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Published on: December 13, 2019
Gangliosides do not affect ABC transporter function in human neuroblastoma cells
Anne-Jan Dijkhuis1, Karin Klappe, Willem Kamps
1Department of Cell Biology, Section of Membrane Cell Biology, University Medical Center Groningen, 9713 AV Groningen, The Netherlands.
Abstract:
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.
Insights
This study found that gangliosides do not significantly impact multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1) in neuroblastoma cells, challenging previous hypotheses.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Glucosylceramide synthase (GCS) has been implicated in multidrug resistance (MDR).
- Gangliosides, products of GCS, were hypothesized to modulate the activity of ATP-binding cassette (ABC) transporters involved in MDR.
- This study investigates the role of gangliosides in P-glycoprotein (Pgp) and multidrug resistance-related protein 1 (MRP1) mediated drug resistance.
Purpose of the Study:
- To challenge the hypothesis that gangliosides modulate ABC transporter activity in MDR.
- To investigate the effect of ganglioside depletion on Pgp- and MRP1-mediated drug efflux in human neuroblastoma cells.
Main Methods:
- Two human neuroblastoma cell lines expressing functional Pgp or MRP1 were used.
- Ganglioside content was depleted using GCS inhibitors D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ).
- Ganglioside levels were quantified using equilibrium radiolabeling, cholera toxin binding, and mass analysis. Efflux activity was assessed via FACS analysis, and cellular sensitivity to vincristine was determined by MTT assay.
Main Results:
- GCS inhibitors efficiently depleted ganglioside content in both cell lines.
- Ganglioside depletion resulted in marginal and opposing effects on Pgp- and MRP1-mediated efflux.
- These effects were significantly less pronounced than those observed with established Pgp and MRP1 inhibitors.
- Cellular sensitivity to vincristine was only slightly enhanced by t-PPPP, while NB-dNJ had no effect.
- MRP1 expression and localization remained unaffected by ganglioside depletion.
Conclusions:
- Gangliosides are not relevant modulators of ABC transporter-mediated multidrug resistance in neuroblastoma cells.
- The study challenges the proposed role of gangliosides in regulating Pgp and MRP1 activity.
- Findings suggest alternative mechanisms may be responsible for GCS-related MDR.
