Ganglioside GD3 enhances apoptosis by suppressing the nuclear factor‐κB‐dependent survival pathway

Abstract

SPECIFIC AIM Glycosphingolipids have emerged as cell death effec-tors because of their role in apoptosis signaling. Be-cause cell survival reflects a balance between death and survival pathways and because the nuclear factor-␬B (NF-␬B) is known to downregulate apoptosis in re-sponse to diverse stimuli, our study examined the role of ganglioside GD3 (GD3) and related structural ana-logs, including short-chain ceramide analogs—for ex-ample, ceramide C2 (C2)— on NF-␬B regulation, mito-chondrial reactive oxygen species (ROS) generation, and survival of cultured rat hepatocytes. PRINCIPAL FINDINGS 1. Mitochondrial ROS formation is common for both C2 and GD3 Incubation of hepatocytes with C2 and GD3 (0 –50 ␮M) resulted in a similar dose-dependent peroxide forma-tion. It is interesting that, despite this, hepatocytes displayed a selective susceptibility to GD3 treatment, which was reflected by a decreased survival (36Ϯ5% vs. 92Ϯ7% for GD3 and C2 at 50 ␮M for 12 h), which was accompanied by apoptotic features. Inhibitors of mito-chondrial electron flow at complexes I and II prevented both peroxide formation and the GD3-induced loss of cell viability, which highlights the relevance of the complex III-induced ROS generation in the killing of hepatocytes by GD3. Other cell-permeable, short-chain ceramides, N-hexanoyl (C6)-or N-octanoyl(C8)-sphin-gosine, were equally effective as C2 in this event. Furthermore, various GD3 analogs, including GluCer, LactCer, and GM1, mimicked the stimulating effect of GD3 on ROS formation; whereas Lyso-GluCer was ineffective, which indicates the requirement of fatty acids for this event. Thus, the N-fatty acyl-sphingosine moiety determines the structural requirement of C2 and GD3 for mitochondrial stimulation of ROS gener-ation, whereas the presence of sugar residues is dispens-able for this event. 2. NF-␬B inactivation is a selective feature of GD3