天然物及化學合成物在急性骨髓性血癌之抗癌機制研究

Abstract

急性骨髓性血癌(Acute myeloid leukemia, AML)是造血系統惡性腫瘤，其特性為患者基因異常或基因表徵改變造成造血幹細胞不正常增生、停止分化，使淋巴球或骨髓衍生的細胞功能異常。患者基因突變、基因表現失調、患者年齡或疾病進程都可能影響患者的預後。傳統化學療法易使正常血球幹細胞被殺死造成嚴重的骨髓性抑制作用或產生抗藥性，造血幹細胞移植(Hematopoietic stem cell transplantation，HCT)用於緩解後治療或進行異體骨髓移植，但受限於捐贈者來源或產生免疫排斥作用，使AML治療成效不佳。因此，發展新治療方式縮短療程或增加緩解率有其必要性。本論文將分三部份探討血癌細胞分子機轉，分別以薯蕷皂苷(dioscin)、FTY720類緣物(SPS8)及強心配醣體(reevesioside F)為實驗材料，觀察這三個化合物對於血癌細胞株HL-60、Jurkat或CCRF-CEM之抗癌作用機制，評估其治療AML 的可行性。Dioscin引發細胞死亡是透過促進Fas/FasL、IGFBP1/2/3的表現，使caspase-3/-8/-9活化，造成粒線體功能失調與DNA片段化。此外，dioscin可增加細胞核C/EBPα的表現，讓骨髓芽母細胞分化成不同的血球細胞。SPS8對HL-60抗細胞增生效果與FTY720相似，SPS8可活化caspase及自噬作用促進細胞凋亡。在基因轉錄層次觀察到SPS8會影響ceramide/sphingosine synthesis訊號傳遞路徑中p38 MAPK標的基因表現。SPS8在體外實驗不具抑制shpingosine kinase-1的活性，在動物試驗也不影響免疫活性。Reevesioside F所引起的抗細胞增生活性與Na+/K+-ATPase α3離子幫浦存在與否相關。Reevesioside F可快速抑制survivin表現，造成cytochrome c釋出與粒線體膜電位流失，進而活化下游caspase訊號傳遞，其中又以caspase-3所媒介的訊號傳遞路徑對血癌細胞凋亡的貢獻最顯著。由上述實驗結果瞭解化合物對血癌之作用機轉，期望可提供血癌新的治療標靶與發展策略。

Acute myeloid leukemia (AML) is a malignant clonal disorder of the hematopoietic system characterized by an enormous heterogeneity of acquired genetic and epigenetic changes in hematopoietic precursor cells and by abnormal proliferation, decreased cell death as well as blocked differentiation, and malfunction of lymphocytes and myeloid-derived cells.The prognosis of treatment depends on genetic mutation, deregulated expression of genes, disease progression and age of patients. Traditional chemotherapy in AML patients typically causes severe myelosuppression because of the killing of normal hematopoietic cells. Hematopoietic stem cell transplantation (HCT) is another modality used in the postremission treatment of AML or allogenetic bone-marrow transplantation, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Thus, new agents to treat AML are urgently required to shorten the treatment or enhance remission rate. Dioscin, SPS8 (an FTY720 analogue) and reevesioside F (a cardenolide glycoside) are employed to investigate the anti-leukemic mechanisms of leukemic cell lines HL-60, Jurkat, and CCRF-CEM. Hopefully, new cellular targets or mechanisms can be discovered for strategy planning against AML after the thesis completion.Dioscin-induced cell death was found to upregulate the expression of Fas, FasL, IGFBP 1/2/3 and active caspase-3, -8 and -9, which facilitated mitochondria dysfunction and DNA fragmentation. Additionally, dioscin increased nucleus expression of C/EBPα enabling differential differentiation of promyeloid blast into leukocytes. SPS8 exhibited equipotent antiproliferative efficiency to FTY720 against HL-60 cells. Further investigation indicated that SPS8 caused cell apoptosis through activation of caspases and autophagy. Transcriptomic profiling further revealed that SPS8 treatment upregulated p38 MAPK target genes and interfered with genes involved in ceramide/sphingosine synthesis. In contrast to FTY720, SPS8 did not modify sphingosine kinase-1 activity. Furthermore, SPS8 did not affect immune activity in an in vivo study. Reevesioside F induced anti-proliferative activity that was highly correlated with the expression of Na+/K+ATPase α3 subunit. Reevesioside F induced a rapid down-regulation of survivin protein, followed by release of cytochrome cfrom mitochondria and loss of mitochondrial membrane potential (∆Ψm).The loss of ∆Ψm and mitochondrial damage were responsible for the activation of caspases, and the amplification of caspase-3-mediated signaling pathway might contribute largely to the execution of apoptosis in leukemic cells. Taken together, the results of the present study have identified the signaling pathways responsible for cell death of leukemia cells. These findings may provide insight into new cellular targets and mechanisms in developing therapeutic strategy in treating leukemia.