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. Author manuscript; available in PMC: 2010 Sep 1.

Published in final edited form as: Clin Cancer Res. 2009 Aug 18;15(17):5404–5413. doi: 10.1158/1078-0432.CCR-09-0365

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.

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.