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研究生: 游雅竹
Ya Chu Yu
論文名稱: 1. 抑制肺癌腫瘤細胞增生的新穎小分子藥物篩選及其反應機制2. 玫瑰樹鹼對肺癌細胞誘導Akt及p53核移動及生長抑制
1.Identification of A Novel Small Molecule Compound That Induced Apoptotic Cell Death in Human Lung Cancer Adenocarcinoma Cells 2.The induced p53 and Akt nuclear translocation and growth inhibition by ellipticine in human lung cancer cells
指導教授: 方剛
Fang, Kang
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2012
畢業學年度: 101
語文別: 中文
論文頁數: 95
中文關鍵詞: 藥物篩選細胞自噬細胞凋亡p53玫瑰樹鹼Akt
英文關鍵詞: Drug screen, autophagy, apoptosis, p53, ellipticine, Akt
論文種類: 學術論文
相關次數: 點閱:189下載:2
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  • 1.本論文使用肺癌細胞株為模式,自42個化學合成有機小分子,利用細胞生長速率測試 (MTT assay),篩選出在低濃度下可以降低細胞生長速率之藥物。其中oxazoline衍生物MZ-01-059能夠有效在5 μM下降低A549細胞的生長速率。次由細胞群落分析證實MZ-01-059可以抑制癌細胞的增生。為了確認非小細胞肺癌的生長率下降是否與細胞凋亡相關,實驗再利用propidium iodide染色後DNA,進行後流式細胞分析,觀察細胞週期變化。實驗結果顯示MZ-01-059會讓具有野生型p53之細胞株A549及H460產生細胞凋亡。其中以H460細胞株較為顯著;但對不具有p53之H1299細胞,則會停滯在S及G2/M週期。另由二維流式細胞儀分析結果,確立H460肺癌細胞主要是以晚期細胞凋亡 (late apoptosis) 方式降低細胞存活率。由西方轉漬法鑑定與細胞凋亡相關的蛋白,發現MZ-01-059處理後細胞,會活化腫瘤抑制基因p53,此外也會使pro-caspase-3以及poly ADP ribose polymerase切割;而LC3-II (microtubule-associated protein 1 light chain 3) 在A549及H1299細胞株會增加,因此推斷這兩種細胞是先以細胞自噬保護細胞,再產生細胞凋亡,證實這個藥物所引發細胞凋亡與細胞自噬相關。本論文結果也顯示MZ-01-059能夠在低濃度下誘導活化在非小細胞肺癌引發不同程度的細胞凋亡,且與p53活化有關。

    2.拓樸異構酶II抑制劑玫瑰樹鹼 (ellipticine) 為具有抑制癌細胞生長的抗癌藥物,本實驗室過去研究指出藥物可經由活化p53,使人類非小細胞肺癌A549細胞株凋亡。本論文持續探討細胞生長因子Akt對於p53進入細胞核的影響,研究使用轉殖p53質體至H1299的穩定細胞株 (HW16)。但當加入外源Akt會增加ellipticine對細胞生長的抑制效應,並讓sub G1週期細胞數目上升,而Akt及p53會被共同誘導移入細胞核內,DNA修補酶PARP也受到剪切。但將AktS473位點突變後,誘導產生的細胞凋亡會被抑制;而AktT308位點突變後抑制能力則較不明顯。Ellipticine所引發的細胞凋亡也與細胞自噬的形成有關,加入Akt後細胞內LC3-I轉換成LC3-II的比例增加,但當AktS473位點突變後,所誘導的細胞自噬也會降低。因此本研究顯示ellipticine所誘導產生的細胞凋亡是與Akt及p53移動至細胞核及細胞自噬形成有關,且AktS473位點的磷酸化對於藥物引發的細胞凋亡具有重要性。

