臺灣博碩士論文加值系統

摘要（第一部分）

卡瓦胡椒素B (Flavokawain B，FKB)是卡瓦萃取物中一種天然存在的植物化學物質，已經有許多文獻指出卡瓦胡椒素B具有抗癌的功效。然而，卡瓦胡椒素B對人類胃癌的抗癌作用及其作用分子機制尚未明確闡明，因此本研究利用體內與體外實驗，探討卡瓦胡椒素B在人類胃癌中，如何誘導細胞週期停滯，以及造成細胞自噬的抗癌分子機制。
研究發現，卡瓦胡椒素B對人類胃癌細胞株(AGS/NCI-N87/KATO-III/ TSGH9201)具有較顯著的促進死亡與抑制生長作用，對人類成纖維細胞株Hs738.St/Int和大白鼠初代肝細胞只具有輕度的抑制細胞生長，不具有細胞毒性與促進死亡的作用。並且在人類胃腺癌細胞株AGS中，卡瓦胡椒素B經由上調Beclin-1/Bcl-2蛋白比例，促進酸性囊狀胞器(Acidic vesicular organelles，AVOs)和GFP-LC3斑點的形成，促進GFP-LC3蛋白轉換成GFP-LC3-I與GFP-LC3-II以及增加內生性自噬蛋白LC3-I與LC3-II (Light Chain 3-I ; Light Chain 3-II)的表現量，同時抑制ATG4B蛋白?〞漪﹞A最後Sequestosome 1 (p62/SQSTM1)在進行自噬降解的途徑中被降解，因此減少自噬的抑制蛋白mTOR，進而促進細胞的自噬。但是卡瓦胡椒素B無法造成凋亡下游路徑的凋亡蛋白Proaspase-3的裂解，同時使用凋亡的抑制劑半胱天冬?“簏s劑(Z-VAD-FMK)也無法回復細胞的存活率，因此無法誘導細胞凋亡。預先加入早期自噬的抑製劑3-Methyladenine (3-MA)和晚期自噬的抑制劑Chloroquine diphosphate salt (CQ)，以及使用RNA干擾(RNA interference，RNAi)的系統將LC3基因沉默(shLC3)，研究發現可以顯著逆轉回復人類胃腺癌細胞株AGS的存活率，因此卡瓦胡椒素B誘導人類胃腺癌細胞株AGS過度的產生自體吞噬，使細胞內容物過度的降解，造成細胞引發自噬性死亡，而不是通過凋亡造成細胞死亡。
卡瓦胡椒素B促進人類胃腺癌細胞株AGS產生大量的活性氧化物累積，因此預先加入活性氧化物的抑制劑N-Acetyl-L-cysteine (NAC)，即使在加入卡瓦胡椒素B，研究發現可以顯著的逆轉回復人類胃腺癌細胞株AGS的存活率與抑制細胞自噬性的死亡，因此活性氧化物被確定是細胞自噬的誘發因素。此外，卡瓦胡椒素B促進人類胃腺癌細胞株AGS產生大量活性氧化物的累積，進而上調JNK1/2信號路徑，造成細胞週期停滯在G2/M期，因此細胞增殖停止。有趣的是，在HER-2/neu基因高表達的人類胃癌細胞NCI-N87與AGS中，因為誘導細胞自噬，造成細胞自我分解代謝的過程增高，因此造成HER-2/neu、PI3K/AKT和mTOR總蛋白與磷酸化蛋白數量減少，同時mTOR蛋白的減少可以更加成促進細胞誘導自噬，因此過度的自噬現象造成細胞死亡。將人類胃腺癌細胞株AGS轉染促凋亡蛋白Bax表達載體，發現在促凋亡蛋白Bax過度表達之下，卡瓦胡椒素B可以促進細胞凋亡，同時減弱細胞自噬。推測卡瓦胡椒素B無法誘導促凋亡蛋白Bax的活化，導致人類胃腺癌細胞株AGS無法啟動細胞凋亡，造成細胞自噬取代細胞凋亡，因此，促凋亡蛋白Bax的活化是卡瓦胡椒素B誘導人類胃癌細胞凋亡的重要關鍵蛋白。
體內研究結果證明，卡瓦胡椒素B誘導無胸腺裸鼠(Nude mice)的腫瘤自噬作用，有效抑制人類胃腺癌細胞株AGS異種移植(Xenograft Model)無胸腺裸鼠的腫瘤生長，並且延長無胸腺裸鼠的存活率。值得注意的是，在卡瓦胡椒素B的誘導下，LC3基因沉默(shLC3)減弱卡瓦胡椒素B誘導人類胃腺癌細胞株AGS-shLC3異種移植無胸腺裸鼠的腫瘤自噬信號蛋白的表現量，因此回復無胸腺裸鼠的腫瘤生長與腫瘤重量。從體外和體內研究獲得的結果，證實卡瓦胡椒素B可以作為預防和治療人類胃腺癌AGS有效的天然抗癌藥物。

