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研究生:鄒曉彤
研究生(外文):Hsiao-Tung Tsou
論文名稱:AC10 及其主成分抑制小鼠黑色素瘤細胞B16F1 和B16F10 之抗腫瘤功效:Wnt/β-catenin signaling pathway 的機制調控
論文名稱(外文):Anti-tumor Activity of AC10 and its major compound Against Murine Melanoma Cells Through the Modulation of Wnt/β-catenin Signaling Pathway
指導教授:楊新玲楊新玲引用關係
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:營養學系碩士班
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:256
中文關鍵詞:AC-10Wnt/β-catenin pathway細胞凋亡細胞週期轉移
外文關鍵詞:Antrodia CamphorataWnt/β-catenin pathwaymelanomametastasisApoptosiscell cycle
相關次數:
  • 被引用被引用:2
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黑色素瘤(melanoma)為皮膚惡性腫瘤的一種,惡性度最高,容易轉移,對西方人而言是皮膚疾病中發病率佔第一位的死亡原因,因黑色素瘤對化學治療及放射線治療的效果不佳,又伴隨著高度的轉移性;再者,目前對於治療黑色素瘤 (melanoma) 藥物的研究尚未完善;若能尋找到輔助癌症治療的天然食材,是當前重要的課題。
Wnt/β-catenin 訊息途徑可調控許多細胞的進程,包括增生(proliferation)、分化(differentiation)、存活(survival)、細胞凋亡(apoptosis)及細胞的移行(motility)。約有30%的黑色素瘤可觀察到Wnt/β-catenin訊息途徑異常活化的現象,因此抑制Wnt/β-catenin 訊息途徑也成為治療黑色素瘤的策略之一。
樟芝(Antrodia camphorata;AC) 為台灣常見的傳統中藥,這種特有的蕈類生長於牛樟樹上,先前文獻指出樟芝具有抗癌、抗發炎及調節免疫等功效。在第一部分的實驗利用樟芝發酵液(AC-10),作用在小鼠黑色素瘤細胞B16F1及B16F10,其IC 50皆約為80ug/mL。在細胞凋亡分析上,AC-10具有劑量效應誘發細胞凋亡的能力,並伴隨p53的蛋白表現增加,cytochrome c 釋出至細胞質、BAX/Bcl-2 的ratio 提高以及caspase-3 、caspase-9以及PARP的裂解。在Wnt/β-catenin調控路徑之下,利用免疫螢光染色法發現AC-10可抑制β-catenin入核表現,並且降低核質β-catenin的蛋白表現;再同時給予AC-10及MG132(proteasome inhibitor),則會使β-catenin的蛋白表現量回復,並利用SB216763(GSK3β抑制劑) 得知AC-10降解β-catenin為GSK3β依賴型(GSK3β-dependent)。但由RT-PCR實驗得知,AC-10 並不改變β-catenin mRNA 表現。而同時給予Cycloheximide (CHX, protein synthesis inhibitor)及AC-10可縮短β-catenin
蛋白質降解的半衰期,此現象說明了AC-10造成β-catenin的減少原因為加速蛋白質的降解作用。同時也利用Reporter assay觀察到AC-10可抑制β-catenin調控的轉錄活性,導致下游標的基因如c-myc、cyclin D1、survivin、MMP-9及VEGF的減少。Flow cytometry分析AC-10會使黑色素細胞瘤皆停滯在G1期,並連帶的抑制細胞週期蛋白cyclin D1、CDK4;增加p27、p21的表現。在細胞轉移部分上,細胞刮傷試驗(wound healing assay)以及細胞侵襲試驗(invasion assay)測定發現AC-10可降低癌細胞遷移的表現,並連帶的使MMP-9、MMP-2及VEGF表現量減少。
在第二部分的實驗中,另以AC-10純化後產物AC-0進行體外試驗觀
察到AC-0也具有抑制β-catenin及促進細胞凋亡表現,由RT-PCR實驗得知,AC-0同樣不會改變β-catenin mRNA表現,而同時給予CHX及AC-0卻無法加速β-catenin蛋白質的降解,因此推測AC-0雖無法加速降解,但令β-catenin減少仍為蛋白降解作用。利用SB216763(GSK3β抑制劑)得知AC-0降解β-catenin為GSK3β非依賴型(GSK3β-independent);並利用免疫沉澱法(IP)來探討GSK3β與β-catenin之交互作用,同樣也證明了GSK3β的非依賴型。
又以體內實驗(裸鼠腫瘤模式)證實AC-0確實可以抑制裸鼠植入B16F10誘發之腫瘤大小、重量、及體積,並且經免疫組織染色分析、腫瘤蛋白分析得知β-catenin及其target gene表現也被抑制。
總結本研究得知AC-10及其主成分AC-0可藉由抑制Wnt/β-catenin訊
息路徑,而達到抑制腫瘤生長及轉移的效用。

Melanoma is the most serious form of skin cancer. Aberrant activation of Wnt/β-catenin signaling cascade has been observed in approximately
one-third of melanomas, indicating that modulation of Wnt/β-catenin activation might be a novel strategy for melanoma treatment. The
downstream targets of Wnt/β-catenin signaling pathway including c-Myc, cyclinD1, MMPs, and survivin, which are regulating number of cellular
functions, such as proliferation, differentiation, survival, apoptosis and invasion. Antrodia camphorata, a well known medicinal mushroom in Taiwan that has been used as Chinese folk medicine for many years.
