10 Two weeks after tumor cell injection, animals with established tumors were randomized to three groups (n = 3) that received daily i.p. injections of FTY720 or OSU-2S at 5 mg/kg, or vehicle for 42 days and were imaged weekly. All animal use was done in accordance with protocols approved by The Ohio State University Institutional Animal Care and ABT-199 order Use Committee. A tissue microarray (TMA) containing both HCC and non-neoplastic liver tissues was constructed from archival paraffin-embedded tissue samples as described.15 The TMA was immunostained for PKCδ and expression

was evaluated in 163 HCC and 71 non-neoplastic liver samples using a semiquantitative scoring system (0 = negative; 1 = weak; 2 = moderate; 3 = strong). Differences among group means were analyzed for statistical significance using one-way analysis of variance followed MDV3100 supplier by the Neuman-Keuls test for multiple comparisons. Differences in the proportions of PKCδ-positive HCC and non-neoplastic liver samples were analyzed by Fisher’s exact test. Differences were considered significant at P < 0.05. Analysis was performed using GraphPad InStat for Windows (GraphPad Software, San Diego, CA). Based on our finding that the antitumor effect of FTY720 in HCC cells was mediated through PKCδ activation, we hypothesized that these two pharmacological activities, i.e., immunosuppression and anti-proliferation, could be

dissociated via structural modifications. Thus, FTY720 was used as scaffold to establish a small focused compound library for lead identification. Among more than 20 derivatives examined, OSU-2S (Fig. 1A) lacked immunomodulatory activity, yet exhibited higher potency than FTY720 in inducing apoptotic death in HCC cells, thereby providing a proof-of-concept of our hypothesis. FTY720 acts as a prodrug that undergoes

SphK2-catalyzed phosphorylation to mediate its immunomodulatory function.16 Radiometric analysis, followed by TLC, revealed that, in contrast Ribonucleotide reductase to FTY720, OSU-2S was not phosphorylated by recombinant SphK2 (Fig. 1B). Although not a SphK2 substrate, OSU-2S’s phosphate derivative, p-OSU-2S, was synthesized to test its ability vis-à-vis p-FTY720, FTY720, and OSU-2S to facilitate the internalization of S1P receptors, a key mechanism underlying FTY720-mediated immunomodulation,17 in S1P1 receptor-overexpressing Huh7 cells. Immunocytochemical analysis indicated a profound effect of FTY720 and p-FTY720 on receptor internalization and clustering in the cytoplasm, whereas OSU-2S or p-OSU-2S failed to show any appreciable effect (Fig. 1C). To confirm these results, the effects of these agents on T-lymphocyte homing in vivo were evaluated in CD2F1 mice. Treatment with FTY720 for 6 hours, even at 1 mg/kg, caused a precipitous drop (≥75%) in the number of T lymphocytes in peripheral blood, whereas OSU-2S exerted no appreciable effect at 1 and 2 mg/kg and a modest decrease at 5 mg/kg (Fig. 1D).