摘要（第二部分）

乳癌是女性最常被診斷的惡性腫瘤之一，乳癌的轉移和復發是乳癌患者死亡的主要原因，雖然目前乳癌的治療方式，已經可以有效的控制乳癌的惡化情形，但仍然無法有效治愈大多數的晚期乳癌患者。
天然食藥用真菌香杉芝(Antrodia salmonea)具有許多生物活性成分，越來越多的證據表明，香杉芝表現出廣泛的抗癌功效，然而，香杉芝對乳腺癌細胞自噬、細胞週期調節和乳腺癌腫瘤抑制的機制作用的研究仍然知之甚少。因此，本研究利用體內與體外實驗，探討香杉芝在體外培養的人類三陰性乳腺癌細胞株MDA-MB-231中，如何誘導細胞週期停滯，以及造成細胞自噬與細胞凋亡的潛在分子機制，並且利用在皮下注射人類三陰性乳腺癌細胞株MDA-MB-231的無胸腺裸鼠(Nude mice)模型中，研究體內抗腫瘤的機制作用。
研究發現，香杉芝經由抑制細胞週期蛋白A (Cyclin A)和細胞週期蛋白B1 (Cyclin B1)和細胞分裂週期蛋白2 (Cell division cycle protein 2，CDC 2)，造成人類三陰性乳腺癌細胞株MDA-MB-231細胞週期停滯在G2/M期，因此細胞增殖停止。此外，預處理活性氧化物的抑制劑N-Acetyl-L-cysteine (NAC)能有效抑制香杉芝誘導的細胞週期停滯，這也表明活性氧化物的累積與接連的G2/M細胞週期停滯，可能是香杉芝促進人類三陰性乳腺癌細胞株MDA-MB-231的死亡與抑制細胞增殖的主要原因，同時香杉芝在人類三陰性乳腺癌細胞株MDA-MB-231中，可以降低環氧合??(Cycloxygenase-2，COX-2)的活化，經由預處理N-Acetyl-L-cysteine (NAC)可以顯著逆轉回復環氧合??(COX-2)的產生。
香杉芝在人類三陰性乳腺癌細胞株MDA-MB-231中，經由增加DNA片段化(DNA fragmentation)，上調Bax/Bcl-2蛋白比例，同時參與內生性的粒腺體(Mitochondrial)信號路徑和外生性的死亡受體(Death-receptor)信號路徑，因此誘導細胞晚期的凋亡。並且經由減少phospho-mTOR (Ser2448)的自噬抑制蛋白，上調Beclin-1/Bcl-2蛋白比例，促進GFP-LC3斑點和酸性囊狀胞器(Acidic vesicular organelles，AVOs)的形成，促進GFP-LC3蛋白轉換成GFP-LC3-I與GFP-LC3-II以及增加內生性自噬蛋白LC3-I轉換成LC3-II的比例，同時增加自噬蛋白?，TG7的活化，最後Sequestosome 1 (p62/SQSTM1)在進行自噬降解途徑中被降解，因此誘導細胞的自噬。值得注意的是，預先處理N-Acetyl-L-cysteine (NAC)可以顯著抑制香杉芝誘導活性氧化物的產生，進而降低香杉芝誘導的粒腺體功能障礙、細胞自噬和細胞凋亡。
在人類三陰性乳腺癌細胞株MDA-MB-231中，使用凋亡的抑制劑半胱天冬?“簏s劑(Z-VAD-FMK)能夠抑制香杉芝所誘導的細胞自噬，減少自噬蛋白LC3-I轉換成LC3-II的比例與降低誘導自噬的酸性囊狀胞器(Acidic vesicular organelles，AVOs)形成。同樣的，使用早期自噬的抑製劑3-Methyladenine (3-MA)和晚期自噬的抑制劑Chloroquine diphosphate salt (CQ)能夠減少香杉芝所誘導的細胞凋亡，減少DNA片段化(DNA fragmentation)與抑制促凋亡蛋白Caspase-3的活化，因此促進凋亡與自噬交互作用的方式為共同合作的關係，共同造成細胞誘導死亡途徑，進而造成自噬性細胞死亡(Autophgic cell death)與細胞凋亡(Apoptosis)。
體內研究結果證明，香杉芝誘導人類三陰性乳腺癌裸鼠皮下移植瘤的細胞週期停滯在G2/M期，進而引發腫瘤的凋亡與自噬作用，這或許是延緩無胸腺裸鼠(Nude mice)的腫瘤發生率、抑制腫瘤的生長、降低腫瘤的重量和抑制腫瘤轉移與侵襲的原因。從體外和體內研究獲得的結果，證實香杉芝可以作為預防和治療人類三陰性乳腺癌MDA-MB-231有效的天然抗癌藥物。

