吳茱萸鹼衍生物對人類卵巢癌細胞之毒性及機制探討

The Mechanism of Evodiamine Derivatives Induced Cytotoxicity of Ovarian Carcinoma cells

蔡湘萍(Hsiang-Ping Thsai)

指導教授 ： 林俊茂 博士

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00250

摘要

拓撲異構酶(topoisomerase)參與DNA拓撲結構的改變，許多抗腫瘤化療藥物皆以拓撲異構酶為作用標靶。喜樹鹼(camptothecin, CPT)是已用於臨床治療的拓撲異構酶I (TopoI)抑制劑，但其抗藥性及嚴重副作用阻礙其應用性。本研究室先前發表一從吳茱萸植物所萃取出的吳茱萸鹼(evodiamine, EVO)，藉由將DNA與TopoI停留在TopoI-DNA cleavage complex而造成TopoI poison。在本研究中，我們探討吳茱萸之有效成分EVO及吳茱萸次鹼(RUT)對於人類卵巢癌細胞A2780之細胞毒性是否透過對TopoI的抑制作用。首先我們以拓撲異構酶分子嵌合模式模擬分析(TopoI Docking Study)預測藥物結合活性，並以表面耦合電漿分析儀及DNA解螺旋膠體分析佐證in vitro下， EVO及RUT可作為DNA intercalator以抑制拓撲異構酶活性。在細胞中，我們觀察這兩個藥物是否會依循TopoI poison模式造成細胞死亡。我們發現EVO可有效抑制人類卵巢癌細胞A2780增生，而RUT之細胞毒性相對較弱。以siRNA降低細胞中TopoI表現量，結果EVO對於A2780細胞的毒殺效果受到抑制，而RUT則無明顯差異。另外，EVO及RUT皆會造成A2780細胞 DNA damage，其中EVO降低細胞中自由態TopoI而RUT則反之。由以上結果推測，EVO係藉由作用在TopoI以造成對卵巢癌細胞A2780之細胞毒性。而RUT則可能經由其他不同機制。最後我們以EVO及RUT處理CPT抗藥株細胞A2780R2000，結果發現兩者皆具有毒殺效果，其中RUT之IC50更達0.85μM，這顯示在將來治療對於CPT產生抗藥性的癌細胞時，EVO及RUT會是個具有潛力的治療藥物。

關鍵字

吳茱萸鹼 ； 拓撲異構酶表面耦合電漿分析

並列摘要

The regulation of DNA topology by topoisomerase I (TopoI) is essential for DNA replication, transcription, and recombination events. Camptothecins (CPTs) are family of compounds that specifically target TopoI. These compounds ‘poison’ TopoI by stabilize reversible TopoI–DNA cleavage complex and prevent the religation of DNA. TopoI activity is elevated in various cancer cells, thus TopoI has been identified as an important therapeutic target in cancer chemotherapy. However drug resistance is the main problem in the successful treatment of cancer. Our previous studies have shown that evodiamine (EVO), a major constituent of Chinese herb evodiae fructus, exhibited anti-tumor activities by inhibit TopoI activity. In our present study, we demonstrated EVO and rutaecarpine (RUT) are strong topoisomerase inhibitors by using TopoI Docking Study, SPR assay and TopoI relaxation assay in vitro. EVO exhibited dose-dependent cytotoxicity of A2780 cells whereas RUT was less cytotoxicity than EVO. EVO showed decreasing cytotoxicity in siRNA-knockdown cells, but not for RUT. Both EVO and RUT induced DNA tailing in the Comet assay and elevation of γ-H2AX levels. EVO induced TopoI depletion in a dose-dependent manner while RUT increased TopoI level in the same treatment. Furthermore, we compared the cytotoxic effect of EVO and RUT in CPT resistance cell line A2780R2000. Both EVO and RUT were able to inhibit A2780R2000 cell growth. These results suggested that EVO and RUT are potential chemotherapeutic agents in the treatment of CPT resistant cancer cells.

並列關鍵字

Evodiamine ； topoisomerase I ； Surface plasmon resonance

參考文獻

1. Muggia FM, Braly PS, Brady MF, Sutton G, Niemann TH, Lentz SL, Alvarez RD, Kucera PR, Small JM: Phase III randomized study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or IV ovarian cancer: a gynecologic oncology group study. J Clin Oncol 2000, 18:106-115.

2. Paclitaxel plus carboplatin versus standard chemotherapy with either single-agent carboplatin or cyclophosphamide, doxorubicin, and cisplatin in women with ovarian cancer: the ICON3 randomised trial. Lancet 2002, 360:505-515.

3. Ozols RF, Bundy BN, Greer BE, Fowler JM, Clarke-Pearson D, Burger RA, Mannel RS, DeGeest K, Hartenbach EM, Baergen R: Phase III trial of carboplatin and paclitaxel compared with cisplatin and paclitaxel in patients with optimally resected stage III ovarian cancer: a Gynecologic Oncology Group study. J Clin Oncol 2003, 21:3194-3200.

4. Covens A, Carey M, Bryson P, Verma S, Fung Kee Fung M, Johnston M: Systematic review of first-line chemotherapy for newly diagnosed postoperative patients with stage II, III, or IV epithelial ovarian cancer. Gynecol Oncol 2002, 85:71-80.

5. ten Bokkel Huinink W, Gore M, Carmichael J, Gordon A, Malfetano J, Hudson I, Broom C, Scarabelli C, Davidson N, Spanczynski M, et al: Topotecan versus paclitaxel for the treatment of recurrent epithelial ovarian cancer. J Clin Oncol 1997, 15:2183-2193.