title: Ceramide induces the dephosphorylation and inhibition of constitutively activated Akt in PTEN negative U87mg cells.
abstract: In the present study, treatment of the PTEN negative U87MG human glioblastoma cell line with C2-ceramide resulted in a dose- and time-dependent decrease in the constitutive phosphorylation of Akt at threonine 308 and serine 473. The C2-ceramide induced dephosphorylation of Akt correlated with a 90-95% reduction in the Akt kinase activity. Exposure to C2-ceramide did not affect the basal or PDGF activated levels PtdIns-3,4-P(2) and PtdIns-3,4,5-P(3), indicating PI3-K activity was not inhibited. Additionally, treatment of cells with the PI3-K inhibitor wortmannin and C2-ceramide resulted in an enhanced rate of Akt dephosphorylation versus either agent alone. Finally, treatment of cells with the phosphatase inhibitors okadaic acid or calyculin A prevented the C2-ceramide induced dephosphorylation and inhibition of Akt activity. These data demonstrate the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in U87MG cells and implicate the activation of ceramide activated protein phosphatase, rather than decreased PI3-K activity, as the mechanism of inhibition.
authors.affiliation: Department of Cancer Research, Lilly Corporate Center, Indianapolis, Indiana, 46285, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Zinda
authors.forename: C J
authors.initials: CJ
authors.lastname: Vlahos
authors.forename: M T
authors.initials: MT
authors.lastname: Lai
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D051059:PTEN Phosphohydrolase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004791:Enzyme Inhibitors; D008387:Marine Toxins; C064769:N-acetylsphingosine; D010080:Oxazoles; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C060980:phosphatidylinositol 3,4-diphosphate; D019319:Okadaic Acid; D013912:Threonine; D012694:Serine; C059041:calyculin A; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D013110:Sphingosine; D000077191:Wortmannin