植物化合物於前列腺癌化學預防與抗發炎之研究

Abstract

前列腺癌（prostate cancer），又稱攝護腺癌。在美國，前列腺癌則是男性僅次於皮膚癌第二常見的癌症。亞洲國家男性罹患前列腺癌的比例比西方國家要低很多，但是移民美國的亞裔第二代，其罹患前列腺癌的比例仍高於居在亞洲國家的人民，其中重要的差異在生活型態的不同，西方的飲食中富有飽合脂肪及含有相對較低的維生素和微量元素。因此，可見飲食習慣與環境因子似乎與前列腺癌有某種程度的關聯性。 雄性激素受體（Androgen receptor, AR）在前列腺癌的生長中扮演極重要的角色。前列腺癌病患經抑制男性賀爾蒙療法（androgen-ablation therapy）後，雖然有一定的療效但多數病患會再度復發，而這種復發的癌症至今仍沒有有效的治療方法。近來研究指出這種復發的癌症與雄性激素受體（Androgen receptor, AR）有著密切的關係，此種復發的腫瘤不需要依賴男性賀爾蒙就能生長而且通常伴有AR基因增幅（Amplification）或過度表現（Overexpression）的現像。因此，近來前列腺癌的研究著重在如何降低AR表現及調控AR所涉及的訊息傳遞。 在本文的第二章中，我們發現類黃酮化合物—luteolin可以有效抑制前列腺癌細胞的生長。由於已有文獻指出若抑制AR的表現則會抑制細胞生長及使細胞走向細胞凋亡。因此，接下來探究在處理luteolin情形下是否會促進前列腺癌細胞細胞凋亡的發生並進一步檢查AR的表現是否受到影響。結果發現luteolin確實能夠促進前列腺癌細胞細胞凋亡，同時也發現AR的蛋白質表現會受到抑制而且會隨著處理luteolin濃度提高和時間增加而有抑制增加的狀況。有趣的是在同樣的處理下AR的mRNA抑制就不如蛋白質來的明顯，因此我們推論luteolin抑制AR的表現中，轉譯後機制可能扮演較重要的角色。接著我們利用蛋白質合成抑制劑（cycloheximide），蛋白解體抑制劑（proteasome inhibitor, MG132）以及免疫共同沈澱法(coimmunoprecipitation)等實驗，證明了luteolin抑制雄性激素受體的表現是透過影響AR與其保護蛋白—熱蛋白90（Heat shock protein 90, Hsp 90）的結合，AR一但失去Hsp 90的保護，雄性激素受體則會進一步被proteasome辨識並進一步裂解。最後我們也想了解在動物體內luteolin是否仍有抑制腫瘤生長的作用，我們將前列腺癌細胞移植到一免疫系統抑制的小鼠，長出腫瘤後腹腔注射給予luteolin，結果前列腺癌細胞在小鼠體內的生長也受到luteolin的抑制。 除了基因上的變化之外，慢性發炎也是許多癌症的起因，包括食道癌、胃癌、肝癌、大腸癌、膀胱癌和前列腺癌等。文獻指出前列腺反覆發炎或慢性發炎，有可能導致前列腺癌。在化學預防臨床試驗結果也發現：長期使用aspirin或是非類固醇抗發炎藥，可適度的降低前列腺癌風險。有鑑於此，在本文的第三章則是以內毒素所刺激的巨噬細胞當成發炎反應實驗模型來尋找新的或更有效的抗發炎藥物，未來可以提供已做癌症預防或治療的另一種選擇。 在第三章的第一節，我們先探討茶多酚甲基化衍生物的抗發炎作用。已有許多證據顯示茶多酚EGCG具有抗發炎、抗氧化、抗腫瘤等等的作用，其作用除EGCG直接作用外還有可能會經由其生物化學轉化衍生物產生這些作用。文獻指出甲基化的EGCG（O-methylated EGCG）較EGCG有較佳的生物活性如抗過敏效果，而抗發炎作用是否也是如此？在此針對O-methylated EGCG的抗發炎作用進行研究。