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標題: | 探討第二型拓樸異構酶亞型專一性藥物及其對生理機能的影響 Identify the topoisomerase II isozyme-specific targeting agents and investigate their biological responses |
作者: | Huang-Ling Hsu 許凰鈴 |
指導教授: | 李財坤(Tsai-Kun Li) |
關鍵字: | 拓樸異構?β亞型, TOP2β, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 第二型拓樸異構酶(TOP2)可以調控DNA的拓樸構型,因此對於DNA生理功能的執行非常重要,臨床有許多針對第二型拓樸異構酶的抗癌藥物如:etoposide (VP-16)和doxorubicin(DOX),TOP2會利用其活性中心的酪胺酸與DNA產生共價鍵結,造成暫時性的DNA斷裂,這個酵素-DNA共價複合體又被稱為切割複合體,此類的藥物會穩定此復合體進而促進癌細胞中DNA斷裂的累積,而引發癌細胞的死亡,我們把這類的藥稱作”TOP2 poison”。另一種針對TOP2的藥物“TOP2 catalytic inhibitor”則是作用在酵素催化週期的其他步驟,此類的藥物並不會引起DNA斷裂並且可以拮抗TOP2 poison的作用。人類細胞中擁有α及β兩種第二型拓樸異構酶亞型,α亞型會在快速分裂中的細胞中大量表現,為抗癌藥物主要的作用目標;而β亞型在細胞各個週期均有表現,主要與特定基因的表現有關。Etoposide 雖然已廣泛的用於多種癌症的化療,但這類藥物會引起染色體易位以及再復發的副作用,另外doxorubicin也廣泛地使用在臨床但受限於它所造成的心臟毒性,近幾年的研究都指出這些副作用和β亞型所造成的DNA斷裂有關,因此我們篩選了一些蒽醌衍生物,目標是找到具有α亞型專一性的TOP2 poison,希望提升毒殺癌細胞的能力並且減少副作用,另一個目標是發展出β亞型的TOP2 catalytic inhibitor去拮抗TOP2 poison造成的β亞型DNA斷列,進而達到減少副作用的效用。DNA 嵌合物是作用在TOP2的一種機制且通常是蒽醌衍生物,首先對這些蒽醌衍生物進行細胞毒性測試,發現這些化合物毒性並沒有特別的高,並且沒有顯著的引起DNA的斷裂,因此推斷這些化合物比較有潛力發展成TOP2 catalytic inhibitor,接著在In Vitro(胞外)分析TOP2 relaxation 活性受這些合成物抑制的情況,去探討作用在TOP2的潛力並挑選β亞型專一性抑制的化合物,結果發現化合物CL-14偏向抑制TOP2β活性。接著分析對TOP2 poison的拮抗作用,發現CL-14和etoposide及doxorubicin一起作用會拮抗其誘發的DNA斷列(~15~20%),確認此拮抗作用是不是具有TOP2β專一性,利用純化的TOP2α及TOP2β進行胞外(In Vitro)DNA 斷裂分析,結果顯示CL-14和etoposide反應有偏向拮抗TOP2β造成的DNA 斷裂, 由以上結果我們推斷CL-14具有潛力發展成TOP2β專一性抑制藥物並且可以用在臨床去去預防TOP2 poison造成的副作用,在RNA干擾(RNAi)實驗發現CL-14和etoposide及doxorubicin作用後顯著的在TOP2α缺失的細胞拮抗TOP2β造成的DNA 斷裂,再者,胞內(In Vivo)生化實驗證明CL-14偏向拮抗etoposide誘發之TOP2β可切性複合體(TOP2β-cleavable complex, TOP2βcc)的形成。CL-12, CL-13和CL-14在結構上具有高度相似性,然而,它們並沒有TOP2β的專一性,因此去分析其結構如何造成亞型的專一性,可提供發展更具效用的臨床抗癌藥物之重要資訊。綜合來說,我們發現CL-14有潛力成為TOP2β專一性的拮抗藥物,可用在臨床去預防etoposide及doxorubicin引發的副作用,提升化學治療的效用。 Topoisomerase II (TOP2) plays crucial roles in cells and TOP2-targeting drugs are effective anticancer drugs but with side-effects. There are two types of TOP2-targeting agents: (i) Poisons stabilize TOP2 cleavable complex (TOP2cc) and induce DNA break; (ii) Inhibitors only interfere with catalytic activity and antagonize the poisoning action. In human cells, the alpha (α) and beta (β) isozymes share similar enzymatic action but play differential functions. Etoposide (VP-16), an active TOP2 poison, induces 2nd malignancies. Notably, TOP2β is mainly responsible for the VP-16-induced DNA sequence rearrangement and carcinogenesis as well as the doxorubicin-induced cardiotoxicity. We screened anthracenedione derivatives and identified potential TOP2 isozyme-specific poisons and/or inhibitors. First, we used comet assay to detect DNA breakage and found anthracenedione derivatives didn’t significantly induce DNA damage responses. The inhibitory ability of TOP2 activity was assessed by an in vitro relaxation assay. We then selected compound CL-14 that preferentially inhibits TOP2β relaxation activity for further study and found that CL-14 only induced few DNA break, but antagonized (~15~20%) TOP2-mediated DNA damage induced by VP-16 as well as doxorubicin. TOP2β-mediated DNA cleavage, but not TOP2α, induced by VP-16 in vitro was also antagonized by CL-14. Together, we suggested that CL-14 is a potential TOP2β-specific inhibitor, which is clinically helpful in preventing TOP2-targetinig side-effects. In agreement with above notion, our data also revealed that CL-14 still antagonized etoposide and doxorubicin-induced DNA break in TOP2α knockdown cells. Furthermore, we also found CL-14 could preferentially antagonize VP-16-induced TOP2β-cleavable complex formation in both trapping and In vivo complex of enzyme assays. CL-12 and CL-13, though sharing a similar structure with CL-14, but didn’t show the isozyme specific inhibition. A structure-based study has also been initiated to unravel the molecular mechanism(s) underlying this isozyme-specific action. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56346 |
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