Figure 1.
Phosphorylation and mutational status of genes that encode components of the RTK/Ras/PI3K pathway.
Nine ovarian clear cell adenocarcinoma (OCCA) and a control (Cntl) cell line (immortalized epithelial cells from ovarian endometrioma) were lysed in cell lysis buffer and analyzed by western blotting. In general, most of the OCCA cell lines displayed higher levels of phosphorylation of Akt (Thr308) and its downstream targets (GSK3β, FOXO 1/3a, 4EBP1 and S6) than the respective levels of phosphorylation in the control line. The abundances and levels of phosphorylation of c-MET (Tyr1234/1235), HER2 (Tyr1221/1222), and HER3 (Tyr1289) were also evaluated. The mutational status of PIK3CA, PTEN, and K-Ras is shown for each cell line.
Figure 2.
Inhibition of cell proliferation by DS-7423 and rapamycin.
(A) Cell viability for each cell line was analyzed using the methyl thiazolyl tetrazolium (MTT) assay 72 h after treatment with DS-7423 or rapamycin at the doses indicated. The data were normalized relative to the value of the control cells. In all nine cell lines, DS-7423 suppressed cell proliferation more robustly than rapamycin when both were used at higher doses. (B) IC50 values for DS-7423 in seven ovarian serous adenocarcinoma (OSA) cell lines (left) were compared with those of nine OCCA cells (right). Four of seven OSA cells had IC50 values >100 nM, which is higher than that of any OCCA cells.
Figure 3.
Inhibition of PI3K/mTOR signaling by DS-7423 and rapamycin in ovarian clear cell adenocarcinoma cell lines.
(A) Immunoblotting of total protein extracts from OCCA cells (OVISE and OVMANA) treated with DS-7423 at concentrations ranging from 0 to 2,500 nM. (B) Immunoblotting of total protein extracts from OVISE cells treated with rapamycin at concentrations ranging from 0 to 2,500 nM.
Figure 4.
Flow cytometric analysis of the cell cycle in cancer cells treated with DS-7423, and in vivo demonstration of the anti-tumor effect of DS-7423 in nude mice.
(A) Cells (5×105) were seeded in the presence of 10% serum and treated with DS-7423 for 48 h at doses of 9.8 nM, 256 nM, or 2,500 nM. DS-7423 blocked OCCA cell cycle progression into the S phase in a dose-dependent manner. The relative size of the sub-G1 population was increased in six of the cell lines (left) but was not affected in the remaining three cell lines (right). (B) Subcutaneous xenograft tumors in athymic BALB/c mice were established following the injection of OCCA cells of either the TOV-21G (left) or RMG-I (right) cell lines. Mice were treated daily (5–7 days per week) at the indicated doses of DS-7423 (1.5, 3, or 6 mg/kg, 8–10 days). Each treatment group contained five mice. Estimated tumor volumes (upper graphs) and body weight losses (BWL) (lower graphs) were shown in the two OCCA cells. Tumor volumes were calculated by the formula {(major axis)*(minor axis)2/2} mm3. Groups were compared at the end of treatment. Points, mean; bars, standard deviation (SD); *p<0.05.
Figure 5.
DS-7423–mediated induction of apoptosis in ovarian clear cell adenocarcinoma cell lines.
(A) All nine OCCA cells were treated with DS-7423 at 156 or 2,560 nM for 48 h, and apoptotic cell proportion was evaluated using annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining, followed by analysis using flow cytometry. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± standard deviation (SD). (B) The apoptotic cells were calculated using flow cytometry by counting the cell population in the right boxes. The example shown (OVISE cells) is representative of the results obtained for all the cell lines tested. (C) The proportion of cells rendered apoptotic by exposure to DS-7423 at 156 nM and 2,560 nM was significantly higher in OCCA cells without mutations in TP53 than in OCCA cells that carry mutations in TP53. (D) Cleaved poly(ADP-ribose) polymerase (PARP) induction was evaluated by immunoblotting in OVISE cells. OVISE cells were treated with DS-7423 at 156 nM for the times indicated (left) or for 4 h at the doses indicated (right).
Figure 6.
Induction of the phosphorylation of TP53 at Ser46 and the accumulation of transcripts of the genes targeted by TP53, which participate in TP53-mediated apoptosis.
(A) Immunoblotting in OVMANA cells treated with DS-7423 at the indicated doses. Phosphorylation levels of MDM2 were inversely associated with p-TP53 at Ser46, but not with p-TP53 at Ser15. (B) Semi-quantitative RT-PCR in OVMANA and OVISE cells treated with DS-7423 at the indicated doses. Both p53AIP1 and PUMA were induced by DS-7423. CT, untreated (negative) control; IR, irradiation at 14 Gy (positive control). GADD45 was induced in OVISE, but not in OVMANA cells. (C) Quantification of the semi-quantitative RT-PCR in (B). Each experiment was repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (D) Effect of TP53 knockdown on apoptosis induction by DS-7423. TP53 was knocked down by two independent siRNAs specific to TP53 (siRNA1 and 2) in OVISE cells, which do not carry any mutation in TP53. The apoptotic cell population was evaluated using annexin-V staining, as described in Figure 5. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (E) Suppression of TP53 expression by siRNAs was confirmed by immunoblotting. (F) Effect of TP53 knockdown on cell proliferation by DS-7423 in MTT assay of OVISE cells. TP53 was knocked down by a siRNA1 specific to TP53 and MTT assay was subsequently performed as in Figure 2. Knockdown of TP53 diminished the anti-proliferative effect caused by DS-7423 on OVISE cells. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05 (G) TP53 expression plasmid (0.1 µg/µL) was cotransfected with pp53 TA Luc (0.25 µg/mL) plasmid into ES-2 cells mutated in TP53. The addition of DS-7423 increased the relative luciferase activity of TP53 in a dose-dependent manner. The experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± SD. *p<0.05.