2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657410摘要：FTY720經磷酸化後會展現強的免疫抑制能力並經由調控sphingosine 1-phosphate receptor (S1PR)做為治療multiple sclerosis的藥物。近年的研究發現，FTY720也可經由調控S1PR-相關 或不相關的路徑來達到抗腫瘤（包括抗血癌)作用。FTY720的結構基礎為芳香環基上連接有 aminodiol及疏水長鏈官能基。磷酸化態FTY720能強效的來調控SIPRs並降低發炎及免疫反 應，雖然其具有抗癌潛力，但因會調控S1P1而造成劑量相關性的心搏變慢的副作用，而尚無 法在抗癌上有臨床用途。也因此，設計並發展類緣化合物來避開S1PR及免疫抑制作用，但達 到強效的抗血癌活性，是一種嶄新的抗癌發展策略。初期成果我們從共同主持人處獲得一系 列以triazole為基礎的類緣化合物，以MTT作用方法分析得知，SPS-8是其中最強且抗血癌（包 括抗人類急性前骨髓性血癌)作用能力最好的化合物。以DAPI及免疫組織化學染色法分析得 知，SPS-8會造成DNA斷裂，並且會造成粒線體膜電位流失，再則，SPS-8會引起Puma-a及 Puma-P蛋白表現顯著增加，Puma是BH3-only的Bcl-2蛋白家族成員，會造成p53-相關或p53-不相關的細胞调亡作用。值得注意的是，FTY720會抑制sphingosine kinase 1 (SK1)活性以及 造成a-tubulin磷酸化作用，但SPS-8則不會有此作用，意味著FTY720會抑制免疫反應，但 SPS-8則不會，此外，SPS-8會引起快速的PKC-G蛋白表現增加及自噬作用。特殊目的我們期 望在完成本三年計晝後，能詳細了解這些類緣化合物，在體外腫瘤細胞及體内動物模式的抗急 性前骨髓性血癌的作用機轉，以及針對有潛力的化合物做好臨床前的研究發展。第一年的目的 為詳細了解訊息傳遞路徑，特別是研究出SPS-8對於三種急性前骨髓性血癌細胞株HL-60、 NB4、MR2中有功能的細胞作用標的。因為SPS-8的作用強度及選擇性尚有待改進，所以在 第一年及第二年前半年，共同主持人將分別合成30個及20個衍生物，以利第二年針對有功能 的細胞作用標的做試驗，瞭解其作用強度及選擇性。此外，也會合併臨床藥物做併用試驗，並 研究其作用機制。第三年則會以皮下植入異體腫瘤細胞(急性前骨髓性血癌細胞)及原位瘤的兩 種動物模式，來研究潛力化合物的體内作用效果，此外，也將探討體内作用的機制。<br> Abstract: FTY720, after phosphorylation by sphingosine kinase, displays potent immunosuppressive activity serving as a therapeutic agent for multiple sclerosis through modulation of sphingosine 1-phosphate receptor (S1PR). Recent studies show that FTY720 may induce anticancer activity, including anti-leukemia, through both S1PR-dependent and independent signaling pathways. Structural basis of FTY720 is an aminodiol on an aromatic moiety with a hydrophobic chain. Phosphorylated FTY720 potently regulates SIPRs, reducing inflammation and immune response. In spite of potential anticancer effect, it cannot be clinically used because of dose-limiting bradycardia effect via modulating S1P1. The development of FTY720 analogues toward anti-leukemia without S1PR effect and immunosuppressive activity is a novel strategy. Preliminary data We obtained a series of triazole-based FTY720 analogues. SPS-8 showed competitive activity to FTY720 against leukemia cells, including human acute promyelocytic leukemia (APL, e.g., HL-60) using MTT colorimetric assay. SPS-8 induced DNA fragmentation by both DAPI and immunohistochemical staining and caused the loss of mitochondrial membrane potential. Furthermore, SPS-8 caused a significant up-regulation of Puma-a and Puma-P, which are BH3-only members capable of inducing both p53-regulated and p53-independent apoptosis pathways. Notably, FTY720 but not SPS-8 inhibited sphingosine kinase 1 (SK1) activity and induced a-tubulin acetylation, indicating that FTY720 other than SPS-8 might inhibit immune response. Furthermore, SPS-8 caused a quick up-regulation of PKC-Z protein expression and autophagic activity. Specific goals We anticipate that the FTY720 analogues can be studied thoroughly after the achievement of the three-year project to understand clear anti-APL mechanism in both in vitro and in vv models and to make a good pre-clinical development of potential compounds. The specific goals in the first year are to clearly identify the signaling cascades, in particular the functional targets, to SPS-8 action in three APL cell lines, including HL-60, NB4 and MR2 cells. Because the potency and selectivity of SPS-8 needs further improved, more than 30 derivatives of compounds will be simultaneously synthesized in this year and the other 20 compounds in first half of the second year. The specific goals in the second year are to examine the potency and selectivity of the derivatives on the functional cellular targets. Moreover, combination treatment of potential derivatives with clinical therapeutic drugs will be examined and the signaling cascades will be studied. In the third year, a xenograft tumor model subcutaneously inoculated with APL cells and an orthotopic xenograft tumor model will be created for the determination of in vivo efficacy of potential compounds. Besides, the functional signals involved in the mechanism will be examined.