臺灣博碩士論文加值系統

為了開發天然藥物於癌症治療上的應用，本實驗室由粗毛金星蕨的甲醇萃取物中，分離出新的黃酮類化合物protoapigenone。在細胞毒殺活性篩選結果中顯示出protoapigenone對人類卵巢癌細胞 (MDAH-2774, SKOV3)、子宮頸癌細胞株 (HeLa, C33A) 、乳癌細胞株 (T47D, MCF-7)及前列腺癌細胞株 (LNCap) 具有明顯的細胞毒殺效果。特別的是，protoapigenone對於正常人類乳腺細胞 (MCF-10A) 以及卵巢上皮細胞 (HOSE6-3, HOSE11-12) 的毒殺效果很低，這結果顯示protoapigenone可能對於抑制人類癌細胞具有選擇性。因此，我們進而探討protoapigenone對女性特有癌症“卵巢癌”及男性特有癌症“前列腺癌”的毒殺作用與機轉。
以protoapigenone對癌細胞的細胞週期以及細胞凋亡的影響為研究方向，由實驗結果得知protoapigenone對卵巢癌細胞以及前列腺癌細胞在作用的前期 (PS外翻的細胞/ Annexin-V/FITC positive cells) 或後期 (造成DNA斷裂的細胞/ TUNEL positive cells) 皆能顯著的引起癌細胞走向細胞凋亡之自殺死亡機制。所引起細胞凋亡的機制乃藉由調控BcL-2蛋白家族，進而活化caspase-3的活性，促使DNA修復蛋白Poly (ADP-ribose) polymerase (PARP) 切裂而無法進行DNA修復作用。另一方面，protoapigenone也藉由影響CDK2、Cyclin B1、Cdc25C蛋白活性表現，而將卵巢癌細胞以及前列腺癌細胞的細胞週期停滯於S及G2/M，因而抑制了癌細胞的不正常增生。在前列腺癌細胞中，研究成果證實p38 MAPK及JNK1/2的活性在protoapigenone對於前列腺癌細胞所引起的毒殺機制中佔著舉足輕重的角色。
進而於活體動物實驗結果中， 顯示protoapigenone在裸鼠動物實驗上確實能有效地抑制卵巢癌腫瘤以及前列腺癌腫瘤的生長。此有效的抑制腫瘤生長作用乃與protoapigenone能引起的細胞凋亡有關。分析裸鼠動物之血液生化值，得知protoapigenone對於治療後的活體動物之肝、腎及造血功能並沒有產生顯著性的不良副作用。
由研究的初步成果證實protoapigenone在in vitro及in vivo的實驗皆具有顯著且低毒性的抑制癌細胞生長效果，開啟了protoapigenone具有潛力成為一個抗卵巢癌及前列腺癌的化合物之發展性。目前的研究成果，已經申請台灣、美國以及歐、日等國之專利，以朝植物藥新藥開發為目標。

In order to develop the application of nature products on cancer therapy, we isolated a novel flavonoid, protoapigenone, from the methanol extract of Thelypteris torresiana (Gaud.) Alston (Thelypteridaceae). The cytotoxic results indicated that protoapigenone showed the significantly cytotoxic activities toward human ovarian cancer cell lines (MDAH-2774, SKOV3), human cervical cancer cell lines (HeLa, C33A), human breast cancer cell lines (T47D, MCF-7) and human prostate cancer cell line (LNCap). Compared the data with immortalized human breast cancer cell line (MCF-10A) and ovarian epithelial cell lines (HOSE6-3, HOSE11-12), it had less cytotoxic activity. These results suggested that protoapigenone has the selective cytotoxicity against human cancer cells. This drawn our interests to investigate the anti-cancer effect and molecular mechanism of protoapigenone on ovarian cancer cells and prostate cancer cells.
The aim of this study is detecting the effect of protoapigenone on cell cycle progression and apoptotic induction in cancer cells. The results showed that protoapigenone significantly induced cancer cells go through apoptosis in early (Annexin-V/FITC positive cells) and late period (TUNEL positive cells). Protoapigenone regulated the BcL-2 family, and then induced caspase-3 activity which cleaved the DAN repair protein Poly (ADP-ribose) polymerase (PARP). On the other hand, protoapigenone also arrested cancer cells at S and G2/M phases by regulating the activation and expression of CDK2, Cyclin B1 and Cdc25C on ovarian and prostate cancer cells. In prostate cancer, the activity of p38 MAPK and JNK1/2 play the critical roles in protoapigenone-induced cell death.
In vivo study, the results showed that protoapigenone suppressed the growth of ovarian and prostate cancer cells without significant hepatotoxicity, nephrotoxicity and hematological toxicity. Protoapigenone actually induced cancer cells go through apoptosis in protoapigenone-treated tumors.
Our results first indicated that protoapigenone may be developed a promising chemotherapeutic agent for ovarian or prostate cancer in the future. The results of our research have filed Taiwan、US、Japan and Europe patent to develop as new botanical drugs.

