臺灣博碩士論文加值系統

牛樟芝(Antrodia camphorate)是台灣本土種的真菌，在民間中草藥使用上，據信具有治療各種疾病的功效。最近的研究報告已證實，牛樟芝能夠誘導各種腫瘤細胞發生細胞凋亡的現象。在這篇的研究中，我們證實牛樟芝的乙醇萃取物及其經由高壓液相層析分離(HPLC)的第6分離層(Fr-6)對人類非小細胞肺癌A549細胞的生長具有抑制效果，並且也說明了它的抗癌活性機制。
在HPLC分離層中，從第5到第7分離層對於人類肺癌A549細胞生長皆具有抑制效果，其中第6分離層的抑制效果是最為顯著的。細胞凋亡分析的數據顯示出Fr-6藉由觸發粒線體的細胞凋亡路徑及內質網壓力而誘導細胞發生細胞凋亡。西方墨點法的實驗結果證實，Fr-6可能藉由降低calpain蛋白質小的次單位(調控的次單位)表現量來活化了粒線體的細胞凋亡路徑及內質網壓力。Fr-6也可能因抑制Erk活化的路徑，抑制了人類肺癌A549細胞的增生，並且Erk活化的路徑被抑制，可能是Fr-6減少了galectin-1蛋白質的表現量及影響H-Ras與細胞膜的結合所造成。此外，Fr-6也造成RhoGDI-a蛋白質表現量的減少，因此活化了c-Jun 的N端激酶(JNK)的路徑，導致癌細胞走向細胞凋亡路徑。Fr-6也可能造成了真核細胞轉譯啟動因子5A (eIF-5A)蛋白質表現量的降低，而這個蛋白質具有成為癌症研究目標的潛能。總結來說，從牛樟芝乙醇萃取物所分離出來的Fr-6是有可能成為開發治療癌症新藥的潛能。

Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrated that A. camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities.
Three HPLC fractions, fractions 5 to 7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6 induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated the mitochondrial pathway and ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression and mislocalization of GFP-H-Ras(12V). Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor a (RhoGDI-a) expression and subsequently activated c-Jun NH2-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the expression level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target. Finally, Fr-6 may be the attractive target of new drug for cancer therapy.

Abstract (in Chinese)……………………………………………………………………....................iv
Abstract………………………………………………………………………………….............................v
Acknowledgement……………………………………………………………………….........................vi
Table of contents………………………………………………………………………......................vii
List of tables………………………………………………………………………….........................ix
List of figures………………………………………………………………………….........................x
Abbreviations…………………………………………………………………………..........................xi
Chapter 1 Introduction…………………………………………………………………....................1
1.1 Background and purpose……………………………………………………......................3
1.2 Apoptosis in cancer………………………………………………………….......................5
1.2.1 Mitochondrial regulation of apoptosis……………....................5
1.2.2 Death receptor pathway……………………………………………………....................6
1.2.3 Endoplasmic reticulum stress-induced apoptosis…………………………......7
1.3 Galectin-1………………………………………………………………….............................8
1.4 Rho GDI dissociation inhibitor α (RhoGDI-α).....................9
1.5 Human calcium-dependent protease (calpain)……………………………...........9
1.6 Human eukaryotic translation initiation factor 5A (eIF5A)…………….10
Chapter 2 Materials and methods……………………………………………………...............12
2.1 Cell lines…………………………………………………………………............................14
2.2 Preparation of the ethanol extract of A. camphorate cultivated by solid-state fermentation………………………………………………………………................14
2.3 Separation of the ethanol extract of A. camphorate by high performance liquid chromatography (HPLC)……………………………………………..........14
2.4 MTT assay………………………………………………………………….............................15
2.5 Analysis of Fr-6 by Gas chromatography/mass spectrometry (GC/MS)............................................................15
2.6 SubG1 cell analysis of Fr-6-treated human A549 cells……………………...16
2.7 Annexin V/PI staining of Fr-6-exposed human A549 cells………………...16
2.8 Assessment of DNA fragmentation……………………………………….................16
2.9 Western blotting………………………………………………………….........................17
2.10 Transfection of GFP-H-Ras(12V) and laser scanning confocal microscope assay………………………………………………………………………........................18
2.11 Focus formation assay…………………………………………………….....................18
2.12 Soft agar assay……………………………………………………………........................19
2.13 Wound scratch assay………………………………………………………......................19
Chapter 3 Results…………………………………………………………………….......................21
3.1 Separation of the ethanol fraction of A. camphorate by HPLC…………23
3.2 The inhibitory activities of HPLC fractions against human A549 cell…….............................................................23
3.3 Fr-6 could evoke human A549 cell apoptosis……………………………..........23
3.4 Fr-6 caused the apoptosis in human A549 cell by triggering the mitochondrial pathway and ER stress……………………………………..................24
3.5 The impact of Fr-6 on the expressions of four tumor-related genes………...........................................................25
3.6 The effect oh Fr-6 on the membrane localization of H-Ras(12V)……26
3.7 Fr-6 inhibited the transformation of human A549 cells……………………..27
3.8 Fr-6 affected the migration capability of human A549 cells……………27
Chapter 4 Discussion…………………………………………………………………......................28
References……………………………………………………………………………............................34

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