Abstract (Part I)

Flavokawain B (FKB), a naturally occurring chalcone in kava extracts, has been reported to possess anticancer activity. However, the effect of FKB on gastric cancer remains unclear. We examined the in vitro and in vivo anticancer activity and autophagy involvement of FKB and determined the underlying molecular mechanisms.
FKB is potently cytotoxic to human gastric cancer cells (AGS/NCI-N87/ KATO-III/TSGH9201) and mildly toxic towards normal (Hs738.St/Int) cells and primary mouse hepatocytes. FKB induced AGS cell death was characterized by autophagy, not apoptosis, as evidenced by increased LC3-I and LC3-II, GFP-LC3 puncta and acidic vesicular organelles (AVOs) formation, without resulting procaspase-3 cleavage. FKB further caused p62/SQSTM1 degradation, mTOR downregulation, ATG4B inhibition, and Beclin-1/Bcl-2 upregulation. Silencing autophagy inhibitors 3-MA/CQ and RNAi (shLC3) significantly reversed the FKB induced cell death of AGS cells.
FKB triggered ROS generation and ROS inhibition by NAC pretreatment diminished FKB induced cell death, which indicated ROS mediated autophagy in AGS cells. Furthermore, FKB induces G2/M arrest and alters cell cycle proteins through ROS-JNK signaling. Interestingly, FKB induced autophagy is nonassociated with the suppression of HER-2 and PI3K/AKT/mTOR signaling cascades. FKB inhibits apoptotic Bax expression, and Bax-transfected AGS cells exhibit both apoptosis and weak autophagy; thus, FKB inactivated Bax results in apoptosis inhibition.
In vivo data demonstrated that FKB effectively inhibited tumor growth, prolonged the survival rate, and induced autophagy in AGS-xenografted mice. Notably, silencing of LC3 attenuated FKB induced autophagy in AGS-xenografted tumors. FKB may be a potential chemopreventive agent in the activation of ROS mediated autophagy of gastric cancer cells.

Abstract (Part II)

