倍半萜天然物及合成物雙胜肽衍生物在人類荷爾蒙不依賴型轉移性前列腺癌細胞的作用機轉探討

前列腺癌是男性癌症中最常被診斷的癌症之一，同時也在男性癌症死亡率中排名第五位。每年全世界約有1,112,000個新診斷為前列腺癌的病例以及308,000個因前列腺癌而死亡的病例。前列腺癌已然成為男性健康及生命的一大威脅。目前臨床上治療前列腺癌，主要有手術治療、放射線治療、荷爾蒙療法、化學藥物療法以及疫苗治療。然而，當疾病病程進展到後期前列腺癌，成為荷爾蒙不依賴型轉移性前列腺癌，其死亡率大幅提高並且尚未有能治癒的藥物，因此是目前急需解決的一大困境。在本論文中，以發展能對抗人類荷爾蒙不依賴型轉移性前列腺癌的藥物為主軸，以第一章天然物zerumbone和第二章合成物雙胜肽化合物為個論，分別探討其在人類荷爾蒙不依賴型轉移性前列腺癌細胞的抗癌機制。在本論文第一章中，探討天然物zerumbone在人類荷爾蒙不依賴型轉移性前列腺癌細胞的抗癌活性作用機轉。Zerumbone的結構屬於倍半萜類，是熱帶植物Zingiber zerumbet Smith的主要活性成分。本研究發現，zerumbone在人類荷爾蒙不依賴型轉移性前列腺癌細胞株PC-3以及DU-145均有抑制生長的作用。將細胞以胸腺嘧啶阻斷方式(thymidine block)使細胞週期同步於S期。以流式細胞儀偵測細胞週期變化，發現zerumbone使細胞停滯在G2/M期。此外，zerumbone會增加MPM2蛋白表現量，顯示mitotic phosphoproteins增加，藉此得以推定zerumbone引起mitotic arrest。在細胞內微小管聚合試驗的結果顯示，zerumbone會抑制微小管聚合。同時，在共軛焦螢光顯微鏡下觀察到，zerumbone使細胞形成單極紡錘體。因此，推斷zerumbone作用於微管蛋白以及微管，抑制微管聚合作用。以流式細胞儀偵測粒線體膜電位的變化，結果發現，zerumbone造成細胞粒線體膜電位喪失，使粒線體損害。我們利用fluo-3/AM染色來偵測細胞內鈣離子含量。結果顯示，zerumbone使細胞內鈣離子濃度明顯上升。Zerumbone亦使calpain I的蛋白表現量增加，同時，與內質網壓力相關的指標蛋白，GRP-78以及CHOP/GADD153的蛋白表現量，在zerumbone處理後亦顯著增加。由此可知，zerumbone引起內質網壓力的活化。Zerumbone造成caspase 3、caspase 9、caspase 7以及PARP-1的活化，由以上結果顯示zerumbone抑制微管聚合，引起內質網壓力以及粒線體損害，導致最後造成細胞凋亡作用。第二章探討合成物雙胜肽衍生物KUD983在人類荷爾蒙不依賴型轉移性前列腺癌細胞的抗癌活性作用機轉。在本研究中發現，KUD983對人類前列腺癌細胞株PC-3、DU-145以及LNCaP均有抑制生長的作用。因此，我們進而探究KUD983人類荷爾蒙不依賴型轉移性前列腺癌細胞中引起的訊息傳遞機制。細胞經PI染色，以流式細胞儀分析，結果發現，KUD983會引起細胞週期停滯在G0/G1期，進而引起細胞凋亡。由西方墨點法結果可知，KUD983會抑制cyclin D1、cyclin E以及Cdk4的蛋白質表現，由此再度證明KUD983會引起細胞週期的改變。此外，c-myc對於cyclin E/Cdk2和cyclin D1/Cdk4複合物的調控扮演重要的角色，KUD983能明顯抑制c-myc蛋白表現，證明c-myc對於KUD983抑制細胞增生有關鍵性的作用。KUD983亦會抑制細胞中重要的能量代謝途徑PI3K/Akt及mTOR pathway。由實驗結果可知，KUD983會引起LC3II的蛋白表現量增加、由流式細胞儀可偵測到AVO-positive cells增加以及在共軛焦螢光顯微鏡下可觀察到LC3 puncta表現。由上述結果可知，除了細胞凋亡作用外，KUD983亦會引起細胞自噬作用。由我們的研究結果可知，在人類前列腺癌治療上，KUD983在未來具有極大的潛力。

