Figure 1.
Cytotoxicity of BPR0L075 in parental and paclitaxel-resistant ovarian cancer cells.
(A) Proliferating human ovarian cancer cells (SKOV-3, OVCAR-3, and A2780-1A9) and paclitaxel-resistant sublines (SKOV-3-TR, OVCAR-3-TR, and 1A9-PTX10) were treated with BPR0L075 (red circle) or paclitaxel (blue square) at 3–15 nM concentrations for 4 days and the cytotoxicity was evaluated by SRB assay. Fraction of cell survival relative to controls represents mean ± SD of 12 determinations. (B) Summary of the P-gp expression, βIII-tubulin expression, and sensitivity of paired ovarian cancer cell lines to paclitaxel, doxorubicin, and BPR0L075 treatment. Numbers in parenthesis represents the degree of resistance (x-fold), expressed as the ratio of the IC50 values as compared with the corresponding parental lines. All experiments were repeated three times.
Figure 2.
BPR0L075 is not a substrate for efflux transporters.
(A) Western blot analysis of the P-gp and βIII-tubulin expressions in the paired ovarian cancer cell lines. Pan β-tubulin and β-actin were loading controls. (B) LC/MS/MS analysis of intracellular BPR0L075 levels, with LC chromatogram showing the retention time of internal standard [1-(1H-indol-3-ylcarbonyl)-1H-imidazole] and BPR0L075 as 2.2 and 2.8 min, respectively. The mass transitions of m/z 212→144 for internal standard and m/z 342→195 for BPR0L075 were monitored for quantification. (C) The intracellular BPR0L075 levels in the paired ovarian cancer cell lines. Cells were incubated with 20 nM BPR0L075 for 6 hours, then cells were washed, harvested, and lysed for LC/MS/MS measurement. Bars represent mean ± SD of three independent tests. No statistically significant difference was observed (P>0.05, student t-test).
Figure 3.
BPR0L075 induces concentration- and time-dependent cell cycle arrest in both parental and paclitaxel-resistant ovarian cancer cells.
OVCAR-3 and OVCAR-3-TR cells were treated with ethanol vehicle or BPR0L075 (10–100 nM) for 24 hours or 10 nM BPR0L075 for indicated durations (0–48 hours), stained with propidium iodide, and analyzed by flow cytometer. The cell cycle profiles are presented as three-dimensional overlay. The X-axis shows the intensity of propidium iodide fluorescence, which indicates cellular DNA content in different cell cycle phases. The Y-axis represents the cell counts; and z-axis shows the concentration or time points of BPR0L075 treatment. 2N, cells residing in the G0–G1 phase of cell cycle; 4N, cells in the G2 phase or mitosis. BPR0L075 induces significant G2/M arrest followed by appearance of a sub-G1 population in both parental and resistant cells, with polyploid cells (arrows) only in the resistant cells. Results are representative of three independent experiments.
Figure 4.
BPR0L075 treatment disrupts microtubule networks in parental and paclitaxel-resistant ovarian cancer cells.
Coverslips containing OVCAR-3 and SKOV-3 parental and paclitaxel-resistant cells were exposed to medium containing ethanol vehicle or 20 nM of BPR0L075 for 18 hours. The cells were fixed, coverslips were then stained with anti-α-tubulin antibody (AlexaFluor 488, green) for microtubules and 4,6-diamidino-2-phenylindole (DAPI, blue) for cell nuclei, and observed by Olympus IX81 fluorescence microscope. The control parental cells showed typical radial microtubule arrays. BPR0L075 treatment disrupted microtubule cytoskeleton in both the parental and resistant ovarian cancer cells. White arrows show the giant, multinucleated cells characteristic of mitotic catastrophe. Scale bar, 10 µm.
Figure 5.
Western blot analysis of the relative protein expressions in the parental and paclitaxel-resistant ovarian cancer cell lines after BPR0L075 treatment.
BPR0L075 treatment (10–100 nM for 24 hours) induced changes in cyclin B1, BubR1, MPM-2, and survivin expressions in OVCAR-3/OVCAR-3-TR cells (A) and SKOV-3/SKOV-3-TR cells (B). BPR0L075 treatment (10 nM) induced changes in PARP, BCL-XL, and BCl-2 proteins at different time (0–72 hours) in OVCAR-3/OVCAR-3-TR cells (C) and SKOV-3/SKOV-3-TR cells (D). 50 µg of the protein lysate was loaded on the SDS-PAGE and experiments were repeated and representative blots are shown.
Figure 6.
BPR0L075 induces PARP cleavage and DNA fragmentation in parental cells, but not in the paclitaxel-resistant ovarian cancer cells.
Western blot analysis of the PARP, caspase-2, and caspase-3 after BPR0L075 (10–100 nM) treatment for 24 hours in the OVCAR-3/OVCAR-3-TR cells (A) and SKOV-3/SKOV-3-TR cells (B). β-actin was loading control. (C) DNA ladder assays in cells treated with 10 nM BPR0L075 for 72 hours. Genomic DNA was subjected to 1% agarose gel electrophoresis. The gel was stained with ethidium bromide and the DNA bands were visualized under an ultraviolet transilluminator. (D) Effect of caspase-3 inhibitor on BPR0L075 induced cytotoxicity. Cells were pretreated with caspase-3 inhibitor (20 µM Z-DEVD-FMK) or negative control inhibitor (20 µM Z-FA-FMK) for 24 hours before incubation with 10 nM BPR0L075 for additional 72 hours. On completion of the incubation, the cell viability was determined by SRB assay. No statistically significant difference of cytotoxicity was observed for each pair of cell lines (P>0.05, student t-test). Data represent mean ± SD.