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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物醫學研究所 > 依資料類型分類 > 碩博士論文 >  靈菌紅素在人類乳癌細胞中引發內質網壓力造成細胞凋亡

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152680

標題: 靈菌紅素在人類乳癌細胞中引發內質網壓力造成細胞凋亡
Essential role of ER stress-induced apoptosis in the anti-cancer effect of prodigiosin on human breast cancer cells
作者: 潘慕芸
Pan, Mu-Yun
Contributors: 張嘉哲
Chia-Che Chang 張嘉哲
生物醫學研究所
關鍵字: 靈菌紅素;內質網壓力;細胞凋亡
Prodigiosin;ER stress;Apoptosis
日期: 2012
Issue Date: 2013-11-18 11:05:02 (UTC+8)
Publisher: 生物醫學研究所
摘要: 內質網 (Endoplasmic Reticulum, ER) 是一個控制蛋白質合成、摺疊以及成熟很重要的胞器,當內質網中有許多不完全摺疊的蛋白質累積,就會產生內質網壓力(ER Stress)而對細胞的存活產生威脅,在無法恢復正常的情形則會促使細胞進行細胞凋亡路徑。Prodigiosin (PG)是由鏈黴菌屬 (Streptomyces) 等類微生物的二級代謝物提煉的紅色色素,先前的研究已指出PG具有抗癌的作用,可有效誘發多種癌細胞的細胞凋亡,然而其中的機制至今仍尚未釐清。在本研究中,我們探討 ER stress 在 PG 之抗癌活性中所扮演的角色。我們觀察到在 PG 的處理下,乳癌細胞株 MCF-7、MDA-MB-231與 T-47D 細胞中的內質網壓力之指標蛋白 GRP78、及 CHOP在蛋白層次與 mRNA表現層次均有明顯的增加,同時,內質網壓力之下游三條路徑IRE1、PERK及ATF6皆被活化,顯示 PG 確實可誘發 ER stress。此外,PG誘發之細胞凋亡的現象以及 BCL2 被抑制的情形在 CHOP knockdown 之後顯著降低,顯示 ER stress 在 PG 之抗癌活性中扮演必要的角色。另一方面,我們發現 PG 可藉由活化CHOP引子而提升CHOP之表現量,且PG處理後可促進 phospho-eIF2α 產生以及 JNK 活化。此外,在MCF-7細胞株中大量表現dominant-negative eIF2α、BCL2 overexpression 或處理 JNK-specific inhibitor SP600125後發現均可明顯降低 PG 所提升之CHOP蛋白表現量,同時細胞的存活率也有顯著回升。綜合以上的結果,我們證實 PG 透過引發 ER stress進而活化 PERK 與 JNK 路徑提昇CHOP 之表現並抑制BCL2使細胞走向凋亡的途徑。
Endoplasmic Reticulum (ER) is the site for synthesis and folding of secretory proteins. Disturbance in the homeostasis of protein folding triggers unfolding protein response (UPR) and causes ER stress, ultimately leading to apoptosis if unresolved. Prodigiosin (PG) is a secondary metabolite of Serratia and Streptomyces and has been identified as a potential anti-cancer agent, mostly owing to induction of apoptosis selectively in some malignant cell lines. The mechanisms of the proapoptotic effect of prodigiosin remain elusive. In this study, we presented evidence supporting the role of ER stress in the anti-cancer effect of PG on human breast cancer cell lines. Quantitative real-time RT-PCR analysis and immunoblot analysis indicated that ER stress markers GRP78 and CHOP are dramatically induced by PG in MCF-7, MDA-MB-231 and T-47D cell lines, suggesting that PG induces ER stress. Furthermore, we found that the three pathway initiated by ER stress IRE1, PERK and ATF6 have been activated upon PG treatment, as evidenced by Ser724 phosphorylation of IRE1, XBP1 mRNA splicing, Thr183/Tyr185 dual phosphorylation of JNK, Ser51 phosphorylation of eIF2α and induction of ATF6 transcriptional activity, confirming that PG is an ER stresser. Notably, knockdown of CHOP by RNAi evidently decreased levels of PARP cleavage and cytotoxicity induced by PG, illustrating CHOP is the essential role of ER stress-induced apoptosis in PG’s anti-cancer effect. In addition, functional blockade of eIF2α and JNK by overexpession dominant-negative eIF2α and JNK-specific inhibitor SP600125 in MCF-7 cell, respectively, markedly suppressed PG-induced CHOP up-regulation. Moreover, we found prodigiosin-induced BCL2 suppression in a CHOP-dependent manner, and BCL2 overexpression greatly enhanced the survival of PG-treated cells. Taken together, we conclude that induction of ER stress-induced apoptosis is essential for the anti-cancer effect of PG, and PG engages PERK and JNK signaling pathways to up-regulate CHOP expression, leading to the induction of apoptosis by reducing BCL-2.
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