    1.The most common form of non-small-cell-lung-cancer (NSCLC) occurs among non-smokers in women. Because of the drug resistance, there is always a need to identify new therapeutic agents against this disease. Based on screening assay from the stock chemicals, we are identifying whether the oxazoline derivatives are potential cytotoxic drugs in human lung cancer epithelial cells through mononuclear cell direct cytotoxicity assay (MTT assay). After screening a total of more than 42 compounds, one of the synthetic chemicals (MZ-01-059), effectively suppressed growth in human adenocarcinoma in three lung cancer cell lines with different p53 genotypes, (A549 (p53+/+), H460 (p53+/+) and H1299 (p53-/-). The result was further confirmed by colony formation assay. However, the apoptotic death by (MZ-01-059) was detected only in A549 and H460 cells, but not in H1299 cells as measured by flow cytometry. The results indicated tumor suppressor p53 is essential for drug activity. In addition, MZ-01-059 activated caspase-3 and poly(ADP-ribose) polymerase cleavages in both A549 cells and H460 cells. The findings demonstrate that the candidate drug induces the anticancer effects through apoptotic cell death in A549 and H460 cells that is regulated by p53.

    2.Topoisomerase II inhibitors, ellipticine and its analogues, were reported promising anticancer drugs due to its antineoplastic effects. Tumor suppressor p53 plays an important role in DNA damage-induced apoptosis. The phosphatidylinositol 3-OH-kinase-Akt pathway inhibits p53-mediated transcription and apoptosis, while the Akt substrate MDM2, an ubiquitin ligase for p53, plays a pivotal role in regulation of the stability of p53. Previously, we showed that ellipticine-induced cytotoxicity in non-small-cell-lung-cancer (NSCLC) was achieved through autophagy and apoptotic death as a result of Akt-modulation. Nucleus translocation of p53 and Akt and recruitment of autophagosome were found in A549 cells. In this study, we further demonstrated that Akt-related cell death also occurred in p53-deficient cells with stable expression of exogenous p53 ( HW16). The cell death phenotype was ameliorated when transfected with dominant-negative AKTS473A construct. On the other hand, the apoptotic phenotype was also be reverted by AktT308A with less dominant effect. Akt and p53 were both translocated into nucleus during apoptosis in ellipticine -treated HW16 cells. Our work demonstrated that Akt and p53 nuclear translocation are essential in elliptpcine- induced apoptosis in HW16 cells.

    1.英文摘要 5 一.緒論 7 1.文獻評述 7 (1)藥物篩選模式 7 (2)肺癌種類 8 (3)臨床上治療肺癌藥物的發展 9 (4)腫瘤抑制基因p53 9 (5)細胞凋亡 14 (6)細胞自噬 15 (7)細胞自噬與細胞凋亡 17 (8)細胞自噬與癌症的發生 18 2.研究目標 20 二.材料與方法 21 1.細胞培養 21 2.小分子化學藥物 21 3.細胞生長率分析 22 4.群落形成測定 (colony forming assay) 23 5.以PI (propidium iodide)染色觀察細胞週期分布 23 6.以PI和Annexin V雙染色觀察細胞凋亡 24 7.蛋白質之萃取 25 8.蛋白質定量 26 9.西方轉漬法 26 10.統計 28 三.結果 29 1.以A549肺癌細胞株進行細胞生長率試驗之篩檢 29 2.由群落形成鑑定MZ-01-059可以抑制非小細胞肺癌的生長速率 30 3.MZ-01-059使具有內生性p53非小細胞肺癌產生細胞凋亡 30 4.以西方轉漬法檢測MZ-01-059所引發細胞凋亡的路徑分子機轉 32 四.討論 34 五.參考文獻 37 六.圖 44 2. 英文摘要 68 圖目錄 69 一.緒論 70 1.文獻評述 70 (1)抗癌藥物Topoisomerase II抑制劑 70 (2)Akt訊息傳遞路徑 71 (3)p53與MDM2的拮抗效應 73 (4)過去研究結果 74 2.研究目標 75 二.材料與方法 76 1.細胞培養 76 2.轉殖重組基因載體 76 3.細胞計數 77 4.核蛋白質的萃取 77 三.結果 79 1.玫瑰樹鹼 (ellipticine) 對HW16生長速率及細胞週期分佈的變化。 79 2.外源Akt影響ellipticine引發p53核移動。 80 3.Ellipticine對HW16細胞自噬分子LC3的變化 81 四.討論 83 五.參考文獻 86

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