Ovarian cancer tissue microarrays were stained with anti-phospho-mTOR antibody. A, representative photographs of ovarian tissue microarray cores of (i) clear cell carcinoma (CCC), and (ii) serous adenocarcinoma (SAC). Magnifications: ×100, and ×400 (inset). B, phospho-mTOR immunoreactivity was scored semiquantitatively from zero to strong, as described in “Materials and Methods.” Representative photos of weak, moderate, and strong staining are shown. Magnifications: ×100, and ×400 (inset). C, histogram indicating immunostaining profile. Proportion indicates proportion of tumors examined. Phospho-mTOR expression was more frequent and stronger in intensity in CCCs than in SACs. D, histogram indicating the expression of phospho-mTOR according to clinical stage.

A, analysis of phospho-mTOR status in four ovarian CCC cell lines. CCC cells were serum-starved overnight, after which the expression of phospho-mTOR was determined by Western blot analysis. Actin expression was used as a loading control. B, sensitivity of CCC cells to RAD001. CCC cells were treated with the indicated concentrations of RAD001 in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (*, significantly different from control; p<0.01). C, RAD001 attenuates the phosphorylation of p70S6K in vitro. KOC7C and RMG1 cells were treated with 10 nM of RAD001 for 6 h in thepresence of 5% FBS. Cells were harvested, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with anti-phospho-p70S6K (Thr³⁸⁹), anti-p70S6K, anti-phospho-AKT (Ser⁴⁷³), anti-AKT, or anti-b-actin antibodies. Blots were scanned and quantified by densitometry. The density of phospho-p70S6K or phospho-AKT was normalized to the corresponding density of bands for total p70S6K or AKT, respectively, and the results are depicted as “Relative P-AKT” or “Relative P-TOR” values. D.(i), RAD001 induced an increase in the percentage of cells in G1 phase. RMG1 and KOC7C cells were treated with or without 10 nM RAD001 for 48 h. Cell cycle analyses were performed with a fluorescence activated cell sorter (FACS)can as described in the “Materials and Methods.” (ii, iii), RAD001 does not induce autophagy. KOC7C and RMG1 cells were treated with 10 nM of RAD001 for 48 h in the presence of 5% FBS. (i) Cells were harvested, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with anti-LC3B or anti-β-actin antibodies. (ii), Cells were fixed, stained with anti-LC3B antibodies, and viewed with a fluorescence microscope.

Athymic nude mice were inoculated s.c. with KOC7C cells or RMG1 cells. When the tumors reached an average size of about 50 mm³, mice were treated with placebo or 2.5 mg/kg RAD001 twice a week for 4 wks. A and C, appearance of subcutaneous tumors.B and D, graphs depicting weekly tumor volumes (mm³) for each treatment group. Points, mean; bars, SD. *, p< 0.05, significantly different from placebo-treated mice. **, p< 0.01, significantly different from placebo-treated mice.

A and B, establishment of cisplatin-resistant variant cell lines. Cisplatin-resistant sublines were established as described in “Materials and Methods.” A, cisplatin-sensitive parental (KOC7C and RMG1) and cisplatin-resistant variant (KOC7C-CR and RMG1-CR) cells were treated with the indicated concentrations of cisplatin in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (*, p< 0.05, **, p<0.01). B, effect of cisplatin on the cleavage of PARP in cisplatin-sensitive parental and cisplatin-resistant variant cell lines. KOC7C, KOC7C-CR, RMG1 and RMG1-CR treated with 10 μM cisplatin or 10 nM RAD001 for 24 h. Cells were harvested, and then lysates were subjected to Western blotting using anti-PARP or anti-b-actin antibody. C, activation of AKT/mTOR signaling in cisplatin-sensitive parental and cisplatin-resistant variant cells in vitro. KOC7C, KOC7C-CR, RMG1 and RMG1-CR cells were serum starved overnight. Cells were harvested, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with anti-phospho-mTOR (Ser²⁴⁴⁸), anti-mTOR, anti-phospho-AKT (Ser⁴⁷³), anti-AKT or anti-b-actin antibodies. AKT, phosphor-AKT and β-actin are from the same membrane and 8% gel, mTOR and phosphor-mTOR were from a separate 6% gel.Blots were scanned and quantified by densitometry. The density of phospho-AKT or phospho-mTOR bands was normalized to the corresponding density of total AKT or mTOR bands, respectively, and the results are shown as “Relative P-AKT” or “Relative P-TOR” values. D, enhanced sensitivity to RAD001 in cisplatin-resistant CCC cells in vitro. Cisplatin-sensitive parental (KOC7C and RMG1) and cisplatin-resistant variant (KOC7C-CR and RMG1-CR) cells were treated with the indicated concentrations of RAD001 in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (**, p<0.01). E, cisplatin-resistant variant (KOC7C-CR and RMG1-CR) cells were treated with the indicated concentrations of cisplatin in the presence or absence of 10 nM of RAD001 for 72 h. Cell viability was assessed by MTS assay.

Athymic nude mice were inoculated s.c. with KOC7C-CR cells or RMG1-CR cells. When the tumors reached an average size of about 50 mm³, mice were treated with placebo or 2.5 mg/kg RAD001 twice a week for 4 wks. A and C, appearance of subcutaneous tumors. B and D, graphs depicting weekly tumor volumes (mm³) for each treatment group. Points, mean; bars, SD. **, p< 0.01, significantly different from placebo-treated mice.