五沒食子酸葡萄醣酯抑制癌細胞生長與苦味酸引發細胞凋亡機制之研究

五沒食子酸葡萄醣酯（pentagalloylglucose, 5GG）為單寧酸（tannic acids）之最小單元，普遍存在各種藥用植物中，為一強效且具專一性的NADPH去氫酶（NADPH dehydrogenase）與黃嘌呤氧化酶（xanthine oxidase）抑制劑。在本實驗室過去的研究中指出，5GG會透過時間與濃度依賴性行為來抑制caspase-3的活性，進一步造成人類血癌細胞株HL-60的細胞凋亡（apoptosis）。另外，本實驗室的研究亦發現5GG能降低人類乳癌細胞株MCF-7中cyclin-dependent kinase（CDK）2和4的活性，增加CDK抑制蛋白p27Kip與p21Cip的蛋白質含量，從而抑制乳癌細胞的細胞週期，使其停留在G1期。本研究中，指出5GG能夠在試管中（in vitro）抑制純化20S與26S蛋白酶體（proteasome）的活性，亦可分別在Jurkat T細胞萃取液與活的Jurkat T細胞中，抑制26S蛋白酶體中似乳糜胰蛋白酶的活性（chymotrypsin-like activity）。由於p27Kip與p21Cip的正常代謝是維持細胞週期進行的要素之一，且p27Kip與p21Cip的代謝是透過26S蛋白酶體來調控。據此，以cycloheximide處理與活體中（in vivo）pulse-chase labeling實驗，分析發現5GG會藉由擾亂p27Kip與p21Cip在人類Jurkat T細胞中的正常分解，使p27Kip與p21Cip的半衰期延長，進而間接地促使Jurkat T細胞的細胞週期停留在G1期。而分別對Jurkat T細胞處理5GG或26S蛋白酶體之專一性抑制劑MG132，皆會造成ubiquitin標示蛋白在Jurkat T細胞中的累積。除此之外，5GG亦會增加蛋白酶體受質Bax在Jurkat T細胞中的穩定性，造成細胞色素c（cytochrome c）釋放到細胞質，導致細胞走向細胞凋亡。因此，本研究發現5GG影響人類血癌細胞細胞週期的新機制：5GG會作為一個蛋白酶體抑制劑，來抑制蛋白酶體所調控的生理路徑。苦味酸（hop bitter acids）分離自啤酒花（Humulus lupulus L.）萃取物，主要由

關鍵字

蛋白酶細胞凋亡；苦味酸；五沒食子酸葡萄醣酯；體

並列摘要

Pentagalloylglucose (5GG), which contains five gallate moieties linked with a glucose core by ester bond, is natural hydrolysable tannin and widely exists in many medical plants. Previous studies indicated that 5GG is a potent and specific inhibitor of NADPH dehydrogenase or xanthine oxidase. Our previous work showed that 5GG can induce human leukemic cell HL-60 undergoing apoptosis through mitochondria- and caspase-3-mediated pathway and block cell cycle progression at G1 phase in human breast caner cell line MCF-7 through inhibition of kinase activities of cyclin-dependent kinases (CDK) 4 and 2, and induction of cyclin-dependent kinase inhibitors (CKIs) p27Kip and p21Cip; yet little is known of the mechanism(s) by which 5GG induces these effects. Herein, we indicated that 5GG could potently and selectively inhibit the activities of purified 20S and 26S proteasome in vitro, 26S proteasome in Jurkat T cell lysates and downregulate the chymotrypsin-like activity of 26S proteasome in intact Jurkat T cells. The turnover of CKIs p27Kip and p21Cip necessary for cell cycle progression mediated by proteasome degradation was disrupted by treatment of human Jurkat T cells with 5GG as shown by cycloheximide treatment and in vivo pulse-chase labeling experiments, and this effect was in tight connection with suppressing proliferation of Jurkat T cells at G1 phase. Additionally, inhibition of proteasome by 5GG as well as by well-established proteasome inhibitor, MG132, resulted in accumulation of ubiquitin-tagged proteins in Jurkat T cells. Furthermore, addition of 5GG in Jurkat T cells also enhanced the stability of proteasome substrate, Bax, and raised the consequent cytochrome c release and apoptosis. Taken together, our results provide a new insight into the molecular mechanism underlying mitogenic cell cycle control down-regulated by 5GG through proteasome-mediated pathway in human leukemic cells and imply that 5GG appears to be a proteasome inhibitor. Hop bitter acids, purified from hops (Humulus lupulus L.), mainly comprise

並列關鍵字

pentagalloylglucose ； apoptosis, ； hop bitter acids

參考文獻

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五沒食子酸葡萄醣酯抑制癌細胞生長與苦味酸引發細胞凋亡機制之研究

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