Anti-proliferative effect of amitozyn in different cell lines.

<p>Cells were treated with Am at indicated concentration for 72 h as described in Material and methods. Cells viability was determined by trypan blue exclusion.</p

General characterization of amitozyn effect on HeLa cells.

<p>(A) Cells were exposed to different concentrations of Am for 24 and 48 h and the percent of 2N and 4N cells in three independent experiments was estimated by FACScan and plotted in graph shown in the lower panel. ▪- percent of 2N cells after 24 h treatment; □ - percent of 4N cells after 24 h treatment; • - percent of 2N cells after 48 h treatment; - percent of 4N cells after 48 h treatment. (B) Kinetics of Am effect on HeLa cells. Cells were exposed to 125 µg/ml of Am for periods from 0 to 72 h and analyzed by FACScan. The percent of 2N and 4N cells from three independent experiments was plotted in graph shown at the bottom. ▴ - 2N cells in control; Δ- 4N cells in control; ♦ - 2N cells after treatment with Am; ◊ - 4N cells after treatment with Am. (C) FACScan analysis of mitotic cell ratio after Am treatment. The HeLa cells were exposed for 24 h to different Am concentrations, fixed with methanol, stained with PI and MPM2-FITC and analyzed by FACScan (upper panel). The average mitotic index of three independent experiments was plotted in the graph (lower panel). The Western blot inset shows the MPM2 level at indicated time points. (D) Reversibility of Am effect. The HeLa cells were synchronized in the G1/S phase by double thymidine block and released by medium addition (top row), exposed to Am (250 µg/ml) for 12 h with subsequent release in medium for 12 and 24 h (second row) or released in Am (250 µg/ml) during the entire experiment (last row). Cells were analyzed by FACScan as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057461#s2" target="_blank">Materials and methods</a>.</p

Perturbation of cell signalling and mitotic checkpoint activation in HeLa cells upon Am treatment.

<p>HeLa cells were treated with 250 µg/ml Am for 12, 24 and 48 h. As a control (C) we used untreated cells grown for 48 h. Cell lysates were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057461#s2" target="_blank">Materials and methods</a>, and the level of indicated proteins was analyzed by Western blot. Actin was used as loading control.</p

Effect of amitozyn on cell morphology and polymerization of tubulin <i>in vitro</i>.

<p>HeLa cells were treated with 0, 15, 30, 60, 125, 250 µg/ml amitozyn for 8 h, fixed and stained with anti-tubulin Ab (green) and PI (red). A and B, the mitotic phenotypes of HeLa cells observed after amitozyn and chelidonine (Chel) treatment, respectively. C and D, ratios of different mitotic phenotypes in cell population after Am and Chel treatment, respectively. Percent of cells with <b>white</b> - normal mitotic spindle; <b>grey</b> - abnormal mitotic spindle of type I; <b>shaded</b> - abnormal mitotic spindle of type II; <b>black</b> - abnormal mitotic spindle of type III. (E) Interphase cell observed in the presence of amitozyn and chelidonine. HeLa cells were exposed to amitozyn and chelidonine at indicated concentrations, fixed and stained with anti-tubulin Ab. Drug concentration is shown in µg/ml. (F) Effect of amitozyn on tubulin polymerization <i>in vitro</i>. Tubulin (60 µM) was polymerized for 10 min at 37°C in the presence of 0–500 µg/ml amitozyn as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057461#s2" target="_blank">Materials and methods</a>.</p

Structure of amitozyn and celandine alkaloids.

<p>Structure of amitozyn and celandine alkaloids.</p

Effect of Am on cell viability.

<p>(A) HeLa cells were treated with Am for 72 h as described in Material and Methods and cell viability was measured by trypan blue exclusion. The cytotoxic effect (B) of Am was estimated after indicated periods using the LDH kit. Average data of three independent experiments are presented. (C) MESSA cells and multidrug resistant MESSA Dx5 cells were treated either with Am or with paclitaxel (D) for 72 h and cell viability was measured as described above.</p

Effect of amitozyn on DNA integrity.

<p>(A) Double-strand breaks analyzed by DNA comet assay (left column) and by immunofluorescence microscopy (right column). Top row - untreated HeLa cells (C), second row – cells treated with 5 µg/ml etoposide (positive control), last row – cells treated with 250 µg/ml amitozyn for 12 h. The tail in the comet assay and histone γ-H2AX visualized by immunofluorescence microscopy shows the presence of DNA double-strand breaks. (B) Percentage of cells containing double-strand breaks revealed by DNA comet assay.</p

Effect of roscovitine and AZ 3146 on the spindle checkpoint in HeLa cells blocked by Am.

<p>Time lapse (A) and western blot analysis (B) analysis of mitotic cells progression after treatment with roscovitine and AZ 3146. HeLa cells were synchronized by double thymidine block and released in the drug free medium. After 6 h cells were treated with 250 µg/ml Am. 5 h later the DMSO (control), roscovitine (10 µM) and AZ 3146 (4 µM) were added. Another cell portion was released in a drug-free medium (wash panel). At indicated timepoints cells were trypsinized and cell lysate was analysed by western blot as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057461#s2" target="_blank">Materials and methods</a>. Actin was used as loading control.</p

Activation of apoptotic markers and modulation of pRb level upon Am application.

<p>HeLa cells were treated with 250 µg/ml Am for 12, 24 and 48 h. As a control (C) we used untreated cells grown for 48 h. Cell lysates were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057461#s2" target="_blank">Materials and methods</a>, and the level of activated caspase-9, caspase-3 and cleaved PARP (A), and total pRb and pRb phosphorylated at Ser 780 (B) was analyzed by Western blot. β-tubulin and actin were used as loading controls.</p

Growth inhibition expressed in IG₅₀ values of amitozyn for different cell lines.

<p>Cells were exposed to 0–500 µg/ml Am. At 72 h cells were harvested by trypsinization and the ratio of viable cells was estimated by trypan blue exclusion. The IG₅₀ value was estimated from cell viability plots and presents the average value of three independent experiments.</p