目 錄
中文摘要………………………………………………………1

英文摘要………………………………………………………3
第一章 緒論
第一節 研究動機與背景……………………………………6
第二節 文獻回顧
一、蕨類植物的保健功效………………………………………9
二、Protoapigenone介紹………………………………………10
三、卵巢癌概觀…………………………………………………11
四、前列腺癌概觀………………………………………………13
五、腫瘤的形成、特性與治療…………………………………18
六、細胞週期與調控方式………………………………………19
七、細胞凋亡療法………………………………………………22
八、細胞凋亡路徑………………………………………………24
九、訊息傳遞在細胞凋亡中的角色……………………………27
第二章 研究設計與目標………………………………………29
一、細胞株選……………………………………………………30
二、評估Protoapigenone於癌症治療的指標…………………30
三、動物實驗……………………………………………………31
第三章 實驗材料與方法
一、植物取得、Protoapigenone純化及配製…………………32
二、細胞培養 (Cell culture)…………………………………33
三、細胞增生試驗 (XTT proliferation assay) ……………34
四、型態學觀察 (Morphological hange) ……………………34
五、細胞存活測驗 (Cell survival assay) …………………36
六、細胞群落分析 (Clonogenic a ssay) ……………………36
七、 Annexin V/FITC細胞凋亡分析…………………………37
八、TUNEL細胞凋亡分析……………………………………40
九、細胞週期及sub-G1分析…………………………………42
十、 SB203580及SP600125之抑制劑處理………………42
十一、specific siRNA抑制p38 MAPK及JNK1/2表現……43
十二、西方點墨法 (immunoblotting) ………………………44
十三、裸鼠動物實驗 ( In vivo assay) ……………………45
十四、免疫組織染色 (Immunohistochemicl staining) ……47.
十五、統計 (Statistical analysis) …………………………48
第四章 Protoapigenone引起卵巢癌細胞MDAH-2774及
SKOV3細胞週期停滯與細胞凋亡作用的機轉探討………49
第一節 前言
一、黄酮類化合物(flavonoids) ………………………………50
二、Protoapigenone對婦科癌細胞株的療效………………51
第二節 結果
一、Protoapigenone抑制卵巢癌細胞增生與細胞群落形成
二、Protoapigenone抑制卵巢癌細胞週期的進行…………52
三、Protoapigenone引起卵巢癌細胞進行凋亡作用………56
四、Protoapigenone與cisplatin的共同處理在
MDAH-2774所產生的加成效果………………………65
五、Protoapigenone在活體實驗中，藉由產生凋亡作用
而抑制卵巢癌腫瘤的生長……………………………67
第三節 討論………………………………………………72
第五章 Protoapigenone引起前列腺癌細胞LNCap細胞週期
與細胞凋亡作用的機轉探討…………………………………77
第一節 前言
一、絲裂原活化蛋白激酶……………………………………78
二、JNK/SAPK路徑……………………………………………79
三、p38 MAPK路徑……………………………………………80
四、SP600125…………………………………………………81
五、SB203580…………………………………………………83
第二節 結果
一、Protoapigenone抑制前列腺癌細胞株LNCap的增生……85
二、Protoapigenone引起前列腺癌細胞株LNCap產生凋
亡作用……………………………………………………87
三、Protoapigenone抑制前列腺癌細胞株LNCap細胞週
期的進行…………………………………………………90
四、Protoapigenone活化前列腺癌細胞株LNCap中
p38 MAPK及JNK1/2活性………………………………92
五、p38 MAPK及JNK1/2在protoapigenone所引起的
生物活性(細胞凋亡與細胞週期停滯) 上的角色…………96
六、Protoapigenone在活體實驗中，藉由活化p38 MAPK
及JNK1/2，進而產生凋亡作用而抑制前列腺癌腫瘤
的生長情形…………………………………………………100
第三節 討論………………………………………………………104
第六章 結論與展望……………………………………………109
第七章 論文著作發表…………………………………………117
第八章 參考文獻………………………………………………120

