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研究生:張郁涓
研究生(外文):Yu-Chuan Chang
論文名稱:甲基蓮心鹼針對FAK及S6K訊息路徑引發腦瘤自噬作用及抑制轉移作用
論文名稱(外文):Neferine induced autophagy and inhibited migration through FAK and S6K1 signaling pathways in brain tumor
指導教授:翁慶豐翁慶豐引用關係
指導教授(外文):Ching-Feng Weng
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
論文頁數:48
中文關鍵詞:甲基蓮心鹼膠質母細胞瘤神經母細胞瘤FAKS6K1自體吞噬細胞凋亡
外文關鍵詞:NeferineGliomaNeuroblastomaFAKS6K1autophagyapoptosis
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Focal adhesion kinase (FAK)及human p70 ribosomal S6 kinase (S6K1)蛋白,是調控癌細胞轉移增生重要的蛋白,文獻指出FAK和S6K1過度表現於多種癌症中。本實驗利用分子對接,將60種天然化合物結構與FAK (PDB ID: 2AL6) 和S6K1 (PDB ID:3A60)結構做結合,選出其中分數最高的甲基蓮心鹼 (Neferine) 為研究目標。以甲基蓮心鹼處理C6膠質母細胞瘤細胞及IMR32神經母細胞瘤細胞利用細胞活性、細胞週期、西方轉印法及傷口癒合等方法,探討抑制調控癌細胞轉移的FAK蛋白及調控癌細胞增生的S6K1蛋白的效果,使癌細胞啟動細胞凋亡(Apoptosis)及自體吞噬(Autophagy)的功效。結果顯示出甲基蓮心鹼有效抑制C6膠質母細胞瘤細胞及IMR32神經母細胞瘤細胞的細胞活性,並使細胞週期停滯在G2/M期,在細胞處理甲基蓮心鹼後,抑制調控細胞增生及轉移的蛋白質, FAK和S6K1及其磷酸化的表現量,且調控自體吞噬的LC3蛋白質及調控細胞凋亡的cleaved caspase 3及cleaved PARP表現量明顯上升,顯示出甲基蓮心鹼會啟動癌細胞細胞凋亡及自體吞噬的機制。並且藉由傷口癒合試驗更進一步證明,處理甲基蓮心鹼後,確實會抑制C6膠質母細胞瘤細胞的細胞轉移。綜合以上結果顯示甲基蓮心鹼能有效抑制FAK及S6K1,降低癌細胞的生長及轉移,並能有效的誘導癌細胞啟動細胞凋亡及自體吞噬的機制,有成為抗癌藥物的潛力。
Focal adhesion kinase (FAK) and human p70 ribosomal S6 kinase (S6K1), non-receptor protein tyrosine, have been overexpressed in many types of tumors and play an important role of cell signaling pathways including cell migration, proliferation, viability, and cell survival. This study was aimed to screen the novel and specific inhibitors of FAK and S6K1 from natural compounds via molecular docking and further investigate the underlying mechanism of action. The 3D structures of FAK (PDB ID: 2AL6) and S6K1 (PDB ID: 3A60) were employed to do docking with 60 natural compounds. Based on their high affinity and energy interaction, top one docking score compound Neferine from Nelumbo Nucifera Gaertn was selected and further validated the anti-tumor effect in C6 rat Glioma and IMR32 human Neuroblastoma cells. FAK (2AL6)-ligands interaction analysis indicated that H-bond with residues Arg86, Arg125, and Tyr251 while S6K1 (3A60)-ligands interaction analysis indicated that H-bond with residues Arg95, Gly100, and Asp236. Neferine showed a potential effect on cell viability by MTT assay and arrest the cell cycle at G2/M phase by flow cytometer. The protein levels of FAK, pFAK, S6K1, and pS6K1 were decreased, which are the key regulators cell migration and proliferation, respectively. Neferine induced the autophagy through Beclin-1 and LC3 pathway, and induced the apoptosis through caspase 3 and PARP. Furthermore, Neferine would inhibit the cell migration on C6 cells by wound healing assay. Taken altogether, this study suggests that Neferine could be a potential inhibitor of FAK and S6K1 for anti-tumorigenesis and anti-metastasis.
Contents
摘要……….. I
Abstract….. III
Contents…. V
Table contents VII
Figure contents IX
Abbreviations XI
Introduction 1
Materials and methods 5
1.1 Chemicals and antibodies 5
1.2 Molecular modeling and docking 5
1.3 Cell culture 6
1.4 Cell viability assay 6
1.5 Cell cycle analysis 7
1.6 Western blotting 7
1.7 Wound healing assay 8
1.8 Statistical analysis 8
Results…… 9
1.1 Molecular docking 9
1.2 Cell viability of Neferine and Temozolomide in C6 and IMR32 cells 9
1.3 Neferine and Temozolomide mediated cell cycle of C6 cells 10
1.4 Effects of Neferine and Temozolomide on FAK and pFAK protein levels in C6 and IMR32 cells 10
1.5 Effects of Neferine and Temozolomide on S6K1 and pS6K1 protein levels in C6 and IMR32 cells 10
1.6 Effects of Neferine and Temozolomide on Autophagic related proteins in C6 and IMR32 cells 11
1.7 Effects of Neferine and Temozolomide on Apoptotic related proteins in C6 cells 11
1.8 Effects of Neferine and Temozolomide on the migration of C6 cells. 11
Discussion… 13
Conclusion… 17
Future work 19
References.. 21

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