Perifosine inhibits Akt phosphorylation and induces cytotoxicity in MM cells. (A) Baseline phosphorylation of Akt, STAT3, and ERK in MM cell lines and tumor cells from MM patients assessed by Western blotting. Lane 1 indicates MM.1S; 2, MM.1R; 3, U266; 4, INA-6; 5, RPMI8226; 6, LR5; 7, Dox40; 8, OPM1; 9, OPM2; and 10 to 12, patient tumor cells. (B) MM.1S cells were cultured with perifosine (10 μM) for the indicated periods. Whole-cell lysates were subjected to Western blotting using anti–p-PDK1, anti–p-Akt, anti-Akt, anti–p-FKHRL1, anti–p-GSK3α/β, anti–p-MEK, anti–p-ERK, anti-ERK2, and α-tubulin Abs. (C) MM.1S cells were cultured with perifosine (1-10 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, and anti-ERK2 Abs. (D) MM.1S cells were cultured with perifosine (5 μM) for 6 hours prior to stimulation with IL-6 (20 ng/mL). Whole-cell lysates were immunoprecipitated with anti-Akt Ab. The immunoprecipitates were washed and subjected to in vitro kinase assay according to manufacturer's protocol. (E) MM.1S (▪), MM.1R (□), U266 (▴), INA-6 (▵), RPMI8226 (•), LR5 (○), Dox40 (♦), OPM1 (⋄), and OPM2 (*) cells were cultured with perifosine (1.5-100 μM) for 24 hours (i) and 48 hours (ii). (F) MM.1S cells stably transfected with Myr-Akt (▪) and control construct (•) were cultured with perifosine (0.6-10 μM) for 24 hours. Whole-cell lysates from control and Myr-Akt–transfected cells were subjected to Western blotting using anti–p-Akt and Akt Abs. (G-H) Freshly isolated tumor cells from 4 patients with MM (G) and PBMCs from 3 healthy donors (H) were cultured with increasing doses of perifosine for 48 hours. Cytotoxicity was assessed by MTT assay (E-H); data represent mean (± SD) of quadruplicate cultures.