1 Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2 Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time-and concentration-dependent manner. FTY720 also induced Bad (Ser 136 ) and ribosomal p70S6 kinase (p70 S6k ) (Thr 389 ) dephosphorylation. 3 FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3 0 -kinase (PI 3-kinase) pathway inhibition. 4 FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5 Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70 S6k , suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6 OA partially inhibited FTY720-induced caspase-3 activation. 7 PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8 Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria. British Journal of Pharmacology (2003) 138, 1303 -1312. doi:10.1038/sj.bjp.0705182 Keywords: FTY720; Akt; PKB; P70 S6k ; Bad; PP2A; apoptosis; leukemia cells Abbreviations: CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; OA, okadaic acid; p70 S6k , ribosomal p70 S6 kinase; PDGF, platelet-derived growth factor; PDK, phosphoinositide-dependent kinase; PDK1, phosphoinositide-dependent kinase 1; phospho-, phosphorylated; PI(3,4)P 2 , phosphatidylinositol 3,4-diphosphate; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-triphosphate; PI 3-kinase, phosphatidylinositol 3 0 -kinase; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PP, protein phosphatase; pRb, retinoblastoma protein; SGK, serum and glucocorticoid-inducible kinase; DC m , mitochondrial membrane potential

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FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) α. We compared the effects of vanadate to PFTα and PFTµ, an inhibitor of transcriptionindependent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcriptionindependent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. Cancer Res; 70(1); 257-65. ©2010 AACR.