Fig 1.
The effects of olaparib, triapine, and cediranib combinations on the growth of subcutaneous SKOV3 xenografts in mice.
Athymic nude mice were inoculated s.c with SKOV3 cells. After 3 days, mice were randomly assigned to 4 groups (n = 3) and treated i.p. with vehicle, cediranib (0.75 mg/kg), the olaparib (50 mg/kg)-triapine (10 mg/kg) combination, and cediranib plus the olaparib-triapine combination daily for a continuous 6-week period (day 3 to 45). (A) Tumor size was measured every 2–3 days. Data are means ± SE. p values were determined by the Wilcoxon matched-pairs signed test compared with the control and between treatment groups. (B) Tumor tissue was excised from mice, photographed, and weighted in the end of the experiment. One largest tumor in control and cediranib groups is not shown. Data are means ± SE. p values were determined by the one-way ANOVA with the Dunnett’s multiple comparison test compared with the control. (C) Representative images of H&E-stained section of tumor tissue from respective treatment groups are shown.
Fig 2.
The effects of olaparib, triapine, and cediranib combinations on the growth of subcutaneous OVCAR3 xenografts in mice.
Athymic nude mice were inoculated s.c with OVCAR3 cells. After 10 days, mice were randomly assigned to 6 groups (n = 5) and treated i.p. with vehicle, cediranib (0.75 mg/kg), olaparib (50 mg/kg) plus cediranib, triapine (10 mg/kg) plus cediranib, olaparib plus triapine, and olaparib plus triapine plus cediranib daily for consecutive 6 weeks (day 10 to 52). (A) Tumor size was measured every 2–3 days. Data are means ± SE. p values were determined by the Wilcoxon matched-pairs signed test compared with control and between groups. (B) Tumor tissue was excised from mice, photographed, and weighted in the end of the experiment. Data are means ± SE. p values were determined by the one-way ANOVA with the Dunnett’s multiple comparison test compared with the control. (C) Representative images of H&E-stained section of tumor tissue from each treatment group are shown. (D) The body weight of mice was measured every 2–3 days. The means of body weight for all treatment groups are shown.
Fig 3.
Characterization of intraperitoneal EOC cell lines and xenografts.
(A) Expression of BRCA2 in PEO1/4 cells and PEO1/4ip cells. Total protein was isolated from cells and subjected to western blot analysis for BRCA2 protein. HSC70 protein was used as a loading control. BRCA2 wild type and mutant bands are shown. (B, C) Sensitivity of PEO1/4 cells and PEO1/4ip cells to olaparib and paclitaxel. Cells were treated with various concentrations of olaparib or paclitaxel for 72 hr. MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SE. (D, E) SCID-Beige mice were inoculated i.p. with PEO1ip or PEO4ip cells. After 3 days, mice were randomly assigned to 2 groups (n = 4) and treated i.p. with vehicle and olaparib (50 mg/kg) daily for 6 weeks (day 3 to 45). The body condition score (BCS) of mice bearing PEO1ip xenografts was monitored and the abdominal circumference of mice bearing PEO4ip xenografts was measured every 2–3 days to determine the endpoint and the Kaplan-Meier survival curve. p values were determined by the Mantel-Cox test compared with the control.
Fig 4.
The effects of olaparib, triapine, and cediranib combinations on peritoneal progression of PEO4ip xenografts and the survival time of mice.
SCID-Beige mice were inoculated i.p. with PEO4ip cells. After 3 days, mice were randomly assigned to 8 groups (n = 5) and treated i.p. with vehicle, single, double, and triple combinations consisting of cediranib (0.75 mg/kg), olaparib (50 mg/kg), and triapine (10 mg/kg) daily for two 3-week periods with a 2-week treatment-free period in between (day 3 to 24 and day 38 to 59). The abdominal circumference was measured and the body condition was monitored every 2–3 days. (A, B) Ascitic fluid smears on ThinPrep slides showed three-dimensional malignant cell clusters with papillary arrangement. (C) High-power microscopic view showed cells with cytological features consistent with ovarian high-grade serous carcinoma. (D) Peritoneal progression of PEO4ip xenografts leading to ascitic development was determined and expressed as a percent increase in abdominal circumference at day 62. Data are means ± SE. p values were determined by the one-way ANOVA with the Dunnett’s multiple comparison test compared with the control. Tumor-free control is the group of mice (n = 2) without implantation of PEO4ip cells. (E, F) The Kaplan-Meier survival curve was determined using a 50% increase in abdominal circumference and BCS2 as the endpoint. p values were determined by the Mantel-Cox test compared with the control. Olap, olaparib; Ced, cediranib; Triap, triapine.
