臺灣博碩士論文加值系統

組蛋白(Histone)是真核細胞內重要的蛋白質之一，其主要存在於細胞核內的染色質中，目前有許多的證據顯示，人體中不正常的組蛋白修飾(histone modifications)可能參與著細胞的癌化過程。其中，組蛋白的修飾作用包括了乙醯化、甲基化及磷酸化等，這些修飾作用可被用於調控細胞內的基因表現及功能，因此組蛋白修飾作用被認為是重要的外基因(epigenetic)變化指標之一。一般而言，不正常的組蛋白修飾作用大多是因為組蛋白修飾酵素(histone-modifying enzymes)活性異常所引發的，且此不正常的修飾作用可能會影響腫瘤發生相關基因(tumorigenesis-related genes)的活化及表現，進而造成腫瘤的形成及發展。
天然物在現今新藥的開發研究上所扮演的角色已經愈來愈重要，因此本實驗室利用萃取技術，從天然物中萃取出許多種類的化合物並分析其生化活性，其中發現squamocin及calactin對於多種人類癌細胞株具有高度的細胞毒性。因此本篇研究將分別探討squamocin與calactin對於人類癌細胞株的抑制作用以及其對於組蛋白修飾酵素基因和蛋白質表現量的影響。由實驗的結果中發現，calactin可以藉由調控組蛋白修飾酵素aurora B, Set8, Msk1, Pcaf及Gcn5的表現而影響了H3K9, H3K18及H3K23的乙醯化、H3S10及H3S28的磷酸化及H4K20的甲基化，而squamocin也可以藉由降低aurora B及Msk1的活性來減少H3S10及H3S28的磷酸化程度。此外，此二種化合物最終皆會抑制癌細胞的增生、造成細胞生長週期的停滯、並使得癌細胞走向細胞凋亡。綜合上述結果，本研究從天然物中所萃取之化合物squamocin及calactin可藉由調控組蛋白修飾作用讓癌細胞在外基因層面上發生改變造成癌細胞死亡，而提出一個新的抗癌觀點。

Histone modifications in tumorigenesis are increasingly recognized as important epigenetic factors leading to cancer. The covalent modifications of the N-terminal tail domains, such as acetylation, methylation, and phosphorylation, are recognized as crucial epigenetic marks that modulate gene expression and genomic function. Aberrant histone modifications may be caused by improper activities of histone-modifying enzymes, leading to inappropriate expression of tumorigenesis-related genes.
Since nowadays the natural products play more and more important roles in drug discovery. In this study, we found that squamocin (isolated from Annonaceae family) and calactin (isolated from Asclepiadaceae family) showed strong cytotoxic activities against various human cancer cell lines. We demonstrated that calactin had the ability to modulate the expressions of histone modification enzymes such as aurora B, Set8, Msk1, Pcaf and Gcn5, which alter the levels of H3K9, H3K18, and H3K23 acetylation, H3S10 and H3S28 phosphorylation, and H4K20 methylation. On the other hand, squamocin could also modulate the phosphorylation levels of H3S10 and H3S28 in association with the downregulation of aurora B and pMsk1 expressions. In addition, both of the two compounds could inhibit the proliferation of cancer cells, arrest the cell cycle, and subsequently lead cells to apoptosis. This study is the first to show that calactin and squamocin affect epigenetic alterations by modulating histone modification, providing a novel view of the antitumor mechanism of these compounds.

