Phospholipidosis and down-regulation of the PI3-K/PDK-1/Akt signalling pathway are vitamin E inhibitable events associated with 7-ketocholesterol-induced apoptosis

Abstract 

Among the oxysterols accumulating in atherosclerotic plaque, 7-ketocholesterol (7KC) is a potent apoptotic inducer, which favours myelin figure formation and polar lipid accumulation. This investigation performed on U937 cells consisted in characterizing the myelin figure formation process; determining the effects of 7KC on the PI3-K/PDK-1/Akt signalling pathway; evaluating the activities of vitamin E (Vit-E) (α-tocopherol) on the formation of myelin figures and the PI3-K/PDK-1/Akt signalling pathway and assessing the effects of PI3-K inhibitors (LY-294002, 3-methyladenine) on the activity of Vit-E on cell death and polar lipid accumulation. The ultrastructural and biochemical characteristics of myelin figures (multilamellar cytoplasmic inclusions rich in phospholipids and 7KC present in acidic vesicles and the reversibility of these alterations) support the hypothesis that 7KC is an inducer of phospholipidosis. This oxysterol also induces important changes in lipid content and/or organization of the cytoplasmic membrane demonstrated with merocyanine 540 and fluorescence anisotropy, a loss of PI3-K activity and dephosphorylation of PDK-1 and Akt. It is noteworthy that Vit-E was able to counteract phospholipidosis and certain apoptotic associated events (caspase activation, lysosomal degradation) to restore PI3-K activity and to prevent PDK-1 and Akt dephosphorylation. When Vit-E was associated with LY-294002 or 3-methyladenine, impairment of 7KC-induced apoptosis was inhibited, and accumulation of polar lipids was less counteracted. Thus, 7KC-induced apoptosis is a PI3-K-dependent event, and Vit-E up- and down-regulates PI3-K activity and phospholipidosis, respectively.

Abbreviations: AO, acridine orange, DMSO, dimethylsulfoxide, DPH, 1,6-diphenyl-1,3,5-hexatriene, FLICA, fluorochrome-labelled inhibitor of caspases, 7KC, 7-Ketocholesterol, MC540, merocyanine 540, 3MA, 3-methyladenine, MDC, monodansylcadaverine, NR, Nile Red, PDK-1, 3′Phosphoinositide-regulated kinase-1, PI3-K, phosphoinositide 3-kinase, PBS, phosphate-buffered saline, PI, propidium iodide, PP2A, protein phosphatase 2A, TMSE, Trimethylsilylethanol, TPBS, 0.1% Tween 20, phosphate buffered saline, Vit-E, vitamin E

Keywords: Akt, PI3-K, 7-ketocholesterol, Phospholipidosis, Vitamin E