2′-Hydroxy C16-Ceramide Induces Apoptosis-Associated
Proteomic Changes in C6 Glioma Cells
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Abstract
Ceramide is a bioactive sphingolipid
involved in regulation of
numerous cell signaling pathways. Evidence is accumulating that differences
in ceramide structure, such as <i>N</i>-acyl chain length
and desaturation of sphingoid base, determine the biological activities
of ceramide. Using synthetic (<i>R</i>)-2′-hydroxy-C16-ceramide,
which is the naturally occurring stereoisomer, we demonstrate that
this ceramide has more potent pro-apoptotic activity compared to its
(<i>S</i>) isomer or non-hydroxylated C16-ceramide. Upon
exposure to (<i>R</i>)-2′-hydroxy-ceramide, C6 glioma
cells rapidly underwent apoptosis as indicated by caspase-3 activation,
PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel
proteomics analysis identified 28 proteins whose levels were altered
during the initial 3 h of exposure. Using the list of 28 proteins,
we performed a software-assisted pathway analysis to identify possible
signaling events that would result in the observed changes. The result
indicated that Akt and MAP kinase pathways are among the possible
pathways regulated by (<i>R</i>)-2′-hydroxy-ceramide.
Experimental validation confirmed that 2′-hydroxy-ceramide
significantly altered phosphorylation status of Akt and its downstream
effector GSK3β, as well as p38, ERK1/2, and JNK1/2 MAP kinases.
Unexpectedly, robust phosphorylation of Akt was observed within 1
h of exposure to 2′-hydroxy-ceramide, followed by dephosphorylation.
Phosphorylation status of MAPKs showed a complex pattern, in which
rapid phosphorylation of ERK1/2 was followed by dephosphorylation
of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2.
These data indicate that (<i>R</i>)-2′-hydroxy-ceramide
regulates multiple signaling pathways by affecting protein kinases
and phosphatases with kinetics distinct from that of the extensively
studied non-hydroxy-ceramide or its unnatural stereoisomer