Akt targeting promotes the radiosensitizing effect of rapamycin in <i>non-responder</i> cells.

<p><b>(A)</b> Cells were treated with MK2206 (A549: 5 μM; H460: 2.5 μM) for 1 h, followed by treatment with rapamycin (100 nM) for 2 h. Control cells received the appropriate concentrations of DMSO. Thereafter, P-Akt (S473, T308) and P-Akt2 (S474) levels were analyzed in protein samples by Western blotting. Blots were stripped and incubated with anti-Akt1 or anti-Akt2 antibodies. The densitometry data represent the mean ratio of phosphorylated Akt1 to Akt1 based on 2 biologically independent experiments; the control conditions were normalized to 1. The densitometry values for phospho-Akt2 represent the mean ratio of phospho-S474 to Akt2 normalized to 1 for the control condition. For colony formation assays, 24 h after pre-plating, cells were treated with MK2206 (A549: 5 μM; H460: 2.5 μM) for 1 h, followed by treatment with rapamycin (100 nM) for 2 h. Control cells received the appropriate concentrations of DMSO. The cultures were irradiated after rapamycin treatment and incubated for colony growth. Data represent the mean SF ± SD of 18 data from three biologically independent experiments in A549 cells and of 12 data from two biologically independent experiments in H460 cells. In <i>non-responder</i> A549 cells, asterisks indicate a significant difference between the radiosensitizing effect produced by the combination of MK2206 and rapamycin compared to the effects of single treatment with rapamycin alone (**, P < 0.01; ***, P < 0.001). <b>(B)</b> Cells were plated in culture dishes and were transfected with non-target-siRNA (siCON) or AKT1-siRNA (siAKT1) after 24 hours at concentrations of 50 nM (A549) and 150 nM (H460). At the indicated time points after transfection, Akt1 knockdown efficiency was tested by Western blotting. GAPDH was used as a loading control. Densitometry values represent the mean ratio of Akt1 to GAPDH from three independent experiments; the control conditions were normalized to 1. In parallel, 2 days after transfection, cells were trypsinized and seeded for clonogenic assays. Twenty-four hours later, cells were treated with rapamycin (100 nM) for 2 hours, followed by irradiation with single doses of 0 to 4 Gy. The clonogenic assay was performed as described in <i>Materials and Methods</i>. Data represent the mean SF ± SD of four biologically independent experiments (21 data) in A549 cells and 6 parallel experiments in H460 cells. In <i>non-responder</i> A549 cells, asterisks indicate a significant difference between the radiosensitizing effect produced by the combination of AKT1-siRNA with rapamycin compared to the effects of single treatment with rapamycin alone (***, P < 0.001). DMF: Dose modification factor.