Isolation of active compound from CACF.

<p>A. Flow chart of bioassay guided isolation of <i>Centratherum anthelminticum</i>. B. HPLC chromatogram of the fraction of CACF-A of the chloroform extract of <i>C. anthelminticum</i>.</p

Photo of <i>Centratherum anthelminticum (L.)</i> seeds.

<p>Photo of <i>Centratherum anthelminticum (L.)</i> seeds.</p

Vernodalin induces cell cycle arrest at G0/G1 stage.

<p>MCF-7 and MDA-MB-231 cells were treated with indicated dosages of verdonalin for 24 hours. Cells were ethanol-permeabilized and stained with propidium iodide before subjected to flow cytometry analysis. Representative figures of cell cyle distribution (G0/G1, S, and G2/M) showing accumulation of vernodalin-treated cells in G0–G1 stage. Data were mean ± SD of two independent experiments.</p

CACF inhibits MCF-7 cells proliferation in a time- and dose-dependent manner.

<p>(A) MCF-7 cells were treated with control DMSO, various concentrations (0.195, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50 µg/ml) of CACF or anti-cancer drug doxorubicin for 24 hours. Cell viability was determined by MTT assays. (B) Real-time cell proliferation was measured using xCELLigence Real-Time Cellular Analysis (RTCA) system. MCF-7 cells were treated with DMSO (control), indicated concentration of CACF or doxorubicin (DOX) and normalized cell index for 3 consecutive treatment days was shown. Data were mean ± SD. Arrow showing time-point of CACF administration.</p

Vernodalin reduces expression of pro-survival molecules.

<p>MCF-7 and MDA-MB-231 cells were treated with control DMSO, standard drug doxorubicin (12.5 µg/ml) or various concentrations of vernodalin (3.125, 6.25, 12.5 µg/ml). Western blot showing the expression levels of the pro-survival molecules Bcl-2 and Bcl-xL in untreated and treated breast cancer cells. β-actin served as a loading control. Decreased Bcl-2 and Bcl-xL protein levels were observed upon doxorubicin or vernodalin treatment. Data were representative of at least two similar experiments.</p

Morphological assessment of CACF-treated MCF-7 cells.

<p>(A) Representative figures of MCF-7 cells were treated with CACF for 12 hours. Cells were stained with apoptosis marker annexin V (green) and nucleus marker Hoechst 33258 (blue). Histogram shows mean fluorescence intensities of annexin V in MCF-7 cells treated with various concentration of CACF. Data were mean ± SD, *<i>P</i><0.05. (B) Representative figures of cytoskeletal F-actin formation in control or CACF-treated MCF-7 cells. Cells were fixed, stained with DY544-phalloidin (red) and Hoechst 33258 (blue) after treated with 6.25 µg/ml CACF or solvent DMSO for 12 hours. Histogram shows mean fluorescence intensities of phalloidin in MCF-7 cells treated with various concentration of CACF. Data were mean ± SD, *<i>P</i><0.05. (C). Representative figures of MCF-7 cells treated with DMSO (control), 6.25 or 12.5 µg/ml of CACF for 24 hours. Cells were also treated with a standard drug doxorubixin (DOX) as positive control of apoptosis induction. Cells were stained with Hoechst 33258 dye (blue). All images were visualized and captured using Cellomic HCS array scan reader (objective 20 ×).</p

Vernodalin induces apoptosis in human breast cancer cells.

<p>(A) Flow cytometry analysis of MCF-7 and MDA-MB-231 cells treated with 3.125, 6.25 and 12.5 µg/ml verdonalin for 24 hours. Representative figures showing population of viable (annexin V- PI-), early apoptotic (annexin V+ PI-), late apoptotic (annexin V+ PI+) and necrotic (annexin V- PI+) cells. (B) Bar chart showing increased proportion of early and late apoptotic cells after vernodalin administration. Data were mean ± SD of two independent experiments. (*<i>P</i><0.05).</p

Vernodalin mediates ROS production.

<p>(A) MCF-7 or MDA-MB-231 cells were treated with DMSO (control) or indicated concentration of vernodalin for 12 hours. Live cells were stained with DHE dye (green) before cells were fixed and stained with Hoechst 33258 (blue). Images were acquired using Cellomic HCS array scan reader (objective 20 ×). Representative figures (control or 6.25 µg/ml vernodalin-treated) were shown. (B) Bar chart showing average fluorescence intensities of DHE dye in the nucleus. Data were mean ± SD of fluorescence intensity readings representative of three independent experiments. (*<i>P</i><0.05).</p

Vernodalin inhibits invasion of MDA-MB-231 human breast cancer cell line.

<p>Real-time cell invasive assay. MDA-MB-231 cells were seeded into upper chamber of CIM plates coated with matrigel. Lower chamber were filled with medium with FCS or medium only. Cells were treated with DMSO (control) or indicated concentrations of vernodalin and continuously monitored for 16 hours. Increased cell migration to lower chamber resulted in higher normalized cell index. Data were mean ± SD from two independent experiments.</p

Chemical structure of vernodalin (1) and 12,13-dihydroxyoleic acid (2).

<p>Chemical structure of vernodalin (1) and 12,13-dihydroxyoleic acid (2).</p