However, in addition to side effects common to immunomodulatory therapy, FTY720 was reported to cause cardiovascular complications, macular edema, and brain inflammation,4 presumably the result of interactions with more than one S1P-receptor subtype.8 Previously, we demonstrated that FTY720 induces learn more apoptosis in HCC cells through the reactive oxygen species (ROS)-dependent activation of protein kinase C (PKC)δ.7 Dissociation of the apoptosis-inducing activity of FTY720 from its S1P receptor agonist activity provides a basis for its pharmacological

exploitation to develop a novel class of antitumor agents. Here, we report the development of a nonimmunosuppressive FTY720 analogue, OSU-2S [(S)-2-amino-2-(4-[(6-methylheptyl)-oxy]phenethyl)pentan-1-ol], which exhibits higher in vitro and in vivo potency than FTY720 in suppressing HCC cell

growth through PKCδ signaling. CA-Akt, constitutively active Akt; DAPI, 4,6-diamidino-2-phenylindole; DCFDA (5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate); DMEM, Dulbecco’s modified Eagle medium; DMS, N,N-dimethylsphingosine; PD-0332991 ic50 DPI, diphenyleneiodonium; FBS, fetal bovine serum; GST-π, glutathione S-transferase-π; HA, hemagglutinin; HCC, hepatocellular carcinoma; Hep3B-luc, luciferase-expressing Hep3B; i.p., intraperitoneal; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; PARP, poly (ADP-ribose) polymerase; p-FTY720, phosphorylated FTY720; PKCδ, protein kinase Cδ; PP2A, protein phosphatase 2A; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; siRNA, small interfering RNA; shRNA, short hairpin RNA; SphK2, sphingosine kinase 2; TLC, thin-layer chromatography; TMA, tissue Cytidine deaminase microarray. Details about reagents, their commercial sources, and experimental procedures are provided in the Supporting Information. The HCC cell lines, Hep3B, PLC5 and Huh7, and primary nonmalignant human hepatocytes were used in this study. FTY720 was synthesized as described,9 and p-FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Synthesis of OSU-2S and phosphorylated OSU-2S (p-OSU-2S) will be described elsewhere. Various polyclonal and monoclonal antibodies were used for western

blotting, immunocytochemical, and flow cytometric analyses. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays as previously reported.10 For assessment of apoptosis, treated cells were stained with Annexin V-Alexa Fluor 488 and propidium iodide according to the vendor’s protocols. For caspase-3 activity, cells were incubated with the fluorogenic caspase-3 substrate (Ac-DMQD)2-Rh110 for 20 minutes. ROS production was detected using the fluorescence probe 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) as described.7 Data were analyzed by ModFitLT V3.0 software program. Immunoblotting of biomarkers in cell lysates and tumor tissue homogenates was performed as reported.