包曼-伯克抑制劑對MCF7乳癌細胞株的蛋白酶體活性以及細胞增生之抑制作用;紅茶茶黃素對癌細胞的蛋白酶體活性以及細胞增生之抑制作用

Inhibitory Effects of Bowman-Birk Inhibitor on Proteasome Activity and Cell Proliferation in MCF7 Breast Cancer Cells;Inhibitory Effects of Black Theaflavins on Proteasome Activity and Cell Proliferation in Cancer Cell Lines

陳郁雯(Yu-Wen Chen)

指導教授 ： 林仁混

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.00264

摘要

中文摘要
近年來，蛋白酶抑制劑(protease inhibitor)發展為預防癌症的藥物，在釵h蛋白酶抑制劑中，Bowman-Birk inhibitor (BBI) 是由大豆純化出的蛋白質(分子量約8 kDa)，具有抑制胰蛋白(trypsin)和凝乳蛋白(chymotrypsin)的能力。科學家發現大豆的攝取能有效地減少癌症的發生率，是因為大豆成分中的BBI能抑制carcinogenesis，而BBI目前已經進入人類phase Ⅱa的臨床試驗；然而，BBI抑制carcinogenesis的精確機制並不是很清楚。在本篇研究中，我們發現BBI能專一性地抑制人類乳癌細胞MCF7中蛋白酶體(proteasome)的chymotrypsin-like活性。BBI抑制蛋白酶體的作用與ubiquitinated蛋白質和已知的蛋白酶體受質- cyclin-dependent kinse inhibitor (CDKI)，p21Cip1/WAF1和p27Kip1的累積有密切的關連性，同時伴隨著參與調控細胞週期的cyclins- cyclin D1以及cyclin E有下降的情形，造成細胞週期停滯在G1/S phase。此外，BBI能有效地抑制與細胞增生有關的訊息傳遞途徑- extracellular signal-related kinases (ERK1/2)的活化，而且利用phosphatase抑制劑或是轉錄作用(transcription)抑制劑都能抑制BBI對ERK活性的抑制作用；這是由於BBI抑制蛋白酶體的弁遄A而導致同樣是蛋白酶體受質-MAP kinase phosphatase-1 (MKP-1)有累積的現象。根據以上的結果，我們發現BBI治療癌症的機制可能是因為BBI具有抑制蛋白酶體弁鄋滲鄐O，導致調控在細胞週期的G1/S phase的CDKIs上升以及cyclins下降，同時BBI會累積MKP-1以抑制ERK1/2的活化，最終阻止MCF7細胞的增生。本篇研究結果為第一個發現BBI可作為蛋白酶體抑制劑而能有效地運用在治療乳癌，並且更進一步提供證據證明適量地攝取大豆的確能有效地抑制癌症的發生。
中文摘要
蛋白酶體抑制劑(proteasome inhibitor)近年來發展為預防及治療癌症的藥物。本篇研究主要利用純化的20S或26S 蛋白酶體以及不同癌細胞中的26S proteasome來探討茶多酚(tea polyphenols)，其中包括茶黃素theaflavin (TF1)、theaflavin-3-gallate (TF2a)、theaflavin-3’-gallate (TF2b)、theaflavin-3,3’-digallate (TF3)以及兒茶素(-)-epigallocatechin-3-gallate (EGCG)是否可做為蛋白酶體抑制劑。由實驗結果發現，在測試的茶多酚中，茶黃素比兒茶素EGCG更能有效地抑制癌細胞的生長，尤其是對人類血癌U937細胞(IC50 value of 6μM)的抑制效果最有效，同時也能抑制純化或是癌細胞中的蛋白酶體chymotrypsin-like的活性。此外，在茶黃素中，以TF3對蛋白酶體抑制劑引發細胞凋亡(apoptosis)有抗藥性的人類乳癌MCF7細胞中的蛋白酶體chymotrypsin-like和peptidyl glutamyl peptide hydrolase (PGPH)活性有較明顯地抑制效果；因此，由實驗結果發現茶黃素抑制蛋白酶體的活性會導致MCF7細胞停止生長。更進一步，我們比較一些多酚類對蛋白酶體的抑制作用，發現具有酯鍵(ester bond)的化合物較能有效地抑制蛋白酶體chymotrypsin-like的活性。有趣的是，這些具有酯鍵(ester bond)的化合物也同時具有galloyl group(s)，而且gallic acid 和n-propyl gallate 也都能抑制蛋白酶體chymotrypsin-like的活性。因此，除了酯鍵之外，我們推測galloyl結構對茶多酚能有效抑制蛋白酶體活性的作用是另一個重要的決定因子。

