Are sphingolipids important in the cell death process in pancreatic cancer cells?

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Apoptosis is now recognized as a major pathway by which cytotoxic agents induce death of cells. Attenuation of this response in cancer cells contributes to a drug resistant phenotype and therefore, agents that facilitate apoptosis should improve therapeutic efficacy. Ceramide, a precursor to several bioactive lipids, and itself an intracellular signaling molecule, has a central role in both apoptotic and mitogenic pathways. Administration of water soluble ceramides to cancer cells in vitro, and other methods of increasing intracellular ceramide, can promote apoptosis. On the other hand, reducing intracellular ceramide levels in these cells leads to drug resistance. However, little is known of sphingolipid signaling in cancer cells and cancer stem cells. We analyzed five pancreatic cancer cell lines for stem cell surface marker expression. Panc1 cells, which express high levels of CD24 and CD44, were chosen for further studies of chemotherapy resistance. We found that these cells are highly resistant to chemotherapy and preferentially undergo senescence in response to gemcitabine (GEM), as judged by the increased expression of β-galactosidase activity. The inclusion of sphingomyelin (SM), at non-toxic concentrations, in the media, synergized with gemcitabine and promoted apoptotic cell death while excluding senescence. Panc1 cells responded to C₈-ceramide in the media by undergoing senscence at low (20 µM) concentrations and cell death at higher concentrations (IC₅₀ = 45 µM). We also examined the ability of SM to enhance GEM efficacy in the Panc1 s.c. tumor xenograft model. Panc1 tumors grown in SCID mice to 0.1-0.2 cm³ were treated as follows: GEM (6 mg i.p. once weekly for 3 weeks), SM (10 mg i.p. every day for 5 days/week x 3 weeks), GEM + SM (as for each drug individually with the GEM dose given with the third SM injection). These regimens were repeated for a second cycle after a 1-week rest. The results indicate that this tumor model is highly resistant to clinically-relevant doses of GEM. Additionally, there was no effect of SM when used as a single agent. However, the combination of GEM with SM resulted in a significant inhibition of tumor growth (P<0.01 compared to GEM alone), with resulting improvement in survival. Median survival times were 38.3 days (untreated), 38.8 days (SM), 39.8 days (GEM) and 54.7 days (GEM + SM combination) (P<0.01 for the comparison of the combination group to GEM alone). Our results indicate that manipulating the sphingolipid content of cells that express surface markers associated with stem cell-like behavior in vitro and in vivo, can increase the chemosensitivity of those cells. These data suggest that targeting the sphingolipid content of pancreatic cancer cells might lead to improved outcome, especially if cancer stem cells are affected. (This work was supported in part by grant CA92723 from the U.S. Public Health Service.)