結果發現在測試的三種茶多酚甲基化衍生物（(-)-Epigallocatechin-3-O-(3-O-methyl)gallate (3'Me-EGCG), (-)-Epigallocatechin-3-O-(4-O-methyl)gallate (4'Me-EGCG) and (-)-4'-methyl epigallocatechin-3-O-(4-O-methyl)gallate (4',4'-diMe-EGCG)）中以3'Me-EGCG的抗發炎作用最為明顯，在抑制一氧化氮（nitric oxide, NO）的產生或是降低可誘發型一氧化氮合酶（induced nitric oxide synthase, iNOS ）表現均是如此。由於茶飲是除水之外消耗最大宗的飲品，所以我們也進一步分析茶飲及生茶葉中是否有茶多酚甲基化衍生物的存在而其含量又為何？在此我們利用高效率液相層析儀分析茶飲及生茶葉，結果發現4',4'-diMe-EGCG並不存在茶飲及生茶葉中。在生茶葉中則是以台灣三峽的青心柑仔茶葉含有較高的3'Me-EGCG和4'Me-EGCG。而比較綠茶，烏龍茶，紅茶及普洱茶飲則發現台灣龍井綠茶飲中有較高3'Me-EGCG和4'Me-EGCG的含量，烏龍茶次之，紅茶及普洱茶則未發現。 在第三章的第二節則是探討另一植物化學因子—類固醇生物鹼（steroidal alkaloids）的抗發炎作用。steroidal alkaloids其結構上近似類固醇的前趨物，常用來合成人造類固醇（anabolic steroids）、類固醇賀爾蒙（steroidal hormones）及腎上腺皮質類固醇（corticosteroids）等類固醇藥劑的材料。而在此我們仍以內毒素所刺激的巨噬細胞作為發炎反應實驗模型，研究steroidal alkaloids對於一些發炎中介者（inflammation mediators）的影響及機制探討。在測試的二種steroidal alkaloids（tomatidine and soalsodine）中，以來自番茄的類固醇生物鹼—tomatidine的抗發炎作用較為明顯。Tomatidine除可以有效抑制NO的產生及降低iNOS表現之外，亦可抑制前列腺素E2（PGE2）的產生和環氧化酶2（Cyclooxygenase-2, COX-2）表現。接著，進一步探究Tomatidine抑制發炎反應的機制，NF-kappa B 為啟動促進發炎之蛋白質表達的一種轉錄因子，實驗發現tomatidine可以經由抑制I-kappa B的磷酸化和I-kappa B的裂解，進而減少NF-kappa B核移位及其活性。另外絲裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)參與發炎反應的訊號傳遞，而tomatidine可以藉由其成員之一的c-Jun N-terminal kinase (JNK) pathway來抑制c-jun 磷酸化以及另一重要轉錄因子Octamer-2的表現，進而影響發炎中介者（inflammation mediators, 如iNOS及COX-2）的表現。 植物化學因子在疾病預防上扮演很重要的角色，然而其作用機制及其分子標的等等還不是很清楚。在本文中採用了許多植物化學因子，如類黃酮化合物、茶多酚甲基化衍生物及類固醇生物鹼進行癌症化學預防的研究，由結果得知：類黃酮化合物—luteolin可有效抑制前列腺癌細胞的生長。而內毒素所引發的發炎模式中，茶多酚甲基化衍生物—3'Me-EGCG及類固醇生物鹼—tomatidine均能降低的發炎中介者的表現。這些結果也說明了植物化學因子在疾病預防上的作用機制及其分子標的，期望藉由本文的研究結果，未來能夠提供癌症化學預防臨床試驗一些資訊，使癌症化學預防能有更多的選擇。

