臺灣博碩士論文加值系統

肺癌是國人最重要的癌症致死原因。肺癌病人通常在腫瘤切除後五年內死於癌症復發或腫瘤轉移，大部分接受化學治療的肺癌患者，常常因為對傳統化療藥物產生抗性而治療失敗，這些傳統化療藥物的副作用也對病人造成極大的痛苦，因此需要新藥的發展以提升肺癌患者的治癒率。近年來COX-2的抑制劑，celecoxib，它的結構修飾物OSU03013，在攝護腺、卵巢癌、乳癌等，已經被研究有抗癌的效果，並且是以AKT的訊息傳遞路徑來達到抑制攝護腺癌之生長。因此，本研究目標即是探討OSU03013在肺癌細胞之毒殺作用及其細胞學鑑定，之後利用二維電泳、質譜分析等蛋白質體學的方法找尋藥物的目標及影響蛋白，並分析這些蛋白/訊息傳遞路徑與細胞生長調控的關係。
在肺癌細胞株A549、CL1-1、H1435的IC50測試實驗中，本研究發現OSU03013具有高度細胞毒殺作用，而此藥物對於肺正常細胞並沒有此現象，所以我們認為它是一個治療肺癌很有潛力的藥物。在細胞學鑑定實驗中，我們發現OSU03013會造成細胞週期停滯在間期一 (Gap 1, G1 arrest) 的現象；OSU03013在肺癌細胞同時也藉由內質網壓力效應去引發細胞凋亡 (apoptosis)。在蛋白質體學的實驗中，我們發現此藥物在肺癌細胞之目標蛋白包含了cAMP-dependent protein kinase inhibitor β form (PKIB, 激酶抑制蛋白)、數種G proteins (G蛋白)、數種Heat-shock proteins (熱休克蛋白)、Antioxidant enzymes (去氧化蛋白)、及其他調控細胞生長、代謝的蛋白；這些蛋白有許多皆以Western blot (西方點墨法) 確認。由於OSU03013在肺癌細胞中因為PKIB的過度活化，我們預測其下游蛋白cAMP-dependent protein kinase (PKA) 的蛋白表現量在處理藥物後會下降，以抑制PKA的訊息傳導路徑，其中一條路徑抑制了Wnt/-catenin活性，所以抑制了肺癌細胞生長；而並非如同在攝護腺癌中，是透過AKT傳導路徑來抑制癌細胞的生長。本研究為首篇在肺癌細胞中偵測OSU03013藥物之抑制癌細胞潛力，及其抑癌分子機轉之研究。

Purpose: Lung cancer is the leading cause of cancer death in many countries including Taiwan. The majority of lung cancer patients receiving the chemo-therapy often fail because of drug resistance. In addition, patients suffer from side effects of current chemotherapies. Therefore, developing the new therapeutic drugs is important and can greatly improve the cure rate of the lung cancer. Background: OSU03013 is a modified compound from celecoxib, which is a COX-2 inhibitor used for arthritis treatment. OSU03013 has been shown to also act as an anti-cancer drug for prostate, breast, and ovary cancer though inhibition of AKT-mediated singling in cell and animal models. Study design: To investigate whether OSU03013 can be a potential drug for lung cancer treatment and to identify the molecular targets for OSU03013, we (1) screened the A549, CL1-1, and H1435 lung cancer cell lines for their IC50 treated with OSU03013, (2) investigated the cell cycle after OSU03013 treatment by flow cytometry, (3) studied the mechanism of cell apoptosis related to cytotoxicity effects of OSU03013, (4) identified target/effector proteins of OSU03013 by two-dimensional electrophoresis and mass-spectrometry (2DE-MS), and (5) confirmed the selected proteins by Western blot analysis. Results: The cytotoxicity of the potential anti-cancer drug OSU03013 was more efficient than the traditional drugs such as cisplatin in A549, CL1-1, and H1435 lung cancer cell lines. OSU03013 caused cell cycle arrest in G1 phase and apoptosis through endoplasmic reticulum stress. Target and effector proteins identified by 2DE-MS including cAMP-dependent protein kinase inhibitor β form (PKIB), several G proteins, some heat-shock proteins, few antioxidant enzymes, and several proteins controlling the cell growth and metabolism. Many of them were confirmed by the Western blot analyses. Conclusion: The proteins which were up- or down-regulated after OSU03013 treatment, including proteins involved in cell growth controls, signal pathways, and damage response pathways. For example, PKIB is up-regulated after OSU03013 treatment, it affects the cAMP-dependent protein kinase A pathway to inhibit the cell growth through inhibition of Wnt/-catenin pathway, which often over-activation in lung cancer. More cell biology and protein functional analyses will help us to gain insights of molecular mechanisms for the anti-cancer effects of OSU03013.

壹、中文摘要……………………………………………………… 1
貳、英文摘要……………………………………………………… 3
叁、緒論…………………………………………………………… 5
一、研究背景…………………………………………………… 5
1. 肺癌治療現況…………………………………………… 5
2. 新穎抗癌藥物OSU03013……………………………… 5
3. 細胞 apoptosis 的分子機制…………………………… 7
4. 藥物標的蛋白的研究…………………………………… 8
二、研究目的……………………………………………………… 13
肆、研究材料與方法………………………………………………… 14
一、研究材料…………………………………………………… 14
1. 肺癌細胞株……………………………………………… 14
2. Celecoxib的結構修飾物OSU03013…………………… 14
二、研究方法…………………………………………………… 15
1. 細胞培養………………………………………………… 15
2. 藥物處理之細胞毒性分析……………………………… 15
3. 細胞週期分析…………………………………………… 16
4. phosphatidylserine (PS) 染色以偵測早期細胞凋亡…… 16
5. 細胞株蛋白質萃取……………………………………… 17
6. 蛋白質定量……………………………………………… 17
7. 蛋白質體學……………………………………………… 18
8. 西方點墨 (Westernblot)………………………………… 21
9. 模擬分子對接 (molecular docking)……………………… 22
伍. 結果……………………………………………………………… 23
1. 藥物OSU03013處理肺癌細胞株A549、CL1-1及H1435… 23
的細胞形態
2. 利用藥物OSU03013處理正常肺細胞株MRC5及肺癌…… 23
細胞株A549、CL1-1及H1435 並探討其藥物毒殺性
3. OSU03013導致細胞週期在間期一停滯 (Gap 1, G1 arrest)… 23
及早期的細胞凋亡 (early apoptosis)
4. 肺癌細胞株A549、H1435、CL1-1處理OSU03013引發… 24
內質網壓力效應（endoplasmic reticulum, ER stress）
5. 利用蛋白質體學方法來尋找藥物處理細胞後的目標……… 25
及影響蛋白
6. 藥物OSU03013與PKA的分子對接 (Molecular Docking)… 26
陸、討論……………………………………………………………… 28
柒、結論與未來工作………………………………………………… 33
捌、附表……………………………………………………………… 35
玖、附圖……………………………………………………………… 42
拾、參考文獻………………………………………………………… 55
拾壹、附 錄………………………………………………………… 61

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