Department of Neurology, Juntendo University School of Medicine; Bunkyo, Tokyo, Japan.
Autophagy. 2011 Feb;7(2):176-87. doi: 10.4161/auto.7.2.14074. Epub 2011 Feb 1.
Caffeine is one of the most frequently ingested neuroactive compounds. All known mechanisms of apoptosis induced by caffeine act through cell cycle modulation or p53 induction. It is currently unknown whether caffeine-induced apoptosis is associated with other cell death mechanisms, such as autophagy. Herein we show that caffeine increases both the levels of microtubule-associated protein 1 light chain 3-II and the number of autophagosomes, through the use of western blotting, electron microscopy and immunocytochemistry techniques. Phosphorylated p70 ribosomal protein S6 kinase (Thr389), S6 ribosomal protein (Ser235/236), 4E-BP1 (Thr37/46) and Akt (Ser473) were significantly decreased by caffeine. In contrast, ERK1/2 (Thr202/204) was increased by caffeine, suggesting an inhibition of the Akt/mTOR/p70S6K pathway and activation of the ERK1/2 pathway. Although insulin treatment phosphorylated Akt (Ser473) and led to autophagy suppression, the effect of insulin treatment was completely abolished by caffeine addition. Caffeine-induced autophagy was not completely blocked by inhibition of ERK1/2 by U0126. Caffeine induced reduction of mitochondrial membrane potentials and apoptosis in a dose-dependent manner, which was further attenuated by the inhibition of autophagy with 3-methyladenine or Atg7 siRNA knockdown. Furthermore, there was a reduced number of early apoptotic cells (annexin V positive, propidium iodide negative) among autophagy-deficient mouse embryonic fibroblasts treated with caffeine than their wild-type counterparts. These results support previous studies on the use of caffeine in the treatment of human tumors and indicate a potential new target in the regulation of apoptosis.
咖啡因是最常摄入的神经活性化合物之一。咖啡因诱导细胞凋亡的所有已知机制都通过细胞周期调节或 p53 诱导起作用。目前尚不清楚咖啡因诱导的细胞凋亡是否与其他细胞死亡机制（如自噬）有关。本文通过 Western blot、电子显微镜和免疫细胞化学技术显示，咖啡因通过增加微管相关蛋白 1 轻链 3-II 的水平和自噬体的数量来增加自噬。磷酸化的 p70 核糖体蛋白 S6 激酶（Thr389）、核糖体蛋白 S6（Ser235/236）、4E-BP1（Thr37/46）和 Akt（Ser473）被咖啡因显著降低。相反，咖啡因增加了 ERK1/2（Thr202/204），表明 Akt/mTOR/p70S6K 途径被抑制，ERK1/2 途径被激活。虽然胰岛素处理磷酸化 Akt（Ser473）并导致自噬抑制，但咖啡因的添加完全消除了胰岛素处理的效果。U0126 抑制 ERK1/2 并不能完全阻断咖啡因诱导的自噬。咖啡因以剂量依赖的方式诱导线粒体膜电位降低和凋亡，而用 3-甲基腺嘌呤或 Atg7 siRNA 敲低抑制自噬则进一步减弱了这种作用。此外，在用咖啡因处理的自噬缺陷型小鼠胚胎成纤维细胞中，早期凋亡细胞（ Annexin V 阳性，碘化丙啶阴性）的数量减少，而其野生型细胞则没有。这些结果支持了以前关于咖啡因治疗人类肿瘤的研究，并表明在调节细胞凋亡方面存在一个新的潜在靶点。
