br Blocking Mitotic Exit Reduces Chromosome Numbers
Blocking Mitotic Exit Reduces Chromosome Numbers in Mitotically Arrested Ovarian Cells
The ability to assess chromosome abnormalities based on the analysis of metaphase chromosome spreads can reveal genetic disorders in EPZ-6438 that have survived the drug treatment. The cell line OVCAR-3 is aneuploid human female, with chromosome counts in the sub- to near triploid range . OVCAR-3 cells were treated for 24 hours with single agents (2.5 nM paclitaxel, 20 nM BI6727) followed by 10 μM proTAME for 24 hours and thereafter transferred to compound-free medium for 14 days followed by examination of metaphase spreads of the surviving cells (Figure 5, A and B). The mean number of chromosomes
DMSO nM Pac
DMSO.5 nM Pac
D nMPac BI6727 proTAME TAME
Figure 3. Induction of apoptosis in ovarian cancer cells following drug treatment. (A) OVCAR-3 cells were incubated with 2.5 nM paclitaxel, 20 nM BI6727, 10 μM proTAME, or combinations thereof. Caspase-3/7 activity in whole cell lysates was measured 48 hours posttreatment using the Caspase-Glo 3/7 Assay. Each bar graph represents the mean value ± SEM (n=3). Apoptosis was validated (B) by measuring the sub G0/G1 fractions or (C) by PE Annexin V staining. Measurements were statistically significant by two-tailed Student's t test (*P ≤ .05; **P ≤ .01,
***P ≤ .001). Each bar graph represents the mean value ± SEM (n=3). (D ,upper and lower panel) Whole cell lysates of OVCAR-3 cells were analyzed evaluating marker proteins for mitochondrial-mediated apoptosis. Endogenous levels of PARP, cleaved PARP, BCL-XL pS62, BCL-2, Caspase-3, cleaved-Caspase-3, pHistone H3, PLK1, Cyclin B1, MCL-1, Cyclin A, Securin, and β-Actin were determined by immunoblotting.
per cell 63.37 ± 8.75 in the OVCAR-3 population increased in cells that survived the 48-hour treatment with 2.5 nM paclitaxel to 72.76 ± 17.63, with 20 nM BI6727 to 99.86 ± 21.37, and with 2.5 nM paclitaxel/ 20 nM BI6727 to 121.80 ± 20.08. Cells that survived the 48-hour triple treatment showed very similar chromosome numbers (65.04 ± 7.43) compared to DMSO-treated cells (Figure 5C). Chromosome spreading in SKOV-3 cells showed again that cells treated with paclitaxel/BI6727/proTAME had very similar chromosome num-bers compared to untreated cells (Supplementary Figure S6, A-C), supporting the view that the treatment with proTAME counteracts the chromosomal instability in ovarian cancer cells induced by paclitaxel and/or BI6727.
Evaluation of the Treatment-Associated Toxicity in Normal Primary Mammalian Cells
Next, we evaluated the correlation between drug treatment and growth of primary human cells. Remarkably, the cell growth of fibroblasts was robust in single, double, and triple treatments over 96 hours, suggesting that the toxicity of the agents at least at the concentrations used in our experiments is very low in primary fibroblasts (Supplementary Figure S7).
Blocking Mitotic Exit Inhibits the Growth of Mitotically Arrested Primary, Patient-Derived Ovarian Cancer Cells
To study the physiological relevance of our experiments, we extended our study to a primary ovarian cancer cell culture established
from tumor specimen derived from patients diagnosed with ovarian cancer. For all single agents (paclitaxel N0.1 nM, BI6727N 8 nM, proTAME N10 μM), a dose-dependent decrease in the viability of primary ovarian cancer cells was observed (Figure 6A). Within the
range of tested concentrations, proTAME exerted the most intense inhibitory effect. Increasing concentrations in the range of 0-100 nM paclitaxel was not sufficient to reduce the growth of primary cells by 50%. In combinatorial approaches including 100 nM BI6727, the
release in fresh
Thymidine 20 h
medium for 5 h
compounds live microscopy up to 48 h
Mitosis duration in hours
The addition of proTAME to primary cells treated with the two mitotic inhibitors paclitaxel and BI6727 in long-term experiments (6 days) augmented the growth inhibition significantly (P b .001) (Figure 6C). The analysis of the Caspase 3/7 activity supported the observation from long-term cultures that the triple treatment exerts the most intense apoptosis compared to the double or single treatments (P b .05) (Figure 6D). Results with 3D cultures revealed a significant increase of dead cells by the combinatorial treatment including proTAME compared to single and double treatments (P b .05) (Figure 6E).