癌細胞中調控CCT-β表現程度對伴侶蛋白折疊與抗藥性之影響

Abstract

近年來，對於癌症的運作機制已經越來越被研究透徹，治療方式也持續地有突破性的發展。然而，對於各式抗癌藥物，在長時間的治療下，癌症細胞所出現的抗藥性，仍然是癌症治療上難以突破的癥結點。Chaperonin containing t-complex polypeptide 1 (CCT) subunit β (CCT-β) 被發現在許多不同種類的癌症以及具抗藥性的癌症細胞中，都有過量表現的狀況(Lin et al., 2009)。因此在本論文中，我主要的目的即為觀察過量表現的CCT-β在癌症細胞中的特性，為此，我首先建立了CCT-β過量表現以及CCT-β抑制表現的兩種穩定細胞株，以觀察CCT-β在癌症細胞中所造成的影響。 首先檢視的，是CCT的蛋白質折疊能力。CCT的兩種受質actin與tubulin，是細胞骨架actin filament與microtubule不可或缺的組成物。在CCT-β過量表現的穩定細胞株中，actin與tubulin的兩種亞型均發現有顯著增加的情形，顯示過量表現的CCT-β能夠增加actin與tubulin的折疊及成熟。另一方面，CCT-

Chaperonin containing t-complex polypeptide 1 (CCT) is a molecular chaperonin that facilitates the protein folding process in eukaryotic cytosol. The expression level of the CCT-β subunit was found elevated in a variety of drug-resistant tumor types (Lin et al., 2009). In this study, the main purpose is to examine the characteristics of up-regulating CCT-β in cancer cells. Initially, the classic substrates of CCT, actin and tubulin, were examined. Overexpression of CCT-β resulted in significantly elevated expression of both molecules, while knockdown of CCT-β gave no apparent difference. Therefore, up-regulation of CCT-β in cancer cells promotes more vigorous cytoskeleton formation. Next, other subunits of CCT were examined. In both CCT-β overexpress and knockdown stable clone, as well as paclitaxel-resistant cells, some but not all of the eight subunits were found to have synchronous changes with CCT-β in final protein amount. This phenomenon could be explained by the sub-assemblies during the complete CCT complex formation. Abnormally high level of CCT-β in cancer cells may hence break the balance of CCT subunits and affect the folding efficiency. On the other hand, expression level of CCT-β also correlated with the phosphorylation of Elk1, a transcription factor which is usually activated by MAPK family member p38. CCT-β up-regulation was found to induce the multi-drug resistance in cancer cells and stable clone, and the expression of multidrug-resistant protein (MDR1) was induced by CCT-β up-regulation. RNA interference experiments revealed that Elk1 knockdown effectively repressed MDR1 expression and drug resistance in CCT-β up-regulated cells. Interestingly, CCT-β seemed able to affect the phosphorylation level of Elk1, indicating that MDR1 expression and drug resistance in cancer cells may be controlled by MAPK signaling and stress-induced CCT-β. Moreover, a recently screened compound called R379603 showed significant apoptosis effects on CCT-β up-regulated cancer cells. R379603 led to CCT-β up-regulated cell death by introducing ER stress in cells, causing caspase activation and apoptosis. Since this is the route bypassing MDR1-related drug resistance, R379603 may therefore be a promising candidate for treating patients with elevated CCT-βand MDR1 expression. Overall, these studies uncovered two possible roles of CCT-β in CCT-β up-regulated cancer cells: enhancing folding efficiencies of cytoskeleton molecules, and inducing multi-drug resistance. Since CCT-β may be leading CCT assembly and substrate folding, the importance of CCT-β should be further investigated.