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系統識別號 U0026-1611201509173900
論文名稱(中文) 蟲草素誘發MA-10小鼠萊式腫瘤細胞凋亡之細胞與分子機制研究
論文名稱(英文) The cellular and molecular mechanisms of cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 104
學期 1
出版年 104
研究生(中文) 潘博雄
研究生(英文) Bo-Syong Pan
學號 S58001080
學位類別 博士
語文別 英文
論文頁數 72頁
口試委員 指導教授-黃步敏
召集委員-楊倍昌
口試委員-王應然
口試委員-鄭宏祺
口試委員-何元順
口試委員-潘敏雄
口試委員-郭靜娟
中文關鍵字 蟲草素  睾丸癌  MA-10 萊氏腫瘤細胞  細胞凋亡  CB17/SCID 小鼠 
英文關鍵字 Cordycepin  Testicular cancer  MA-10 Mouse Leydig tumor cell  Apoptosis  CB17/SCID Mice 
學科別分類
中文摘要 在台灣地區從1999至2008年間的睾丸癌每10萬人發生率人口從0.85增加到1.54人。而治療睪丸癌的方式是使用放射線和手術切除與化療來同時進行。然而,此種化療治療方式不僅產生許多的副作用並嚴重的影響著生活機能品質。因此,近年來人們著重於試圖開發出較低副作用的取代化療藥物來改善病人的癒後情形。蟲草素,也稱為3'-脫氧腺苷,為冬蟲夏草中所特有的物質。它具有廣泛的生物效應,包括刺激固醇類生合成,消炎作用,抑制血小板聚集,並具有抗癌作用。在我們先前的研究中,已經初步證實蟲草素能促使睪丸癌細胞走向凋亡路徑。在許多的癌症形成和發展過程之中,p38細胞分裂素活化蛋白激酶(MAPKs)訊息傳遞路徑在調節細胞的存活和死亡過程中,扮演著相當重要的角色。然而在萊式腫瘤細胞中,p38 MAPKs對於調節其細胞凋亡的機制並未十分清楚。在本篇研究當中,我們發現使用蟲草素給予在MA-10小鼠萊式腫瘤細胞之後,會藉由調節p38 MAPK和PI3K/AKT訊息傳遞路徑來促使細胞凋亡。當蟲草素給予在MA-10、TM4和NT2/D1細胞中,會明顯地降低細胞存活率,在適當濃度處理之下,並不會引起正常小鼠萊式細胞的死亡。在訊息傳遞的路徑探討研究之中,我們發現蟲草素會增加細胞內活性氧化物(ROS)的含量,活化caspase-3和裂解PARP。並在流式細胞儀中的Annexin V陽性結果分析發現,蟲草素引起細胞死亡的方式為細胞凋亡。SB203580 為一種p38 MAPKs的抑制劑,可以明顯的阻止由蟲草素所引起MA-10細胞凋亡現象。此外,將蟲草素與PI3K的抑制劑woetmannin或是自噬的抑制劑3-MA共同處理在MA-10細胞中,會明顯的增加細胞死亡率。此外,我們也發現在蟲草素引起MA-10細胞凋亡的過程中,會活化p53、p21和TGFß路徑並降低CDK2的活性。我們在嚴重複合型免疫缺乏小鼠的異體腫瘤生長實驗當中,給予蟲草素治療之後,可以明顯抑制腫瘤生長。在本篇的研究結果,我們證實蟲草素具有高度專一性的經由活化p38 MAPKs路徑去促使MA-10細胞凋亡。
英文摘要 The incidence of testicular cancer between 1999 and 2008 in Taiwan from 0.85 increased to 1.54 per population of hundred thousand people, and radical orchidectomy combined with chemotherapy is the common protocol to treat testicular and Leydig cell cancers. However, these drugs used for chemotherapy could cause drug toxicity, side effects and serious impacts on the quality of life. Thus, people more focus on developing lower side effect chemotherapy drugs to improve prognosis in recent years. Cordycepin, also called 3’-deoxyadenosine, has been identified as a major bioactive metabolite in Cordyceps sinensis. It possesses a wide range of biological effects, including stimulation on steroidogenesis, anti-inflammatory effect, inhibition of platelet aggregation, and anti-cancer effect. We have found that cordycepin could induce testicular tumor cell apoptosis. The p38 MAPKs play important roles in the regulation of balance between cell survival and cell death on the development of various cancers. However, the roles of p38 MAPKs regulating apoptotic effects on Leydig tumor cells remain unclear. In the present study, we showed that cordycepin (3-deoxyadenosine) selectively induced apoptosis in MA-10 mouse Leydig tumor cells through regulating the p38 MAPKs and PI3K/AKT signaling pathways. Cordycepin reduced viability in MA-10, TM4, and NT2/D1 cells, but not cause cell death of primary mouse Leydig cells on moderate concentration. Cordycepin increased reactive oxygen species (ROS) levels, which is associated with the induction of apoptosis as characterized by positive Annexin V binding, activation of caspase-3, and cleavage of PARP. Inhibition of p38 MAPKs activity by SB203580 significantly prevented cordycepin-induced apoptosis in MA-10 cells. Co-treatment with wortmannin or the autophagy inhibitor 3-methyladenine (3-MA) elevated levels of apoptosis in cordycepin-treated MA-10 cells. Moreover, cordycepin activated p53, p21 and TGFß; and downregulated CDK2. The antitumour activity of cordycepin-treated MA-10 cells was significantly distinct in severe combined immunodeficiency (SCID) mice in vivo. These results suggested that cordycein is a highly selective treatment to induce MA-10 cells apoptosis via p38 MAPKs signaling.
