Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin
Inhibition of AMPK-dependent autophagy enhances in vitro antiglioma effect of simvastatin
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DOI：
10.1016/j.phrs.2011.08.003
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发表时间：
2012-01-01
影响因子：
9.3
通讯作者：
Trajkovic, Vladimir
中科院分区：
文献类型：
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作者：
Misirkic, Maja;Janjetovic, Kristina;Trajkovic, Vladimir
研究了自噬在羟甲基戊二酰辅酶A（HMG-CoA）还原酶抑制剂辛伐他汀诱导的胶质瘤细胞死亡中的作用。通过免疫印迹分析自噬的诱导和自噬调节信号通路的激活。流式细胞术/荧光显微镜用于评估自噬相关的细胞内酸化和凋亡标志物（磷脂酰丝氨酸暴露、DNA片段化和半胱天冬酶激活）。采用结晶紫法、MTT法和LDH释放法测定细胞活力。辛伐他汀处理U251和C6胶质瘤细胞系引起的自噬溶酶体样胞质内酸性囊泡的外观。U251细胞中自噬的诱导通过自噬体相关的LC 3-II和促自噬beclin-1的上调以及选择性自噬靶点p62的下调来证实。辛伐他汀诱导AMP活化蛋白激酶（AMPK）及其靶Raptor的活化，同时下调Akt的活化。哺乳动物雷帕霉素靶蛋白（mTOR）。一个主要的AMPK/Akt下游靶点和一个主要的负性自噬调节因子，及其底物p70 S6激酶1也被辛伐他汀抑制。甲羟戊酸，HMG-CoA还原酶酶活性的产物，AMPK siRNA或AMPK的药理学失活与化合物C抑制，而Akt（10-DEBC盐酸盐）和mTOR（雷帕霉素）的抑制剂模拟自噬诱导辛伐他汀。用巴弗洛霉素A1、3-甲基腺嘌呤和LC 3 β shRNA抑制自噬，以及用化合物C或AMPK siRNA抑制AMPK，显著增加辛伐他汀处理的U251细胞的凋亡死亡。这些数据表明，AMPK依赖性自噬反应的抑制可能使胶质瘤细胞对他汀类药物诱导的凋亡敏感。(C)2011爱思唯尔有限公司保留所有权利。
The role of autophagy, a process in which the cell self-digests its own components, was investigated in glioma cell death induced by the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting drug simvastatin. Induction of autophagy and activation of autophagy-regulating signalling pathways were analyzed by immunoblotting. Flow cytometry/fluorescent microscopy was used to assess autophagy-associated intracellular acidification and apoptotic markers (phosphatidylserine exposure, DNA fragmentation and caspase activation). Cell viability was determined by crystal violet, MTT or LDH release assay. Simvastatin treatment of U251 and C6 glioma cell lines caused the appearance of autophagolysosome-like intracytoplasmic acidic vesicles. The induction of autophagy in U251 cells was confirmed by the upregulation of autophagosome-associated LC3-II and pro-autophagic beclin-1, as well as by the downregulation of the selective autophagic target p62. Simvastatin induced the activation of AMP-activated protein kinase (AMPK) and its target Raptor, while simultaneously downregulating activation of Akt. Mammalian target of rapamycin (mTOR). a major AMPK/Akt downstream target and a major negative autophagy regulator, and its substrate p70 S6 kinase 1 were also inhibited by simvastatin. Mevalonate, the product of HMG-CoA reductase enzymatic activity, AMPK siRNA or pharmacological inactivation of AMPK with compound C suppressed, while the inhibitors of Akt (10-DEBC hydrochloride) and mTOR (rapamycin) mimicked autophagy induction by simvastatin. Inhibition of autophagy with bafilomycin A1, 3-methyladenine and LC3 beta shRNA, as well as AMPK inhibition with compound C or AMPK siRNA, markedly increased apoptotic death of simvastatin-treated U251 cells. These data suggest that inhibition of AMPK-depenclent autophagic response might sensitize glioma cells to statin-induced apoptotic death. (C) 2011 Elsevier Ltd. All rights reserved.