Breast cancer is the most prevalent malignancy observed in women. Approximately one-third of all women with breast cancer develops metastases and ultimately dies due to the effects of the disease. Although some current therapies have shown promise against breast cancer, but still no effective cure for majority of patients in the advanced stages.
Antrodia salmonea (AS) possesses a number of bioactive components. Increasing evidence suggests that fruiting body of AS exhibits a wide range of biological activities. In the present study, we examined the effects of AS on cell cycle arrest and the underlying molecular mechanisms involved in autophagic cell death and apoptotic cell death in vitro in MDA-MB-231 cells and on tumor regression in vivo using an athymic nude mice model.
Antrodia salmonea (0–200 μg/mL) treatment significantly induced G2/M cell cycle arrest in MDA-MB-231 cells by reducing the levels of cyclin A, cyclin B1 and CDC2 proteins. In addition, N-acetylcysteine (NAC) pretreatment prevented AS induced G2 cell cycle arrest, indicating that ROS accumulation and subsequent cell cycle arrest might be a major mechanism of AS induced cytotoxicity and inhibited cell growth. Further, AS treatment decreased COX-2 expression and AS treatment recovery COX-2 was significantly reversed by NAC pretreatment in MDA-MB-231 cells.
AS induced late apoptosis through increased DNA fragmentation, dysregulated Bax/Bcl-2 ratio and both mitochondrial and death receptor signaling pathways. AS induced autophagy was evidenced via increased LC3-II/LC3-I ratio、ATG 7 activation, decreased p-mTOR, vanished p62/SQSTM1 expressions, GFP-LC3 puncta, Acidic vesicular organelles (AVOs) formation and upregulated Beclin-1/Bcl-2 ratio in MDA-MB-231 cells. Interestingly, blockade of AS induced ROS production by NAC pretreatment substantially attenuated AS induced autophagy, mitochondrial dysfunction and apoptosis.
Inhibition of apoptosis by Z-VAD-FMK suppressed AS induced autophagicdeath (decreased LC3-II/LC3-I ratio and AVOs), Similarly, inhibition of autophagy by 3-methyladenine/chloroquine diminished AS-induced apoptosis (decreased DNA fragmentation and caspase-3) in MDA-MB-231 cells, Therefore, Autophagy and apoptosis cooperating to induce cell death.
The in vivo study results revealed that AS treatment was effective in terms of delaying the tumor incidence and reducing the tumor growth in MDA-MB-231 xenografted nude mice. Confirmed that AS significantly modulated the xenografted tumor progression as demonstrated by induction of cell cycle arrest, apoptosis and autophagy. Antrodia salmonea could be an anticancer agent for human breast cancer.