Fig 5.
The effects of olaparib, triapine, and cediranib combinations at different dose levels on peritoneal progression of PEO4ip xenografts and the survival time of mice.
SCID-Beige mice were inoculated i.p. with PEO4ip cells. After 1 day, mice were randomly assigned to 4 groups (n = 5) and treated i.p. with vehicle, the olaparib (135 mg/kg)-cediranib (5 mg/kg) combination, and the same combination with triapine at 1.7 mg/kg or 3.3 mg/kg daily for a continuous 5-week period (day 1 to 36). The abdominal circumference was measured and the body condition was monitored every 2–3 days. (A) The representative appearance of mice from control (right) and triapine plus olaparib plus cediranib (left) groups at day 62 are shown. (B) The Kaplan-Meier survival curve was determined using a 50% increase in abdominal circumference and the attainment of BCS2 as the endpoint. p values were determined by the Mantel-Cox test compared with the control and between treatment groups. (C) The body weight of mice was measured every 2–3 days. The means of body weight for treatment groups are shown. Olap, olaparib; Ced, cediranib; Triap, triapine.
Table 1.
Dose equivalency between human and mouse.
Fig 6.
The effects of cediranib on AKT signaling and the sensitivity of BRCA2-wild type and mutated EOC cells to olaparib and triapine in vitro.
PEO1 and PEO4 cells were treated with 1.25 μM cediranib, 0.75 μM triapine, or both drugs for 1 hr and then treated with various concentrations of olaparib for 72 hr. (A) MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SD. (B) EOB was calculated to determine the effects of the combinations of cediranib, triapine, and olaparib on cell survival at all data points. EOB < 0 indicates antagonism. EOB = 0 indicates additivity. EOB > 0 indicates synergism. (C) PEO1 and PEO4 cells were treated with cediranib at 1.25, 2.5, and 5 μM for 24 hr. Western blot analysis was performed to determine the protein levels of phospho-AKT (Ser473), AKT, mTOR, phospho-S6 (Ser235/236), and S6. (D) PEO1 and PEO4 cells were serum-starved and then treated with 50 μM olaparib in the absence and presence of 5 μM cediranib for indicated time periods. Western blot analysis was performed to determine the protein levels of phospho-AKT (Ser473), phospho-FoxO1 (Ser248), and HSC70.
Fig 7.
Effects of cediranib on olaparib-induced apoptosis and cell cycle progression in EOC cells.
(A) PEO1 and PEO4 cells were pre-treated with 0, 2.5, and 5 μM cediranib for 1 hr and then treated with 0, 5, and 10 μM olaparib in the continuous presence of cediranib. After 24 and 48 hr of incubation, cells were lysed, and native protein was obtained to determine caspase 3/7 activity. The protein concentration of lysates was also determined to normalize the caspase 3/7 activity in each sample. The level of apoptosis is expressed as the fold change in the normalized caspase 3/7 activity with respect to the vehicle-treated control in each cell line. (B) PEO1 and PEO4 cells were treated with 0, 2.5, and 5 μM cediranib for 24 hr. Western blot analysis was performed to determine the protein levels of phospho-Rb (Ser780), cyclin A, p27 Kip1, and HSC70. (C) PEO1 and PEO4 cells were treated with 5 μM cediranib for 24 and 48 hr. Cells were pulse-treated with 10 μM EdU for 1 hr prior to flow cytometric analysis. G1, S, G2 populations were gated to show the percentage of cells in each phase. Data are means ± SD. *, p < 0.01; #, p < 0.05, compared with the 0 hr controls. (D) The AKT signaling pathway leads to blockade of the FoxO1-p27 Kip1-mediated apoptosis and G1 arrest. Cediranib inhibits olaparib-induced AKT activity, thereby enhancing the efficacy of olaparib (and triapine) against EOC. Olaparib also induces apoptosis possibly through FoxO1-p27 Kip1 and unknown mechanisms that remain to be determined.