Safingol (L-threo-sphinganine) induces autophagy in solid tumor cells through inhibition of PKC and the PI3-kinase pathway.
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.
Coward
Jesse
J
Laboratory of New Drug Development, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Ambrosini
Grazia
G
Musi
Elgilda
E
Truman
Jean-Philip
JP
Haimovitz-Friedman
Adriana
A
Allegood
Jeremy C
JC
Wang
Elaine
E
Merrill
Alfred H
AH
Schwartz
Gary K
GK
D000165:Acridine Orange; D001343:Autophagy / Q000187:drug effects*; D002452:Cell Count; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002853:Chromatography, Liquid; D005455:Fluorescent Antibody Technique; D006801:Humans; D009369:Neoplasms / Q000201:enzymology* / Q000473:pathology* / Q000648:ultrastructure; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013107:Sphingolipids / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D053719:Tandem Mass Spectrometry
D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D013107:Sphingolipids; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D011493:Protein Kinase C; D000165:Acridine Orange; D013110:Sphingosine; C005682:safingol