五沒食子酸葡萄糖酯抑制人類前列腺癌細胞轉移之研究

Abstract

part1 根據行政院衛生署的統計資料，惡性腫瘤為台灣人民死亡原因的首位，其中，前列腺癌分居國人癌症死亡的第八位及男性癌症死亡的第七位，而且，前列腺癌的罹患率有逐年增加的趨勢。前列腺癌導致死亡的主因，在於施以荷爾蒙療法治療後，復發的前列腺癌產生了對荷爾蒙療法的抵抗性，形成非荷爾蒙依賴型的前列腺癌，此病灶的特性是極度具侵犯性，常常會轉移到身體的其他組織或器官，造成器官衰竭而死亡，目前對此症仍是束手無策。骨骼是前列腺癌轉移的眾多目標器官之一，經由前人的研究指出，前列腺癌轉移到骨骼上的機制，受到了許多生長因子與細胞激素的調節，此外，matrix metalloproteinases (MMPs) 也被發現在前列腺癌細胞的轉移上具有促進的功能，因此，若能抑制這些因子對前列腺癌細胞的作用，將有助於改善或治療荷爾蒙非依賴性的前列腺癌細胞的轉移。在本篇論文中，我們發現表皮生長因子可以誘導荷爾蒙非依賴性的前列腺癌細胞株PC-3在體外的轉移試驗中大量的轉移，而五沒食子酸葡萄糖酯 (5GG) 則能抑制表皮生長因子對PC-3細胞轉移的促進作用。進一步對PC-3細胞產生能分解胞外基質的酵素做檢驗，發現MMP-9可以受到表皮生長因子的調控，而5GG則能抑制表皮生長因子所促進的MMP-9表現。為了更進一步檢視5GG的作用機制，我們探討了與表皮生長因子相關的訊息傳遞路徑，結果發現，調控MMP-9表現的轉錄因子能夠被5GG抑制，c-jun N-terminal kinase 2 (JNK2) 也能被5GG所抑制，於是我們檢視最上游的表皮生長因子受體，結果發現此受體也會受到5GG的影響而降低其表現，使用抑制劑避免表皮生長因子受體被分解則能回復JNK2的磷酸化，這些結果顯示表皮生長因子可能是經由JNK2來控制MMP-9的表現，而5GG則是抑制在最上游的受體來達到抑制癌細胞轉移的效果。 part2 近年來，外來生物入侵所造成的災害正逐漸受到重視中，無論是農業的損失或是自然環境的破壞都造成相當大的損失，因此，對於外來物種的防治已經到了刻不容緩的地步。其中，原本由日本人引進台灣準備作為食材的非洲大蝸牛，由於適應力強、繁殖力旺盛，已經廣泛的分布在台灣各處了，他們最大的危害在於啃食作物的幼葉、嫩芽，造成作物生長遲滯、產量下降。由於使用化學藥品殺螺的成效並不顯著，而且容易造成環境污染，因此，目前科學家正嘗試使用天然的方法來達到抑制螺類生長的效果。苦茶渣溶液是農民傳統上用來抑制螺類的方法，其特徵是專一性高且可自然分解。本篇研究的目的即在利用科學的方法檢視苦茶渣做為殺螺劑的可行性及原理。我們發現苦茶渣溶液能有效的抑制蝸牛的行動力，並進而造成蝸牛的死亡。觀察其死亡時的特徵，可以發現其螺肉呈內縮脫水狀，推測可能是由於苦茶渣刺激其體表過度分泌黏液，使其失去水分而脫水死亡。接下來我們檢視苦茶渣溶液的細胞毒性，發現其只有在高濃度時才能造成細胞死亡，顯示其可能不是經由殺死蝸牛體表的細胞達到使蝸牛死亡的效果。另外，由於兒茶素是茶類中含量相當豐富的物質，因此我們也檢測了苦茶渣溶液中總兒茶素的含量，結果發現其兒茶素的含量也不高。追溯前人關於可殺螺的植物的研究，發現很多都將結果導向皂素的作用，也就是認為皂素洗去了蝸牛體表的黏液，使其脫水死亡。關於苦茶渣中所含皂素的相關研究還沒有相當清楚，因此未來可能的研究重點將放在苦茶渣皂素的定性實驗中。

PART1 Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of death in men in western world. Androgen-independent prostate cancer (AIPC), an untreatable and fatal malignancy, almost invariably develops subsequent to androgen ablation therapy in early stage prostate carcinoma. At present, there is no effective therapy for it. The metastasis of prostate cancer to bone is associated with a substantial bone matrix turnover. Matrix metalloproteinase-9 (MMP-9) plays roles in both normal bone remodeling and invasion and metastasis of prostate cancer. Bone matrix turnover and tumor cell proliferation are linked to each other in a positive feedback cycle that can be disrupted by inhibition of MMP activity. Additionally, EGF induces MMP-9 expression in many prostate cancer cells and its cognate receptor, EGFR, is proposed to be involved in the development of androgen-independent prostate cancer (AIPC). Penta-O-galloyl-β-D-glucose (5GG), a kind of galloylglucose, contains one glucose and esterified with five gallic acid on five hydroxyl groups of the glucose. It has been shown to exert several biological activities including anti-oxidation, induction of apoptosis and metastasis inhibition. In this study, we demonstrated that EGF-induced MMP-9 expression was inhibited by 5GG. Subsequently, we seek to figure out the signal transduction pathways involved in 5GG-mediated MMP-9 down-regulation under EGF stimulation. We found that NF-κB nuclear translocation was reduced. Therefore, we examined the signal transduction pathways that may be involved in regulating NF-κB nuclear translocation. We found that 5GG inhibited JNK1/2 phosphorylation but maintained Akt phosphorylation with minimal effect on ERK1/2 and p38 under EGF induction. Next, we checked the phosphorylation status of EGFR. Surprisingly, we found that 5GG down-regulated not only EGFR phosphorylation but also its protein level under EGF stimulation. By using proteasome and lysosome inhibitors, we demonstrated that this phenomenon resulted from 5GG-mediated EGFR protein degradation. Inhibition of MMP-9 and EGFR expression by 5GG may help reduce the frequency of prostate cancer bone metastasis and the development of AIPC. These findings make 5GG a therapeutic candidate for prostate cancer. Part2 The invading foreign species in Taiwan have caused tremendous losses in agriculture and economy. They also destroy the established ecology of Taiwan. One of these invading species is the African Giant snail, which eats up new leaves and buds of the crops. Efforts have been made to prevent the spread of snails but little effect is seen. In this study, we tried to investigate the conventional, naturally degradable oil tea pomace in inhibiting the activity of snails. We found that both two kinds of oil tea pomace water extracts were capable of killing snails, causing their over-secretion of surface mucus, thus dehydrated to death. We also found that these oil tea pomace water extracts were cytotoxic only at higher doses. Finally, we examined the total amount of phenols in these oil tea pomace water extracts. We found that they didn’t contain much amount of phenols, suggesting that the molluscicidal activity of oil tea pomace water extracts was not resulted from phenols. Overall, these information provided us some insights into the molluscicidal activity of oil tea pomace. However, the exact active component in oil tea pomace water extracts remained to be clarified, one possibility was the saponins.