Ceramide-mediated Macroautophagy Involves Inhibition of Protein Kinase B and Up-regulation of Beclin 1

Abstract

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C 2 -ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B 1 . Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C 2 -ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C 2 -ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifendependent up-regulation. Tamoxifen and C 2 -ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.Macroautophagy or autophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long lived proteins and organelles (1-3). Autophagy starts with the formation of a multilayer membrane-bound autophagosome that sequesters fractions of the cytoplasm (4, 5). In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed (6, 7).The physiological importance of autophagy during starvation has been primarily highlighted in rat liver (8) and then in different cell types (reviewed in Refs. 9 and 10). At the same time, the term autophagic cell death or type II programmed cell death (PCD II) has been introduced (11) to describe a cell death different from apoptosis or type I programmed cell death (PCD I) (reviewed in Refs. 12 and 13). The recent progress made in characterization of the molecular mechanism controlling autophagy has brought a renewal of interest for this process (14). There is now evidence for the role of autophagy during development (15-17), in the life span extension (15,18), and in disease such as cancer (19,20), neurodegenerative disease (21, 22), and myopathies (23,24).A family of autophagy-related genes discovered in yeast and almost integrally conserved in all eucaryotic phyla controls the formation of the autophagosome (25). Two conjugation systems (Atg5p-Atg12p and Atg8p lipidation) are involved in...

Implications of galactocerebrosidase and galactosylcerebroside metabolism in cancer cells

“…49 However, this finding remains controversial, and many authors still attribute the induction of the apoptosis to the applied ceramide molecule. 16,40,50 Scarlatti et al postulate a pathway due to inactivation of proteinkinase B and activation of becelin1 by ceramide leading to apoptosis. 50 …”

“…16,40,50 Scarlatti et al postulate a pathway due to inactivation of proteinkinase B and activation of becelin1 by ceramide leading to apoptosis. 50 …”

Ceramide triggers an NF-κB-dependent survival pathway through calpain

“…C2 ceramide was shown to trigger growth arrest in a number of cell types, [6][7][8] and different types of cell death such as apoptosis, 9-11 necrosis 12,13 and autophagocytosis. 14 All these findings make ceramide appealing for cancer treatment, notably a Phase II clinical trial to study the effectiveness of ceramide cream in treating women who have cutaneus breast cancer is going on (sponsored by the National Cancer Institute). Given the potential importance of ceramide as an anticancer drug, it is fundamental to investigate whether it can activate any parallel survival pathway, as reported for other drugs.…”

Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes

“…8,9 MCF-7 cells can also be induced to undergo autophagy and cell death by treatment with the antiestrogen tamoxifen, which increases the intracellular ceramide level and eliminates the inhibitory effect of class-I PI3K pathway. 10 Expression of death-associated protein kinase and its death-related protein kinase-1 in MCF-7 and various other cell lines can induce autophagy and caspase-independent cell death. 11 When caspase-8 was inhibited and the atg7 and beclin-1 expression knocked down by RNA interference, death through autophagy was blocked in mouse L929 cells.…”