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Selumetinib suppresses AKT phosphorylation in AZD8055-treated BRAF mutant cells.

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posted on 2012-07-10, 00:34 authored by Alan L. Ho, Elgilda Musi, Grazia Ambrosini, Jayasree S. Nair, Shyamprasad Deraje Vasudeva, Elisa de Stanchina, Gary K. Schwartz

A, uveal melanoma cell lines produced distinct biochemical responses to AZD8055 and selumetinib exposure. Cells were treated with the indicated drugs or vehicle (denoted by “-”) for 24 hours and Western blots were then performed. Total AKT was used as a loading control. Of note, selumetinib alone inhibited S6K1 phosphorylation in BRAF cells and to a lesser extent in GNAQ cells (lane 1 versus 3 in Blot #2). Given how effectively AZD8055 inhibited S6K1 phosphorylation, though, it is unlikely that this selumetinib effect significantly contributed to the impact of the combination in BRAF cells. B, IGF-1R phosphorylation increased with AZD8055 treatment and was suppressed by selumetinib in the BRAF mutant cell line OCM1A. OCM1A cells were treated as described in A, and cellular lysates were created and analyzed with phosphorylated receptor tyrosine kinase (RTK) antibody array blots. The blots reflect the phosphorylation status of 42 RTKs. Duplicate spots in the corners of each blot are positive controls. IGF-1R duplicate spots are circled. C, inhibition of IGF-1R blocked AKT phosphorylation in OCM1A cells, but did not induce PARP cleavage in combination with AZD8055. Cells were treated with the same drugs and concentrations as detailed in A in addition to the IGF-1R small molecule inhibitor NVP-AEW541 at 1000 nM. Cells were treated for 24 hours before Western blots were performed. Ku70 was used as a loading control. D, IGF-1R inhibition with NVP-AEW541 failed to induce apoptosis in combination with selumetinib in OCM1A cells. Cells were treated with drugs for 48 hours and analyzed by flow cytometry for DNA content. The percentages of sub-G1 cells were quantified. Results are the mean of two independent experiments. Error bars, SE.


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