Figure 6.

Biological effects of MEK inhibitors in combination with PI3K inhibitors and camptothecin. A, FACS-based analysis was used to determine percentage of MDAMB231 cells in G₁, S, and G₂ phases of the cell cycle at 72 h after treatment with the single drugs and with their combinations at indicated doses. Whereas PI3K inhibitors PIK90 and PI103 do not affect cell cycle distribution, their addition to MEK inhibitors CI1040 or U0126 results in complete G₁ arrest. Columns, mean of duplicates; bars, SD. For each duplicate, 40,000 cells were acquired. The result was confirmed in two independent experiments. B, Representative Western blot analysis of MDAMB231 cells treated with the indicated drugs and their combinations for 4 or 24 h. The cells were treated with drugs in full serum conditions and harvested at 4 and 24 h after drug addition for protein and RNA isolation. C, cyclin D1 gene expression changes induced by the same drug treatments as in panel B: 1, control; 2, CI1040 (5 µmol/L); 3, PIK90 (5 µmol/L); 4, PI103 (0.5 µmol/L); 5, CI1040 + PI103; 6, CI1040 + PIK90; 7, CI1040 + PIK90 + rapamycin; 8, PIK90 + rapamycin; 9, rapamycin (5 nmol/L). Gene expression was analyzed by Taqman assay; the results are averaged from duplicates. Relative cyclin D1 expression is presented as the percentage of that of control (untreated) cells. D, G₁ arrest induced by MEK inhibition rescues the cells from apoptosis induced by cytotoxic agent. MDAMB231 cells were treated with camptothecin (2.5 µmol/L) for 48 h, which resulted in 20% of apoptotic cells. Pretreatment of cells with CI1040 for 1 h reduced the amount of apoptotic cells, whereas pretreatment of cells for 24 h completely eliminated apoptosis induced by camptothecin. Columns, mean of duplicates; bars, SD. For each duplicate, 40,000 cells were acquired. The results were confirmed in two independent experiments.