探討乙醯化微管蛋白在歐洲杉醇抗藥性前列腺癌細胞之分子角色

Chemoresistant Role of Acetyl-tubulin in Docetaxel-Resistant Prostate Cancer Cells

吳竑麟(Hong-Lin Wu)

指導教授 ： 侯自銓

高雄醫學大學/醫學院/生物化學研究所/碩士(2011年)

摘要

歐洲杉醇(docetaxel)為基礎的化學療法，普遍被認為是一項對前列腺癌有效的治療。很不幸的，關於歐洲杉醇在臨床治療上，經常遇到許多不良的副作用，其中包括抗藥性。因此，探討歐洲杉醇所產生抗藥性之相關分子是現今重要的課題。先前研究發現，許多癌細胞中微管蛋白(tubulin)的改變，可能引起歐洲杉醇抗藥性，但是其詳細機轉仍然不清楚。因此本篇研究，探討乙醯化微管蛋白(acetyl-tubulin)在前列腺癌細胞所產生歐洲杉醇抗性的關聯。利用人類前列腺癌細胞(PC3)與歐洲杉醇抗藥株(PC/DX)進行研究，藉由西方墨點法觀察乙醯化微管蛋白、alpha-tubulin、beta-tubulin、gamma-tubulin和beta III-tubulin 在抗藥株中高度表現，以及乙醯化微管蛋白、alpha-tubulin、beta-tubulin和 gamma-tubulin 表現量隨著歐洲杉醇篩選濃度增加而上升。當 PC3 以不同濃度及時間的歐洲杉醇處理之下，會增加乙醯化微管蛋白的表現。在前列腺癌抗藥性病患組織檢體中也發現 乙醯化微管蛋白的過度表現。並觀察微管蛋白去乙醯化酵素--組蛋白去乙醯酶6 (histone deacetylase 6, HDAC6)，其基因與蛋白質的表現量在抗藥株中明顯下降。更進一步，使用 HDAC6 siRNA 抑制 HDAC6，導致乙醯化微管蛋白表現上升，經不同濃度的歐洲杉醇處理，利用細胞存活率分析(MTT assay)，發現 PC3 對歐洲杉醇產生抗性。另外，發現可調節微管(microtubule)的蛋白質 kinesin (KIF5B、KIF2C)以及 stathmin (STMN1) 的表現量在抗藥株中明顯上升，有可能是導致抗藥性的原因之一。並且利用微管聚集化實驗觀察抗藥株中的乙醯化和 KIF2C 分佈於微管上，以及 PC3 處理歐洲杉醇誘導微管上的乙醯化增加，並且提高KIF2C 與微管的結合。除此之外，PC3 在表皮生長因子(epidermal growth factor, EGF)的處理下，活化表皮生長因子接受器(epidermal growth factor receptor, EGFR)，並誘導乙醯化微管蛋白的表現，同時也可以降低對歐洲杉醇的細胞毒性；以及抑制抗藥株的 EGFR 則會減少乙醯化微管蛋白的表現。根據上述實驗，我們發現乙醯化微管蛋白在歐洲杉醇抗藥性前列腺癌細胞扮演重要角色，並且可藉由EGFR訊號傳遞路徑調控。此外KIF2C可能是促進抗藥性的因子，而他們之間是如何調控，則需要做進一步的研究。

關鍵字

前列腺癌 ； 歐洲杉醇 ； 抗藥性 ； 乙醯化微管蛋白

並列摘要

Docetaxel-based chemotherapy has generally been considered as one of the effective treatments for prostate cancer. Unfortunately, clinical treatment with docetaxel often encounters a number of undesirable side effects, including drug resistance. Therefore, it has become essential to identify molecular events that may be associated with the development of docetaxel resistance. Tubulin isoforms have been previously examined for their resistant ability to docetaxel in many cancers, but the real mechanisms remained unclear. In this study, we evaluated the feasibility of employing docetaxel as a cytotoxic agent for PC3 cells and to examine the role of acetyl-tubulin in docetaxel-resistant prostate cancer. Human prostate cancer cell lines(PC3) and docetaxel-resistant cell subclones(PC/DX), to investigate the expression of acetyl-tubulin, alpha-tubulin, beta-tubulin, gamma-tubulin and beta III-tubulin were significantly higher expression in PC/DX cells than in parental PC3 cells and up-regulation of acetyl-tubulin, alpha-tubulin, beta-tubulin and gamma-tubulin expression with different concentrations of docetaxel PC/DX cells by western blotting analysis. The expression of acetyl-tubulin was gradually increased by docetaxel in a dose- and time-dependent manner in PC3 cells. In the docetaxel-resistant prostate cancer patient tissue samples were also up-regulation of acetyl-tubulin. Histone deacetylase 6, a deacetyl enzyme of tubulin, mRNA and protein levels were significantly decreased in PC/DX than in PC3 cells. Further, we used siRNA to inhibit HDAC6 expression. Up-regulation of acetyl-tubulin in HDAC6 knockdown PC3 cells became more resistant to docetaxel. In addition, we also found up-regulation of kinesin (KIF5B、KIF2C) and stathmin (STMN1) in the PC/DX cells that may be as a function of docetaxel-resistant. Microtubule have more acetylation and KIF2C in the PC/DX cells, and tubulin acetylation also stimulates KIF2C binding to microtubules. Moreover, up-regulation of acetyl-tubulin protein expression after recombinant epidermal growth factor treatment and reducing docetaxel cytotoxicity in PC3 cells, and inhibition of EGFR in the PC/DX cells to cause down-regulation of acetyl-tubulin. This study highlights the role of acetyl-tubulin in docetaxel-resistant prostate cancer cells and that may be regulated by EGFR signaling pathway. Moreover, KIF2C may be required for this resistant factor. The detailed mechanisms for which will be further explored.

並列關鍵字

prostate cancer ； docetaxel ； chemoresistance ； acetyl-tubulin

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