SAHA diminished Akt/mTOR/eIF4E-BP/eIF4E signal pathway, and a PI3K inhibitor, LY294002, decreased cyclin D1 protein levels in MCL cells. (A) Jeko1 cells were cultured with SAHA (5 μM) for the indicated times. Levels of phospho-Akt, Akt, phospho-mTOR, phospho-eIF4E-BP, eIF4E-BP, eIF4E, and active eIF4E (bottom; see “Materials and methods, Immunoprecipitation and Western blot analysis”) were detected by Western blot analysis. (B) 2 MCL lines (Jeko1 and SP49) were treated with the PI3K inhibitor, LY294002 (50 μM) for 4 hours. Levels of cyclin D1 were determined by Western blot analysis. β-Actin was used as an internal control. (C) Jeko 1 cells were treated with the mTOR inhibitor RAD001 (200 nM) for 4 hours. Levels of cyclin D1, Akt, phospho-Akt, eIF4E-BP, and phospho-eIF4E-BP were detected by Western blot analysis. (D) 2 additional MCL lines (SP49, SP53) and a prostate cancer cell line PC3, in which Akt/mTOR/eIF4BP pathway is active because the PI3K inhibitory protein (PTEN) is deleted, were treated with SAHA (5 μM, 7 hours). Phosphorylation status of Akt and eIF4E-BP as well as whole Akt and eIF4E-BP levels were examined by Western blot. SAHA treatment decreased phosphorylated Akt and eIF4E-BP, whereas total levels of these proteins were not affected.