Taxol-induced ceramide generation and apoptosis in human breast cancer cells
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Purpose: Taxol has emerged as a valuable antimitotic chemotherapeutic agent, particularly in advanced breast and ovarian cancers. Although much is known about cytotoxic mechanisms, the effectiveness of Taxol cannot be solely explained by microtubular interaction. This study was undertaken to determine whether ceramide generation plays a role in Taxol-induced apoptosis. Methods: Hormone-independent MDA-MB-468 and hormone-dependent MCF-7 breast cancer cell lines were employed, and ceramide metabolism was characterized using [3H]palmitic acid as lipid precursor. Results: Exposure of cells to Taxol resulted in enhanced formation of [3H]ceramide. Ceramide increased nearly 2-fold in MDA-MB-468 cells exposed to 50 nM Taxol, and more than 2.5-fold in MCF-7 cells exposed to 1.0 µM Taxol. These concentrations mirrored the EC50 (amount of drug eliciting 50% cell kill) for Taxol in the two cell lines. Use of cell-permeable C6-ceramide as a medium supplement revealed that MDA-MB-468 cells were 20-fold more sensitive to ceramide than MCF-7 cells (P<0.001). Ceramide was generated as early as 6 h after exposure to Taxol in MDA-MB-468 cells, whereas the earliest signs of apoptosis were detected 12 h after treatment, and by 24 h the apoptotic index was six times that of untreated cells. Both fumonisin B1, a ceramide synthase inhibitor, and L-cycloserine, a serine palmitoyltransferase inhibitor, blocked Taxol-induced ceramide generation, whilst sphingomyelin levels remained unchanged, indicating a de novo pathway of ceramide formation. L-Cycloserine reduced Taxol-induced apoptosis by 30% in MDA-MB-468 cells and totally blocked Taxol-induced apoptosis in MCF-7 cells. Conclusions: These results suggest that Taxol-induced apoptosis is, in part, attributable to ceramide and sphingoid bases. This is of relevance to drug mechanism studies, as ceramide is a known messenger of apoptosis. Clinical use of Taxol with ceramide-enhancing agents may maximize cytotoxic potential.
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