運用蛋白質體學技術探討Bortezomib之 抗癌機轉

Studies on the anti-cancer mechanisms of Bortezomib by proteomic techniques

謝永信(Yung-Hsin Hsine)

指導教授 ： 梁有志

臺北醫學大學/醫學院/醫學科學研究所/碩士(2008年)

摘要

蛋白酶體是存在於真核細胞內的一種酵素複合體，它可以選擇性分解細胞中的未折疊好的蛋白質。Bortezomib是第一個被FDA所認可的蛋白酶體抑制劑，目前已用於復發性與具抗性的多發性骨髓瘤病人治療上。之前的研究顯示，Bortezomib具有抑制腫瘤細胞生長與促進癌細胞凋亡的功能，主要是藉由阻斷NF-κB路徑。然而Bortezomib引起癌細胞凋亡的分子機制仍然不清楚。在此研究中，我們運用蛋白質體學相關技術，探討Bortezomib抑制Burkitt’s lymphoma細胞生長的機制。Burkitt’s lymphoma通常會有c-myc過度表現的現象。首先，我們發現Bortezomib可以抑制人類Burkitt’s lymphoma細胞株CA46與Daudi細胞的增生與促進其細胞的凋亡，並且隨著藥劑量的增加而越顯著。此外，以西方墨點法分析，隨著Bortezomib藥劑濃度的增加，可以抑制二細胞株c-myc蛋白的表現。藉由二維電泳技術分析，發現在CA46細胞中，Bortezomib會改變97個蛋白質的表現，進一步以MALDI-Q-TOF鑑定出16個差異性蛋白質，其中有13個蛋白質表現上升，3個蛋白質表現下降。在Daudi中發現Bortezomib會改變60個蛋白質的表現，進一步以MALDI-Q-TOF鑑定出21個差異性蛋白質，其中有14個蛋白質表現上升，7個蛋白質表現下降。藉由文獻及資料庫的記載，這些由Bortezomib所改變的蛋白質與細胞內多種生理功能相關，包括未折疊蛋白反應、RNA修飾、蛋白質的運輸與生合成、細胞凋亡及訊息傳遞等。這些結果指示Bortezomib誘導的細胞死亡，除了阻斷NF-κB訊息路徑，尚受到許多其它分子的調控。

關鍵字

蛋白酶體抑制劑 ； 淋巴瘤 ； 細胞凋亡 ； 蛋白質體學 ； 二維電泳

並列摘要

The proteasome is an abundant multi-enzyme complex that provides the main roles for selective degradation of intracellular unfolding proteins in eukaryotic cells. Bortezomib (PS-341) is a proteasome inhibitor which first approved by FDA to enter clinical trails in relapsed/refractory multiple myeloma patients. Previous studies have demonstrated that Bortezomib inhibited tumor cells proliferation and induced apoptosis through blocking NF-κB pathway. However, the detail mechanism of Bortezomib on the cancer cells apoptosis is still not well understood. In this study, we have used proteomic techniques to investigate the effects of Bortezomib on the human Burkitt’s lymphoma CA46 and Daudi cells, which overexpress c-myc oncogene. First, we found that Bortezomib significantly inhibited both cells proliferation and induced both cell apoptosis in a dose dependent manner. In addition, Bortezomib dose-dependently inhibited c-myc expression in both cells by Western blot. By two-dimensional electrophoresis analysis, we found that Bortezomib significantly altered 97 protein levels in CA46 cells, and in which 16 proteins were identified by MALDI-Q-TOF. Among 16 proteins, 13 proteins were found to increase and 3 proteins to decrease after Bortezomib treatment. In Daudi cells, Bortezomib significantly altered 60 proteins levels, and in which 21 proteins were identified by MALDI-Q-TOF, including 14 up-regulated proteins, and 7 doen-regulated proteins. All of those proteins have been reported to be associated with multiple cellular functions, including unfolding protein response (UPS), RNA processing, protein targeting and biosynthesis, apoptosis, and signal transduction. Our results suggest that Bortezomib-induced tumor cell apoptosis might mediate several other molecules besides NF-κB.

並列關鍵字

Bortezomib ； proteasome inhibitor ； proteomic ； apoptosis ； 2DE ； PS-341

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