抗藥性癌症細胞中CCT-β表現調控機制之研究

Regulatory mechanism of CCT-β expression in multidrug resistant cancer cell lines

周憶安(Yi-An Chou)

指導教授 ： 梁博煌

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10144

摘要

癌症的研究在近年來蓬勃發展，治療方式也日新月異的在突破，然而隨著治療方式、藥物的研發和使用，癌症細胞卻開始產生抗藥性，造成治癒上的困難。Chaperonin containing t-complex polypeptide 1 subunit β (CCT-β) 被發現在抗藥性的癌症細胞中，有過量表現的情形 (Lin et al., 2009)。因此在本論文中，我先比較兩種癌症細胞和其具有抗藥性的細胞中CCT-β的表現量，發現在兩種抗藥性癌症細胞中都可以觀察到CCT-β有過量表現的情形。另外，當增加癌症細胞中CCT-β的表現量時，則可以提高對paclitaxel的抗性。相對的，此蛋白質的表現量被抑制，抗藥性癌症細胞對於paclitaxel敏感性有回復的現象。由此可知CCT-β與癌症細胞的抗藥性的產生之間相關性的存在。進一步分析CCT-β的上游基因，發現三種特別的轉錄因子－Sp1、 CREB 和Elk1 的DNA結合位置，這些轉錄因子在先前的研究中已被證實與癌症的發生或進程有關。而將這些結合位突變後，可明顯觀察到luciferase reporter system表現量下降，表示Sp1和Elk1會調控CCT-β在抗藥性癌症細胞中轉錄作用的進行。另外，之前的研究指出在癌症細胞中，有些訊號傳遞路徑會藉由將Elk1磷酸化以提升其與DNA結合的能力並促進特定基因的轉錄表現。分析過後的確可以在抗藥性癌症細胞中觀察到磷酸化的Elk1顯著增加。又由於Elk1已被證明為MAPK 的受質之一，而MAPK有三種磷酸酶，分別是ERK、JNK 和p38，在使用了相對的抑制劑進行實驗後，發現只有在p38的活性被抑制的情況下，可觀察到Elk1磷酸化的情形減少和CCT-β的表現量降低。另一方面，亦觀察到被認為與癌症細胞抗藥性有關的MDR1，當p38訊號傳遞路徑被阻斷時，其表現量亦跟著下降。
總括而論，本篇研究嘗試去釐清抗藥性癌症細胞中，使CCT-β表現量增加的詳細分子機制，發現藉由p38訊號傳遞路徑活化Elk1後會增加其與基因的結合力，可能因而促進CCT-β轉錄作用的進行，增加在細胞中的表現量，同時此路徑亦會調節MDR1的表現量。因此可以進一步去探討在抗藥性癌症細胞中誘發p38 訊號傳遞路徑的分子，同時，更可進一步探討p38 訊號傳遞路徑調控MDR1表現的機制，和分析CCT-β與MDR1之間的關係，或許更能解釋造成癌症細胞產生抗藥性的原因，再設計出更有效的標靶治療藥物。

關鍵字

CCT-β ； 抗藥性癌症細胞 ； 轉錄因子 ； 轉錄調控 ； 訊號傳遞路徑 ； 多重抗藥性蛋白

並列摘要

Chaperonin containing t-complex polypeptide 1 subunit β (CCT-β) is a molecular chaperone that facilitates protein folding in eukaryotic cytosol, and the expression is elevated in a variety of drug-resistant tumor types (Lin et al., 2009). In this thesis, I first identified the expression of CCT-β was up-regulated in two drug-resistant cancer cells compared to their parental cancer cells. Besides, transfecting CCT-β in cancer cells increased the resistance to paclitaxel. In contrast, transient knockdown of CCT-β by siRNA restored the sensitivity to paclitaxel in drug-resistant cancer cells. Furthermore, promoter analysis revealed that Sp1 and Elk1 are critical for activation of the CCT-β promoter, which were reported deregulation in cancer cells and related with cancer initiation or progression. After mutating these transcription factor binding sites, luciferase reporter assay using MES-SA/Dx-5 and 7-TR showed down-regulated expression, demonstrating that transcription of the gene encoding the β subunit of CCT is regulated by Sp1 and Elk1 in drug-resistant cancer cells. Moreover, previous studies have reported phosphorylation of Elk1 increased DNA-binding ability and promoted the transcription of specific genes. Elk1 has been proved to be a substrate of MAPK. There are three subfamily of MAPK: ERK, JNK, and p38. Treatment of different inhibitors, respectively, revealed that only inhibition of the p38 signaling pathway by SB 203580 was sufficient to reduce phosphorylation of Elk1, further affecting the expression of CCT-β in time- or dose-dependent manner.　Otherwise, MDR1, which is thought to be the major reason for drug resistance, was also down-regulated by inhibition of p38 pathway. Therefore, blocking p38 pathway not only down-regulates the expression of CCT-β but also MDR1.
Overall, these studies elucidate the possible mechanisms underlying the up-regulation of CCT-β in drug-resistant cancer cells, transcription of the CCT-β regulated by phosphorylation of Elk1 under the control of p38 signaling pathway, and simultaneous regulation of MDR1 expression. More studies are needed to find out an unknown factor that induces this pathway to mediate the transcription of CCT-β and MDR1, and the correlation between CCT-β and MDR1. These may serve as the therapeutic targets to develop drugs for treating multidrug-resistant cancers.

並列關鍵字

CCT-β ； transcriptional regulation ； Sp1 ； Elk1 ； mitogen-activated protein kinase ； multidrug resistance protei

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