Gangliosides do not affect ABC transporter function in human neuroblastoma cells.
Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells.
Dijkhuis
Anne-Jan
AJ
Department of Cell Biology, Section of Membrane Cell Biology, University Medical Center Groningen, 9713 AV Groningen, The Netherlands.
Klappe
Karin
K
Kamps
Willem
W
Sietsma
Hannie
H
Kok
Jan Willem
JW
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D045744:Cell Line, Tumor; D002518:Ceramides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005732:Gangliosides / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D002518:Ceramides; D004791:Enzyme Inhibitors; D005732:Gangliosides; D017485:1-Deoxynojirimycin; C059896:miglustat; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase