title: Glucosylceramide synthase inhibitors sensitise CLL cells to cytotoxic agents without reversing P-gp functional activity.
abstract: Malignant B-cells from most chronic lymphocytic leukaemia (CLL) patients over-express MDR1 encoded P-glycoprotein (P-gp) multidrug efflux pump. Inhibition of glucosylceramide (GC) synthesis has been shown in cell lines to correlate with the expression and function of P-gp and sensitise cancer cells to cytotoxic agents. We investigated the hypothesis that reducing intracellular GC levels will reduce P-gp expression in malignant cells from CLL patients. We studied the ability of glucosylceramide synthase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1) and N-nonyl-deoxygalactonojirimycin (OGB-2) to sensitise CLL cells to conventional cytotoxic drug 2-chlorodeoxyadenosine (CdA) and the cytostatic drugs chlorambucil and fludarabine. The effect on P-gp activity was analysed using the calcein-AM accumulation assay where a multidrug activity factor (MAF) of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. The P-gp over-expressing cell line CEM-VLB showed a MAF value of 96.4 with the P-gp inhibitor Z.3HCL, which fell to 15.7 after co-incubation with OGB-1 and 45.9 with OGB-2. The IC(50) for vincristine fell from >10 microg/ml to 55.5 ng/ml in the presence of OGB-2. In P-gp(+ve) peripheral blood mononuclear cells from three normal volunteers, the mean MAF values for Z.3HCL, OGB-1 and OGB-2 were 23.86, 1.83 and 16.2 respectively. In 9/13 CLL samples the mean P-gp functional activity was 22.15 and P-gp was over-expressed in 12/13 samples. However, the MAF value with OGB-1 and OGB-2 was <10. Nevertheless, sensitisation in CLL cells was observed by a reduction in the IC(50) in the presence of OGB-1 and OGB-2 with the conventional drugs. We conclude that although GCS inhibitors sensitize CLL cells to cytotoxic and cytostatic drugs, they do not appear to have any effect on P-gp functional activity.
authors.affiliation: Department of Haematology, University College London Medical School, Royal Free Campus, London, UK.
authors.forename: Gareth
authors.initials: G
authors.lastname: Gerrard
authors.forename: Terry D
authors.initials: TD
authors.lastname: Butters
authors.forename: Kanagasabai
authors.initials: K
authors.lastname: Ganeshaguru
authors.forename: Atul B
authors.initials: AB
authors.lastname: Mehta
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000368:Aged; D000369:Aged, 80 and over; D001402:B-Lymphocytes / Q000187:drug effects / Q000201:enzymology; D045744:Cell Line, Tumor; D002699:Chlorambucil / Q000494:pharmacology; D003603:Cytotoxins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005562:Formazans / Q000378:metabolism; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors*; D006801:Humans; D020128:Inhibitory Concentration 50; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D008956:Models, Chemical; D015394:Molecular Structure; D013778:Tetrazolium Salts / Q000378:metabolism; D014740:Vidarabine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003603:Cytotoxins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005562:Formazans; D013778:Tetrazolium Salts; C085925:calcein AM; D002699:Chlorambucil; D017485:1-Deoxynojirimycin; C079782:MTT formazan; C090092:migalastat; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D014740:Vidarabine; C024352:fludarabine