Prostate cancer is the most common cancer in American men and the second leading cause of cancer death in this population. It is well known that there are significant differences in the incidence of certain types of cancer in particular countries or regions of the world. The incidence of clinically significant prostate cancer is vastly different between Western and East Asia countries. A difference points to factors in the ‘Western life-style’ fostering prostate cancer. One candidate is a diet rich in saturated fat and relatively low in vitamins and micronutrients from fruit and vegetables.

Androgen receptors play a critical role in regulating the growth, proliferation and progression in prostate carcinoma and androgen-refractory prostate cancer. Recent studies have suggested that prostate cancer cell proliferation is inhibited by AR downregulation. Hence, a therapy approach are focus on how to inhibiting AR expression and/or blocking the AR-mediated signaling. In chapter 2, our aim was to investigate luteolin, a flavonoid, affects AR expression and function in prostate cancer cells and xenografts. Luteolin inhibited LNCaP cell growth and the expression of androgen regulated PSA genes. Moreover, luteolin treatment resulted in repressing androgen-dependent trans-activation of AR by inhibiting AR nuclear translocation. Western blot analysis demonstrated that AR protein expression was inhibited by luteolin in dose- and time-dependent manners. Luteolin decreased the association of AR and heat shock protein 90 (Hsp90), which in turn induced AR degradation through proteasome-mediated pathway in ligand-independent manner. Our results also demonstrate that luteolin suppressed LNCaP xenograft tumor growth, PSA secretion, and AR protein expression in severe combined immunodeficient (SCID) mice. Recurrent or chronic inflammation has been implicated in the development of many human cancers, including those of the esophagus, stomach, liver, large intestine, urinary bladder and prostate. In clinical chemopreventive trials, using aspirin or NASIDs could suppress the incidence of many cancers, such as colon cancer, breast cancer, and prostate cancer. Hence, our study used the LPS-stimulated macrophage as an inflammation model to investigate the effect of O-methylated EGCG derivatives and steroidal alkaloids on anti-inflammation effects, and find more potent and other valuable compounds for anti-inflammation disorders. Tea polyphenols have been reported to act in ways that are antioxidative, antimutagenic effects, anticarcinogenic, and antiallergic activity. (-)-Epigallocatechin-3-gallate (EGCG), a major tea polyphenol, undergo substantial biotransformation to different derivatives that includes the methylated compounds. Recent researches have showed that the O-methylated derivatives of EGCG have more effectively compared with EGCG on biological impacts. In the part 1 of chapter 3, we compare the three O-methylation derivatives of EGCG ((-)-Epigallocatechin-3-O-(3-O-methyl)gallate (3'Me-EGCG), (-)-Epigallocatechin-3-O-(4-O-methyl)gallate (4'Me-EGCG) and (-)-4'-methyl epigallocatechin-3-O-(4-O-methyl)gallate (4',4'-diMe-EGCG)) to EGCG on the anti-inflammatory effects. We found that 3'Me-EGCG has higher inhibitory effect on the nitric oxide generation and iNOS and COX-2 expression as compared with EGCG, while 4'Me-EGCG and 4',4'-diMe-EGCG were less effective. In addition, we have investigated the composition of the three O-methylated EGCG derivatives, 3'Me-EGCG, 4'Me-EGCG, and 4',4'-diMe-EGCG in tea leaves by HPLC. We found that different content of these O-methylated EGCG show in various fresh tea leaves. Neither fresh tea leaves nor commercial tea leaves could detect the 4',4'-diMe-EGCG. Higher levels of 3'Me-EGCG and 4'Me-EGCG were detected in Chinshin-Kanzai (a species of Camellia Sinensis) cultivated in the mountain area of Sansia, Taipei County, Taiwan. Also, these O-methylated EGCG were found to be higher in autumn and winter than spring and summer. The young leaves (apical bud and the two youngest leaves) were found to be richer in these O-methylated EGCG than old leaves (from the tenth to the fifth leaf). In fermentation level, the amount of O-methylated EGCG is higher in unfermented longjin green tea than semifermentated oolong tea. However, the fermentated black tea and puerh tea did not find these O-methylated EGCG.

In the part 2 of chapter 3, our researches have used steroidal alkaloid as agents in order to find new compounds for anti-inflammation disorders. The structures of steroidal alkaloids, tomatidine and soalsodine, were similar as anabolic steroids, steroidal hormones, and corticosteroids. Inducible nitric oxide synthase and cyclooxygenase-2 are important enzymes that mediate inflammatory processes and associated with inflammatory diseases and cancer. However, the molecular mechanisms of anti-inflammatory effects of these steroidal alkaloids were not clear. Our results were indicated that tomatidine has more potent anti-inflammatory action as compared to solasodine and diosgenin. Tomatidine could decrease LPS-stimulated production of nitrite oxide (NO) and prostaglandin (PG) via inhibition of iNOS and COX-2 expression. Tomatidine inhibited the LPS-induced iNOS and COX-2 expression through suppression of I-kappaB α phosphorylation and NF-kappaB nuclear translocation, and JNK pathway in turns inhibited the c-jun phosphorylation and Oct-2 expression. Recent studies demonstrate that phytochemicals can protect humans against diseases and how phytochemicals interfere with this mechanism is still unclear. In this dissertation, our results demonstrated that luteolin, a naturally flavnoid, could inhibited the cell growth in prostate cancer cells and xenografts. The LPS-stimulated macrophage could be use as an inflammation model to investigate the effect of O-methylated EGCG derivatives and steroidal alkaloids on anti-inflammation effects. Our experiments found that 3'Me-EGCG and tomatidine could act as anti-inflammatory agents and may be possible to develop as useful agents for chemoprevention of cancer or inflammatory disorders. However, our preclinical research might promising and ready for further study in clinical chemopreventive trials.