Previous studies have emphasized the role of glucosylceramide (Glu-Cer) synthase in multidrug resistance (MDR) regulation. However, the mechanism by which the inhibition of this enzyme results in increased drug retention and cytotoxicity remains unclear. In this study, we investigated the respective role of ceramide (Cer) accumulation and Glu-Cer derivatives depletion in MDR reversal effect of 1-phenyl-2-decanoylamino-3-morpholino-1-propanolol (PDMP), a Glu-Cer synthase inhibitor. We show here that treatment with PDMP resulted in increased rhodamine 123 (Rh123) retention and potent chemosensitization of P-glycoprotein (P-gp)-expressing cells, including KG1a cells, KG1a/200 cells, K562/138 cells, and K562/mdr-1 cells. Metabolic studies revealed that PDMP induced not only time-dependent Cer accumulation but also reduction of all glycosylated forms of Cer, including Glu-Cer, lactosylceramide (Lac-Cer), monosialo ganglioside (GM3) and disialo ganglioside (GD3). The influence of these metabolites on P-gp function was investigated by measuring Rh123 retention in PDMP-treated cells. P-gp function was found to be stimulated only by the addition of gangliosides in all resistant cell lines, whereas Glu-Cer, Lac-Cer, and Cer had no effect. Moreover, in KG1a/200 cells, GD3 and, to a lesser extent, GM3 were found to phosphorylate P-gp on serine residues. Altogether, these results suggest that, at least in leukemic cells, gangliosides depletion accounts for PDMP-mediated MDR reversal effect, and that gangliosides are important P-gp regulators perhaps through their capacity to modulate P-gp phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Multidrug-resistant cancer cells display elevated levels of glucosylceramide (Lavie, Y., Cao, H. T., Volner, A., Lucci, A.,
Han, T. Y., Geffen, V., Giuliano, A. E., and Cabot, M. C. (1997) J. Biol. Chem. 272, 1682–1687). In this study, we have introduced glucosylceramide synthase (GCS) into wild type MCF-7 breast cancer cells
using a retroviral tetracycline-on expression system, and we developed a cell line, MCF-7/GCS. MCF-7/GCS cells expressed an
11-fold higher level of GCS activity compared with the parental cell line. Interestingly, the transfected cells demonstrated
strong resistance to adriamycin and to ceramide, whereas both agents were highly cytotoxic to MCF-7 cells. The EC50 values of adriamycin and ceramide were 11-fold (p < 0.0005) and 5-fold (p < 0.005) higher, respectively, in MCF-7/GCS cells compared with MCF-7 cells. Ceramide resistance displayed by MCF-7/GCS cells
closely paralleled the activity of expressed GCS with a correlation coefficient of 0.99. In turn, cellular resistance and
GCS activity were dependent upon the concentration of the expression mediator doxycycline. Adriamycin resistance in MCF-7/GCS
cells was related to the hyperglycosylation of ceramide and was not related to shifts in the levels of either P-glycoprotein
or Bcl-2. This work demonstrates that overexpression of GCS, which catalyzes ceramide glycosylation, induces resistance to
adriamycin and ceramide in MCF-7 breast cancer cells.
[Show abstract][Hide abstract] ABSTRACT: The sphingolipid composition and multidrug resistance status of three human neuroblastoma cell lines were established. SK-N-FI cells displayed high expression and functional (efflux) activity of P-glycoprotein, while multidrug resistance-related protein 1 was relatively abundant and most active in SK-N-AS cells. These two cell lines exhibited higher sphingolipid levels, compared to SK-N-DZ, which had the lowest activity of either ATP-binding cassette transporter protein. SK-N-DZ cells also differed in ganglioside composition with predominant expression of b-series gangliosides. In conclusion, these three neuroblastoma cell lines offer a good model system to study sphingolipid metabolism in relation to ATP-binding cassette transporter protein function.
[Show abstract][Hide abstract] ABSTRACT: Resistance to natural product chemotherapy drugs is a major obstacle to successful cancer treatment. This type of resistance is often acquired in response to drug exposure; however, the mechanisms of this adverse reaction are complex and elusive. Here, we have studied acquired resistance to Adriamycin, Vinca alkaloids, and etoposide in MCF-7 breast cancer cells, KB-3-1 epidermoid carcinoma cells, and other cancer cell lines to determine if there is an association between expression of glucosylceramide synthase, the enzyme catalyzing ceramide glycosylation to glucosylceramide, and the multidrug-resistant (MDR) phenotype. This work shows that glucosylceramide levels increase concomitantly with increased drug resistance in the KB-3-1 vinblastine-resistant sublines KB-V.01, KB-V.1, and KB-V1 (listed in order of increasing MDR). The levels of glucosylceramide synthase mRNA, glucosylceramide synthase protein, and P-glycoprotein (P-gp) also increased in parallel. Increased glucosylceramide levels were also present in Adriamycin-resistant KB-3-1 sublines KB-A.05 and KB-A1. In breast cancer, detailed analysis of MCF-7 wild-type and MCF-7-AdrR cells (Adriamycin-resistant) demonstrated enhanced glucosylceramide synthase message and protein, P-gp message and protein, and high levels of glucosylceramide in resistant cells. Similar results were seen in vincristine-resistant leukemia, etoposide-resistant melanoma, and Adriamycin-resistant colon cancer cell lines. Cell-free glucosylceramide synthase activity was higher in lysates obtained from drug-resistant cells. Lastly, glucosylceramide synthase promoter activity was 15-fold higher in MCF-7-AdrR compared with MCF-7 cells. We conclude that selection pressure for resistance to natural product chemotherapy drugs selects for enhanced ceramide metabolism through glucosylceramide synthase in addition to enhanced P-gp expression. A possible connection between glucosylceramide synthase and P-gp in drug resistance biology is suggested.
Molecular Cancer Therapeutics 06/2004; 3(5):633-9. · 5.68 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.