Accumulation of glucosylceramides in multidrug-resistant cancer cells

Multidrug-resistant (MDR) tumors and cancer cell lines demonstrate a wide variety of biochemical changes. In this study we used drug-sensitive wild-type (wt) cancer cell lines and respective MDR subclones, and we demonstrate the accumulation of distinct lipids in MDR cells. These lipids were either absent or present at very low levels in drug-sensitive cells. The compounds, termed lipid-1 and lipid-2, migrated on thin-layer chromatography as a doublet. They could be radiolabeled by incubating MCF-7-AdrR (Adriamycin-resistant) breast cancer cells with [3H]serine, [3H]palmitic acid, or [3H]galactose. Utilization of these precursors by MCF-7-wt cells for synthesis of lipid-1 and -2 was minimal. Two inhibitors of sphingolipid biosynthesis, L-cycloserine and fumonisin B1, prevented the observed accumulation of the lipid compounds. An inhibitor of glucosylceramide synthesis, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, completely abolished the formation of lipid-1 and -2 in MCF-7-AdrR cells and, to a lesser extent, inhibited the formation of lactosylceramides and gangliosides. Utilizing mass spectrometry, the multidrug resistance-associated lipids were further characterized as monoglycosylceramides of two major species that contained either 16-carbon (palmitic) or 24-carbon (lignoceric and nervonic) fatty acids. The carbohydrate head group of glycosylceramides was identified as glucose, not galactose, thus designating the accumulated lipids as glucosylceramides. Incorporation of [3H]palmitic acid into glucosylceramide was strikingly higher (8-10 times) in MCF-7-AdrR cells compared with MCF-7-wt cells. Since the rate of glucosylceramide degradation in MCF-7-AdrR cells was not attenuated, accelerated glycosphingolipid synthesis in MDR cells is suggested. Glucosylceramide also accumulated in KB-V-1, a vinblastine-resistant epidermoid carcinoma but not in KB-3-1, drug-sensitive wt cells. MDR ovarian adenocarcinoma cells (NIH:OVCAR-3) also contained elevated levels of glucosylceramide. Our results demonstrate a correlation between cellular drug resistance and alterations in glucosylceramide metabolism.