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研究生:黃千祐
研究生(外文):Chien-Yu Huang
論文名稱:烏蘇酸對第一介白質-β和腫瘤壞死因子-α所誘發的大鼠腦纖維癌瘤母細胞株轉移之抑制作用;利用AmesTest篩選四種茶類萃取物與五種多酚類化合物對PhIP和2-AAF致突變反應之抑制作用探討
論文名稱(外文):Inhibitory Effects of Ursolic Acid on the Invasion of C6 Glioma Cells Induced by IL-1β or TNF-α?Studies on the Inhibitory Effects of Four Teas and Five Polyphenols on the Mutagenicities of PhIP and 2-AAF by Ames Test
指導教授:林仁混林仁混引用關係
指導教授(外文):Jen-Kun Lin
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物化學暨分子生物學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:106
中文關鍵詞:-九烏蘇酸普洱茶紅茶腫瘤壞死因子基質金屬蛋白酶綠茶烏龍茶第一介白質
外文關鍵詞:Interleukin-1oolong teablack teaursolic acidpu-erh teatumor necrosis factorgreen tea
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PART I: 烏蘇酸 (Ursolic acid,UA;3-β-hydroxy-urs-12-en-28-oic acid)是一種存在於迷迭香(rosemary)中的多酚類化合物,目前已被證實具有抗氧化和抗癌的活性。在本篇研究中,我們利用大鼠腦纖維癌瘤母細胞(C6 glioma cell),研究烏蘇酸對於抑制癌細胞的侵入轉移作用,並深入探討烏蘇酸抑制癌細胞轉移的機制為何。由實驗結果顯示,當大鼠腦纖維癌瘤母細胞與烏蘇酸共同培養一小時後,加入第一介白質-β(IL-1β)和腫瘤壞死因子-α(TNF-α)誘導癌細胞轉移24小時後,可以有效抑制癌細胞的侵入轉移作用,但是烏蘇酸所抑制的癌細胞侵入轉移作用並不會影響細胞的增生或造成細胞的毒性。由於調控癌細胞侵入和轉移主要是透過基質金屬蛋白酶-九對於細胞外基質的活化,因此進一步的實驗也顯示烏蘇酸可隨著濃度增加有效抑制基質金屬蛋白酶-九(MMP-9)的活性和蛋白質的表現情形。目前的研究報告已知大鼠腦纖維癌瘤母細胞中的基質金屬蛋白酶-九的表現是透過轉錄因子NFκB所活化的訊息傳導路徑,因此在本篇實驗中我們也發現,烏蘇酸在第一介白質-β (IL-1β)或腫瘤壞死因子-α(TNF-α)的誘導下,藉由累積大量的IκBα而降低轉錄因子NFκB(p65)進入細胞核內進行目標基因的活化;而IκBα的累積是透過抑制活化上游的IKK而導致下游的IκBα活性下降並造成IκBα的累積作用。根據實驗結果更進一步的顯示,烏蘇酸所抑制IKK的活性是受到PKC-ζ的調控,而PKC-ζ是主要調控轉錄因lNFκB活化基質金屬蛋白酶-九的重要酵素;進來的研究報告指出,PKC-ζ的活化是由於細胞受到第一介白質-β (IL-1β)和腫瘤壞死因子-α(TNF-α)的誘導下,會啟動其受體並引起下游的共同活化因子ZIP/p62,活化PKC-ζ並與之進行交互作用,而造成一連串的訊息傳遞,我們的實驗結果也證明,烏蘇酸的確可以抑制ZIP/p62和PKC-ζ的交互作用。根據以上的結果,我們發現烏蘇酸抑制大鼠腦纖維癌瘤母細胞的侵入轉移作用可能是藉由抑制上游的ZIP/p62和PKC-ζ的交互作用,導致下游的轉錄因子NFκB無法活化基質金屬蛋白酶-九而有效降低癌細胞的侵入轉移作用。本篇研究是首次發現烏蘇酸可以作為治療腦纖維瘤癌的轉移作用,並提供更進一步的證據證明由天然植物迷迭香所萃取的烏蘇酸成分可以有效預防癌細胞的轉移作用。
PART II: 對於預防日常生活所發生的突變作用上,茶與多酚類化合物一直備受重視。在本篇研究中,我們探討利用Ames test的方法篩檢不同品種茶類包括綠茶(green tea)、烏龍茶(oolong tea)、普洱茶(pu-erh tea)、紅茶(black tea)和數種常見的多酚類化合物包括單寧酸tannic acid、gallic acid、rutin、umbelliferone、apigenin、quercetin對於抑制PhIP(2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine)和 2-AAF(2-Acetylaminofluorene)兩種致突變劑的抗突變作用。實驗結果顯示,在低濃度的情況下,綠茶和20年生普洱茶對於抑制PhIP和2-AAF明顯比紅茶、烏龍茶和2年生普洱茶來的有效,進一步比較此兩種年份的普洱茶發現,20年生的普洱茶對於抑制PhIP的抗突變作用比2年生的效果來的更好;但是對於抑制2-AAF的抗突變作用上,兩種都具有明顯的抑制效果。而比較數種多酚類化合物發現,tannic acid、gallic acid、rutin、umbelliferone和quercetin對於抑制PhIP的抗突變作用比apigenin來的有效,但是apigenin和其他多酚類化合物對於抑制2-AAF的抗突變作用則均具有明顯的抑制效果。在這些多酚類化合物中,值得注意的是tannic acid幾乎可以在低濃度的情況下就有可以有效抑制2-AAF的抗突變作用。綜合來說,本篇實驗中證明不同品種與生產方式的茶葉對於抗突變作用上也不盡相同;另外,這些可以抗突變的茶類和多酚類化合物在這些日常生活易受到的致突變作用上也可以作為一個有效的天然預防化學突變的方法之一。
PART I: Ursolic acid (UA; 3-β-hydroxy-urs-12-en-28-oic acid), a constant constituent of Rosmarinus officinalis extracts, is a triterpenoid compound which has been shown to have antioxidant and anticarcinogen properties. In the present study, we found that ursolic acid was able to reduce interleukin-1 beta (IL-1β) or tumor necrosis-alpha (TNF-α-induced rat C6 glioma cell invasion which was examined by a reconstituted basement membrane in a transwell chambers, but the inhibitory effect by ursolic acid did not influence cell proliferation or cause cell cytotoxity. The results analyzed by zymography assay and Western blotting revealed that the activity and expression of matrix metalloproteinase-9 (MMP-9) was abolished by ursolic acid in a dose-dependent manner. Because MMP-9 is the target gene of the transcription factor nuclear factor-kappa B (NF-κB), we further investigated that ursolic acid on the activity of NF-κB. As our expectations, ursolic acid up-regulated the levels of IkappaBalpha (IκBα and attenuated the nuclear translocation of p65. Moreover, the accumulation of IκBα was due to inhibit the phosphorylation of IκBα and IKK which was a kinase responsible for the IL-1β or TNF-αinduced phosphorylation and degradation of IκBα Furthermore, ursolic acid suppressed IL-1β or