay. Error bars represents means ±SEM (n = 10). Symbols (***) represent p value <0.0001. (C), The effects of flavonoids on A549 cell viability were compared. Error bars represent means ±SEM (n = 10). Symbols (***) represent p value <0.0001. The P-values in (A), (B) and (C) were calculated using Student's t-test. (D), Proapoptotic effect of luteolin on HeLa cells. HeLa cells treated with indicated concentrations of luteolin were double stained with PI/Annexin-V allophycocyanin. Apoptosis analysis was performed by flow cytometry.</p

Kinetic parameters of the binding of luteolin to VRK1.

<p>Kinetic parameters of the binding of luteolin to VRK1.</p

Direct interaction of luteolin with VRK1.

<p>(A), Pull-down assay using luteolin-conjugated sepharose 4B beads or control sepharose 4B beads with recombinant VRK1 protein. Purified GST and GST-VRK1 proteins are incubated with indicated beads, and then pull down assay was performed. Each proteins were detected by immunoblotting with GST antibody. (B), Pull-down assay using luteolin conjugated sepharose 4B beads with SH-SY5Y cell lysate. Cell lysate are incubated with indicated beads, and then pull-down was performed. The proteins were detected by immunoblotting with VRK1 antibody. (C), SPR detection for the interaction of luteolin with VRK1. The data were obtained by kinetic titration method with sequentially injection of analytes without regeneration steps. Data for eupatilin and wogonin were obtained by classical methods.</p

NMR titration assay and in silico modeling for interaction of luteolin with VRK1.

<p>(A), NMR titration experiments were performed. Spectrum of chemical shift perturbations versus amino acid residues of the VRK1 protein after binding of luteolin. (B), Mapping of chemical shift perturbations on the VRK1 protein. Most of the perturbed residues (shown in red) are located close to the catalytic domain of VRK1. (C), <i>in silico</i> modeling of the binding mode of luteolin to the VRK1 protein. Luteolin is predicted to fit in the vicinity of the G-loop, catalytic site, and α-C lobe.</p

Inhibitory effects of luteolin on VRK1 kinase activity.

<p>(A) and (B), <i>in vitro</i> kinase activity measurements for VRK1 with BAF (A) or VRK1 with histone H3 (B) were performed by increasing concentrations of luteolin (0.0, 1.0, 10, 50, 100, and 250 µM), and then VRK1 and substrate proteins were stained with silver nitrate. (C), Chemical structures and molecular weights of luteolin, eupatilin, and wogonin. (D) and (E), <i>in vitro</i> kinase assay for VRK1 with BAF were performed by increasing concentrations (0.0, 1.0, 10, 50, 100, and 250 µM) of eupatilin (D) or wogonin (E), and then VRK1 and BAF proteins were stained with silver nitrate. (F) and (G), Quantification of VRK1 auto-phosphorylation (F) or BAF phosphorylation (G) described in (B), (D) and (E). Data in (F) and (G) represent means of three independent experiments ±SEMs.</p

Luteolin-induced cell cycle arrest and nuclear envelope disassembly defects.

<p>(A), Cell cycle analysis was carried out by flow cytometry. HeLa cells transfected with GFP or GFP-VRK1 were treated with luteolin for 24 hours, and 10,000 cells were gated for analysis. Quantitative data are below the histogram plots. (B) and (C), HeLa cells treated with vehicle or luteolin were stained with lamin B antibody to visualize nuclear envelope and with Hoechst 33342 to visualized DNA. Alexa 488 dye-conjugated antibody was used as secondary antibody. The slides were visualized by fluorescence microscopy (B), or confocal laser scanning microscopy (C). (D), Fluorescent staining of VRK1, BAF, and DNA for analysis of the phosphorylation-mediated re-localization of BAF. Cells co-transfected with GFP or GFP-BAF with RFP or RFP-VRK1 were treated with DMSO or 10 µM luteolin for 24 hours.</p