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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^*,, Karin Klappe^*, Willem Kamps, Hannie Sietsma and Jan Willem Kok^1,*

^* Department of Cell Biology, Section of Membrane Cell Biology, University Medical Center Groningen, 9713 AV Groningen, The Netherlands
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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