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 Peyer's 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 Peyer's 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 Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.     

FTY720: a most promising immunosuppressant modulating immune cell functions

FTY720, the pharmacological analog of S1P, acts as an agonist of sphingosine-1-phosphate receptors, resulting in the inhibition of lymphocyte egress from secondary lymphoid tissues and thymocytes from the thymus, peripheral lymphopenia and interfering with normal functions of several other cell types. FTY720 has been clinically tried for transplantation and multiple sclerosis, showing promising protective effects. This review will summarize potential applications and effects of FTY720.     

新型免疫抑制剂FTY720对大鼠胶原诱导性关节炎的抑制作用

目的观察新型免疫抑制剂FTY720对大鼠胶原诱导性关节炎发生和发展的影响。方法以胶原诱导大鼠关节炎模型,从第1次免疫造模开始,大鼠分别予以FTY720(0.1、0.6mg·kg-1)和雷公藤多苷(6、12mg·kg-1)灌胃治疗,连续4周。观察记录大鼠活动和关节炎指数、体重、足底厚度和足爪肿胀;血常规分析大鼠外周血淋巴细胞、单核细胞和中性粒细胞变化;流式分析外周血CD4+和CD8+T淋巴细胞变化;组织学及免疫组织化学分析炎性关节病理改变和滑膜组织中CD4+和CD8+T淋巴细胞的变化。结果 FTY720给药后能抑制大鼠关节炎的发生和发展,明显降低大鼠外周血淋巴细胞特别是CD4+T淋巴细胞数量和滑膜组织中CD4+T淋巴细胞数量。结论 FTY720可有效控制大鼠胶原诱导性关节炎的发生和发展,其作用机制可能是通过抑制外周血CD4+T淋巴细胞浸润到滑膜组织。     

Analysis of the mode of action of a novel immunosuppressant FTY720 in mice.

Accelerated lymphocyte homing and apoptosis have been suggested to contribute to potent immunosuppressive effects of FTY720, however, its main mechanism of action remains to be fully elucidated. Here, we examined the mode of action of FTY720 in mice. FTY720, when given at a single dose of 1 mg/kg, markedly decreased the number of peripheral blood lymphocytes (PBL) but moderately increased the lymphocyte numbers in lymph nodes (LN) and Peyer's patches (PP) in normal mice, as previously observed in rats. However, the sharp decrease in PBL numbers was also observed in aly/aly mice lacking LN and PP, indicating that this phenomenon is not explained by accelerated lymphocyte homing to LN and PP. In addition, the finding that a single administration of FTY720 did not suppress proliferative responses of T cells suggested that the PBL reduction could occur without inhibiting lymphocyte functions. However, when administered at the same dose for 2 weeks, FTY720 induced severe systemic lymphopenia, as well as marked suppression of lymphocyte proliferative responses in normal mice. The same treatment also prolonged skin allograft survival in aly/aly mice. Our results suggest that FTY720 suppresses in vivo immune functions mainly by inducing systemic lymphopenia and also by inhibiting T cell functions.     

一种新的免疫抑制剂FTY720对小鼠心脏移植的救治作用(英文)

AIM: FTY720 is a new synthetic immunosuppressive agent which has a unique mechanism of action and induces long-term graft acceptance in rat and dog allotransplantation as prophylactic administration. The present study investigated whether FTY720 was able to rescue ongoing acute rejection of solid organ transplants in a mouse heterotopic cardiac transplantation model. METHODS: BALB/c hearts were heterotopically grafted in C57BL/6 mice. FTY720, at the doses of 0.5, 1, and 5 mg·kg~(-1)·d~(-1) or vehicle was administered to recipients once daily by oral gavage from d 3 to d 7 after transplantation. Histological changes of grafts, and the lymphocyte number in the peripheral blood and the peripheral lymph nodes were determined on d 5 after transplantation. RESULTS: FRY720 prolonged the median graft survival time dose-dependently and significantly. Histological evaluation revealed less lymphocytic infiltration in cardiac allografts treated with FTY720. Moreover, FTY720 remarkably lowered the number of periph     

Activation of caspases and mitochondria in FTY720-mediated apoptosis in human T cell line Jurkat

FTY720, a novel immunosuppressive drug originally derived from a metabolite from Isaria sinclairii, is known to induce apoptosis in lymphocytes. In this study, we investigated the involvement of caspases and mitochondria in FTY720-mediated apoptosis using Jurkat cells, a human T cell line. Our results indicated that FTY720-induced activation of caspases 2, 3, 6, 8, 9 and 10, whereas caspases 1 and 5 were not activated. We also observed in the FTY720-treated cells a loss of mitochondrial membrane potential, a release of cytochrome c into cytosol and an exposed phosphatidylserine (PS) at the outer surface of the cell membrane. Pretreatment with a peptide inhibitor, benzyloxycarbonyl-Asp-CH2COC-2, 6-dichlorobenzene (Z-Asp-CH2-DCB), prevented apoptosis and externalization of phosphatidylserine, whereas the inhibitor did not prevent the mitochondrial events. This suggests that caspases may play a role downstream of the mitochondrial pathway. Therefore, caspase cascade in FTY720-treated cells may be initiated by activation of mitochondria.     

