AZC-5(Aziridinoquinonylcarbon-5)對DNA烷基化作用能力的研究及其對Hep-2喉癌細胞毒殺作用的探討

Studies on the DNA alkylation ability of AZC-5 and possible mechanism of death effect on Hep-2 cell induced by AZC-5

蔡欣達(Hsin-Da Tsai)

指導教授 ： 林鈺玲

臺北醫學大學/醫學院/醫學科學研究所/碩士(2002年)

摘要

AZC-5 ( bis-Aziridinoquinoylcarbon-5的簡稱)為bis-Aziridinoquinonyl 類的抗癌藥物。而上述藥物是由Aziridinylbenzoquino類的化合物衍生而來。在過去已經有實驗證實此類化合物可以藉由烷基化DNA ，使DNA與化合物結合，而影響DNA雙股螺旋打開，進而影響癌細胞的增殖。本論文所用藥物AZC-5即是有此特性的新合成雙體結構的化合物。由 in vitro 的實驗結果證明AZC-5在2mM~200mM濃度下的確可烷基化DNA，使DNA雙股不易分開為單股。而在細胞毒性及存活率的實驗中 , 利用MTT assay的方法測試細胞的相對存活率及Trypan blue染色計算細胞死亡率。結果發現人類喉癌細胞Hep-2隨著劑量及時間的增加會增加其死亡率 , 並且與正常細胞Skin Fibroblast有明顯差異。Hep-2細胞經AZC-5作用24 hrs.後其IC50=5.5mM。 而在AZC-5造成Hep-2細胞死亡的機制探討上，我們發現在AZC-5作用24 hrs.後低於0.75mM時會arrest在G2/M phase而大於0.75mM至3mM時則使細胞arrest 在G2/M phase ，而當藥物作用到60 hrs.時除了有G2/M arrest外並且可以在floe cytometry儀器的觀察下看到sub-G1 峰的出現，但對於調控細胞週期的重要蛋白 cdc-2 與 cdk-2在總量表現上並沒有明顯變化。 由Hoechst stain結果中發現AZC-5作用60 hrs.後在1.5mM有明顯的apoptotic body , 而1H NMR上所得到的結果也顯示細胞膜磷酯質CH2/CH3的比值隨AZC-5作用時間的增加也顯著的上升。由western blot的結果中看到AZC-5處理Hep-2 cell 24hrs.後, p53蛋白有略微活化增加的現象 , RNA結合物質TIAR蛋白表現量也大幅增加至3倍，抑制apoptosis產生的Bcl-2蛋白表現量也很高 , 在60 hrs.時Bcl-2表現量才下降。

關鍵字

相對存活率 ； 凋亡 ； 烷化

並列摘要

AZC-5 (bis-Aziridinoquinoylcarbon-5) is a 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 AZC-5 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 AZC-5. In the preliminary data, it’s found that the AZC-5 induces cell death of human larynx cancer cell (Hep-2) in a dose-dependent and time-dependent manner. The IC50 of AZC-5 to Hep-2 cell is 5.5 mM by MTT assay. We also found that the AZC-5 makes a lower lethal effect on human skin fibroblast cell at same concentration of Hep-2. According to the results of flow cytometry the AZC-5 induced the Hep-2 cell S phase arrest in 0.75 mM and G2/M phase in 3 mM. The apoptotic signal progression of Hep-2 induced by AZC-5 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 AZC-5.From Western blot results , we found the AZC-5 could induced the Hep-2 cell arrest at G2/M phase at 3 mM .

並列關鍵字

AZC-5 ； alkylation ； western blot ； Hoechst stain ； flow cytometry ； apoptosis ； cell cycle arrest ； 1H NNR

參考文獻

Lusthofa , Nicolaas J. DE Mola , Lambert H.M. Janssena , Richard J.M. Egberinkb , Willem Verboomb and David N. Reinhoudtb Chem.-Biol. Interaction , 76 (1990) 193-209

John A. Hartley, 1Mark D. Berardini , and Robert L. Souhami Analytical Biochemistry 193, 131-134(1991)

Francis G. Blankenberg , Peter D. Katsikis , Richard W. Storrs , Christian Beaulieu , Daniel spielman , James Y. Chen , Louie Naumovski , and Jonathan F. Tait Blood, vol 89 , No 10 (May 15), 1997: pp 3778-3786

Ashkenazi,A.,and Dixit, V.M. Death receptors: signaling and modulation. Science(Washington DC). 281:1305-1308,1998.

Amy C Hobeika, Prem S Subramaniam and Howard M Johnson a Induces the Expression of the Cyclin-dependent Kinase Inhibitor p21 in Human Prostate Cancer Cells Oncogen 1997 (1997) 14, 1165-1170