Takahisa Shinomiya

A novel immunosuppressant, FTY20, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allostransplantation

A new compound with an immunosuppressive property was purified from culture filtrates of Isaria sinclairii and was chemically modified to FTY720. Rat spleen cells incubated with FTY720 demonstrated features characteristic of apoptosis--such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies--by electron microscopy, and genemic DNA fragmentation by agarose gel electrophoresis. When FTY720 was administered in liver-allografted rats at a dose of 0.5 mg/kg from day 1 to day 14 after transplantation, the recipients survived significantly longer than the control group. Pretransplant treatment with 5 mg/kg of FTY720 one day before and on the day of grafting induced a remarkable prolongation of recipient survival, and three of 10 recipients survived for longer than 50 days. Furthermore, administration of FTY720 at 5 mg/kg on days 3 and day 4 after grafting also prolonged survival. In canine kidney allografting, a pretransplant 2-day course of FTY720 at 5 mg/kg prolonged graft survival. Daily administration of FTY720 in combination with CsA resulted in a significant prolongation of graft survival in a synergistic manner. In addition, FTY720 appeared to be nontoxic in canine recipients. These results demonstrated that FTY720, having a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.

A novel immunosuppressive agent FTY720 induced Akt dephosphorylation in leukemia cells

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. Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time‐ and concentration‐dependent manner. FTY720 also induced Bad (Ser136) and ribosomal p70S6 kinase (p70S6k) (Thr389) dephosphorylation. FTY720‐induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3′‐kinase (PI 3‐kinase) pathway inhibition. FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL‐1. BALL‐1 cells were resistant to FTY720‐induced apoptosis. Okadaic acid (OA) inhibited the FTY720‐induced dephosphorylation of Akt and p70S6k, suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. OA partially inhibited FTY720‐induced caspase‐3 activation. PP2A or PP2A‐like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 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.

Immunosuppressant FTY720 Induces Apoptosis by Direct Induction of Permeability Transition and Release of Cytochrome c from Mitochondria

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.

Evidence that FTY720 induces T cell apoptosis in vivo.

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyers patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyers patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyers patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.

T cell selective apoptosis by a novel immunosuppressant, FTY720, is closely regulated with Bcl‐2

A novel immunosuppressant FTY720 caused a significant decrease in peripheral T lymphocytes, but not in B lymphocytes upon oral administration. This decrease was mainly a result of FTY720‐induced apoptosis. In this study, we confirmed FTY720‐induced T cell selective apoptosis using lymphoma cell lines in vitro. Viability loss, DNA fragmentation, Annexin V binding, and caspases activation (caspase‐3, ‐8, and ‐9) were observed in Jurkat cells (T lymphoma cells), but not significantly in BALL‐1 cells (B lymphoma cells). These results indicated that FTY720 selectively induced apoptosis in T cell lymphoma to a greater extent than in B cell lymphoma, a finding that is similar to the result observed when FTY720 was treated with T lymphocytes and B lymphocytes in vitro. FTY720 released cytochrome c from mitochondria in Jurkat intact cells as well as from isolated Jurkat mitochondria directly, but not from mitochondria in BALL‐1 cells nor from isolated BALL‐1 mitochondria. BALL‐1 cells and B cells had more abundant mitochondria‐localized anti‐apoptotic protein Bcl‐2 than did Jurkat cells and T cells. FTY720‐induced apoptosis is inhibited by the overexpression of Bcl‐2, suggesting that the cellular Bcl‐2 level regulates the sensitivity to FTY720.

Coordinate Involvement of Cell Cycle Arrest and Apoptosis Strengthen the Effect of FTY720

A novel reagent, FTY720 (2‐amino‐2‐[2‐(4‐octylphenyl)ethyl]‐l,3‐propanediol hydrochloride), has been shown to induce a significant decrease of lymphocytes and lymphoma cells and is expected to be a potent immunosuppressant and anti‐tumor drug. The decrease in lymphocytes and lymphoma cells is mainly the result of FTY720‐induced apoptosis. FTY720 directly affects mitochondria and induces cell death. Moreover, FTY720 activates protein phosphatase (PP) 2A and affects anti‐apo‐ptotic intracellular signal transduction proteins to attenuate the anti‐apoptotic effect. In this study, we examined the relationship between FTY720‐induced apoptosis and cell cycle regulation. FTY720 induced apoptosis significantly at the GO/G1 phase and caused GO/G1 cell cycle arrest of the human lymphoma cell lines HL‐60RG and Jurkat. Simultaneously, retinoblastoma protein (pRB) was dephosphorylated, suggesting that dephosphorylation of pRB was related to FTY720‐induced GO/G1 cell cycle arrest. Because this dephosphorylation was completely blocked by a specific PP1/ 2A inhibitor, okadaic acid, it appears that FTY720‐activated PP2A is essential for FTY720‐induced cell cycle arrest. FTY720‐induced apoptosis was inhibited by Bcl‐2 overexpression in Jurkat cells, but this did not prevent FTY720‐induced cell cycle arrest, suggesting that the mechanism of FTY720‐induced cell cycle arrest is independent of the mechanism of FTY720‐induced apoptosis. These two independent pathways strengthen the effect of FTY720.

