The Anti-Cancer Mechanism of Evodiamine, Genistein and YC-1 in Human Breast Cancer Cell Lines

癌症是一種成因複雜的慢性疾病，從正常細胞的癌化、癌細胞轉移到癌細胞抗藥性的生成，整個過程不僅由眾多訊息傳遞路徑參與調控，還有許多癌細胞與周圍環境的交互作用而成。本論文將焦點放在乳癌細胞，著重在探索研發的藥物抑制癌細胞生長與促進癌細胞凋亡的作用機轉。 第一篇實驗主要討論中草藥吳茱萸萃取物evodiamine引起人類多重抗藥性乳癌細胞NCI/ADR-RES凋亡的機轉。Evodiamine主要是經由引起細胞內微小管的聚集，造成細胞週期停滯於G2/M期，造成Raf-1蛋白激酶與Bcl-2蛋白磷酸化，使Bcl-2蛋白抑制細胞凋亡的功能喪失，導致細胞死亡。將人類多重抗藥性乳癌細胞NCI/ADR-RES殖入裸鼠背部評估evodiamine在生物體內的活性，evodiamine可以明顯抑制癌細胞的生長，抗癌效果較paclitaxel來的好。實驗證明evodiamine是個具有潛力的lead compound，值得開發用來治療癌細胞具有抗藥性的癌症病患。 第二篇實驗主要探討合併給予日常防癌食品的主成分與治療乳癌的化療藥物，有可能影響化療藥物的作用。我們的實驗發現大豆異黃酮(genistein)會顯著降低微小管結合藥物(paclitaxel與vincristine)所引起的細胞凋亡。大豆異黃酮干擾微小管結合藥物的作用不是影響了藥物與微小管之間的結合或是影響了微小管的動態平衡，而是透過降低了微小管所引起的cyclin B1/cdc2 kinase的表現，進而降低Bcl-2蛋白磷酸化所致。因此genistein可能會降低微小管結合藥物之藥效。 第三篇實驗主要討論benzyl-indazole類化合物YC-1引起人類乳癌細胞MCF-7細胞凋亡的機轉。根據實驗結果，我們認為YC-1引起人類乳癌細胞MCF-7細胞凋亡可能是經由活化轉錄因子E2F1，使下游p53與p73的蛋白累積，並抑制了NF-B的活性，進而引起造成人類乳癌細胞凋亡。此外，YC-1可以抑制血管新生、抑制癌細胞HIF-1的表現及抑制免疫細胞釋放cytokines，再加上YC-1對癌細胞有抑制生長與引起細胞凋亡的作用，我們認為YC-1對於敏感性較差的人類多重抗藥性乳癌細胞NCI/ADR-RES的動物實驗仍有良好的藥效，應是綜合上述作用所致，也因此我們認為YC-1在臨床化學治療的使用極具潛力。Cancer is a chronic disease with complicated factors. There are multiple signaling pathways regulated in carcinogenesis, metastasis and multiple drug resistance. The interactions between cancer and the microenvironment also play important roles in the disease. The present study is focused on the antitumor effects of the developing drugs on inhibiting the proliferation and inducing apoptosis in human breast cancer cells. The first part of this research is to elvaluate the apoptotic mechanism of evodiamine in human multiple drug-resistant breast cancer cells NCI/ADR-RES. Evodiamine induced tubulin polymerization resulted in cell cycle arrest at G2/M phase, Raf-1 kinase and Bcl-2 phosphorylation and leading to cell apoptosis. The in vivo anticancer effects of evodiamine evaluated in tumor xenograft implantation of NCI/ADR-RES cells were superior to that of paclitaxel. Evodiamine therefore represents a highly promising chemotherapeutic lead compound for treatment of human multiple-drug resistant cancer cells. The second part of this research is to study the inversion of chemoprevensive components and chemotherapeutic agents. We found that genistein, the phytoestrogen present in soybeans, specifically inhibited apoptosis induced by tubulin-binding agents, paclitaxel and vincristine. Genistein did not alter the binding of tubulin or the tubulin dynamic. Genistein abrogated apoptosis caused by paclitaxel and vincristine via decreasing cyclin B1/cdc2 kinase expression leading to down-regulation of Bcl-2 phosphorylation. The last part of this research is to study the apoptotic mechanism of YC-1 in human breast cancer cells, MCF-7. According to the results, we hypothesized that YC-1 increased the activity of the transcription factor E2F1 and caused following signaling transduction, p53 and p73 accumulation and inhibition on NF-B activity, resulting in cancer cell apoptosis. Moreover, multiple effects of YC-1, such as anti-angiogenesis, down-regulation of HIF-1 expression and inhibition on cytokines release, contribute to anti-cancer effects on xenograft of human multiple drug-resistant cancer NCI/ADR-RES, eventhough NCI/ADR-RES cells exhibited lower sensitivity to YC-1. Therefore, YC-1 showed a potential therapeutic efficacy in cancer chemotherapy.縮寫表 1 摘要 3 英文摘要 5 緒論 7 研究動機與目的 7 文獻回顧 9 實驗材料與方法 37 第一章 吳茱萸成分EVODIAMINE在人類多重抗藥性乳癌細胞NCI/ADR-RES作用機轉之探討 45 中文摘要 47 英文摘要 48 緒言 49 材料與方法 52 結果 53 討論 58 第二章 GENISTEIN干擾微小管結合藥物引起人類乳癌細胞凋亡之作用機轉 71 中文摘要 73 英文摘要 74 緒言 75 材料與方法 78 結果 79 討論 83 第三章 YC-1引起人類乳癌細胞MCF-7細胞凋亡之機轉探討 95 中文摘要 97 英文摘要 98 緒言 99 材料與方法 104 結果 105 討論 110 總結與展望 123 著作 125 參考文獻 12