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  • br Recently a growing body


    Recently, a growing body of evidence has indicated that three-dimensional (3D) cell culture systems represent more accurately the actual microenvironment where EPZ031686 reside in tissues. Therefore, we tested in a third long-term experiment the cell viability in a 3D multicellular model which is widely used as avascular tumor model for metastasis and invasion research. We could demonstrate that the triple combination has the most intense inhibitory effect compared to the combination paclitaxel/BI6727 and compared to the single agents (Figure 2D).
    Furthermore, we tested the long-term effects in SKOV-3 cells. We observed for all single treatments a weak to moderate inhibitory effect on cell growth over 5 days (Supplementary Figure S4A). The triple combination was significantly more efficient to inhibit the growth compared to the treatment with paclitaxel/BI6727 (P b .001) (Supplementary Figure S4A). Furthermore, the 48-hour treatment of SKOV-3 cells followed by a 10-day observation period showed the most intense inhibition of colony formation using the triple combination compared to double or single treatments (Supplementary Figure S4B, upper and lower panel). Together, different long-term analyses revealed that the triple combination is much more efficient in inhibiting viability and colony formation of different ovarian cancer cell lines compared to paclitaxel or the combination paclitaxel/BI6727.
    APC/C Inhibitors Increase Apoptosis in Mitotically Arrested Ovarian Cancer Cells
    Very low levels of Caspase-3/7 activation in OVCAR-3 cells could be detected after single treatments compared to controls (Figure 3A). This activation could be enhanced by incubation with paclitaxel/BI6727 [2.5 nM paclitaxel/20 nM BI6727 activation to 3.5-fold compared with 2.5 nM paclitaxel (P b .001)] and even further by the triple treatment [20 nM BI6727/2.5 nM paclitaxel/10 μM proTAME activation to 6.8-fold compared with paclitaxel/BI6727 (P b .01)]. The FACS analysis showed a significant increase of the sub G0/G1 peak indicating an apoptotic cell population comparing paclitaxel/BI6727 versus paclitaxel/ BI6727/ proTAME-treated cells (P b .05) (Figure 3B). Furthermore, cells were stained with PE Annexin V/7-AAD and analyzed by FACS. We observed a moderate increase of Annexin V–positive cells after an incubation with paclitaxel for 48 hours (2.5 nM, 12%), BI6727 (20 nM, 11%), or proTAME (10 μM, 10%) compared to controls (DMSO, 7%) (Figure 3C). The co-incubation of cells for 48 hours with 20 nM BI6727 and paclitaxel induced 13% PE Annexin V/7-AAD–positive cells versus 17% positive cells following 2.5 nM paclitaxel/20 nM BI6727/10 μM proTAME (Figure 3C). The examination of apoptosis in SKOV-3 cells based on Caspase-3/7 activation, sub G0/G1 peak, and PE Annexin V/7-AAD staining confirmed our observations from OVCAR-3 cells (Figure S5, A-C) showing that the triple treatment induces significantly more apoptosis in ovarian cancer cell lines compared to single or double treatments (P b .01). 
    We examined members of the antiapoptotic BCL-2 family, which contribute to the regulation of apoptosis during mitotic arrest [48]. In contrast to the treatment with single agents, the combinatorial treatment (paclitaxel/BI6727) prompted elevated levels of BCL-XL phosphory-lation and cleavage of PARP and Caspase-3, respectively, with the highest levels in triple-treated cells (Figure 3D, upper panel). Recent studies describe a pathway involving the protein myeloid cell leukemia 1 (MCL-1), an antiapoptotic member of the BCL-2 family, that couples the timing of mitosis to the induction of apoptosis [48]. PLK1 was shown to regulate the stability of FBW7, promoting the degradation of MCL-1 [49]. While the co-treatment with BI6727/paclitaxel decreased the MCL-1 level, the triple treatment led to further degradation of MCL-1 (Figure 3D, lower panels). High levels of PLK1, Securin, and phopho-Histone H3 in co-treated cells suggest that the inactivation of antiapoptotic BCL-2 family members (downregulation of MCL-1, phosphorylation of BCL-XL) occurs in mitosis (Figure 3D, upper and lower panel). In summary, BI6727, paclitaxel, and proTAME cooperate to activate the mitochondrial pathway, leading to the activation of Caspase-3 (Figure 3, A and D).
    Preventing Mitotic Exit Increases Cellular Death in Mitosis
    To assess the fate of cells following drug treatment in more detail, we analyzed SAC activity by monitoring the mitotic exit. OVCAR-3 cells stably transfected with mCherry-H2B were synchronized in the S-phase, released in media with drugs, and followed by time-lapse microscopy (Figure 4A). While control cells completed mitosis normally (1.1 ± 0.12 hours), paclitaxel significantly increased mitotic duration (17.5 ±
    5.4 hours) (Figure 4, B and C). The combinatorial treatment (paclitaxel/ BI6727) caused a prolonged mitotic arrest (19.0 ± 4.5 hours) (Figure 4, B and C). Remarkably, the triple treatment significantly shortens the time in mitotic arrest compared to paclitaxel (11.5 ± 3.2 vs. 17.5 ± 5.4 hours) (Figure 4, B and C). To shed more light on the cell fate induced by different applications, we tracked the outcome of single cells during mitotic arrest and after mitotic exit. Two percent of DMSO-treated cells died during mitotic arrest; 2% could evade mitosis but died in the next interphase (Figure 4D). Drug treatment had differential effects on the death in mitosis (paclitaxel, 35%; paclitaxel/BI6727, 45%; paclitaxel/BI6727/proTAME 100%) or death in interphase (paclitaxel, 2%; paclitaxel/BI6727, 10%; paclitaxel/BI6727/ pro-TAME 0%), showing that the strong mitotic arrest induced by the triple combination is associated with highly frequent death in mitosis (Figure 4D). The observations revealed that triple-treated cells have a short resting time in mitosis because they die before they can exit mitosis. While treatment with paclitaxel and paclitaxel/BI6727 increased endoreduplication, only the triple combination blocked endoduplication completely, indicating that an increase in genetic complexity is prevented (Figure 4E).