FTY720 enhances TRAIL-mediated apoptosis by up-regulating DR5 and down-regulating Mcl-1 in cancer cells

FTY720, Fingolimod, is a functional antagonist to the sphingosine-1-phoaphate (S1P) receptor and an inhibitor of sphingosine kinase 1. Here, we showed that a combination of FTY720 and TRAIL induced apoptosis in human renal, breast, and colon carcinoma cells. Most importantly, this combination had no effect on normal cells. Furthermore, the combined treatment with FTY720 and TRAIL reduced tumor growth in xenograft models. FTY720 up-regulated death receptor (DR)5 at post-translational level. Knockdown of DR5 markedly blocked apoptosis induced by the combined treatment. FTY720 also inhibited Mcl-1 expression at the post-translational level. Over-expression of Mcl-1 blocked apoptosis induced by FTY720 and TRAIL. Interestingly, phospho-FTY720 and inhibitors of sphingosine kinase failed to enhance TRAIL-induced apoptosis. Thus, FTY720 enables TRAIL-induced apoptosis through up-regulation of DR5 and down-regulation of Mcl-1 in human cancer cells.     

Diffusion tensor imaging detects treatment effects of FTY720 in experimental autoimmune encephalomyelitis mice

Fingolimod (FTY720) is an orally available sphingosine‐1‐phosphate (S1P) receptor modulator reducing relapse frequency in patients with relapsing–remitting multiple sclerosis (RRMS). In addition to immunosuppression, neuronal protection by FTY720 has also been suggested, but remains controversial. Axial and radial diffusivities derived from in vivo diffusion tensor imaging (DTI) were employed as noninvasive biomarkers of axonal injury and demyelination to assess axonal protection by FTY720 in experimental autoimmune encephalomyelitis (EAE) mice. EAE was induced through active immunization of C57BL/6 mice using myelin oligodendrocyte glycoprotein peptide 35–55 (MOG_35–55). We evaluated both the prophylactic and therapeutic treatment effect of FTY720 at doses of 3 and 10 mg/kg on EAE mice by daily clinical scoring and end‐point in vivo DTI. Prophylactic administration of FTY720 suppressed the disease onset and prevented axon and myelin damage when compared with EAE mice without treatment. Therapeutic treatment by FTY720 did not prevent EAE onset, but reduced disease severity, improving axial and radial diffusivity towards the control values without statistical significance. Consistent with previous findings, in vivo DTI‐derived axial and radial diffusivity correlated with clinical scores in EAE mice. The results support the use of in vivo DTI as an effective outcome measure for preclinical drug development. Copyright © 2013 John Wiley & Sons, Ltd.     

FTY720-P 对破骨细胞 EphA2-EphrinA2双向信号通路作用的研究

目的探讨 FTY720-P 对破骨细胞 EphA2-EphrinA2 双向信号通路的影响。方法取小鼠巨噬细胞 RAW264.7，采用地塞米松及 1α，25-二羟维生素 D₃ 诱导分化为破骨细胞，并行抗酒石酸酸性磷酸酶（tartrate resistant acid phosphatase，TRAP）染色鉴定。将诱导培养的破骨细胞分为两组，实验组给予 400 ng/mL 的 FTY720-P 处理，对照组不给予 FTY720-P。培养 48 h 后，取细胞行实时荧光定量 PCR 检测、Western blot 检测以及免疫荧光染色观察，分析细胞中 EphA2、EphrinA2、RhoA，以及骨重建相关蛋白 BMP-2 及 TGF-β₁ 的表达。 结果经 TRAP 染色鉴定，RAW264.7 细胞成功诱导成为破骨细胞。培养 48 h 后，与对照组相比，实验组 EphA2 及 EphrinA2 mRNA 及蛋白相对表达量均明显降低（P<0.05），RhoA 蛋白相对表达量也明显降低（P<0.05）；BMP-2 及 TGF-β₁ mRNA 相对表达量明显增高（P<0.05），蛋白表达增强。 结论FTY720-P 能通过影响破骨细胞 EphA2-EphrinA2 双向信号通路之间的传导下调 RhoA，并促进 TGF-β₁ 和 BMP-2 表达，最终影响破骨细胞的破骨作用。     

Overexpression of the pp32r1 (ANP32C) oncogene or its functional mutant pp32r1Y140H confers enhanced resistance to FTY720 (Finguimod)

pp32r1 (ANP32C) is oncogenic and has been shown to be overexpressed in tumors of the breast, prostate, and pancreas. In this work we show that pp32 family proteins are able to bind to the sphingosine analog FTY720 (Finguimod). Molecular docking studies highlight that a conserved residue F136 is likely to be a key determinant of the FTY720 binding site on the pp32 leucine-rich repeat domain. Transduction of the renal carcinoma cell line ACHN or cervical cancer cell line HeLa with lentivirus expressing the oncogenic family member pp32r1 or a pp32r1Y140H functional mutant illustrated an enhanced resistance to FTY720 induced apoptosis. These findings highlight that certain cancers overexpressing pp32r1 or pp32r1 mutants are likely to demonstrate enhanced resistance to FTY720 treatment.     

黄芪、红景天、FTY720对柯萨奇病毒CVB3诱导小鼠病毒性心肌炎的药效学研究

目的：观察黄芪、红景天、FTY720作为单味药物对小鼠柯萨奇B3（CVB3）所致病毒性心肌炎（VMC）的治疗作用。方法：腹腔接种CVB3病毒液以建立VMC模型。随机分为空白组、VMC对照组、黄芪组、红景天组、FTY720组，观察五组动物在心肌重／体重比值；心肌组织切片（HE染色）；心肌酶（LDH．CK-MB）活力改变差异。结果：在心肌重／体重上，给药组与VMC对照组间无显著性差异（P〉0．05）；显微镜下观察心肌组织切片，VMC对照组局部可见广泛增生纤维组织，小鼠淋巴单核细胞浸润严重。药物干预组仅有淋巴单核细胞浸润，无纤维组织增生，空白对照组无病理性组织变化；LDH的活性测定显示黄芪组、红景天、FTY720组酶活性明显降低与VMC对照组有显著性差异（P〈0．01、P〈0．05）。CK-MB测定结果表明黄芪、红景天、FTY720组与VMC对照组有显著性差异（P〈0．01、P〈0．05）。结论：黄芪、红景天、FTY720对CVB，诱导小鼠急性病毒性心肌炎有一定治疗作用。     

Discovery of APD334: Design of a Clinical Stage Functional Antagonist of the Sphingosine-1-phosphate-1 Receptor

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FTY720对体外培养MCF-7细胞增殖及凋亡影响的研究

目的探讨FTY720对体外培养MCF-7细胞增殖及凋亡的影响。方法体外培养MCF-7细胞,将其培养液中加入不同浓度的FTY720,继续培养48小时,MTT法检测MCF-7细胞的增殖情况;瑞士-姬姆萨染色观察细胞形态变化;流式细胞仪检测FTY720对MCF-7细胞的细胞周期、凋亡率的影响。结果当FTY720浓度为6 400 ng/ml时,体外培养MCF-7细胞的增殖受到明显抑制,抑制率为62.0%（P〈0.05）,而且抑制率呈浓度依赖性;镜下可见细胞出现凋亡的形态;流式细胞仪检测分析显示FTY720可将MCF-7细胞阻滞于G1期,并促进其发生凋亡,凋亡率呈浓度依赖性。结论在体外培养的MCF-7细胞培养液中加入一定浓度的FTY720,能明显抑制该细胞的增殖,调节该细胞周期并诱导其凋亡。