論文目次 Abstract in Chinese..................................I
Abstract.............................................III
Acknowledgement......................................V
Table of Contents....................................VII
List of Figures......................................X
Abbreviations........................................XIII
Chapter 1: Introduction..............................1
1.1. Testicular cancer and Leydig tumor...........1
1.2. Treatment of testicular cancer...............1
1.3. Cordyceps sinensis and Cordycepin............1
1.4. The signaling pathways of cancer development and progression..........................................2
1.4.1. Mitogen Activated Protein Kinases Signaling Pathways.............................................3
1.4.2. Reactive Oxygen Species (ROS).............4
1.4.3. Caspase Cascade Signaling pathways........4
1.4.4. The p53 Signaling Pathways................4
1.4.5. PI3K/AKT/mTOR signaling Pathways..........5
1.4.6. Autophagy Signaling Pathways..............5
1.4.7. The endoplasmic reticulum stress..........6
1.5. The MAPK Signaling Pathway and Steroidogenesis......................................7
1.6. Objectives and hypothesis....................7
Chapter 2: Materials and Methods.....................8
2.1. Chemicals...................................8
2.2. Cell culture................................8
2.3. Isolation of Leydig cells...................9
2.4. MTT cell viability assay....................10
2.5. Lactate dehydrogenase (LDH) release assay................................................10
2.6. Annexin V staining and flow cytometry.......11
2.7. Western blotting............................12
2.8. ROS measurements............................12
2.9. Small interfering RNA (siRNA) design and transient transfection...............................13
2.10. Animal treatment............................14
2.11. Ethics statement............................14
2.12. Immunohistochemistry........................14
2.13. Statistical analysis........................15
Chapter 3: Results...................................16
3.1. Cordycepin induced apoptosis in MA-10 mouse Leydig tumor cells...................................16
3.2. Cordycepin induced MA-10 cell apoptosis through caspase signaling....................................17
3.3. Cordycepin induced apoptosis by activating p38 signaling............................................19
3.4. Cordycepin induced apoptosis and autophagy by regulating PI3K/AKT/mTOR signaling...................20
3.5. Cordycepin led to ROS accumulation..........22
3.6. Cordycepin induced apoptosis by regulating CDK activity through the p53 and TGFβ signaling pathways.............................................23
3.7. Cordycepin inhibited tumor growth in an allograft model of testicular cancer.................23
3.8. Cordycepin induced MA-10 cell apoptosis through ER stress signaling..................................24
Chapter 4: Discussion................................26
Chapter 5: Conclusion................................29
Chapter 6: References................................30
Chapter 7: Figures...................................37
Chapter 8: Appendix..................................71
Chapter 9: Publications..............................72


List of Figures
Figure 1. Cordycepin inhibited testicular tumor cell viability............................................37
