Effects of alkylphospholipids on PEL. (A) Inhibition of PEL cell proliferation induced by miltefosine (right panel) and perifosine (left panel) as measured by MTS assay. Shown in each panel is the absorbance at 490 nm (y-axis) in the absence of drug (gray circles) or increasing, indicated doses of either miltefosine or perifosine, ranging from 10μM to 50μM. Treatment time is represented on the x-axis. Each data point is the average of triplicate or quadruplicate measurements. Error bars represent the SD and, in most cases, are smaller than the symbol. (B) Immunoblot analysis of extracts harvested from indicated PEL cell lines treated with DMSO (vehicle), the indicated drug, or untreated cells (−). Membranes were probed with antibodies raised specifically against the phosphorylated forms of FOXO1, GSK3β, mTOR, S6K, or S6 proteins. Membranes were probed with anti-actin antibody, as a loading control. (C) Tumor progression is delayed in miltefosine-treated mice and significantly delayed in perifosine-treated mice. Mice were treated with 50 mg/kg miltefosine (n = 5), perifosine (n = 5), or vehicle (n = 5) by intraperitoneal injection and followed for 20 days after formation of palpable tumors. Error bars represent the SD for each group of animals. (D) Tumors excised from miltefosine- and perifosine-treated mice display decreased phosphorylation of the ribosomal S6 protein, compared with vehicle controls. Staining is not observed in sections not incubated with primary antibody. Original magnification ×400.