探討MCAK(Mitotic centromere-associated kinesin)在歐洲杉醇抗藥性前列腺癌細胞之分子角色

Chemoresistant Role of Mitotic centromere-associated kinesin In Docetaxel-Resistant Prostate Cancer Cells

吳俊宏(Jyun-Hong 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(2014年)

摘要

歐洲杉醇(docetaxel)為前列腺癌第一線藥物。Docetaxel 在臨床上雖然能有效的延長前列腺癌患者的生存率，但多數患者最後會對docetaxel產生抗藥性，影響其治療效果。因此本篇研究欲探討在人類前列腺癌細胞PC3之docetaxel抗藥株PC/DX是如何使docetaxel造成細胞G2/M phase arrest的機制失效。處理docetaxel後PC3中乙醯化微管蛋白和MCAK (mitotic centromere associate kinesin)的量會隨著劑量提高而有上升的趨勢，在微管聚集化實驗中也發現MCAK在細胞骨架上也聚集上升的現象。根據文獻顯示乙醯化的微管蛋白能促進動力蛋白 (motor protein)的結合，利用PC3與不同濃度的抗藥株PC/DX15、PC/DX25進行研究，發現乙醯化微管蛋白以及MCAK的表現量有隨着抗藥株的上升而增加的趨勢。此結果顯示可能是前列腺癌細胞對docetaxel產生抗藥性原因之一。為了證明MCAK在抗藥株當中的確是扮演一個主要抗藥性的角色，因此利用siRNA方式抑制MCAK蛋白質的表現，利用MTT assay發現細胞的確會恢復對其docetaxel的敏感性。進一步證實在抑制MCAK蛋白質表現後，利用流式細胞儀分析結果得知當抑制MCAK表現量後G2/M phase百分比例比scramble控制組要來得高，因此MCAK在抗藥株中扮演一個G2/M phase transition的角色。而文獻也指出了MCAK主要參與細胞週期的M phase，並且透過許多kinase所調控，像是Aurora B能負向調節MCAK使其座落於中心粒的位置進一步造成細胞分裂，而Plk1(Polo-like kinase 1) 能正向調節MCAK增加其活性。因此我們也發現了隨著抗藥株濃度的增加Aurora B和Plk1有上升的趨勢。因此推測Aurora B與Plk1兩者與細胞G2/M transition是造成docetaxel抗藥性的原因之一。因此轉染GFP-Plk1-WT過量表現Plk1質體與GFP-P1-KD kinase domain點突變Plk1質體，加入不同濃度的docetaxel利用MTT assay發現GFP-Plk1-WT的存活率比GFP-Plk1-KD還要來得高。此結果顯示Plk1可能去調節MCAK的活性進而影響microtubule的分解能力，並與乙醯化微管蛋白共同作用誘導細胞產生抗藥性。

關鍵字

前列腺癌 ； docetaxel抗藥性

並列摘要

Docetaxel is the first-line drug for prostate cancer. It prolongs patient’s lifespan on clinical, but many patients develope docetaxel resistance that cause treatment failure. In this study, we investigated how PC/DX cell lines, docetaxel-resistant subclones of prostate cancer cell line PC3, escape the G2/M phase arrest in cell cycle after docetaxel treatment. Previous studies indicate that acetyl-tubulin promotes the motor protein binding to microtubules. And we also found that after docetaxel treatment, acetyl-tubulin and MCAK (mitotic centromere associate kinesin) were up-regulated in PC3 cells and were both time- and dose-dependent. And MCAK was up-regulated in cytoskeleton. Also, acetyl-tubulin and MCAK were up-regulated in different concentrations of docetaxel-resistant PC/DX cells, and were dose-dependent. We suggested that it probably led to drug resistance. Then, in MTT assay, MCAK knockdown in PC/DX cells increased the survival rates of cells, and also in flow cytometry assay, increased the percentages of G2/M phase after docetaxel treatment. Many previous studies indicate that MCAK present in M phase of cell cycle and is regulated by many kinase, such as Aurora B, which negativity regulate MCAK to the centrosome and lead cell to mitosis, and Plk1, which positively regulate MCAK to increase its activity. In this study, we found that Aurora B and Plk1 were up-regulated in different concentrations of docetaxel-resistant PC/DX cells, and were dose-dependent. We suggested that Aurora B and Plk1 were associated with docetaxel resistance on cell cycle G2/M phase transition. Furthermore, in MTT assays, we found that over expression wild-type Plk1 had higher survival rate than KD Plk-1, which has a mutation in its kinase domain. Taken together, we concluded that in prostate cancer cells, Plk1 might regulate MCAK to affect microtubule depolymerization, and together with tubulin acetylation to induce drug resistance

並列關鍵字

Prostate cancer ； docetaxel-resistant ； MCAK ； Plk1

參考文獻

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