Induction of apoptosis of activated murine splenic T cells by cycloprodigiosin hydrochloride, a novel immunosuppressant

Two types of immunosuppressants, cycloprodigiosin hydrochloride (cPrG) and L-leucyl-L-leucine methyl ester (LeuLeuOMe), both have the ability to selectively inhibit the lysosomal function, and a related compound to cPrG, prodigiosin 25-C, and LeuLeuOMe have been reported to selectively inhibit the T cell function in vitro. We therefore examined the cell-type specificity of cPrG and LeuLeuOMe using murine splenocytes. Concanavalin A (Con A)- and lentil lectin-induced proliferation was suppressed by cPrG more profoundly than lipopolysaccharide-induced proliferation. At the optimal concentration, Con A induced the proliferation of both CD4+ and CD8+ cells, whereas at a supra-optimal concentration Con A induced rather selective proliferation of CD8+ cells. Irrespective of the dose of Con A, CD4+ and CD8+ cells were equally affected by cPrG. In contrast, LeuLeuOMe induced the selective loss of CD8+ cells. cPrG enhanced the apoptosis of murine splenocytes and nylon fiber column-purified T cells cultured in the presence of Con A, as shown by the decrease in cell size and/or DNA fragmentation. Overall, this study revealed that the cell-type specificity of cPrG is different from that of LeuLeuOMe, and that the immunosuppression by cPrG is associated with apoptosis.     

Cadmium-induced apoptosis in murine fibroblasts is suppressed by Bcl-2

We investigated the induction of apoptosis by cadmium in NIH 3T3 murine fibroblasts. Apoptosis was triggered effectively by 10 microM CdCl2 within 24 h, under which conditions cell viability was reduced by 50%. Cadmium-induced apoptosis was demonstrated by both morphological and biochemical analysis. We have shown that cadmium concentrations of 5-20 microM caused nuclear fragmentation. Moreover, internucleosomal DNA fragmentation was evoked by 10-25 microM CdCl2 within 24 h, as detected by the formation of ladder patterns in DNA electrophoresis. Since the induction of programmed cell death occurs together with modifications in the cell cycle, we examined the ability of cadmium to block cell divisions by using a 5-bromo2-deoxy-uridine incorporation assay. Our results indicate that about 40% of treated cells are blocked in G0-G1 phase when exposed to 10 microM cadmium for 27 h. Finally, we addressed the question of whether the effect of cadmium could be prevented by suppressing apoptosis. Over-expression of the anti-apoptotic protein Bcl-2 in NIH 3T3 cells protects against cadmium toxicity, thus suggesting a role for Bcl-2 in the regulation of cadmium-induced apoptosis.     

FTY720, a novel immunomodulator in de novo kidney transplant patients: pharmacokinetics and exposure-response relationship

The pharmacokinetics, safety, and preliminary efficacy of FTY720, a novel immunomodulator, were examined in de novo renal transplant patients. Both noncompartmental and population methods were used to estimate pharmacokinetic estimates in the patients. The steady-state plasma concentrations of FTY720 increased in accordance with maintenance dose level, indicating linearity in clearance and volume of distribution over the 0.25- to 2.5-mg dose range. The pharmacokinetics of FTY720 in de novo renal transplant patients were characterized by the long terminal phase half-life of approximately 200 hours across doses, high volume of distribution (>3000 L), and low clearance (10.8 L/h). The intersubject variation of clearance was 55%, and the intrasubject variation of FTY720 concentrations was 28%. The population analysis revealed significant positive relationships between baseline alkaline phosphatase and clearance, as well as between baseline body weight on apparent volume of distribution. There was no relationship between FTY720 concentrations within a given FTY720 dose cohort and the rate of allograft rejection.     

Potential anti-tumor effects of FTY720 associated with PP2A activation: a brief review

FTY720 (Fingolimod, Gilenya^?) is an FDA-approved immunosuppressant currently used in the treatment of multiple sclerosis. However, a large number of studies over the last few years have shown that FTY720 shows potent antitumor properties that suggest its potential usefulness as a novel anticancer agent. Interestingly, the restoration of protein phosphatase 2A (PP2A) activity mediated by FTY720 could play a key role in its antitumor effects. Taking into account that PP2A inactivation is a common event that determines poor outcome in several tumor types, FTY720 could serve as an alternative therapeutic strategy for cancer patients with such alterations.     

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P1 receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.     

Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance

Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of deltapsim after stimulation of the respiratory state. To evaluate the binding of [14C]paclitaxel to isolated mitochondria, mitochondrial proteins were precipitated yielding 18.6 +/- 2.1 cpm/microg of protein. After stimulation of the respiratory state, binding of [14C]paclitaxel increased up to 163.2 +/- 46.7 cpm/microg of protein. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.     

