Endocrine Abstracts

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has manifold actions which may be involved in its chemopreventive effects on breast cancer growth and progression. Ceramide is a sphingolipid involved in the mammalian stress response and is implicated in the induction of apoptosis. Its role in the mechanism of 4HPR action has not been fully explored. Here we attempt to elucidate the relationship between 4HPR, ceramide production and cell viability. Survival of viable cells was measured by metabolism of a tetrazolium salt in a cell titer proliferation assay and ceramide production was monitored by following incorporation of ³H palmitic acid into the sphingolipid. 4HPR was differentially toxic to two breast cancer cell lines (MCF-7 and MDA MB 231) suggesting different mechanisms of action in the two lines. 4HPR caused a dose-dependent increase in ceramide production in MCF-7 cells which correlated with a decrease in cell survival. The ceramide synthase inhibitor fumonisin B₁ partially reversed this ceramide response but did not affect the cytotoxic response to 4HPR. MDA MB 231 cells were less sensitive to 4HPR and exhibited much smaller changes in ceramide production. Putative modulators of ceramide metabolism caused modest enhancement of ceramide accumulation and cytotoxicity. In MCF-7 cells 4HPR-induced cytotoxicity was dependent on Z-VAD-fmk-sensitive caspase activation whereas the ceramide response was upstream or independent of caspase involvement. The cytotoxicity of 4HPR in MDA MB 231 cells was independent of such caspase action. While offering circumstantial support, the evidence presented for a definitive role for ceramide during 4HPR action remains inconclusive. The differential action of 4HPR in the two cell lines warrants further investigation.