Figure 2. Effect of cordycepin induced apoptosis of MA-10 cells................................................38
Figure 3. Effect of cordycepin induced apoptosis in testicular germ cell tumors..........................39
Figure 4. Cordycepin induced G2/M cell cycle arrest in testicular germ cell tumors..........................40
Figure 5. Cordycepin inhibited human prostate cancer cells viability......................................41
Figure 6. The cell cycle distribution of MA-10 cells................................................42
Figure 7. Cordycepin induced apoptosis of MA-10 cells through caspase signaling............................43
Figure 8. Caspase inhibitor reversed cordycepin-induced caspase cascade in MA-10 cell apoptosis..............44
Figure 9. Effect of caspase-3 signaling pathway in cordycepin-induced cell death in MA-10 cells.........46
Figure 10. Cordycepin induced MAPK signaling activation in MA-10 cells.......................................47
Figure 11. MAPKs inhibitors reversed cordycepin-induced MAPK signaling pathway in MA-10 cells................48
Figure 12. MAPK inhibitors reversed cordycepin-induced cell death in MA-10 cells............................49
Figure 13. Cordycepin induced apoptosis of MA-10 cells by activating p38 signaling pathways....................50
Figure 14. Cordycepin induced apoptosis of MA-10 cells through activating p38 signaling pathways............52
Figure 15. Cordycepin induced apoptosis and autophagy in MA-10 cells by regulating PI3K/AKT/mTOR signaling pathways.............................................53
Figure 16. Effect of wortmanin with or without cordycepin on PI3K/AKT/mTOR signaling pathways of MA-10 cells................................................54
Figure 17. Effect of wortmanin with or without cordycepin on cell viability of MA-10 cells.....................56
Figure 18. Effect of autophagy inhibitor with or without cordycepin on cell viability of MA-10 cells..........57
Figure 19. Cordycepin stimulated ROS accumulation in MA-10 cells.............................................58
Figure 20. N-acetyl-L-cysteine (NAC) pretreatments could inhibit cordycepin-induced ROS generation in MA-10 cells................................................59
Figure 21. Effect of NAC with or without cordycepin on cell viability of MA-10 cells........................60
Figure 22. Cordycepin induced apoptosis of MA-10 cells by regulating CDKs through the p53 pathways.............61
Figure 23. Effects of p53 siRNA on cordycepin-induced p53 signaling in MA-10 cell apoptosis....................62
Figure 24. Cordycepin induced apoptosis of MA-10 cells by regulating TGFβ pathways.............................63
Figure 25. Effect of p53 siRNA with or without cordycepin on cell viability of MA-10 cells.....................64
Figure 26. Cordycepin inhibited tumor growth in an allograft model of testicular cancer.................65
Figure 27. Cordycepin induced apoptosis of MA-10 cells by regulating FoxO signaling pathways...................66
Figure 28. Cordycepin induced apoptosis of MA-10 cells by activating PERK/EIF2α signaling pathways.............68
Figure 29. Cordycepin induced apoptosis of MA-10 cells by activating ATF6 and IRE1 signaling pathways..........69
Figure 30. Schematic representation of cordycepin triggers apoptosis of MA-10 mouse Leydig tumor cells through p38, caspase, and PI3K/AKT/mTOR signaling pathways.............................................70
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