組蛋白去乙醯酶抑制劑用以抑制抗藥性慢性血癌幹細胞生長之研究

Investigation and utilization of Histone Deacetylase Inhibitors to suppress the growth of drug-resistant chronic myeloid leukemia stem/initiating cells

袁莉屏(Li-Ping YUAN)

指導教授 ： 何元順

共同指導教授 ： 葉淇臺

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系所/碩士(2014年)

摘要

Imatinib (Gleevec)是治療慢性骨髓性白血病臨床的第一線標靶藥物，但疾病復發頻繁且容易伴隨著抗藥性的發生。癌症幹細胞學說是近年癌症生物學非常重要之進展，此理論不但提供慢性骨髓性白血病生成之可能原因，更解釋慢性血癌產生抗藥性導致難以治癒之可能機轉。因此，在慢性骨髓性白血病的治療上如何標靶及清除癌症幹細胞是一項很重要之研究議題。我們利用GEO資料庫系統分析發現組蛋白去乙醯?＆a族（HDAC）在CML患者之高度表現與Imatinib抗藥性具有相關性。因此本研究之目的主要探討Imatinib抗藥性與慢性血癌幹細胞生成之關聯及合併HDAC抑製劑來治療Imatinib抗藥性之可能性。首先，我們以K562慢性血癌細胞為模式，利用FACSaria分選並鑑定CD34(+) / CD38(-)細胞具有高度Oct4 ,CD133, β-catenin和Sox2等癌幹細胞基因表現。此外，我們還證實CD34(+) / CD38(-)細胞亦有高度表現HDAC家族和抗藥性基因，推測與Imatinib抗藥性有相關。有趣的是，我們也發現HDAC抑製劑：SAHA可增強Imatinib抑制CD34(+) / CD38(-)細胞生長之能力並且可以藉由調控細胞週期的蛋白（CDK , P18 , P21和P27）和細胞凋亡蛋白（BCL - 2和BAX）的表現增加來誘導CD34(+) / CD38(-)細胞進行凋亡。從機制分析，我們認為合併SAHA及Imatinib治療可以降低細胞內HDAC 4 , 5和cyclin D3的表現，進而提升細胞對Imatinib敏感性增加。藉此研究，我們證實了CD34(+) / CD38(-)細胞具高度表現HDAC基因能力，這種高表達與Imatinib抗藥性有關。加入HDAC抑製劑：SAHA 使得對Imatinib抗藥性的癌症幹細胞對淤Imatinib再度敏感化。這種藥物之組合有可能被用於未來臨床的使用，以減少對Imatinib的抗藥性和CML復發。

關鍵字

慢性骨髓性白血病 ； 抗藥性血癌幹細胞 ； 組蛋白去乙醯酶抑制劑

並列摘要

Imatinib (Gleevec) is the clinical first line agent for treating chronic myeloid leukemia (CML), but disease recurrence are frequent and accompanied with resistance. One of the major contributors for imatinib-resistance is leukemia-initiating cells (LICs). Thus, targeting and eliminating LICs represents an important task for treating CML. Through public GEO database analysis, we have identified that a high expression level of histone deacetylases (HDAC) is associated with imatinib resistance in CML patients. In this study, we aimed to investigate the relevance of Imatinib -resistant cells and CD34 + cells in the generation of CML stem cells and the usage of HDAC inhibitor for possible combination therapy. First, we isolated and identified CD34(+)/CD38(-) cells with high expression of Oct4, CD133, β-catenin, and Sox2. Furthermore, we also demonstrated that high expression of HDAC and drug resistant gene were found in CD34(+)/CD38(-) LICs and associated with their resistance to imatinib. Importantly, the usage of HDAC inhibitor, SAHA, in combination with Imatinib sensitized CD34(+)/CD38(-) LICs and induced apoptosis, shown by increased expression of cell cycle (CDK2, P18, P21, and P27) and apoptotic proteins (BCL-2 and BAX). Mechanistically, the combined treatment reduced HDAC 4, 5 and cyclin D3 protein expression, which led to increased sensitivity toward SAHA and Imatinib. In conclusion, we demonstrated that LICs (CD34+/CD38-) express a high level of HDAC and this high expression was associated with imatinib resistance. Adding HDAC inhibitor, SAHA, re-sensitized imatinib-resistant LICs to imatinib. This combination could potentially be employed for future clinical settings to reduce imatinib resistance and CML recurrence.

並列關鍵字

chronic myeloid leukemia ； drug-resistant chronic myeloid leukemia stem cell ； Histone Deacetylase Inhibitors

參考文獻

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