臺灣博碩士論文加值系統

中文摘要：
攝護腺癌為美國男性罹患最高的癌症並且在癌症致死排名中居第二大位。而在台灣男性的十大癌症排名第五位並且致死率也排名第七位。
近年來包括中草藥治療的另類療法 (Complementary Alternative Medicine, CAM) 逐漸興起，我們嘗試從中草藥中萃取與純化其中的抑癌成分並了解其作用機制。在過去的幾年中，我們與合作的實驗室分別從南柴胡與當歸萃取與純化出一些抑癌的成份。
南柴胡 (Bupleurum scorzonerifolium) 是一種重要的中草藥，在中國、日本和亞洲其他國家中，被當做藥物用在治療流感、登革熱、瘧疾、癌症及月經紊亂等疾病。本實驗室從南柴胡的萃取物中發現了新的純化物異柴胡內酯 (isochaihulactone)，對於肺癌細胞株A549的體外與體內測試皆有良好的抗癌效果。
當歸 (Angelica sinensis)，是傳統中藥，廣泛應用在治療婦女疾病方面，臨床上的用途包括活血、解痙和鎮痛等。在本實驗室先前的研究，從當歸的萃取物中發現了天然純化物正-丁烯基苯酞 (n-butylidenephthalide, BP)，在對抗惡性腦瘤的體外與體內研究上皆能有效抑制癌細胞的生長。
我們在本研究中則進一步探討異柴胡內酯及正-丁烯基苯酞針對攝護腺癌細胞生長之抑制作用，並進一步分析其作用機制。
異柴胡內酯會增加JNK的磷酸化，而前處理JNK的抑制物 (SP600125) 可以減少一部份異柴胡內酯造成的細胞死亡。異柴胡內酯的作用會造成LNCaP細胞週期停滯在G2/M期，伴隨著p53及p21蛋白質的表現量上升，而細胞週期檢查點的cdc25c、cyclin B1及cdc2等蛋白質的表現量下降；我們也觀察到Bcl-2蛋白質的磷酸化程度增加與蛋白酶caspase的活化。異柴胡內酯也誘發了EGR-1與NAG-1蛋白質的表達。前處理JNK的抑制物可以降低異柴胡內酯誘發的NAG-1蛋白質。利用siRNA抑制NAG-1基因的表達則可以部分阻斷異柴胡內酯造成的細胞凋亡。.
正-丁烯基苯酞的處理影響了LNCaP及PC-3人類攝護腺癌細胞株的生長與細胞週期。透過西方點墨法發現JNK訊息傳遞路徑受到正-丁烯基苯酞的活化。正-丁烯基苯酞造成細胞週期停滯在G0/G1期並且伴隨著p21及p27蛋白質的表現量上升，而細胞週期檢查點的cyclin D1及cdk2等蛋白質的表現量下降。我們利用cDNA微陣列的方式來找出正-丁烯基苯酞抑制攝護腺癌細胞生長的機制。結果發現正-丁烯基苯酞誘發了包含GADD153/CHOP在內的內質網相關基因的表達。西方點墨法證實了正-丁烯基苯酞處理LNCaP與PC-3細胞後，誘發了GRP78/BiP、IRE1-?悀哆ADD153/CHOP等蛋白質的表現。為了探討內質網壓力與正-丁烯基苯酞造成細胞死亡之間的關連性，我們利用siRNA抑制IRE1-?捋P GADD153/CHOP的表達，結果發現可以有效降低正-丁烯基苯酞引起的細胞死亡。而利用siRNA抑制JNK基因，可以降低由正-丁烯基苯酞誘發的IRE1與GADD153/CHOP蛋白質表達。結果顯示正-丁烯基苯酞誘發的內質網壓力應該是透過JNK的訊息傳遞路徑。透過異種異位的小鼠腫瘤移植模式也證明了正-丁烯基苯酞能有效抑制攝護腺癌的生長。
總而言之，我們的研究顯示異柴胡內酯可以用來抑制LNCaP攝護腺癌細胞的生長。另一方面，我們的研究顯示正-丁烯基苯酞可以藉由誘發內質網壓力來有效抑制攝護腺癌的生長。

