||A screening-based approach to select drugs for ovarian cancer treatment
||Institute of Oral Medicine
卵巢癌為婦女癌症死亡率的第一名。大部分的卵巢癌患者無法被早期診斷，整體病人的五年存活率低於50%。儘管化療一開始幾乎都可使疾病得到控制，但最後常因抗藥性的產生而死亡。因此，尋找潛在的治療藥物對於惡性卵巢癌的治療是迫切需要的。我們選擇兩個對順鉑（cisplatin）有不同耐受性的卵巢癌細胞株A2780和A2780CP70做為篩選新的治療藥物模組，希望找到有效毒殺抗藥性細胞的藥物。這裡我們利用Cmap資料庫預測以及即時、高通量的篩選系統RTCA，使用一個包含640個經過生物活性且經過美國食品藥物管理局認證的藥物庫來篩選出治療惡性卵巢癌的藥物。我們藉由兩株細胞總共分析篩選了100個藥物，經過實驗分析後最後挑選了MPA和DS候選藥物，此二藥物同時具有抑制抗藥性細胞A2780CP70以及A2780癌細胞的效果。因此我們更進一步透過西方墨點法分析處理藥物後細胞凋亡標記cleaved PARP以及caspase-3活化的表現量。實驗結果得知MPA、DS藥物可以誘導cleaved PARP蛋白表現量上升。此外，我們也觀察了候選藥物對於細胞週期的影響。實驗結果發現MPA藥物造成細胞停滯G1/S phase，而且Sub/G1的細胞比例也有增加。在DS藥物處理下，使A2780及A2780CP70細胞停滯於G2/M。Sub/G1細胞比例些微上升。從實驗結果知道，MPA、DS能抑制細胞的增殖，並會誘導細胞死亡。因此，可以知道MPA與DS是潛在的卵巢癌治療藥物。而這些FDA認證藥物將可以提供惡性卵巢癌患者新的治療策略。
Ovarian cancer is the most lethal gynecologic malignancy. Most patients with ovarian cancer are diagnosed in advanced stages and the overall five-year survival rate for all stages of ovarian cancer is less than 50%. In spite of the high response rate of initial treatment, the majority will relapse and eventually die due to the development of chemoresistance. Therefore, to screen new therapeutic drugs for patients with malignant ovarian cancer is urgently needed. For screening new therapeutic drugs, we used a chemoresistant ovarian cancer cell line pair, A2780 and A2780CP70. We used connectivity map (Cmap) database prediction and a FDA approved drug library, in which 640 diverse compounds have well-characterized bioactivities, to screen therapeutic drugs by a real time and high-throughput screening RTCA system. 100 FDA approved drugs have been screened and we selected the 2 candidate compounds, MPA and DS, which had a good cytotoxicity on A2780CP70 and parental cells. Furthermore, we treated A2780CP70 and A2780 with MPA and DS and analyzed the protein level of cleaved PARP and active form caspase-3, which plays a significant role in apoptosis, by western blot analyses. The result showed that MPA induced the protein level upregulation of cleaved PARP. But, DS didn’t significantly cause apoptotic processes. In addition, we assessed the effect of MPA and DS on cell cycle perturbations. Cell cycle analysis showed that MPA caused A2780CP70 and A2780 cell cycle arrest at G1/S phase, and the amount of cells at Sub-G1 increased. Treatment of DS in A2780CP70 and A2780 cells showed that the cell cycle was arrested at G2/M phase. Our data suggest that MPA and DS inhibited cell growth, and also induced cell death. Thus, MPA and DS are potential therapeutic drugs for ovarian cancer. In the future, these FDA approved drugs could provide a new treatment strategy for patients with advanced ovarian cancer.
第一章 緒論 1
第一節 、卵巢癌(ovarian cancer) 1
第二節 、卵巢癌與癌幹細胞 4
第三節 、老藥新用（Drug repurposing） 6
第四節 、細胞凋亡（Cell Apoptosis） 7
第五節 、細胞週期（Cell cycle） 10
第六節 、Connectivity map（Cmap） 11
第二章 研究動機及架構 13
第一節 、研究動機 13
第二節 、研究架構 14
第三章 實驗材料與方法 15
第四章 實驗結果 36
第五章 討論 51
第六章 結論 58
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