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Originally published In Press as doi:10.1194/jlr.M500518-JLR200 on March 18, 2006

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Journal of Lipid Research, Vol. 47, 1187-1195, June 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

Anne-Jan Dijkhuis*,{dagger}, Karin Klappe*, Willem Kamps{dagger}, Hannie Sietsma§ and Jan Willem Kok1,*

* Department of Cell Biology, Section of Membrane Cell Biology, University Medical Center Groningen, 9713 AV Groningen, The Netherlands
{dagger} Department of Pediatric Oncology and Hematology, Beatrix Children's Hospital, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands
§ Department of Pathology and Laboratory Medicine, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands

Published, JLR Papers in Press, March 18, 2006.

1 To whom correspondence should be addressed. e-mail: j.w.kok{at}med.umcg.nl

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.

Supplementary key words glucosylceramide synthase • D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol • N-butyldeoxynojirimycin • P-glycoprotein • multidrug resistance-related protein 1 • detergent-resistant membrane

Abbreviations: Cer, ceramide; CFDA, 5-carboxyfluorescein diacetate; CSA, cyclosporin A; CTB, cholera toxin, B subunit; DRM, detergent-resistant membrane; FCS, fetal calf serum; GCS, glucosylceramide synthase; HPTLC, high-performance thin-layer chromatography; MDR, multidrug resistance; MRP1, multidrug resistance-related protein 1; MTT, 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide; NB-dNJ, N-butyldeoxynojirimycin; Pgp, P-glycoprotein; Rh123, rhodamine 123; TNE, Tris-NaCl-EDTA buffer; t-PDMP, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; t-PPPP, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol; TRITC, tetramethyl rhodamine iso-thiocyanate


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