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研究生:許凰鈴
研究生(外文):Huang-Ling Hsu
論文名稱:探討第二型拓樸異構亞型專一性藥物及其對生理機能的影響
論文名稱(外文):Identify the topoisomerase II isozyme-specific targeting agents and investigate their biological responses
指導教授:李財坤
指導教授(外文):Tsai-Kun Li
口試委員:詹迺立顧記華黃旭山
口試委員(外文):Nei-Li ChanJih-Hwa GuhHsu-Shan Huang
口試日期:2014-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:97
中文關鍵詞:拓樸異構&;#37238;β亞型
外文關鍵詞:TOP2β
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第二型拓樸異構&;#37238;(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&;#37238;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&;#37238;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&;#33981;&;#37260;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&;#33981;&;#37260;衍生物,首先對這些&;#33981;&;#37260;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.


口試委員審定書……………………………………………………………..... I
致謝………………………………………………………………………………II
中文摘要………………………………………………………………………………………………III
ABSTRACT……………………………………………………………………………………………..V
INTRODUCTION……………………………………………………………………………………. 1
1. DNA Topoisomerase
1.1 Type I and Type II DNA topoisomerase
2. Topoisomerase II as anticancer drugs target
2.1 Mechanisms of inhibiting of TOP2
2.2 TOP2 poisons and inhibitors in clinic uses and unwanted side-effects
3. The distinct roles of TOP2 isozymes in the TOP2-targeting anticancer efficacy
and unwanted side effects
3.1 Identification routes of isozymes-specific target agents
3.2 Anthracyclines and anthracenediones are potential TOP2α-specific poisons or
TOP2β-specific inhibitors
SPECIFIC AIMS ………………………………………………………………...18
MATERIALS AND METHODS ………………………………………………..19
- Chemicals, plasmid and cell lines
- Antibodies and immunoblotting analysis
- Cytotoxicity assay (MTT assay)
- Alkaline single cell gel-electrophoresis (comet) assay
- Lentivirus-based RNA interference (RNAi)
- In vitro TOP2 cleavage assay
- DNA relaxation assay
- In vivo complex of Enzyme (ICE) bioassay
- DNA-unwinding assays
- DNA binding Assays
- In vivo DNA decatenation assay
RESULTS……………………………………………………………………………26
- New anthracenedione derivatives don’t induce DNA damage responses significantly and this fact is consistent with their low ability in cancer cells killing.
- CL-01 and CL-14 preferentially inhibited TOP2β activity in an In Vitro relaxation assay.
- Structure determination of hTOP2βcore-DNA-anthracenedione cleavable complexes using the “drug-replacement” platform technology.
- CL-12, CL-13 and CL-14 did not act like strong DNA intercalator as mitoxantrone and thus exhibiting weak DNA-unwinding ability
- The VP-16 induced TOP2-mediated chromosomal DNA cleavage was antagonized by ICRF-193, CL-13 and CL-14, but not CL-12
- The VP-16 induced TOP2β-mediated DNA cleavage was preferably antagonized by CL-14 in an In Vitro cleavage assay.
- CL-14 preferentially antagonized VP-16-induced TOP2β-cleavable complex formation in trapping assay and in In Vivo complexes of enzyme assay.
- CL-14 still antagonized the VP-16-induced chromosomal DNA breaks in a TOP2α -specific deficient cell line
- CL-13 antagonized VP-16-induced both the TOP2αcc and TOP2βcc formation in trapping and ICE assays
- CL-13 antagonized both the TOP2α- and TOP2β-mediated DNA breaks induced by VP-16 treatment
- CL-14 could also antagonize the doxorubicin-induced TOP2-mediated DNA breaks
- CL-14 preferentially antagonized the doxorubicin-induced TOP2β cc formation in trapping assay
- CL-14 preferentially antagonized the doxorubicin-induced TOP2β-mediated DNA breaks in TOP2α-deficient cells
- The TOP2α isozyme, rather than TOP2β, is the main enzyme involved in the DNA decatenation process and, unlike ICRF-193, CL-14 doesn’t cause any defect in chromosome segregation.
DISCUSSION………………………………………………………………………45
FIGURES AND TABLES…………………………………………………………52
- Table 1: The cytotoxicity of anthracenedione derivatives in HL-60 cells and
intensity of inducing DNA breakage
- Table 2: The TOP2-targeting potential of anthracenedione derivatives and the
preference of isozymes inhibition.
- Figure 1. CL-14 preferentially inhibited TOP2β activity in an In Vitro relaxation
assay.
- Figure 2. Structure of the hTOP2βcore-DNA cleavage complex with Anthracenedione derivatives and etoposide
- Figure 3. CL-12, CL-13 and CL-14 showed the poor DNA unwinding ability.
- Figure 4. CL-12, CL-13 and CL-14 not act as strong DNA intercalating agents.
- Figure 5. CL-12 didn’t show the antagonist effect on TOP2-mediated DNA cleavage induced by VP-16.
- Figure 6. VP-16 induced TOP2-mediated DNA cleavage that was antagonized by CL-13 in high concentration.
- Figure 7. VP-16 induced TOP2-mediated DNA cleavage that was antagonized by CL-14.
- Figure 8. CL-12 and CL-13 didn’t show antagonized effects on isozymes-mediated DNA cleavage induced by VP-16 In Vitro.
- Figure 9. VP-16 induced TOP2β-mediated DNA cleavage that was antagonized by CL-14 In Vitro.
- Figure 10. CL-14 antagonized VP-16-induced TOP2β-cleavable complex formation in trapping assay.
- Figure 11. CL-14 preferentially antagonized VP-16-induced TOP2β-cleavable complex formation in ICE assay.
- Figure 12. CL-14 preferentially antagonized TOP2β-mediated DNA damage induced by VP-16 in TOP2α deficient cells.
- Figure 13. CL-14 preferentially antagonized TOP2β-mediated DNA cleavage in intensity of DNA cleavage manner.
- Figure 14. CL-13 both antagonized VP-16-induced TOP2α and TOP2β-cleavable complex formation in trapping assay.
- Figure 15. CL-13 preferentially antagonized VP-16-induced TOP2α-cleavable complex formation in ICE assay.
- Figure 16. CL-13 preferentially antagonized TOP2α-mediated DNA damage induced by VP-16.
- Figure 17. CL-13 both antagonized TOP2β-mediated and TOP2α-mediated DNA cleavage induced by VP-16 in intensity of DNA cleavage manner.
- Figure 18. Doxorubicin induced TOP2-mediated DNA cleavage that was antagonized by CL-14
- Figure 19. CL-14 preferentially antagonized doxorubicin-induced TOP2β- cleavable complex formation in trapping assay.
- Figure 20. CL-14 preferentially antagonized TOP2β&;#8722;mediated DNA damage induced by doxorubicin.
- Figure 21. TOP2α is mainly involved in DNA decatenation process and CL-14 didn’t resulted in defective chromosome segregation.
RENFERENCES…………………………………………………………………… 91


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