中文摘要.................................................................................... 7
英文摘要.................................................................................... 9
1. 前言................................................................................... 11
1.1 癌症與組蛋白修飾...............................................................11
1.1.1 組蛋白..........................................................................12
1.1.2 組蛋白乙醯化..............................................................13
1.1.3 組蛋白甲基化..............................................................14
1.1.4 組蛋白磷酸化..............................................................16
1.2 Mitogen-activated protein kinase (MAPK)路徑...................17
1.3 細胞週期...............................................................................18
1.4 細胞凋亡...............................................................................21
1.5 研究目的...............................................................................23
2. 材料與方法....................................................................... 24
2.1 細胞培養條件及方法...........................................................24
2.2 細胞存活率的測定(MTT assay) ..........................................24
2.3 RNA萃取(RNA extraction) ..................................................25
2.4 反轉錄聚合酵素鏈鎖反應(reverse transcription polymerase
chain reaction) ......................................................................25
2
2.5 即時定量PCR (real-time quantitative PCR).........................26
2.6 蛋白質萃取(protein extraction) ............................................28
2.7 西方墨點法(western blot analysis).......................................29
2.8 流式細胞儀(flow cytometry)................................................32
2.9 分子對接(Docking)...............................................................32
2.10 數據統計分析( Analysis of data ).........................................33
3. 實驗結果........................................................................... 34
3.1 Squamocin .............................................................................34
3.1.1 Squamocin對於人類癌細胞株之細胞毒殺實驗.........34
3.1.2 Squamocin對人類癌細胞株之組蛋白修飾酵素基因表現
量之影響......................................................................34
3.1.3 Squamocin對人類癌細胞株之組蛋白修飾酵素及組白
修飾蛋白質表現量之影響..........................................35
3.1.4 Squamocin對人類癌細胞株細胞週期之影響.............35
3.1.5 Squamocin對人類癌細胞株細胞凋亡相關蛋白質的影
響..................................................................................36
3.2 Calactin.....................................................................................37
3.2.1 Calactin對於人類血癌細胞株細胞毒殺實驗.............37
3.2.2 Calactin對K562 細胞株之組蛋白修飾酵素基因表現量之
3
影響..............................................................................38
3.2.3 Calactin對K562 細胞株之組蛋白修飾酵素及組蛋白修飾
蛋白質表現量之影響..................................................38
3.2.4 Calactin對K562 細胞株細胞週期之影響...................39
3.2.5 Calactin對K562 細胞株細胞凋亡相關蛋白質的影響40
3.2.6 Calactin可以分子對接(docking)至Top1 之中.............41
4. 討論..................................................................................... 42
4.1 Squamocin .............................................................................42
4.2 Calactin..................................................................................45
4.3 結論.......................................................................................48
5. 圖表說明............................................................................. 49
圖1. DNA與組蛋白所形成之核小體結構...............................49
圖2. Squamocin之結構..............................................................50
圖3. Calactin之結構...................................................................50
圖4. MTT assay分析squamocin對於癌細胞株之細胞毒殺效果
............................................................................................51
圖5. Q-RT-PCR分析squamocin對於癌細胞株之aurora B及
Msk1 基因表現之影響......................................................52
圖6. 西方點墨法分析squamocin對於癌細胞株之aurora B,
4
Msk1, H3S10p及H3S28p蛋白質表現之影響..................53
圖7. 流式細胞儀分析squamocin對於癌細胞株之細胞週期之
影響....................................................................................54
圖8. 流式細胞儀分析squamocin對於癌細胞株之細胞凋亡比
例之影響............................................................................55
圖9. 西方點墨法分析squamocin對於癌細胞株之PARP, pERK,
pJNK以及活化態caspase-3, -8,及-9 蛋白質表現量之影
響........................................................................................56
圖10. MTT assay分析calactin對於K562 細胞株之細胞毒殺效
果......................................................................................57
圖11. MTT assay分析calactin對於Molt-4 細胞株之細胞毒殺效
果......................................................................................58
圖12. Q-RT-PCR分析calactin對K562 細胞株組蛋白修飾酵素基
因表現之影響..................................................................59
圖13. 西方點墨法分析calactin對於K562 細胞株之組蛋白修飾
酵素及組白修飾蛋白質表現量之影響............................60
圖14. 流式細胞儀及西方墨點法分析calactin對於K562 細胞株
之細胞週期之影響............................................................61
圖15. 流式細胞儀分析calactin對於K562 細胞株之細胞凋亡比
5
例之影響..........................................................................62
圖16. 西方墨點法分析calactin對於K562 細胞株Bax, Bcl2,
pERK, PARP及活化態caspase-3, -8,及-9 蛋白質表現之
影響..................................................................................63
圖17. Calactin 可docking至Top1-DNA複合物........................64
圖18. Squamocin誘發癌細胞之細胞週期停滯及凋亡之推測圖
..........................................................................................65
圖19. Calactin誘發癌細胞之細胞週期停滯及凋亡之推測圖66
6. 參考文獻........................................................................... 67
7. 論文................................................................................... 82
7.1 會議論文.................................................................................82
7.2 期刊論文.................................................................................84
8. 論文期刊抽印…………………………...………………….......86
8.1 Squamocin modulates histone H3 phosphorylation levels and
induces G1 phase arrest and apoptosis in cancer
cells…………………………………………………………..86
8.2 16-Hydroxycleroda-3,13-dien-15,16-olide deregulates PI3K
and Aurora B activities that involve in cancer cell apoptosis.96
8.3 Amiloride modulates alternative splicing in leukemic cells and
6
re-sensitizes Bcr-AblT315I mutant cells to
imatinib……………………………………………………..106

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