Abstract

Recent evidence suggests a role for aberrant ceramide levels in the pathogenesis of cancer and chemoresistance and indicates that manipulation of tumor ceramide levels may be a useful strategy in the fight against breast cancer. This study demonstrates that alterations in the degree and position of unsaturation of bonds in the sphingoid backbone of d-erythro-N-octanoyl-sphingosine (Cer) affect the antiproliferative ability of ceramide analogs in breast cancer cells. The most potent analog of Cer we tested is (2S,3R)-(4E,6E)-2-octanoylamidooctadecadiene-1,3-diol (4,6-diene-Cer), which contains an additional trans double bond at C(6)-C(7) of the sphingoid backbone. 4,6-Diene-Cer exhibited higher potency than Cer in tumor necrosis factor (TNF)-α-resistant (IC₅₀ of 11.3 versus 32.9 μM) and TNF-α-sensitive (IC₅₀ of 13.7 versus 37.7 μM) MCF-7 cells. 4,6-Diene-Cer was also more potent than Cer in inducing cell death in MDA-MB-231 and NCI/ADR-RES breast cancer cell lines (IC₅₀ of 3.7 versus 11.3 μM, and 24.1 versus 86.9 μM, respectively). 4,6-Diene-Cer caused a prolonged elevation of intracellular ceramide levels in MCF-7 cells, which may contribute to its enhanced cytotoxicity. Furthermore, treatment of MCF-7 cells with Cer or 4,6-diene-Cer resulted in induction of apoptosis by 8 h via the mitochondrial pathway, as demonstrated by release of cytochrome c, loss of membrane asymmetry (measured by Annexin V staining), and a decrease in the mitochondrial membrane potential. Importantly, both Cer and 4,6-diene-Cer displayed selectivity toward transformed breast cells over nontransformed breast epithelial cells. These data suggest that these and other novel ceramide analogs represent potential therapeutic agents in breast cancer treatment.