Reduced UDP-glucose Levels Are Associated with P-glycoprotein Over-expression in L1210 Cells and Limit Glucosylceramide Synthase Activity

• Published in: Anticancer research 2015-05, Vol.35 (5), p.2627-2634
• Main Authors: Turáková, Katarina, Pavlíková, Lucia, Messingerová, Lucia, Lakatoš, Boris, Breier, Albert, Sulová, Zdena
• Format: Article
• Language: English
• Subjects: 4-Chloro-7-nitrobenzofurazan - administration & dosage; Animals; Apoptosis - drug effects; ATP-Binding Cassette, Sub-Family B, Member 1 - biosynthesis; Cell Line, Tumor; Ceramides - administration & dosage; Drug Resistance, Neoplasm - genetics; Glucosyltransferases - biosynthesis; Humans; Mice; Neoplasms - drug therapy; Neoplasms - genetics; Neoplasms - pathology; Uridine Diphosphate Glucose - biosynthesis
• ISSN: 1791-7530

P-glycoprotein (Pgp) expression in neoplastic cells is known to reduce cell sensitivity to several cytotoxic Pgp substrates. A member of the ABC transporter family, Pgp, represents the most frequently described membrane efflux pump and its expression in neoplastic cells is responsible for multi-drug resistance. Several lines of evidence indicate that the expression and increased function of both Pgp and glucosylceramide synthase (GCS, an enzyme responsible for ceramide pathway de-activation in the regulation of apoptosis progression) enhance the resistance of Pgp-positive cells. Previously, we described a reduction in the uridine diphosphate (UDP)-glucose contents of mouse leukemia cells (R) expressing Pgp due to vincristine selection compared to parental L1210 cells (S). The reduced availability of UDP-glucose as a glucose donor in R cell glycosylation reactions could limit GCS-catalyzed ceramide glycosylation. Consequently, the over-expression of Pgp in Pgp-positive L1210 cells may be associated with reduced ceramide glycosylation. To test this idea, we measured the expression and activities of Pgp and GCS, UDP-glucose levels, cellular uptake of C12-NBD-ceramide (a fluorescent analogue of ceramide) and ceramide-induced cell death in S and R cells. T-cells, another Pgp-positive variant of L1210 cells that express Pgp due to their transfection with a gene encoding human Pgp were also used in this study. We detected significantly reduced levels of C12-NBD-ceramide glycosylation and reduced UDP-glucose contents in Pgp-positive R and T-cells compared to S cells. C12-NBD-ceramide uptake assays revealed nearly identical dynamics of uptake time-dependency curves. The Pgp-positive L1210 variants (R and T) are more sensitive than Pgp-negative S cells to ceramide-induced cell damage, as measured by an fluorescein isothiocyanate-labeled annexin V and propidium iodide apoptosis necrosis kit. Short chain C2-ceramide was more effective at inducing cell damage than ceramide analogues with longer chains. These evidence indicates that the down-regulation of UDP-glucose contents in Pgp-positive L1210 cells is responsible for their collateral sensitivity to ceramide-induced apoptosis.