Previous studies have shown that A. camphorata (AC) possessed greater anti-tumor activity against a variety of tumor cells. However, the anti-tumor efficacy of AC against melanoma was poorly understood. In addition, the currently employing treatment for melanoma is a tough topic, due to the high resistance to radio-and chemotherapy and most the synthetic
chemotherapeutic drugs are volnarable to non-melanoma skin cells.Therefore, the present study, we aimed to investigate the anti-tumor efficacy
of fermented culture broth extracts of A. camphorata (AC-10) and it derived pure compound (AC-0) in murine melanoma cells. The first set of experiment, we observed AC-10 treatment significantly decreased murine melanoma B16F1 and B16F10 cell viability with an IC 50
value of 80ug/mL. The reduction of cell viability is directly correlated with the inhibition of β-catenin and its downstream protein expression.
Immunofluorescence analysis confirmed that AC-10-treatment markedly reduced β-catenin translocation into the nucleolus, and also downregulates
??-catenin-mediated transcriptional activity. Furthermore, MG132 a proteosomal inhibitor that prevent proteosomal degradation of β-catenin,
conversely, GSK3β inhibitor SB216312 also suppressed β-catenin degradation which strongly suggest that β-catenin degradation is GSK3β dependent. In a similar way, AC-10-treatment attenuate GSK3β expression, suggesting that AC-10 may regulate the proteasomal
degradation of β-catenin by GSK3β-dependent nmechanism. Furthermore, flow cytometry analysis showed that AC-10-treatment significantly arrest
G1 to S-phase transition followed by the suppression of cyclin D1 (wnt/β-catenin target gene), CDK4 expression and increased in p27, p21
levels. Moreover, TUNEL assay revealed that AC-10-treatment induce apoptosis in a dose-dependent manner, followed by the disregulation of
BAX/BCL-2 ratio, and the down-regulation of pro-caspase-9, pro-caspase-3 and pro-PARP. Migration and invasion assay shows AC-10 could abate melanoma metastatic ability, through the inhibition of MMP-9, MMP-2,
VEGF expression, which are also wnt/β-catenin target genes. In the second part, AC-0 also found to effectively inhibit Wnt/β-catenin pathway cascades, and suppressed β-catenin-mediated transcriptional
activity. Similar with AC-10, AC-0 treated cell found to decreased melanoma metastasis and augmented apoptotic induction. MG132 and AC-0
treatment prevents β-catenin expression, however, SB216312 failed to prevent GSK3β expression, which suggesting that AC-0-treatment may regulate the proteasomal degradation of β-catenin via GSK3β-independent mechanism. This phenomenon also demonstrated with immunoprecipition assay that AC-0 treated cells decreased the interaction between β-catenin and GSK3β.
In vivo study, AC-0 decreased the growth of B16F10-derived tumors development in the athymic nude mice. The decreased B16F10-derived tumor growth was associated with a down-regulation of Wnt/β-catenin target
genes such as c-myc, cyclin D1, MMP-9. AC-0 treatment also induced B16F10-derived tumors apoptosis in nude mice. In conclusion, our data demonstrated that AC-10 and its major compound AC-0 appreciably modulate Wnt/β-catenin pathway in melanoma cells.
Therefore, we believe AC-10/AC-0 might be a potential chemo-preventive agent for melanoma treatment.