目錄
天然植物對胃癌與乳癌死亡機制探討
第一部分
卡瓦胡椒素B誘導人類胃腺癌細胞自噬之機制探討
摘要 2
Abstract 4
第一章 前言 5
第二章 文獻探討 7
ㄧ、胃癌(Gastric cancer) 8
二、卡瓦(Kava) 11
三、活性氧化物(Reactive oxygen species，ROS) 15
四、自噬(Autophagy) 18
五、細胞週期與癌症 24
第三章 實驗試劑、材料與儀器 25
ㄧ、實驗試劑 26
二、實驗材料 32
三、實驗儀器 33
第四章 實驗方法 36
ㄧ、人類胃癌細胞株、人類成纖維細胞株與大白鼠初代肝細胞之細胞
培養 37
二、卡瓦胡椒素B (Flavokawain B)之配製 45
三、倒立式位相差顯微鏡觀察人類胃癌細胞株與人類成纖維細胞株之
細胞形態(Morphology) 45
四、人類胃癌細胞株與人類成纖維細胞株之細胞存活率(Cell viability)
測定 46
五、大白鼠初代肝細胞之細胞存活率(Cell viability)測定 50
六、人類胃腺癌細胞株之群落形成能力(Colony formation assay) 51
七、人類胃腺癌細胞株之活性氧物質(Reactive oxygen species，ROS)
產量測定法 52
八、利用流式細胞分析儀(Flow cytometry )探討卡瓦胡椒素B
(Flavokawain B)對人類胃腺癌細胞週期之影響 54
九、利用流式細胞分析儀(Flow cytometry)探討卡瓦胡椒素B
(Flavokawain B)對人類胃腺癌細胞自噬(Autophagy)之影響 57
十、利用?B啶橙染色法(Acridine orange staining)探討卡瓦胡椒素B
(Flavokawain B)對人類胃腺癌細胞自噬(Autophagy)之影響 59
十一、利用GFP-LC3 (Green Fluorescent Protein- Light Chain 3)質體轉
染探討卡瓦胡椒素B (Flavokawain B)對人類胃腺癌細胞自噬
(Autophagy)之影響 60
十二、利用BAX過度表達(Overexpression)探討卡瓦胡椒素B
(Flavokawain B)對人類胃腺癌細胞凋亡(Apoptosis)與細胞自噬
(Autophagy)之影響 62
十三、TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end
labeling) 檢測人類胃腺癌細胞凋亡 65
十四、人類胃癌細胞總蛋白質萃取 67
十五、人類胃癌細胞蛋白質定量 69
十六、西方墨點法(Western blot) 71
十七、質體(Plasmid)製備 80
十八、質體抽取(plasmid DNA extraction) 83
十九、RNA interference (shRNA)之細胞實驗 85
二十、無胸腺裸鼠(Nude mice)異種移植腫瘤(Xenograft Tumor )之動物
實驗 92
二十ㄧ、動物組織包埋與切片 95
二十二、動物組織切片蘇木精-伊紅(Hematoxylin-eosin)染色法 95
二十三、動物組織蛋白質萃取 96
二十四、免疫組織化學染色(Immunochemistry Staining，IHC) 98
二十五、無胸腺裸鼠(Nude mice) 異位移植腫瘤(Xenogr a f t
Tumor )之存活率實驗 102
二十六、RNA interference (shRNA)之動物實驗 104
二十七、實驗統計 105
第五章 實驗結果與圖表 106
ㄧ、卡瓦胡椒素B (Flavokawain B，FKB)誘導自噬，顯著促進人類胃
腺癌細胞株AGS的死亡與抑制群落形成能力 107
二、卡瓦胡椒素B (Flavokawain B，FKB)調節Beclin-1 / Bcl-2蛋白 比
例，促進人類胃腺癌細胞株AGS誘導自噬 112
三、卡瓦胡椒素B (Flavokawain B，FKB)透過自噬造成人類胃腺 癌細
胞株AGS死亡，而不是透過凋亡 117
四、卡瓦胡椒素B (Flavokawain B，FKB)促進活性氧化物的累積
，造成人類胃腺癌細胞株AGS自噬性的死亡 124
五、卡瓦胡椒素B (Flavokawain B，FKB)促進過氧化氫(Hydrogen
peroxide，H2O2)的累積與改變鈣離子的動態平衡，造成人類胃腺癌細
胞株AGS的死亡 132
六、卡瓦胡椒素B (Flavokawain B，FKB)造成人類胃癌細胞株NCI-N87
與過度表達Bax蛋白的人類胃腺癌細胞株AGS，同時誘導自噬與凋亡
136
七、卡瓦胡椒素B (Flavokawain B，FKB)促進活性氧化物的累積，進
而上調JNK信號路徑造成細胞週期停滯在G2/M期 142
八、卡瓦胡椒素B (Flavokawain B，FKB)抑制HER-2/neu基因高表達
的胃癌細胞株AGS與NCI-N87的HER-2/neu、PI3K/AKT和mTOR蛋白
的表現 150
九、卡瓦胡椒素B (Flavokawain B，FKB)抑制人類胃腺癌細胞株AGS
異種移植(Xenograft Model)無胸腺裸鼠(Nude mice)的腫瘤生長與延長
無胸腺裸鼠(Nude mice)的存活率 152
十、LC3基因沉默(shLC3)減弱卡瓦胡椒素B (Flavokawain B，FKB)誘
導人類胃腺癌細胞株AGS-shLC3自噬性死亡與回復無胸腺裸鼠(Nude
mice)的腫瘤生長 158
十ㄧ、卡瓦胡椒素B (Flavokawain B，FKB)減弱人類胃腺癌細胞株
AGS-shLC3異種移植(Xenograft Model)無胸腺裸鼠(Nude mice)的腫瘤
自噬蛋白表現 163
第六章 討論 167
第七章 結論 177

