Coward Jesse, Ambrosini Grazia, Musi Elgilda, Truman Jean-Philip, Haimovitz-Friedman Adriana, Allegood Jeremy C, Wang Elaine, Merrill Alfred H, Schwartz Gary K
Laboratory of New Drug Development, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Autophagy. 2009 Feb;5(2):184-93. doi: 10.4161/auto.5.2.7361. Epub 2009 Feb 6.
Safingol, the synthetic L-threo-stereoisomer of endogenous (D-erythro-) sphinganine, is an inhibitor of protein kinase C and sphingosine kinase in vitro, and in some cell types has been implicated in ceramide generation and induction of apoptosis. Utilizing electron microscopy, acridine orange staining, and immunoblot and fluorescent localization studies of the myosin light chain-associated protein (LC3), we determined that safingol induces cell death of an exclusively autophagic character and lacking any of the hallmarks of apoptosis. Safingol inhibited PKCbeta-I, PKC delta and PKC epsilon, and inhibited phosphorylation of critical components of the PI3k/Akt/mTOR pathway (Akt, p70S6k and rS6) and the MAPk pathway (ERK). Inhibition of PI3k with LY294002 or suppression of PKC delta and PKC epsilon with siRNA in HCT-116 cells induced autophagy, though not to the extent caused by safingol. Conversely, activation of PKCs with phorbol 12,13-dibutyrate (PDBu) or transient transfection of a constitutively active form of Akt each reduced safingol's autophagic induction, but not completely, indicating that Akt- and PKC-dependent pathways both contribute partially and independently to safingol-induced autophagy. Accordingly, combining siRNA depletion of PKC epsilon with LY294002 inhibition of PI3k induced autophagy to a degree comparable to safingol. Liquid chromatography, electrospray tandem mass spectrometry analysis indicated that safingol did not elevate levels of any endogenous sphingolipids previously shown to induce autophagy (ceramide, sphingosine-1-phosphate and dihydroceramide); therefore, these effects may be due to safingol per se or another metabolite. Thus, our studies establish that safingol induces autophagy through inhibition of PKCs and PI3k by safingol directly rather than via changes in endogenous sphingolipids.
沙芬戈是内源性（D-赤藓糖型）鞘氨醇的合成L-苏型立体异构体，在体外是蛋白激酶C和鞘氨醇激酶的抑制剂，在某些细胞类型中，它与神经酰胺生成和细胞凋亡诱导有关。利用电子显微镜、吖啶橙染色以及肌球蛋白轻链相关蛋白（LC3）的免疫印迹和荧光定位研究，我们确定沙芬戈诱导的细胞死亡具有独特的自噬特征，且缺乏细胞凋亡的任何标志。沙芬戈抑制PKCβ-I、PKCδ和PKCε，并抑制PI3k/Akt/mTOR途径（Akt、p70S6k和rS6）以及MAPk途径（ERK）的关键成分的磷酸化。在HCT-116细胞中，用LY294002抑制PI3k或用siRNA抑制PKCδ和PKCε可诱导自噬，但其程度不如沙芬戈所致。相反，用佛波醇12,13-二丁酸酯（PDBu）激活PKC或瞬时转染组成型活性形式的Akt均可部分降低沙芬戈的自噬诱导作用，但不完全消除，这表明Akt和PKC依赖性途径均部分且独立地参与沙芬戈诱导的自噬。因此，将PKCε的siRNA缺失与LY294002对PI3k的抑制相结合诱导的自噬程度与沙芬戈相当。液相色谱-电喷雾串联质谱分析表明，沙芬戈不会提高先前显示可诱导自噬的任何内源性鞘脂（神经酰胺、鞘氨醇-1-磷酸和二氢神经酰胺）的水平；因此，这些作用可能归因于沙芬戈本身或另一种代谢产物。因此，我们的研究表明，沙芬戈通过直接抑制PKC和PI3k诱导自噬，而非通过内源性鞘脂的变化。
