Figure 1.
4SC-207 induces a G2/M arrest only in proliferating cells.
(A) Structure of 4SC-207. (B) RKO cells arrested (■) or proliferating (●) were incubated for 72hrs with a range of 4SC-207 concentrations (from 10nM to 100μM) and cell viability was measured using the Alamar Blue assay (viability values were normalized to the DMSO-treated sample set to 100%). The mean of four independent experiments + SEM (standard error of the mean) is plotted in the graph. (C) Proliferating RKO cells were treated with 100nM 4SC-207 or 0.1% DMSO as a control. After 24hrs the cells were stained with PI and the cellular DNA content was determined by flow cytometry.
Figure 2.
4SC-207 induces mitotic delay/arrest.
Synchronized HeLa wt cells were treated at the end of S phase with 4SC-207, nocodazole (10, 30 and 100nM), or 0.1% DMSO and the first mitotic division was followed by time-lapse microscopy. (A) Scatter plot distributions of the mitotic durations for the different treatments from one representative experiment (for each condition 100 cells were counted and each dot represents one cell that completes mitosis in the indicated time). (B) Quantification of the mitotic durations from three independent experiments. Bars represent means + SEM (standard error of the mean). (C) Cells were categorized into four groups according to their fate after mitotic entry: cells that divided normally (‘normal division’, green), cells that died in mitosis (‘mitotic death’, grey), cells that divided into more than two daughter cells or failed to complete cytokinesis (‘aberrant division’, blue), and cells that arrested in mitosis (‘mitotic arrest’, red). Representative images for each category are displayed (scale bar 5μm). The mean of three independent experiments (100 cells per condition per experiment) + SEM is summarized in the bar chart. (D) HeLa wt cells were first thymidine synchronized then treated with 30nM nocodazole, 100nM 4SC-207 or 0.1% DMSO. 9hrs after thymidine release, cells were fixed and immuno-stained for α-tubulin (green) and DNA (Hoechst, red). Representative images are displayed as individual and merged channels (scale bar 5μm). (E) Quantification of spindle morphology from synchronized HeLa cells stably expressing H2B-mCherry/mEGFP-α-tubulin treated with 0.1% DMSO, 50nM nocodazole or 50nM 4SC-207. Spindles were categorized into three groups: bipolar, ball-like and multi-polar. The mean of three independent experiments + SEM is summarized in the bar chart (the total number of spindles for DMSO, nocodazole and 4SC-207 treated cells were, respectively, 138, 125 and 117). Statistical significance between compound treated and DMSO treated cells in panels (B), (C) and (E) was evaluated using the Student’s t-test and indicated with * (p<0.01 for panel (B) and (E), p<0.001 for panel (C)).
Figure 3.
4SC-207 inhibits tubulin polymerization in vitro and in vivo.
In vitro polymerization of tubulin, purified from pig brain, in the presence of 0μM (●), 0.25μM (■), 0.5μM (▲) and 1μM (♦) nocodazole (panel A) or 0μM (●), 0.5μM (■), 1μM (▲) and 2μM (♦) 4SC-207 (panel B). Results show the extent of tubulin polymerization as a function of time (absorbance at 350nm was monitored at 30s intervals). Each data point represents the mean of three independent experiments + SEM (standard error of the mean). For clarity, only even-data points were plotted. Statistical significance between data points of compound treated and DMSO treated samples was determined using the Student’s t-test and highlighted by a vertical rectangle (p<0.01). (C) Representative examples of HeLa wt cells following cold treatment (on ice), addition of 0.1% DMSO, 4SC-207 (500nM) or nocodazole (500nM) and fixed at 0min or 5min after transfer to 37°C. Cells were fixed and immuno-stained for α-tubulin (green) and DNA (Hoechst, red) (scale bar 5μm).
Figure 4.
4SC-207 reduces microtubule growth rates and track lengths.
(A) Time projections (the maximum intensity projections of all time points) of time-lapse sequences of interphase HeLa cells stably expressing EB3-EGFP (see Videos S5, S6 and S7) treated with 0.1% DMSO, 50nM nocodazole or 50nM 4SC-207 (scale bar 5μm). (B) Box plot representing the distributions of average growth speeds of microtubules in interphase HeLa cells stably expressing EB3-EGFP treated with 0.1% DMSO, nocodazole (50 and 100nM) or 4SC-207 (50 and 100nM). For each condition 20 cells were analyzed. (C) Box plot representing the distributions of average microtubule track lengths following the different treatments. For each condition 20 cells were analyzed. Statistical significance between compound treated and DMSO treated cells in panels (B) and (C) was evaluated using the Student’s t-test and indicated with * (p<0.001). In the boxes the median of the distributions is displayed as a thick line and the whiskers extend to Q3+1.5*IQR and Q1-1.5*IQR, were Q1=quartile1, Q3=quartile3, IQR=interquartile range (Q3-Q1). Samples that fall outside the boundaries are considered outliers (open circles).
Figure 5.
4SC-207 inhibits proliferation with an average GI50 of 11nM and promotes tumor growth reduction in vivo.
(A) 4SC-207 was tested on a panel of 50 tumor cell lines from 14 different tissues and the GI50 values are represented as Z-scores, i.e. deviation of actual GI50 from the average GI50 divided by the standard deviation (SD). Negative Z-scores indicate higher sensitivity, positive Z-scores less sensitivity of cell lines towards the compound with respect to the mean GI50. The x-axis is displayed in log scale. (B) Female SCID mice bearing a HCT-15 colon cancer xenograft were treated with indicated concentrations of 4SC-207 (▲ and Δ), Taxol® (×) and control (○). Treatment was started at an average tumor volume of 100mm3 (Day 0) and continued for 3 weeks (treatment of 4SC-207 at 120mg/kg was extended for an additional week). Tumor volume and body weight were measured three times a week (see also Table 3). Significant reduction of tumor volume, indicated by * (two-tailed Mann-Whitney U-test; significance level p<0.05), was observed for treatment Group 3 (120mg/kg 4SC-207) on Day 12 (p=0.048), Day 14 (p=0.031) and Day 19 (p=0.025).