Figure 4.

Inhibition of ERK signaling augments Perifosine-induced cytotoxicity. (A) MM.1S and MM.1R cells were cultured with perifosine (5 μM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-MEK, anti–p-ERK, and anti-ERK1/2 Abs. (B-C) MM.1S (B) and MM.1R (C) cells were cultured for 14 hours with control media and with 2.5 μM, 5 μM, and 7.5 μM perifosine in the absence (□) or presence of 5 μM (light blue bars) or 10 μM (dark blue bars) MEK1/2 inhibitor U0126. Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (D) MM.1S cells were cultured for 8 hours with control media, perifosine (5 μM), U0126 (5 μM), or perifosine (5 μM) plus U0126 (5 μM). Cells were then lysed and subjected to Western blotting using anti–p-Akt, anti-Akt, anti–p-ERK, anti-ERK1/2, anti–p-JNK, caspase-8, and PARP Abs. (E) Freshly isolated tumor cells from MM patients (n = 2) were cultured for 24 hours with control media (□) and with 5 μM () or 10 μM(▪) perifosine in the presence or absence of U0126 (5 μM). Cytotoxicity was assessed by MTT assay; data represent means (± SD) of quadruplicate cultures. (F) MM.1S cells were cultured for 48 hours with control media (□) and with 1.25 μM (light orange bars), 2.5 μM (medium orange bars), or 5 μM (dark orange bars) perifosine with or without U0126 (2.5 μM) and in the presence or absence of BMSCs. Cell proliferation was assessed by [³H]-thymidine uptake; data represent means (± SD) of quadruplicate cultures.