第二部分
香杉芝誘導人類三陰性乳腺癌(MDA-MB-231)
細胞自噬和細胞凋亡之機制探討
摘要 180
Abstract 182
第一章 前言 184
第二章 文獻探討 187
ㄧ、乳癌(Breast cancer) 188
二、香杉芝(Antrodia salmonea) 190
三、凋亡(Apoptosis) 192
四、凋亡(Apoptosis)與自噬(Autophagy)之間的關係 193
五、細胞發炎(Inflammation) 195
第三章 實驗方法 196
ㄧ、人類乳腺癌細胞株與人類正常乳腺上皮細胞株之細胞培養 197
二、香杉芝(Antrodia salmonea)之配製 199
三、倒立式位相差顯微鏡觀察人類乳腺癌細胞株與人類正常乳腺上皮
細胞株之細胞形態(Morphology) 199
四、人類乳腺癌細胞株與人類正常乳腺上皮細胞株之細胞存活率(Cell
viability)測定 200
五、人類乳腺癌細胞株與人類正常乳腺上皮細胞株之活性氧物質
(Reactive oxygen species，ROS)產量測定法 202
六、利用流式細胞分析儀(Flow cytometry )探討香杉芝(Antrodia
salmonea)對人類三陰性乳腺癌細胞週期之影響 204
七、利用?B啶橙染色法(Acridine orange staining)探討香杉芝 (Antrodia
salmonea)對人類三陰性乳腺癌細胞自噬之影響 205
八、利用流式細胞分析儀(Flow cytometry)探討香杉芝(Antrodia
salmonea)對人類三陰性乳腺癌細胞粒腺體膜電位 (Mitochondrial
membrane potential)之影響 207
九、利用流式細胞分析儀(Flow cytometry)探討香杉芝(Antrodia
salmonea)對人類三陰性乳腺癌細胞凋亡之影響 209
十、利用GFP-LC3 (Green Fluorescent Protein- Light Chain 3)質體轉染
探討香杉芝(Antrodia salmonea)對人類三陰性乳腺癌細胞自噬
(Autophagy)之影響 211
十ㄧ、TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end
labeling) 檢測人類三陰性乳腺癌細胞凋亡 212
十二、人類三陰性乳腺癌細胞總蛋白質萃取 213
十三、人類三陰性乳腺癌細胞蛋白質定量 213
十四、西方墨點法(Western blot) 213
十五、無胸腺裸鼠(Nude mice)異位移植腫瘤(Xenograft Tumor)之動物
實驗 214
十六、動物組織包埋與切片 217
十七、動物組織蛋白質萃取 217
十八、免疫組織化學染色(Immunochemistry Staining，IHC) 217
十九、動物組織凋亡分析(Tissue TUNNEL assay) 218
二十、實驗統計 221
第四章 實驗結果與圖表 222
ㄧ、香杉芝(Antrodia salmonea)顯著的抑制人類三陰性乳腺癌細胞株
MDA-MB-231的存活率與造成細胞形態的改變 223
二、香杉芝(Antrodia salmonea)誘導人類三陰性乳腺癌細胞株
MDA-MB-231產生活性氧化物(Reactive oxygen species，ROS)
225
三、香杉芝(Antrodia salmonea) 造成人類三陰性乳腺癌細胞株
MDA-MB-231粒腺體膜電位 (Mitochondrial membrane potential △Ψm）
流失 227
四、香杉芝(Antrodia salmonea) 促進活性氧化物(Reactive oxygen
species，ROS)的累積，造成人類三陰性乳腺癌細胞株MDA-MB-231
的死亡 228
五、香杉芝(Antrodia salmonea)促進活性氧化物(Reactive oxygen
species，ROS)的累積，造成人類三陰性乳腺癌細胞株MDA-MB-231
的G2/M期細胞週期停滯(Cell cycle arrest) 229
六、香杉芝(Antrodia salmonea)促進活性氧化物(Reactive oxygen
species，ROS)的累積，造成G2/M期的細胞週期停滯(Cell cycle arrest)
和降低環氧合??-2 (COX-2)的活性表現 231
七、香杉芝(Antrodia salmonea)促進活性氧化物(Reactive oxygen
species，ROS)的累積，造成人類三陰性乳腺癌細胞株MDA-MB-231
的DNA損傷(DNA damage) 234
八、香杉芝(Antrodia salmonea)誘發人類三陰性乳腺癌細胞株
MDA-MB-231的DNA片段化(DNA Fragmentation) 236
九、香杉芝(Antrodia salmonea)活化粒線體(Mitochondrial)和死亡受體
(Death-receptor)路徑誘導人類三陰性乳腺癌細胞株MDA-MB-231晚
期細胞凋亡 239
十、香杉芝(Antrodia salmonea)促進人類三陰性乳腺癌細胞株
MDA-MB-231自噬相關蛋白(Autophagy Related Protein)的表現 243
十一、香杉芝(Antrodia salmonea) 促進人類三陰性乳腺癌細胞株
MDA-MB-231的GFP-LC3轉換 246
十二、香杉芝(Antrodia salmonea)誘導人類三陰性乳腺癌細胞株
MDA-MB-231形成酸性囊狀胞器（Acidic vesicular organelles, AVOs
）造成自噬性細胞死亡 248
十三、香杉芝(Antrodia salmonea)誘發超氧自由基(superoxide anion，
·O2-)與過氧化氫(Hydrogen peroxide，H2O2)的生成，促進人類三陰性
乳腺癌細胞株MDA-MB-231自噬和細胞凋亡 252
十四、香杉芝(Antrodia salmonea)調節人類三陰性乳腺癌細胞株
MDA-MB-231的Beclin-1/Bcl-2和Bax/Bcl-2蛋白比例，促進細胞自噬和
細胞凋亡 256
十五、香杉芝(Antrodia salmonea)促進自噬與凋亡的共同合作，造成
人類三陰性乳腺癌細胞株MDA-MB-231的死亡 259
十六、香杉芝(Antrodia salmonea)抑制人類三陰性乳腺癌細胞株
MDA-MB-231異種移植(Xenograft Model)無胸腺裸鼠(Nude mice)的腫
瘤生長 262
十七、香杉芝(Antrodia salmonea)降低人類三陰性乳腺癌細胞株
MDA-MB-231異種移植(Xenograft Model)無胸腺裸鼠(Nude mice)的腫
瘤重量 264
十八、香杉芝(Antrodia salmonea)誘發人類三陰性乳腺癌裸鼠皮下移
植瘤的原位凋亡(Situ apoptosis) 266
十九、香杉芝(Antrodia salmonea)誘導人類三陰性乳腺癌裸鼠皮下移
植瘤的凋亡(Apoptosis)、自噬(Autophagy)和G2/M期的細胞週期停滯
，經由免疫組織化學染色分析 268
二十、香杉芝(Antrodia salmonea)誘導人類三陰性乳腺癌裸鼠皮下移
植瘤的凋亡(Apoptosis)、自噬(Autophagy)和G2/M期的細胞週期停滯
，經由西方墨點法(Western blot)分析 270
二十ㄧ、香杉芝(Antrodia salmonea)抑制人類三陰性乳腺癌螢光素??
細胞株MDA-MB-231-Lucifrein異種移植(Xenograft Model)無胸腺裸
鼠(Nude mice)的腫瘤生長 272
第五章 討論 274
第六章 結論 282
參考文獻 284