關鍵字

茶黃素 ； 茶多酚 ； 包曼-伯克抑制劑

並列摘要

Abstract
Protease inhibitors are a class of well-established cancer chemopreventing agents. Among several types of protease inhibitors, the Bowman-Birk inhibitor (BBI), a soybean-derived protein with the well-characterized ability to inhibit trypsin and chymotrypsin activities, has been shown to be an effective suppressor of carcinogenesis and treated in phaseⅡa clinical trial in human. However, the precise mechanisms by which BBI suppresses carcinogenesis are unknown. In this study, we demonstrated that BBI specifically and potently inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo in MCF7 breast cancer cells. Inhibition of the proteasome by BBI is associated with accumulation of ubiquitinated proteins and the known proteasome substrates, p21Cip1/WAF1 and p27Kip1, accompanied with down-regulation of cyclin D1 and cyclin E which led to arrest cell cycle at G1/S phase. Furthermore, BBI suppressed MCF7 cell growth without showing cytotoxicity and had a novel effect on a loss of phoshporylated extracellular signal-related kinases (ERK1/2) when comparison with well-characterized chemopreventive agents. BBI was unable to inactivate ERK1/2 in the presence of a phosphatase inhibitor, sodium orthovanadate, or a transcriptional inhibitor, actinomycin D, suggesting the involvement of a specific phosphatase. We found an induction of dual specific MAP kinase phosphatase-1 (MKP-1) in dose- and time-dependent fashion which was correlated with dephosphorylation of ERK1/2 in BBI-treated MCF7 cells. In addition, BBI exhibited no inhibitory effects on EGF-induced activation of ERK1/2 and Akt. Taken together, we demonstrated that BBI indeed suppressed ERK1/2 activity via up-regulation of MKP-1 mediated by blockade of proteasome function. Our results supported the notion that the inhibition of proteasome activity by BBI is a novel mechanism that might contribute to cancer preventative effects of BBI. This study further provides the evidence that soybean products intake indeed have the potential to advance as chemopreventive agents.
Inhibitors of proteasome are currently emerging as novel cancer preventing and therapeutic agents. To determine whether the tea polyphenols, including theaflavin, theaflavin-3-gallate, theaflavin-3’-gallate, theaflavin-3,3’-digallate (TF3), and (-)-epigallocatechin-3-gallate (EGCG) were potential proteasome inhibitors, we treated purified 20S or 26S proteasome and 26S proteasome in leukemia and cancer cell lines with these compounds. TF3 displayed a potent inhibitory effect on the growth of U937 cells with an estimated IC50 value of 6μM. Furthermore, TF3 had more efficient inhibition on the proteasomal chymotrypsin-like activity of purified proteasome derived from different sources and 26S proteasome in four kinds of cancer cell extracts as compared to those of EGCG. In addition, the theaflavins, especially TF3, inhibited the proteasomal chymotrypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities of 26S proteasome in a concentration-dependent manner in human breast cancer MCF7 cells which have been described to be resistant to apoptosis induced by proteasome inhibitors. These results illustrated that proteasome inhibition by theaflavins lead to anti-proliferation of MCF7 cells. Furthermore, comparing well-characterized polyphenols on proteasome inhibition, the ester bond-bearing compounds, with some exceptions, exhibited more potently inhibitory effects on the proteasomal chymotrypsin-like activity than those without ester bond(s). Interestingly, the gallic acid and n-propyl gallate also had slightly inhibition on proteasome activities. Therefore, we suggest that the galloyl moiety might be the other important structure of polyphenols that contributes to the proteasome inhibition.

並列關鍵字

Bowman-Birk inhibitor ； theaflavins ； tea polyphenols

參考文獻

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