EM011 perturbs spindle architecture and manipulates multipolar mitosis. Panel shows immunofluorescence confocal micrographs of prostate cancer cells stained for microtubules (green, FITC) and DNA (red, PI) upon 25 μM EM011 treatment for noted hours. There was an accumulation of mitotic cells in prometaphase (white arrowheads) with multipolar spindles at 24 hr of drug-exposure. At 48 hr of EM011 treatment, some multinucleate cells (open arrowhead) were evident and apoptotic bodies (white arrows) were visible at 48 and 72 hr.

EM011-induced apoptosis requires activation of spindle assembly checkpoint. Impairment of the spindle checkpoint by knock-down of Mad2 or BubR1 expression by specific siRNAs suppresses EM011-induced mitotic arrest and apoptosis. A. Immunoblot analysis of Mad2 and BubR1 expression in PC-3 cells transfected with luciferase (control), Mad2, or BubR1 siRNAs for 24 hr. Cells transfected with Mad2, BubR1, or control siRNAs for 24 hr were treated with 25 μM EM011 for 0, 24, 48, or 72 hr. B. Histogram-overlays of cell-cycle profiles of 24 hr drug-treated PC-3 cells that were transfected with luciferase (control), Mad2, or BubR1 siRNAs. C. Mitotic cells quantified by a mitosis-specific MPM-2 antibody in 24 hr drug-treated cells (transfected with luciferase (control), Mad2, or BubR1 siRNAs) using dual-color flow cytometry. D. Quantitation of MPM-2 positive cells in luciferase (control), BubR1 or Mad2 siRNAs transfected cells that were treated with EM011 for 0, 24, 48 and 72 hr. Values represent averages and error bars show SD (p < 0.05). E. Histogram-overlays of cell-cycle profiles of luciferase (control), BubR1 or Mad2 siRNA transfected cells that were drug-treated for 72 hr depicting differences in sub-G1 population (indicative of apoptotic cells). F. Quantitation of apoptotic cells (sub-G1) shows that cells with attenuated checkpoint molecules had reduced apoptotic responses at the noted hours (0, 24, 48 and 72) upon drug-treatment. Values represent averages and error bars show SD (p < 0.05).

Bioluminescent imaging shows tumor reduction of PC-3 intratibial xenografts upon EM011 treatment for five weeks. Cold (blue) colors indicate low photon counts whereas hot colors (red) show high counts. A. Top-panel shows representative mice and their tibial bioluminescence indicating progression of tumor growth in control vehicle-treated mice. Bottom-panel shows mice treated daily with 300 mg/kg EM011 by oral gavage. Note that a significant decrease of photon pixels was visible at day 35 of EM011 treatment. A steep upward transition of tumor growth occurs at day 21 in vehicle-treated mice. B. Quantitative evaluation of photon emission, thus, tumor burden. Stars represent significance at p < 0.05, n=10. C. Paraffin-embedded tibial-sections from vehicle-treated and 21 day EM011-treated mice were processed for TUNEL assay to visualize apoptotic cells. In contrast to controls, numerous TUNEL-positive cells were visible in EM011-treated sections. Significant cleaved caspase-3 immunostaining is also evident in sections from drug-treated mice compared to vehicle-treated controls.

EM011 induces activation of mitotic checkpoint. A. Mitotic arrest is mediated by spindle-assembly checkpoint, as visualized microscopically by immunofluorescent staining of BubR1 (green) at 24 hr post-treatment. B. Immunoblot analysis also showed BubR1 accumulation until 36 hr of drug treatment. β-actin was used as a loading control. C. EM011 treatment for 72 hr induces apoptosis in PC-3 cells. Fragmented DNA in the terminal apoptosis stages was quantified using terminal deoxynucleotidyl transferase–mediated bromo-dUTP reaction (TUNEL assay) in EM011-treated cells. After the indicated incubation periods, cells were processed for a flow cytometry-based terminal deoxynucleotidyl transferase-mediated BrdUTP reaction. The addition of BrdUTP to the terminal deoxynucleotidyl transferase reaction labels DNA strand breaks that are then detected by an Alexa-Fluor 488-labeled anti-bromodeoxyuridine antibody. DNA content was determined using propidium iodide (PI, x axis). The number of apoptotic cells is indicated by the number of Alexa-Fluor 488-positive cells (vales on the top of the cytogram). Data from a representative experiment of two experiments.

EM011 treatment induces a mitochondrial-driven caspase-dependent apoptosis. A. Mitochondrial potential drop is observed beginning 24 hr of 25 μM EM011 exposure that progressively peaks at 72 hr as revealed by FACS-analyses using fluorescent-marker, DiOC6(3). B. Quantitation of time-dependent increase in the number of depolarized cells upon 25 μM EM011 treatment. Values represent averages and error bars show SD (p < 0.05). C. Immunoblot analyses for p-Bcl2, total Bcl2, BAX, PUMA, activated caspase-3 and cleaved PARP over time of EM011 treatment. D. In the same time-frame, significant activation of caspase-3 activity also occurs as measured luminogenically. E. Caspase-inhibitor, z-VAD-fmk, along with EM011 treatment for 48 hr significantly reduces sub-G1 population while accumulating polyploid cells (green profiles). Cells treated with EM011 alone for 48 hr (red profiles) show significant apoptotic population as compared to vehicle-treated controls (purple profiles). F. Representative confocal immunomicrographs showing interphase, mitotically-arrested, multinucleated and apoptotic cells. Tubulin (green); DNA (red). These cell phenotypes were observed in different magnitudes upon treatment with drug alone or drug along with caspase inhibitor. The 48 hr co-treated cells show a larger proportion of cells with multipolar mitosis and multinucleation with excess DNA amounts. EM011-alone treated cells show preponderance of apoptotic bodies. G. Quantitation of these different cell phenotypes from random image fields totaling 100 cells in 48 hr PC-3 cells treated with vehicle, EM011, z-VAD-fmk, EM011+ z-VAD-fmk.

EM011 inhibits cellular proliferation and impedes cell-cycle by causing mitotic arrest followed by apoptosis in PC-3, LNCaP, C4-2, and C4-2B cells. A. Plot of the percentage of cell survival versus EM011 concentrations used for the determination of IC₅₀ values. Cells were treated with increasing gradient concentrations of EM011 for 72 hr, and the percentage of cell proliferation at the indicated drug concentrations, compared with vehicle-treated cells, was measured by the standard sulforhodamine B assay as described in Materials and Methods. The IC₅₀ values of EM011 for all four prostate cancer cell types are indicated on the panel. B. The effect of EM011 on the cell-cycle profile of all four prostate cancer cells are shown in a three-dimensional disposition. Cells were harvested for analysis at the indicated times, stained with PI and analyzed by fluorescence-activated cell sorting (FACS) using Cell-Quest Software. The x axis shows the intensity of PI fluorescence, which is indicative of the total DNA content of cells in different cell-cycle phases. The y axis depicts the number of cells in each phase of the cell-cycle and the z axis indicates the time points (0, 24, 48, and 72 hr). Results are representative of three experiments done in triplicate. C. Quantitative representation of mitotic and apoptotic index as a function of time of treatment with 25 μM EM011.