Abstract:
Prostate cancer (PCa) is the most common malignancy in American men and the second leading cause of deaths from cancer. It is the fifth top malignanacy and seventh top cancer mortality in Taiwan men.
Moreover, the Complementary Alternative Medicine (CAM) has been proven to be helpful in cancer therapy attracted us to investigate the anti-tumor potential of several herbal extracts.
Nan-Chai-Hu (Bupleurum scorzonerifolium, BS) is an important Chinese herb in the treatment of influenza, fever, malaria, cancer, and the menstrual disorders in China, Japan, and many otherparts of Asia. Our laboratory have a novel lignan from the extract fraction of BS, isochaihulactone, that has antitumoral activity against lung cancer A549 cells in vitro and in vivo.
Danggui (Angelica sinensis, AS), a traditional Chinese medicine for menopausal symptoms and is recommended as a tonic, hemopoetic, spasmolytic and analgesic drug in clinical practice. In our previous study, n-butylidenephthalide (BP), a compound derived from AS chloroform extract, showed a dramatic antitumoral effect against malignant brain tumors in vitro and in vivo.
Isochaihulactone caused cell cycle arrest at G2/M phase in LNCaP cells, which was correlated with an increase of p53 and p21 levels and downregulation of the checkpoint proteins cdc25c, cyclin B1 and cdc2. Bcl-2 phosphorylation and caspase activation were also observed. Isochaihulactone induced phosphorylation of c-Jun-N-terminal kinase (JNK), and JNK inhibitor partially reduced isochaihulactone-induced cell death. Isochaihulactone also induced the expressions of EGR-1 and NAG-1. Expression of NAG-1 was reduced by JNK inhibitor, and knocking down of NAG-1 inhibited isochaihulactone-induced cell death.
Two human prostate cancer cell lines, LNCaP and PC-3, were treated with BP, and evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest at G0/G1 phase, which was correlated with an increase of p21 and p27 levels and downregulation of the checkpoint proteins cyclin D1 and cdk2. To determine the mechanism of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by treatment with the BP in the LNCaP cells. Several BP-inducible genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. Treatment with BP induced ER stress, as evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-? and GADD153/CHOP in both LNCaP and PC-3 cell lines. We then tested the contribution of ER stress related genes to protect against BP-induced cell death using RNA interference. Blockage of IRE1-? or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Western blot showed the involvement of JNK1/2 signaling pathway. Blockage of JNK1/2 signaling by JNK siRNA resulted in decreased the expression of IRE1 and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. The anti-proliferative effect of BP was further demonstrated in LNCaP xenograft animal model.
Taken together, our study suggests that isochaihulactone can significantly inhibit LNCaP cell growth. In the other aspect, our results suggested that the anti-proliferative effect of BP is facilitated by ER stress in prostate cancer cells.

正文目錄 頁次
第一章 緒論 (Introduction) (1)
第一節 攝護腺 (前列腺)癌現況 (Prostate Cancer, PCa) (1)
第二節 攝護腺癌細胞株 (Prostate Cancer Cell Lines) (3)
第三節 輔助與另類療法
(Complementary and Alternative Medicine, CAM) (4)
第四節 南柴胡 (Bupleurum Scorzonerifolium, BS) (6)
第五節 當歸 (Angelica Sinensis, AS) (7)
第六節 細胞週期 (Cell Cycle) (9)
第七節 細胞凋亡 (Apoptosis) (12)
第八節 干擾RNA與微小RNA (RNAi and MicroRNA) (15)
第九節 內質網壓力 (ER Stress) (17)
第二章 研究目標與方向 (Aims and Goals) (22)
第三章 材料與方法 (Materials and Methods) (25)
第一節 試劑與藥品 (Reagents and Chemicals) (25)
第二節 植物萃取之原理 (Principles of Plants Extraction) (26)

第三節 南柴胡之萃取物及其純化物異柴胡內酯的製備法
(BS-AE and isochaihulactone preparation)

(28)
第四節 當歸之萃取物 (Angelica Sinensis Extract) (30)
第五節 細胞株培養 (Cell Culture) (30)
第六節 細胞存活分析 (Cell Viability Assay) (31)
第七節 膠體電泳及西方點墨法 (Western Blot Analysis) (32)
第八節 細胞週期分析
(Flow Cytometric Cell Cycle Analysis) (33)
第九節 細胞凋亡分析 (Detection of Apoptosis) (34)
第十節 細胞免疫化學染色 (Immunocytochemistry Staining) (36)
第十一節 RNA萃取 (RNA Extraction) (37)
第十二節 聚合酶連鎖反應
(Polymerase Chain Reaction, PCR) (37)
第十三節 siRNA與細胞轉染 (SiRNA and Transfection) (38)
第十四節 動物實驗 (Animal Experiments) (39)
第十五節 統計分析 (Statistical Analysis) (40)
第四章 實驗結果與分析 (Results) (41)
第一部分 異柴胡內酯與攝護腺癌
(Isochaihulactone and Prostate Cancer) (41)
第二部分 正-丁烯基苯酞與攝護腺癌
(n-Butylidenephthalide and Prostate Cancer) (50)
第五章 討論 (Discussion) (58)
第一部分 異柴胡內酯與攝護腺癌
(Isochaihulactone and Prostate Cancer) (58)
第二部分 正-丁烯基苯酞與攝護腺癌
( n-Butylidenephthalide and Prostate Cancer) (66)
第六章 參考文獻 (References) (115)

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