目錄
謝誌............................................................................................................................I
縮寫表 .....................................................................................................................II
中文摘要 ................................................................................................................III
Abstract ....................................................................................................................V
第一章 前言.........................................................................................................1
第二章 文獻探討.................................................................................................3
第一節 樟芝及其主成分介紹.........................................................................4
第二節 皮膚癌介紹.........................................................................................8
第三節 Wnt/β-catenin pathway介紹.............................................................18
第四節 Wnt/β-catenin pathway與細胞凋亡之相關性.................................27
第五節 Wnt/β-catenin pathway與細胞週期之相關性.................................35
第六節 Wnt/β-catenin pathway與細胞轉移之相關性.................................42
第三章 研究動機與假說....................................................................................49
第一節 研究動機...........................................................................................50
第二節 研究假說...........................................................................................51
第三節 實驗設計...........................................................................................52
第四章 材料方法..................................................................................................55
第一節 實驗儀器...........................................................................................56
第二節 實驗材料...........................................................................................58
第三節 實驗方法...........................................................................................64
第一部分AC-10
1. 樟芝發酵液 (AC-10).............................................................................. 64
2. 細胞培養(cell culture).......................................................................64
3. 細胞形態(morphology)之觀察.........................................................66
4. 細胞存活率分析(cell viability).........................................................66
5. 細胞凋亡試驗(TUNEL assay)..........................................................68
6. 蛋白質萃取(protein extraction)........................................................70
7. 西方墨點(western blotting)分析.......................................................73
8. 流式細胞儀分析(flow cytometry)....................................................78
9. 免疫螢光染色(immunofluenrence)................................................80
10. 轉型作用(transformation)...............................................................82
11. 質體抽取(plasmid DNA extraction)................................................84
12. 轉染作用(transfection)....................................................................88
13. 報導基因活性測定(reporter assay).........................................................90
14. 抑制劑之處理..................................................................................92
15. RNA萃取(RNA extraction)..............................................................95
16. 逆轉錄反應(RT-PCR)......................................................................95
17. 細胞群落形成分析(colony formation assay)..................................98
18. 細胞遷移試驗(Wound Healing assay)..........................................99
19. 細胞移行試驗(Transwell invasion assay)......................................100
20. 統計分析.........................................................................................101
第二部分AC-0
1. AC-10主成分(AC-0)...........................................................................102
? 2. 細胞培養(cell culture).......................................................................102
? 3. 細胞形態(morphology)之觀察..........................................................103
4. 細胞存活率分析(cell viability)..........................................................103
? 5. 細胞凋亡試驗(TUNEL assay)............................................................103
6. 蛋白質萃取(protein extraction)..........................................................103
7. 西方墨點(western blotting)分析........................................................103
8. 免疫螢光染色(immunofluenrence)....................................................103
9. 轉染作用(transfection).......................................................................104
10. 報導基因活性測定(reporter assay).........................................................104
11. 抑制劑之處理...................................................................................104
12. 免疫沉澱分析(Immunoprecipitation)..............................................104
13. RNA干擾(RNA interference, RNAi)...................................................106
14. Annexin V staining..............................................................................108
15. RNA萃取(RNA extraction)................................................................109
16. 逆轉錄反應(RT-PCR)........................................................................109
17. 細胞群落形成分析(colony formation assay)....................................110
18. 細胞遷移試驗(Wound healing assay)............................................110
19. 細胞移行試驗(Transwell invasion assay)..........................................110
20. 腫瘤異種移植(Tumor Xenograft study)..............................................110
21. 免疫化學組織染色(immunochemistry staining)................................111
22. 組織細胞凋亡分析(tissue TUNEL assay)..........................................114
23. 組織病理分析(H&E staining).............................................................116
24. 統計分析..............................................................................................117
第五章 實驗結果與圖表..................................................................................118
第一部分 樟芝發酵液(AC-10)對小鼠黑色素瘤細胞B16F1、B16F10之影響
第一節 樟芝發酵液(AC-10)對B16F1、B16F10小鼠黑色素瘤細胞之存活率與生長情形的影響....................................................120
第二節 樟芝發酵液(AC-10)對B16F1、B16F10小鼠黑色素瘤細胞 Wnt/β-catenin pathway之調控....................................121
第三節 樟芝發酵液(AC-10)誘發B16F1、B16F10小鼠黑色素瘤細胞凋亡......................................................................................125
第四節 樟芝發酵液(AC-10)對B16F1、B16F10小鼠黑色素瘤細胞週期影響..............................................................................126
第五節 樟芝發酵液(AC-10)對B16F1、B16F10小鼠黑色素瘤細胞轉移影響..............................................................................127
第二部分AC-10主成分(AC-0) 對小鼠黑色素瘤細胞B16F10之影響
第一節 AC-10主成分(AC-0)對小鼠黑色素瘤細胞B16F10之存活率與生長情形的影響................................................................162
第二節 AC-10主成分(AC-0)對高轉移性小鼠黑色素瘤細胞B16F10
Wnt/β-catenin pathway之調控......................................163
第三節AC-10主成份(AC-0)誘發高轉移性小鼠黑色素瘤B16F10細胞凋
亡...................................................................................167
第四節AC-10主成份(AC-0)對高轉移性小鼠黑色素瘤B16F10細胞轉移
之影響...........................................................................168
第五節 β-catenin siRNA參與AC-10主成份(AC-0) 調控Wnt/β-catenin
pathway之細胞凋亡、細胞增生以及細胞轉移作用...........170
第六節AC-10主成分(AC-0)抑制裸鼠皮下植入小鼠黑色素瘤細胞
B16F10之腫瘤生長................................................................171
第六章 綜合討論.........................................................................................217
第七章 結論...........................................................................................228
第八章 參考文獻...................................................................................233


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