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

Anticancer Res. 2015 May;35(5):2627-34.


Background/aim: 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.

Materials and methods: 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.

Results: 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.

Conclusion: 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.

Keywords: L1210 cells; P-glycoprotein; UDP-glucose; ceramide induced cell death; glucosylceramide synthase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 4-Chloro-7-nitrobenzofurazan / administration & dosage
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis*
  • Animals
  • Apoptosis / drug effects
  • 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


  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • C12-NBD-ceramide
  • Ceramides
  • Glucosyltransferases
  • ceramide glucosyltransferase
  • 4-Chloro-7-nitrobenzofurazan
  • Uridine Diphosphate Glucose