Phytosphingosine induced mitochondria-involved apoptosis.

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Sphingosine, sphinganine, and phytosphingosine are structural analogs of sphingolipids and are classified as long‐chain sphingoid bases. Sphingosine and sphinganine are known to play important roles in apoptosis. In the present study, we examined the phytosphingosine‐induced apoptosis mechanism, focusing on mitochondria in human T‐cell lymphoma Jurkat cells. Phytosphingosine significantly induced chromatin DNA fragmentation, which is a hallmark of apoptosis. Enzymatic activity measurements of caspases revealed that caspase‐3 and caspase‐9 are activated in phytosphingosine‐induced apoptosis, but there is little activation of caspase‐8 suggesting that phytosphingosine influences mitochondrial functions. In agreement with this hypothesis, a decrease in ΔΨm and the release of cytochrome c to the cytosol were observed upon phytosphingosine treatment. Furthermore, overexpression of mitochondria‐localized anti‐apoptotic protein Bcl‐2 prevented phytosphingosine apoptotic stimuli. Western blot assays revealed that phytosphingosine decreases phosphorylated Akt and p70S6k. Dephosphorylation of Akt was partially inhibited by protein phosphatase inhibitor OA and OA attenuated phytosphingosine‐induced apoptosis. Moreover, using a cell‐free system, phytosphingosine directly reduced ΔΨm. These results indicate that phytosphingosine perturbs mitochondria both directly and indirectly to induce apoptosis. (Cancer Sci 2005; 96: 83–92)

Novel tamoxifen derivative Ridaifen-B induces Bcl-2 independent autophagy without estrogen receptor involvement.

Autophagy is a self-proteolysis process in eukaryotic cells that results in the sequestering of intracellular proteins and organelles in autophagosomes. Activation of autophagy progress continued growth of some tumors, instead extensive autophagy induces cell death. In a previous study, we synthesized a novel tamoxifen derivative, Ridaifen (RID)-B. RID-B induced mitochondria-involved apoptosis even in estrogen receptor (ER)-negative cells. Since tamoxifen induces autophagy other than apoptosis, we treated ER-negative Jurkat cells with RID-B in the present study. RID-B treatment induced apoptosis and LC3 and lysosome colocalization, which results in the formation of autolysosomes. Western blotting revealed that LC3 was converted to LC3-I to LC3-II with RID-B treatment, suggesting that RID-B induced autophagy without ER involvement. Moreover, overexpression of the anti-apoptotic protein Bcl-2 suppressed the RID-B-induced cell death, but not the induction of autophagy. These results presumed that RID-B-induced autophagy is independent of Bcl-2, making RID-B-induced autophagy different from RID-B-induced apoptosis. Since Beclin 1 level is unchanged during RID-B treatment, RID-B induced autophagy pathway is Bcl-2/Beclin1 independent noncanonical pathway.

Mechanism of mitochondrial 7A6 antigen exposure triggered by distinct apoptotic pathways: Involvement of caspases

During the early stages of apoptosis, 7A6 antigen is exposed on mitochondria. 7A6 antigen is observed in various cells and is thought to be a specific marker of apoptosis determination. However, the exposure mechanism of 7A6 during apoptosis is poorly understood.

Inulin stimulates phagocytosis of PMA‐treated THP‐1 macrophages by involvement of PI3‐kinases and MAP kinases

Inulin is a polysaccharide that enhances various immune responses, mainly to T and B cells, natural killer cells, and macrophages in vivo and in vitro. Previous reports describe that inulin activates macrophages indirectly by affecting the alternative complement pathway. In this study, we examined the direct effect of inulin on PMA-treated THP-1 macrophages. Inulin treatment did not stimulate the proliferation of THP-1 macrophages at all. However, inulin treatment significantly increased phagocytosis of the polystyrene beads without the influence of serum. Doses of around 1 mg/mL had the maximal effect, and significant progression of phagocytosis occurred at times treated over 6 h. Inulin augmented phagocytosis not only with polystyrene beads but also with apoptotic cancer cells. The inulin-induced phagocytosis uptake was suppressed in Toll-like receptor (TLR) 4 mutated C3H/HeJ mice peritoneal macrophages. Moreover, inulin-induced THP-1 macrophage TNF-α secretion was inhibited using a blocking antibody specific to TLR4, suggesting that TLR4 is involved in the binding of inulin to macrophages. Furthermore, we used specific kinase inhibitors to assess the involvement of inulin-induced phagocytosis and revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, especially p38, participated in phagocytosis. These results suggest that inulin affects macrophages directly by involving the TLR4 signaling pathway and stimulating phagocytosis for enhancing immunomodulation.