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P₁ receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.     

Sulfated polymannuroguluronate, a novel anti-AIDS drug candidate, inhibits T cell apoptosis by combating oxidative damage of mitochondria

Sulfated polymannuroguluronate (SPMG) has entered the phase II clinical trial as the first anti-AIDS drug candidate in China. Herein, we report that SPMG was effective at protecting T lymphocytes against apoptosis. Further studies indicated that SPMG significantly elevated mitochondrial membrane potential (MMP) of T cells; inhibited mitochondrial release of cytochrome c (cyto c) in T cells; enhanced the activities of mitochondrial enzyme complex I, III, and V; and subsequently increased ATP level and ATP/ADP ratio. In addition, SPMG potently suppressed reactive oxygen species (ROS) generation in mitochondria at cellular level and scavenged free radicals in cell-free system. The molecular mechanism underlying the ATP-involved and ROS-dependent antiapoptosis of SPMG is characterized as having been caused by its engagement with mitochondrial import receptor and ADP/ATP carrier in T-cell outer and inner mitochondrial membrane, respectively. All these might shed new light on the understanding of anti-AIDS functions of SPMG by protecting T cells of persons infected with human immunodeficiency virus.     

Survivin和Bcl-2调节槲皮素诱导的A549细胞凋亡

目的研究槲皮素诱导肺腺癌细胞A549凋亡,探讨survivin和Bcl-2在槲皮素诱导A549细胞凋亡中的调节作用。方法分别采用MTT法、荧光染色、流式细胞仪和免疫细胞化学观察了槲皮素对肺腺癌A549细胞增殖、凋亡、细胞周期和蛋白表达的影响。结果槲皮素抑制肺腺癌A549细胞增殖的作用明显,且呈浓度和时间依赖性。形态学检测显示出细胞凋亡的特征变化,槲皮素能使肺腺癌A549细胞周期阻滞于G0/G1期,且A549细胞survivin和Bcl-2蛋白表达同时下降,而caspase-3活性升高。结论槲皮素能诱导A549细胞凋亡,其机制可能是使肺腺癌A549细胞周期阻滞于G0/G1期,并同时下调survivin和Bcl-2蛋白的表达,直接激活caspase-3而诱导A549细胞凋亡。     

Naringenin-induced apoptosis is attenuated by Bcl-2 but restored by the small molecule Bcl-2 inhibitor,HA 14-1, in human leukemia U937 cells

Naringenin, a naturally occurring citrus flavonone, has shown cytotoxicity in various human cancer cell lines as well as inhibitory effects on tumor growth and there is increasing interest in its therapeutic applications. In this study, the effect of ectopic Bcl-2 expression on naringenin-induced apoptosis was investigated. We found that Bcl-2 overexpression markedly protected human leukemia U937 cells from time- and dose-dependent induction of apoptosis by naringenin, as did caspase-3 and caspase-9 inhibitors. Additionally, Bcl-2 overexpression attenuated naringenin-induced Bax translocation and cytosolic release of cytochrome c. Our results also indicated that co-administration of HA14-1 and naringenin increased apoptosis in Bcl-2 overexpressing U937 cells by restoring mitochondrial dysfunction and activation of caspase-9 and caspase-3, as well as by cleavage of poly (ADP-ribose) polymerase. Taken together, these observations indicate that Bcl-2 confers apoptosis resistance to naringenin by inhibiting a mitochondrial amplification step in U937 cells.     

Capsaicin-induced apoptosis is regulated by endoplasmic reticulum stress- and calpain-mediated mitochondrial cell death pathways

Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca²⁺, calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.     

新型反义寡核苷酸F951对HL60细胞Bcl-2基因表达及细胞增殖的抑制作用

目的　研究新型反义寡核苷酸F951对白血病细胞Bcl- 2基因表达及细胞增殖的影响。方法　不同浓度的F951与HL60细胞共培养后,采用台盼蓝拒染法、MTT比色法检测HL60细胞增殖,用流式细胞术和RT -PCR法检测Bcl- 2蛋白及其mRNA表达,DNA片段化分析法检测凋亡细胞。结果　5, 10, 20μmol·L-1F951分别与HL60细胞共孵育1~5d,细胞生长明显受抑,这一抑制作用随F951浓度的升高和作用时间的延长而增强。MTTA值与未处理组相比F951 5, 10, 20μmol·L-1组细胞抑制率分别为: 20 .56%、37 .66%、54 .11%与对照组相比差异均有显著性;F951处理后HL60细胞Bcl 2mRNA水平下降,Bcl- 2蛋白减少;凝胶电泳可见典型的梯形带,且随着浓度提高诱导DNALadder的作用更加明显。结论　F951可下调Bcl -2基因表达,促进细胞凋亡而抑制白血病细胞增殖。