表目錄
天然植物對胃癌與乳癌死亡機制探討
第一部分
卡瓦胡椒素B誘導人類胃腺癌細胞自噬之機制探討
表ㄧ 實驗試劑 26
表二 實驗材料 32
表三 實驗儀器 33
表四 培養人類胃癌細胞株的RPMI 1640培養液之配方 37
表五 培養人類胃癌細胞株的IMDM培養液之配方 38
表六 培養人類成纖維細胞株的DMEM/HG培養液之配方 39
表七 培養大白鼠初代肝細胞的RPMI 1640培養液之配方 40
表八 1X磷酸鹽緩衝液(1X Phosphate buffered saline，1X PBS)之配方 41
表九 Propidium iodide (PI)染劑之配方 55
表十 In Situ Cell Death Detection Kit 65
表十一 Lysis buffer製備之配方 67
表十二 BSA標準品配製 70
表十三 SDS-PAGE製備之配方 72
表十四 6 X Protein loading dye製備之配方 73
表十五 5X Electrode buffer製備之配方 73
表十六 1X Transfer buffer製備之配方 74
表十七 Stripping buffer製備之配方 75
表十八 一級抗體名稱與貨號 75
表十九 二級抗體 77
表二十 FavorPrepTM Plasmid DNA Extraction Maxi Kit 83
表二十一 jetPEIR Transfection Reagent 86
表二十二 shRNA質體 87
表二十三 EnVisionTM FLEX Mini Kit (Dako) 98

第二部分
香杉芝誘導人類三陰性乳腺癌(MDA-MB-231)
細胞自噬和細胞凋亡之機制探討
表二十四 細胞凋亡與自噬性細胞死亡之間的特性關係 193
表二十五 DMEM/F12培養液之配方 197
表二十六 FITC Annexin V Apoptosis Detection Kit之內容物 209

