Simvastatin treatment downregulates Akt phosphorylation and Akt kinase activity and induces apoptosis in LNCaP cells. (A) Cells were incubated with varying doses of simvastatin (sim) in the absence or presence of cholesterol complexes for 16 hours. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to total Akt or S473-phosphorylated Akt (S473-P). (B) LNCaP cells were incubated in the presence of 20 μM simvastatin or vehicle (control) in serum-free medium at 37°C for the indicated times, after which lysates were collected for Western blot and in vitro kinase assay. A GSK3 fusion protein was used as Akt substrate after immunoprecipitation with anti-Akt antibody. Kinase assay eluates were blotted with antibodies to total Akt, phospho-GSK3α/β (p-GSK3α/β), T308-P Akt, and S473-P Akt. (C) Cells in serum-free medium were treated with 20 μM simvastatin in the absence or presence of cholesterol (chol) complexes at 37°C for 12 hours, after which lysates were collected and kinase assay was performed as in B. (D) LNCaP cells were treated for varying times with 20 μM simvastatin. Apoptosis was determined by DNA fragmentation. The means ± SD of triplicate determinations are shown. (E) LNCaP cells were treated with 20 μM simvastatin with or without cholesterol complexes for 12 hours followed by DNA fragmentation analysis (*P < 0.05). (F) LNCaP cells were incubated with 20 μM simvastatin with or without cholesterol complexes for 12 hours, after which lysates were collected for immunoblot with the indicated antibodies. c-Caspase-7, cleaved caspase-7.