TNF-α induced activation of protein kinase C-zeta (PKC-ζ) which plays the key role in the regulation of MMP-9 expression in C6 glioma cells by activating NF-κB. Recent studies indicate that the association of ZIP/p62 with PKC-ζ provides a scaffold for the NF-κB pathway in both the TNF-?or IL-1β cascades. Our data showed ursolic acid potently inhibited the association of ZIP/p62 and PKC-ζ. Taken together, we demonstrated that ursolic acid could efficient inhibit the interaction of ZIP/p62 and PKC-ζ, further suppressed the activation of NF-κB and down-regulation of MMP-9 protein which in part contribute to its inhibitory effects on IL-1β or TNF-α induced C6 glioma cell invasion. These results strongly suggested the potent implication of ursolic acid in chemoprevention and treatment of cancer metastasis and invasion.
PART II: It has been an increasing interest in anticarcinogens and antimutagenicities of edible plants origin in recent years. In this study, we determined that the antimutagenic properties of various tea extracts including green, oolong, pu-erh tea, and black tea and six polyphenols include rutin, quercetin, apigenin, gallic acid, umbelliferone, and tannic acid were examined by Ames test which is a widely applied short-term bacterial assay for detecting DNA damage effect of mutagen. The antimutagenic activity of 20 years old of pu-erh tea and green tea extracts against PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) and 2-AAF (2-Acetylaminofluorene) was greater than those of oolong tea, black tea, and 2 year old of pu-erh tea extracts. Compared with different ages of pu-erh tea products, 20 years old potently suppress mutagenicity against these two mutagens than 2 years old. But both 20 years and 2 years old of pu-erh tea extracts show a strong inhibitory effects against 2-AAF. In addition, tannic acid, gallic acid, rutin, umbelliferone, and quercetin exhibited stronger antimutagenicities against PhIP than apigenin; while all of these polyphenols displayed inhibitory effect against 2-AAF. Among these polyphenols, tannic acid reveals the greatest antimutagenic ability against 2-AAF in a very low concentration (2 μg/plate). Taken together, these results suggest that formation of different metabolites during various stages of tea fermentation may affect antimutagenicities potencies against chemical mutagens of PhIP and 2-AAF. Moreover, teas and polyphenols from fruits and vegetables may be effective chemoprotective agents aganist mutagenicities certain environmental toxic chemicals.
Part Ⅰ: Inhibitory Effects of Ursolic Acid on the Invasion of C6 Glioma Cells Induced by IL-1β or TNF-?
中文摘要…………………………………………………………………7
Abstract …………………………………………………………………9
Introduction …………………………………………………………11
Materials and Methods ………………………………………………16
Results …………………………………………………………………23
Discussion ……………………………………………………………32
References………………………………………………………………39
Figure …………………………………………………………………48
Part Ⅱ: Studies on the Inhibitory Effects of Four Teas and Six Polyphenols on the Mutagenicities of PhIP and 2-AAF by Ames Test
中文摘要 ………………………………………………………………65
Abstract ………………………………………………………………66
Introduction …………………………………………………………68
Materials and Methods ……………………………………………73
Results…………………………………………………………………76
Discussion ……………………………………………………………82
References ……………………………………………………………87
Figures ………………………………………………………………92
Appendix ………………………………………………………………99
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1.Gonzalez de Mejia, E. [The chemo-protector effects of tea and its components]. Arch Latinoam Nutr, 53: 111-118., 2003.
PART II:
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5.Chung, F. L., Schwartz, J., Herzog, C. R., and Yang, Y. M. Tea and cancer prevention: studies in animals and humans. J Nutr, 133: 3268S-3274S., 2003.
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