圖目錄
天然植物對胃癌與乳癌死亡機制探討
第一部分
卡瓦胡椒素B誘導人類胃腺癌細胞自噬之機制探討
Fig. 1 FKB mediates growth inhibition of gastric carcinoma cells through
induction of autophagy. 111
Fig. 2 FKB enhances autophagy as a death mechanism in human gastric
cancer (AGS) cells. 116
Fig. 3 FKB induces cell death via autophagic pathways, not apoptotic
pathways, in AGS cells. 124
Fig. 4 FKB triggers ROS-mediated autophagy in AGS cells. 131
Fig. 5 Effects of ROS modulators on FKB-induced AGS cell death. 135
Fig. 6 Bax transfection promotes both apoptosis and autophagy in
FKB-treated AGS cells. 141
Fig. 7 FKB induces G2/M arrest through ROS-JNK signaling pathways in
AGS cells. 149
Fig. 8 FKB inhibits phosphorylation of HER-2/neu, PI3K/AKT and
mTOR in HER-2/neu-expressing gastric cancer (AGS and NCI-N87)
cells. 151
Fig. 9 In vivo inhibition of AGS-xenografted tumors in nude mice by
FKB treatment. 157
Fig. 10 In vitro and in vivo effects of LC3 silencing on FKB-mediated
autophagy. 162
Fig. 11 Western blot and histochemical analyses of autophagy in AGS
shLC3-xenografted tumors. 166

第二部分
香杉芝誘導人類三陰性乳腺癌(MDA-MB-231)
細胞自噬和細胞凋亡之機制探討
Fig. 1 AS inhibits viability of human breast cancer cells. 224
Fig. 2 AS induces intracellular ROS generation and mitochondrial
dysfunction in MDA-MB-231 cells. 226
Fig. 3 AS induces intracellular ROS generation and mitochondrial
dysfunction in MDA-MB-231 cells. 227
Fig. 4 AS induces intracellular ROS generation and mitochondrial
dysfunction in MDA-MB-231 cells. 228
Fig. 5 AS induces G2/M arrest in MDA-MB-231 cells. 230
Fig. 6 The effects of AS on cell-cycle regulatory proteins and COX-2
expression in MDA-MB-231 cells. 233
Fig. 7 The effects of NAC on AS-induced PARP cleavage in
MDA-MB-231 cells. 235
Fig. 8 AS triggers apoptotic DNA fragmentation in MDA-MB-231 cells.
238
Fig. 9 AS-induced late apoptosis is mediated through activation of
mitochondrial and death-receptor pathways. 242
F i g . 10 AS t r igger s au tophag y s ignal i n g mo lecu les in
MDA-MB-231 cells. 245
Fig. 11 AS promote conversion of GFP-LC3 in MDA-MB-231 cells.
247
Fig. 12 AS induces acidic vesicular organelle (AVOs) formation in
MDA-MB-231 cells. 251
Fig.13 AS triggers superoxide radical production and activates autophagy
and apoptosis in MDA-MB-231 cells. 255
Fig. 14 AS increases the ratio of Beclin-1/Bcl-2 and Bax/Bcl-2 in
MDA-MB-231 cells. 258
Fig. 15 Interaction between AS-induced apoptosis and autophagy of
MDA-MB-231 cells. 261
Fig. 16 Time-course effects of AS on MDA-MB-231 xenograft growth in
nude mice. 263
Fig. 17 In vivo inhibition of MDA-MB-231 xenograft tumors by AS
treatment. 265
Fig. 18 In situ apoptosis using TUNEL analysis in MDA-MB-231
xenografted tumors. 267
Fig. 19 Immunohistochemical analysis for caspase-3, LC3B, and cyclin
B1 in MDA-MB-231 xenografted tumors. 269
Fig. 20 AS inhibits MDA-MB-231 xenograft growth in nude mice
through induction of apoptosis- and autophagy-related proteins by
Western blotting. 271
Fig. 21 AS treatment suppressed tumor growth in living
MDA-MB-231-luciferase cells injected nude mice.

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2.期刊論文

王厄舟、潘勝利和張淑芳，卡瓦胡椒化學成分及藥理活性研究發展，
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