關鍵字

前列腺癌；細胞凋亡；細胞自噬； zerumbone ； β-雙胜?

並列摘要

Prostate cancer is one of the most common diagnosed cancers and is the fifth leading cause of death in men. There is annual number of 1,112,000 new diagnosed cases and 308,000 deaths due to prostate cancer in the world. Accordingly, prostate cancer is a big threat for health and life in men. The major treatments for prostate cancer in clinical include surgery, radiotherapy, hormone therapy, chemotherapy and vaccine. However, if the disease progresses to castration-resistant prostate cancer, the mortality rises dramatically and the efficacy of the treatment still needs to be improved. Development of the novel agents is an urgent challenge for hormone-refractory metastatic prostate cancer (HRMPC). The aim of the study is to identify the mechanisms of compounds against HRMPC cells. In the first chapter, we have investigated the anticancer mechanism of a nature product, zerumbone, in hormone-refractory metastatic prostate cancer cells. Zerumbone, a cyclic sesquiterpene, is the active component of tropical plant, Zingiber zerumbet Smith. The data demonstrated that zerumbone induced antiproliferative effect in PC-3 and DU-145 cells. Besides, it induced cell arrest at G2/M phase in cells after thymidine synchronization. Zerumbone also increased the protein expression of MPM2, revealing zerumbone-induced mitotic arrest. The result of microtubule assembly assay showed that zerumbone inhibited microtubule assembly. Confocal immunofluorescence microscopic examination showed monopolar spindles in the zerumbone-treated cells suggesting that zerumbone targeted tubulin/microtubule and inhibited microtubule assembly. Furthermore, zerumbone induced the loss of mitochondrial membrane potential. Detection of intracellular Ca2+ levels by fluo-3/AM staining showed that zerumbone induced a significant increase of intracellular Ca2+ concentration. Moreover, zerumbone increased the protein expression of calpain I, GRP78 and CHOP/GADD153, and cleavage of caspase 3, caspase 9, caspase 7 and PARP-1 suggesting the induction of endoplasmic reticulum (ER) stress and caspase cascades. Taken together, the data indicate that zerumbone induces caspase-dependent apoptotic cell death through inhibition of microtubule assembly, ER stress induction and mitochondrial damage. The second part is to identify the apoptotic mechanism of synthetic β-dipeptide derivative, KUD983. KUD983 inhibited cell proliferation in human prostate cancer cell lines, PC-3, DU-145 and LNCaP. Using thymidine block treatment, it induced cell cycle arrest at G0/G1 phase and subsequent apoptosis associated with the down-regulation of several related proteins including cyclin D1, cyclin E and Cdk4, and the de-phosphorylation of RB. Furthermore, KUD983 decreased the levels of total protein and nuclear protein of c-myc, a key player in regulating cyclin E/Cdk2 and cyclin D1/Cdk4 complexes, indicating the central role of c-myc in KUD983-mediated antiproliferative effect. KUD983 also inhibited PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways which regulate multiple cellular functions. In addition to cell apoptosis, KUD983 also induced cell autophagy by the detection of increased LC3II expression, AVO-positive cells and LC3 puncta formation. The data suggest that KUD983 is an anticancer β-dipeptide against HRMPCs through the inhibition of c-myc, PI3K/Akt and mTOR/p70S6K/4E-BP1 pathways, leading to antiproliferative effect and induction of apoptosis and autophagy in HRMPCs.