Perifosine induces JNK/caspase-dependent MM cell apoptosis. (A) MM.1S and RPMI8226 cells were cultured with perifosine (5 and 10 μM) for 24 hours. Cells were then subjected to cell-cycle profiling by PI staining and flow cytometry. Percentage indicates sub-G1 phase cells. (B) MM.1S cells were cultured with perifosine (5 and 10 μM) for 8 hours. (C) MM.1S cells were also cultured with perifosine (10 μM) for 6 and 12 hours. Cells were then lysed and subjected to Western blotting using caspase-8, caspase-9, and PARP Abs. (C) MM.1S cells were cultured for 24 hours with perifosine (5 and 7.5 μM) in the presence of control media (□), and with 25 μM of Z-IETD-FMK (▧), Z-LEHD-FMK (▧), or Z-VAD-FMK (▪). (D) MM.1S cells were cultured with perifosine (10 μM) for the indicated periods. Cells were then lysed and subjected to Western blotting using anti–p-p38 MAPK, anti-p38 MAPK, anti–p-JNK, anti-JNK, and α-tubulin Abs. (E) MM.1S cells were cultured with perifosine (5 μM and 10 μM) for 8 hours, in the presence or absence of SP600125 (10 μM). Cells were then lysed and subjected to Western blotting using anti–p-JNK, anti-JNK, and caspase-8 Abs. (F) MM.1S cells were cultured for 24 hours with control media (□), or with 2.5 μM (▦), 5 μM (▦), and 7.5 μM(▪) perifosine, in the presence or absence of SP600126 (10 μM). (G) MM.1S cells were transiently transfected with control (•) or JNK2 siRNA expression plasmid (▪). Whole-cell lysates were subjected to Western blotting using anti-JNK2 and α-tubulin Abs. (H) MM.1S cells were cultured for 24 hours with control media (□) or perifosine (5 and 7.5 μM) in the presence or absence of control media (□), 200 nM (▦), or 400 nM (▪) SCIO469. Cytotoxicity was assessed by MTT assay; data represent mean (± SD) of quadruplicate cultures.
