br Furthermore antiapoptotic BCl XL and MCL have
Furthermore, antiapoptotic BCl-XL and MCL-1 have been shown to be important mitotic survival factors, and cell showing an elevated BCL-XL level are demonstrated to be less responsive to taxane-based therapy . In our study, we showed that the triple combination could achieve two very important results: 1) to increase the proapoptotic phosphorylation of BCL-XL (pS62) and 2) to reduce the levels of MCL-1 based on an FBW7/PLK1-based mechanism. By acting on BCL-XL and MCL-1, the arrested GW3965 lose the prosurvival signaling rapidly (within 16 hours in our experiments), which quickly triggers apoptosis and explains the reduced time in mitotic arrest and quick death in mitosis according to our time-lapse experiment.
The clinical relevance of our data was shown in experiments using primary ovarian cancer cells from different patients (Figure 6, Supplementary Figure S8). While the triple combination induced
Figure 4. Combinatorial treatment of mitotic OVCAR-3 cells with proTAME promotes death in mitosis and blocks endoreduplication.
(A) Treatment schedule for time-lapse microscopy. Cells were first incubated with 2 mM thymidine to arrest them in the S-phase. After releasing the cells in fresh medium for 5 hours, drugs were added to cells and time-lapse microscopy was started up to 48 hours. (B) Representative time-lapse of OVCAR-3 cells expressing mcherry-H2B treated as in (A) with 1 nM paclitaxel, 1 nM paclitaxel/10 nM BI6727, or 1 nM paclitaxel/10 nM BI6727/10 μM proTAME (from t = 0 hour to t = 48 hours). Scale bar: 10 μm. (C) Duration of mitosis in OVCAR-3 cells treated with 1 nM paclitaxel, 1 nM paclitaxel/10 nM BI6727, or 1 nM paclitaxel/10 nM BI6727/10 μM proTAME was assessed by time-lapse microscopy. Mitotic duration of each cell in the different treatment groups (n=40) is quantitated in the bar graph. (D) Rate of cells exhibiting death in mitosis (DiM) or death in interphase (DiI) observed in the different treatment groups in percentage. (E) Endoreduplication rate observed in the different treatment groups in percentage (***P ≤ .001).
DMSO Paclitaxel BI6727
number of cells
Figure 5. Analysis of chromosomal aberrations in drug-treated ovarian cancer cells. (A) Following the incubation with single (2.5 nM paclitaxel, 10 nM BI6727, 10 μM proTAME), double, or triple agents for 48 hours and subsequent release into fresh medium for 14 days, metaphase spreads of treated OVCAR-3 cells were prepared, and the chromosomes were stained with Hoechst. Scale bar: 20 μm (B) The histogram plotting of the distribution of chromosome numbers at day 14 is shown. (C) Quantification of the number of chromosomes in 500 OVCAR-3 cells incubated in the presence of different agents for 48 hours. Measurements were statistically significant by two-tailed Student's t test (***P b .001). Each bar graph represents the mean value ± SEM (n=3).
pronounced apoptosis in primary ovarian cancer cells, the induction of apoptosis was very low in primary normal human cells, suggesting a favorable therapeutic window for this combinatorial approach (Supplementary Figure S7).
Unequal chromosome distribution during cell division as a mechanism that contributes to chromosomal instability (CIN) is a feature associated with many types of cancer. CIN results from errors in DNA replication, DNA repair, or chromosomal mis-segregation
primary ovarian cancer (primary tumor)
nM Paclitaxel (72 hours)
15 µM proTAME proTAME
DMSO Pac Pac/proTAME
Figure 6. Blocking mitotic exit sensitizes ovarian cancer patient-derived primary cells to paclitaxel. (A) Primary tumor cells were treated with increasing concentrations of paclitaxel, BI6727, or proTAME or combinations. (B) The cell viability was determined after 72 hours or
(C) over a period of 6 days using the Cell Titer-Blue Cell Viability Assay. (D) After treatment for 48 hours Caspase 3/7 activity was measured using the Caspase-Glo 3/7 assay. (E) 3D cultures grown out of primary tumor cells were treated (5 nM paclitaxel, 50 nM BI6727, 15 μM proTAME, or combinations thereof), cells were stained, and fluorescence intensities of dead cells were determined. Measurements were statistically significant by two-tailed Student's t test (*P ≤ .05). Each bar graph represents the mean value ± SEM (n=3).