圖 表 目 錄
Figure1-1：粗毛金星蕨植物型態與protoapigenone結構式………8
Figure1-2：Two death pathways—an extrinsic pathway
and an intrinsic pathway……………………………25
Figure1-3：Mitogen-activated protein kinase cascades……28
Figure2-1：研究設計與目標………………………………………29
Figure3-1：Apoptosis - the programmed death of a cell…35
Figure3-2：Annexin-V assay……………………………………38
Figure3-3：TTUNEL assay………………………………………41
Figure3-4：種植癌細胞至裸鼠皮下……………………………46
Table4-1：Cytotoxicity of protoapigenone on various cancer cells lines and immortalized non-cancer epithelial cells determined by XTT cell proliferation assay.53
Figure4-1：The effect of protoapigenone on colony
formation.....................................55
Figure4-2：The effect of protoapigenone on cell cycle
distribution of ovarian cancer cells…………………………57
Figure4-3：Proteins involved in S and G2/M progression
were modulated by protoapigenone. ………………………58
Figure4-4：The cytotoxicity of protoapigenone on different
cell cycle………………………………………………………60
Figure4-5：Protoapigenone induced apoptotic death in
ovarian cancer cells. …………………………………62
Figure4-6：Proteins involved in apoptosis pathway were
regulated by protoapigenone…………………………64
Figure4-7：Combined protoapigenone and cisplatin
treatment generated synergistic cytotoxic effect……66
Figure4-8：Protoapigenone suppressed MDAH-2774
ovarian tumor growth in nude mice xengraft assay…68
Table 4-2：(A) Body weight (BW) profile, (B) complete blood
count and biochemical profile for the nude mice
after treatment with protoapigenone for 7 weeks……69
Figure4-9：The cleaved PARP expression in ovarian tumor
tissues …………………………………………………71
Figure4-10：Schematic diagram showing the proposed
mechanisms of activation of protoapigenone in
MDAH-2774 and SKOV3 ovarian cancer cells………76
Figure 5-1：SP600125 structure……………………………………82
Figure 5-2：SB203580 structure……………………………………83
Figure5-3：Effect of protoapigenone on inhibiting the growth of LNCap cells………………………………………………86
Figure5-4：Induction of apoptosis by protoapigenone in LNCap
cells………………………………………………………89
Figure5-5：Effect of protoapigenone on cell cycle distribution of LNCap cells………………………………………91
Figure5-6：Protoapigenone enhanced the activation of p38
MAPK and JNK1/2 in LNCap cells……………………93
Figure5-7：SB203580 and SP600125 reversed the
protoapigenone- induced activation of p38 MAPK
and JNK1/2 and growth inhibition in LNCap cells.…95
Figure5-8：Effect of p38 MAPK inhibitor SB203580 and JNK1/2
inhibitor SP600125 on protoapigenone-induced
apoptosis and cell cycle arrest………………………97
Figure5-9：Effect of specific siRNAs for p38 MAPK and JNK1/2
on protoapigenone-induced apoptosis and cell
cycle arrest………………………………………………99
Figure5-10：Protoapigenone suppressed the xenograft tumor
growth by increasing p38 MAPK and JNK1/2
activation……………………………………………………102
Table 5-1：Body weight (BW) profile and complete blood count
and biochemical profile for the nude mice after
treatment with protoapigenone for 5 weeks…………103

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