吳茱萸鹼透過對第一型DNA拓撲異構酶之 抑制活性毒殺癌細胞之機制探討

Topoisomerase I inhibition mediates evodiamine cytotoxicity on cancer cells

林惠元(Hui-Yuan Lin)

指導教授 ： 林俊茂

臺北醫學大學/醫學院/醫學科學研究所/碩士(2009年)

https://doi.org/10.6831/TMU.2009.00066

摘要

用來調節DNA 鬆懈的第一型DNA拓撲異構酶(Topo I)對於DNA的複製、轉錄和重組是不可或缺的一角。在過去的研究顯示，許多癌細胞中Topo I的活性都特別的高，因此Topo I便被視為癌症治療的重要標的。CPT是已知的Topo I標靶藥物，但細胞對之產生抗藥性的問題仍是個尚未解決的難題。先前的研究指出，一種從吳茱萸植物所萃取出的吳茱萸鹼(EVO)，在多種癌細胞中證實具有抗癌的效果，然而它對Topo I的抑制影響尚未明瞭。在我們這次的研究中，我們便去探討EVO作用在癌細胞中的Topo I的詳細機轉。我們發現EVO會抑制MCF-7細胞生長並對之有細胞毒性(IC50=5.66μM) ，而隨著時間和劑量的增加，EVO活化p53、抑制Topo I的活性以及降低Topo I的蛋白表現的程度則更為明顯。利用流式細胞儀分析細胞週期後也發現，細胞在加入EVO後會停滯在G2/M期。再進一步使用siRNA knockdown Topo I，發現EVO對MCF-7細胞的細胞毒性下降了，最後還發現EVO對A2780R2000 CPT抗藥株細胞具有毒殺效果。由以上這些結果顯示，EVO是個具有潛力的Topo I抑制劑，這代表著在將來治療抗CPT的癌細胞時，EVO會是個高度令人期待的替代治療藥物。

關鍵字

第一型DNA拓撲異構酶吳茱萸鹼

並列摘要

The regulation of DNA relaxation by Topo I is essential for DNA replication, transcription, and recombination events. Topo I activity is elevated in various cancer cells, thus Topo I has been identified as an important therapeutic target in cancer chemotherapy. CPT is the representative drug that targets Topo I, however drug resistance is still the main problem in the successful treatment of cancer. Previous studies demonstrated that EVO, a major constituent of Chinese herb evodiae fructus, exhibited anti-tumor activities on several cancers, but its inhibition effects on Topo I remained unclear. In our present study, the action mechanisms of EVO on the Topo I in cancer cells were explored. EVO inhibited the growth and increased cytotoxicity of MCF-7 cells (IC50=5.66μM). EVO activated p53 and inhibited Topo I activity and decreased Topo I protein level in a time and dose dependent manner. The flow cytometric cell cycle analysis of EVO treated cells indicated a block of G2/M phase. After knockdown of Topo I by topoisomerase I siRNA, EVO showed decreasing cytotoxicity to MCF-7 cells. We also found that EVO had cytotoxic effect in A2780R2000 CPT resistance cell lines. These results suggested that EVO is a potent Topo I poison, and therefore represents a highly promising chemotherapeutic agent in the treatment of CPT resistant cancer cells.

並列關鍵字

Topoisomerase I ； Evodiamine

參考文獻

Arbuck SG, Takimoto C. 1995, 9-Aminocamptothecin. The sixth conference on DNA topoisomerases in therapy.

Barton J Bradbury and Michael J Pucci, 2008, Recent advances in bacterial topoisomerase inhibitors, Current Opinion in Pharmacology 8:574–581

Boonsong A, Curran S, McKay JA, Cassidy J, Murray GI, McLeod HL , 2002, Topoisomerase I protein expression in primary colorectal cancer and lymph node metastases. Hum Pathol 33:1114–1119

Burden DA, OsheroV N, 1998, Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim Biophys Acta 1400:139–154

Champoux JJ. 2001, DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–413.