化合物AZ-4與粒線體電子傳遞鏈complex I抑制劑對肺癌細胞的抗癌作用

中文摘要研究指出aziridinylbenzoquinone類化合物具有抗癌活性，結構中之aziridine具有基化去氧核糖核酸，進而抑制去氧核糖核酸複製的能力。本論文所使用之抗癌藥物為全合成化合物AZ-4，其結構上帶有兩個aziridine環，並且分別位於兩個以-(CH2)-O-(CH2)-O-(CH2)-O-(CH2)-O-(CH2)-架橋連接的naphthoquinone上。AZ-4對非小型肺癌細胞H460的LD50為1.25mM，且AZ-4對H460的細胞毒性會隨著濃度與時間的增加而變強。以流氏細胞儀觀察細胞週期的變化可發現，AZ-4濃度為0.125mM-0.5mM時，會使細胞停留在G2/M時期；G2/M時期的相關蛋白Cdc2的表現約會減少50 %，但CyclinB的表現則不會隨著AZ-4濃度的增加而有明顯的變化。而以流氏細胞儀觀察細胞週期sub-G1則可發現，AZ-4濃度為1mM和2mM時，會使H460走向程式化凋亡，相同濃度於Hoechst染色的實驗中也可發現凋亡小體的出現；於NMR的實驗中亦觀察到CH2/CH3會上升。電子傳遞鏈對細胞的生存扮演著非常重要的角色，粒線體內膜上電子傳遞鍊的五個complexes，各自負責電子傳遞或是產生ATP，提供生命體活動所需之能量。其中NADH:ubiquinone oxidoreductase，是電子傳遞鏈的第一個complex，簡稱為complex I。1998年Fang等人的研究中指出，當complex I被抑制時，間接也會抑制負責合成多氨類的酵素：ornithine decarboxylase(ODC)的活性。帶正電的多氨類可以穩定帶負電的去氧核糖核酸，幫助去氧核糖核酸的複製，加速細胞的生長；因此一旦抑制ODC，使多氨類的生合成減少，便能阻礙去氧核糖核酸的複製，抑制癌細胞的生長。本論文的研究主題主要是探討抑制癌細胞粒線體complex I的活性，降低癌細胞的增生速率，再配合抗癌藥物AZ-4，希望能降低AZ-4的使用劑量。目前已知具有最佳抑制效果的complex I抑制劑是rotenone，但是它對正常細胞的毒性很強，因此rotenone不能用在抗癌治療上。Pterulone是一種從真菌中所發現的complex I抑制劑，對細胞的毒性低，但是抑制效果稍差(IC50 = 36mM)，於是我們利用有機合成的方式，改變pterulone的官能基，使它與complex I active site的結合更加緊密，變成更好的complex I抑制劑。目前合成得到的最好之pterulone 衍生物為trimethyl benzyl ether(IC50 = 8.5mM)，和最強的complex I抑制劑：rotenone(IC50 = 0.5nM)仍有很大的差距。為了證實本論文的想法是否正確，於是先以complex I抑制劑：rotenone降低癌細胞的生長速率，觀察是否可以進一步降低抗癌藥物AZ-4的使用劑量；應此我們先以complex I抑制劑：rotenone抑制肺癌細胞(H460)生長，配合自行合成出的抗癌藥物：AZ4，觀察是否在rotenone的作用下，可以降低對非小型肺癌細胞(H460)的使用劑量。

關鍵字

肺癌；粒線體；電子傳遞鏈；細胞凋亡；細胞週期

並列摘要

英文摘要 AZ-4 is kind of anti-cancer drug like bis-aziridinoquinonyl thiaters. It’s a new compound that developed from aziridinylbenzoquinone drug. In the previous experiments, the ability of DNA alkylation by aziridinylbenzoquinone compounds had been confirmed. It’s used to disturb DNA replication and decrease the survival rate of cancer cell. We synthesis a new bis-type anti-cancer drug AZ4 by the different chain length and composed of linker. From the results, we confirmed the ability of DNA alkylation and the cross-link with DNA by AZ4. In the preliminary data, it’s found that the AZ4 induces cell death of human lung cancer (H460) in a dose-dependent and time-dependent manner. The IC50 of AZC-5 to H460 cell is 5.5 mM by MTT assay. We also found that the AZ4 makes a lower lethal effect on human skin fibroblast cell at same concentration of H460. According to the results of flow cytometry the AZ4 induced the Hep-2 cell S phase arrest in 0.75 mM and G2/M phase in 3 mM. The apoptotic signal progression of H460 induced by AZ4 included the increase the ratio of CH2/CH3 from cell membrane phospholipid motion in a dose- & time-dependent manner as determined by 1H NMR analysis. Besides, data from Hoechst staining also revealed that a lot of apoptotic bodies could be found at 60 hrs. after treatment by AZ4.From Western blot results , we found the AZ4- could induced the H460 cell arrest at G2/M phase at 3 mM . Ⅳ

並列關鍵字

lung cancer ； mitochondria ； electron transport chain ； apoptosis ； cell cycle

參考文獻

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國際替代計量

化合物AZ-4與粒線體電子傳遞鏈complex I抑制劑對肺癌細胞的抗癌作用

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