抗有絲分裂藥物已被報導具有抗腫瘤生成的能力，其中Combretastatin A-4 (CA-4)被認為是最具有抗癌活性的藥物之一，其特色是擁有抗微管聚合的特性並可抑制腫瘤血管的新生。烯雙炔類藥物是藉由模擬CA-4的結構而達到具有抗癌活性的新型藥物，先前的研究已經指出LO-OMe與LO-NH2對於不同的癌細胞株具有高度的細胞毒殺性，但仍未有此藥物對於神經母細胞瘤細胞株SH-SY5Y的抗癌活性之報告。因此，本研究將探討CA-4其衍生物對於神經母細胞瘤細胞株SH-SY5Y的毒殺效力，並進一步深究其抗癌機制。經細胞存活試驗得知LO-OMe與LO-NH2處理48小時的IC50的濃度分別為3.2 μM與2.1 μM。LO-OMe與LO-NH2處理均導致自噬作用標幟蛋白LC3-II增加，且p62(亦稱為sequestosome, SQSTM1)蛋白有堆積的現象。此外在GFP-LC3報導H4細胞株實驗中，新型烯雙炔藥物之處理皆能顯著誘導細胞中含綠螢光量點數量之增加，且螢光量點之大小也有提升現象，顯示此藥物可能會誘導細胞自噬體之累積。另一方面，我們先前的研究也指出LO-OMe與LO-NH2會造成SH-SY5Y細胞週期停滯在G2/M時期，而此類抗微管藥物已被證實能有效誘發計畫性細胞凋亡。在LO-OMe與LO-NH2處理的SH-SY5Y細胞中可偵測到活化型Caspase-3，並造成其下游cleavaged PARP的含量顯著提升，這些數據顯示藥物處理促使SH-SY5Y走向計畫性細胞凋亡路徑。綜合前述實驗數據，我們得知這兩種新型的烯雙炔藥物會誘發SH-SY5Y細胞中自噬體之累積，而由p62蛋白堆積的現象則指出細胞自噬體可能無法順利被送達溶酶體降解，我們認為可能因新型烯雙炔藥物具有抑制微管聚合之活性所致。當細胞無法藉由自噬作用抵禦微環境的壓力便會誘發計畫性細胞凋亡，因而促使這類的新型烯雙炔藥物具有毒殺癌細胞之活性，但其詳細的分子訊息機轉有待進一步的深入探討。

Antimitotic drug, combretastatin A-4 (CA-4) is a natural plant extract exhibits powerful anticancer and anti-angiogenesis activity by targeting microtubule formation. CA-4 enediyne derivatives, such as LO-OMe and LO-NH2 were shown to possess potent cytotoxicity in various cancer cell lines except human neuroblastoma cell. Here, we further investigate the cytotoxic effects of LO-OMe and LO-NH2 on neuroblastoma SH-SY5Y cells. Our viability assay demonstrated that LO-OMe and LO-NH2 mediated 50 percent killing of SH-SY5Y cells at a concentration of 3.2 and 2.1 µM, respectively. Cell lysates of drug treated cells were collected for analyzing markers of cellular autophagy and apoptosis using antibodies against LC3, p62, caspase-3, cleaved PARP. The results indicated a dose-dependent increase of LC3-II/LC3-I ratio and p62 protein upon drug treatments. In addition, consistent effects were observed in GFP-LC3 reporter H4 cell line. Both compounds elicited the increase in the number and the size of GFP-LC3 puncta, indicating an accumulation of autophagosomes in reporter cells. Our previous data showed LO-OMe and LO-NH2 treatment led to cell cycle arrest at G2/M. In addition, LO-OMe and LO-NH2 also significantly unregulated the level of active caspase-3, which resolved in the increase of cleaved-PARP. In summary, our results showed that LO-OMe and LO-NH2 induced the increase of autophagosomes. The accumulation of p62 indicated an inhibition in the fusion between autophagosome and lysosome, which might be due to the interference of microtubule polymerization exerted by these compounds. Unresolved cellular stress then resulted in programmed cell death of SH-SY5Y neuroblastoma cells. However, the molecular details awaits further investigation.

目錄
論文審定書 i
致謝 ii
中文摘要 iii
英文摘要 iv
前言 - 1 -
第一章:抗有絲分裂試劑(Antimitotic agents) - 1 -
1.微管與抗有絲分裂試劑 - 1 -
2.微管聚合抑制劑(Microtubule-destabilizing agents，MDAs) - 3 -
3.微管聚合穩定劑(Microtubule-stabilizing agents，MSAs) - 4 -
4.Combretastatin A-4 - 4 -
5.新型烯雙炔藥物(Enediyne) - 5 -
第二章:細胞自噬(Autophagy) - 7 -
1.細胞自噬的緣起 - 7 -
2.細胞自噬的分子機制 - 8 -
3.微管、微管相關蛋白MAPL3B與細胞自噬 - 11 -
4.泛素相關蛋白P62(SQSTM1)與細胞自噬 - 13 -
第三章:細胞凋亡(Apoptosis) - 15 -
1.細胞凋亡 - 15 -
2.細胞凋亡途徑 - 16 -
3.Bcl-2家族蛋白 - 17 -
4.Caspase蛋白酶 - 18 -
5.PARP(Poly ADP ribose polymerase) - 19 -
研究動機 - 21 -
材料與方法 - 22 -
新型烯雙炔藥物 - 22 -
SH-SY5Y細胞培養與製備 - 22 -
細胞存活率試驗 - 23 -
蛋白質萃取 - 23 -
蛋白質定量 - 24 -
西方墨點法 - 25 -
共軛焦顯微鏡樣品製備 - 26 -
實驗結果 - 28 -
CA-4及其烯雙炔衍生物對SH-SY5Y細胞毒殺性 - 28 -
CA-4及其烯雙炔衍生物能夠誘發H4細胞自噬 - 29 -
CA-4及其烯雙炔衍生物能夠誘發SH-SY5Y細胞自噬 - 29 -
CA4及其烯雙炔演生物能夠誘發SH-SY5Y細胞凋亡 - 30 -
討論與總結 - 32 -
附錄 - 42 -
引用文獻 - 48 -

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