2′-Hydroxy C16-Ceramide Induces Apoptosis-Associated Proteomic Changes in C6 Glioma Cells

Abstract

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that difference in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determines the biological activities of ceramide. Using synthetic (R) 2′-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or nonhydroxylated C16-ceramide. Upon exposure to (R) 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 hours 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 (R) 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 hr 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 (R) 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.

Fatty acid 2-hydroxylation inhibits tumor growth and increases sensitivity to cisplatin in gastric cancer

“…FA2H-regulated suppression of GLUT1 in gastric cancer cells may induce oxidative stress [40], which contributes to the observed inhibition of tumor growth. Alternatively, incorporation of 2-OHFAs into ceramides may alter ceramide regulation of electron transport chain and ROS generation [41,42]. However, whether this potential regulation of free radicals and the resulting lipid peroxides is involved in the ( R )-2-OHFA-induced apoptosis requires extensive investigation in the future.…”

“…There is evidence to demonstrate that FA2H and racemic 2-OHFA improve sensitivity to Elisidepsin (PM02734), a synthetic plasma membrane-disrupting cyclodepsipeptide drug in cancer treatment in vitro and in vivo , presumably by regulating membrane drug interaction [15,16]. The resulting ( R )-2-hydroxy FAs (( R )-2-OHFAs) are incorporated into sphingolipids that induce apoptosis at significantly lower concentrations as compared to non-OH counterparts in C6 glioma cells, suggesting that FA2H and ( R )-2-OHFAs induce specific proapoptotic signaling although this remains little studied [17]. Moreover, FA2H silencing facilitates cell growth and inhibits cAMP-induced cell cycle exit and growth arrest in D6P2T Schwannoma cells [18], indicating diverse functions of FA2H in regulating signaling pathways as related to cell proliferation.…”

2′-Hydroxy ceramide in membrane homeostasis and cell signaling

“…Szulc et al showed that synthetic 2R′ -hydroxy-C6-ceramide showed stronger growth inhibitory activity against MCF7 cells compared to the 2′S isomer or non-hydroxy-C6-ceramide (Szulc et al, 2010), though the mechanism of the growth inhibition was not determined. Using synthetic 2′R - and 2′S -hydroxy-C16-ceramide, we demonstrated that proapoptotic activity of 2′R isomer was much more potent compared to the 2′S isomer or non-hydroxy C16-ceramide (Kota et al, 2013). Treatment with 2′R -hydroxy-C16-ceramide induced rapid dephosphorylation of Akt and MAP kinases, while the 2′S isomer and non-hydroxy C16-ceramide did not induce dephosphorylation at the same concentrations.…”

Mass Spectrometric Analysis of Sphingomyelin with N‐α‐Hydroxy Fatty Acyl Residue in Mouse Tissues

“…For example, forcibly changing the ceramide composition in cells by overexpression or silencing the ceramide synthases shows that long‐chain ceramide is pro‐apoptotic, while ceramide with very long chain FA is anti‐apoptotic (Grösch et al, 2012). It is also reported that ceramide with hFA potently induces apoptosis compared to that with non‐hydroxylated FA (Kota et al, 2013). In this regard, hSM detected in intestinal mucosa and skin may serve as precursor of a pro‐apoptotic ceramide with high potency.…”