Influence of Ceramide Metabolism on P-Glycoprotein Function in Immature Acute Myeloid Leukemia KG1a Cells

Abstract

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 timedependent Cer accumulation but also reduction of all glycosylated forms of Cer, including Glu-Cer, lactosylceramide (LacCer), 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 GluCer, 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.The anthracycline daunorubicin (DNR) is widely used in the treatment of acute myeloid leukemia (AML). In first line therapy, DNR is most often administered in association with the antimetabolite cytosine arabinoside. Although the DNR/ cytosine arabinoside combination induces a relatively high rate of complete remission, the high incidence of relapse indicates that this regimen is ineffective in completely eradicating leukemic progenitors. This clinical observation connotes the inherent chemoresistance of immature myeloid leukemic cells. Recent studies have phenotypically characterized leukemic stem cells as CD34 ϩ CD38 Ϫ cells (Lapidot et al., 1994). This discrete cellular compartment proceeds to limited differentiation and provides the blast cells in terminal division, which represent the vast majority of the malignant cell population in AML patients. Among different mechanisms responsible for the chemoresistance of immature leukemic cells to DNR, the multidrug resistance (MDR) phenotype may play an important role. Indeed, CD34 expression has been correlated with both the mdr-1 gene and P-glycoprotein expression (P-gp) (Campos et al., 1992). Moreover, further studies have shown that CD34 ϩ P-gp ϩ AML cells present higher P-gp-mediated drug efflux capacity than CD34 Ϫ P-gp ϩ AML cells (Leith et al., 1995) and that leukemic progenitors display more efficient P-gp than blast cells in terminal division (Demur et al., 1998)...

Gangliosides do not affect ABC transporter function in human neuroblastoma cells

“…Our results are in contrast with those of Plo et al (23), who show modulation of Pgp activity by the gangliosides GD 3 and GM 3 in acute myeloid leukemia cells. In their study, they used only PDMP to reduce ganglioside content and showed that an z50% reduction is sufficient to exert an effect on Pgp activity, whereas in our study, a stronger reduction of ganglioside content by either of two different GCS inhibitors was without effect.…”

“…In their study, they used only PDMP to reduce ganglioside content and showed that an z50% reduction is sufficient to exert an effect on Pgp activity, whereas in our study, a stronger reduction of ganglioside content by either of two different GCS inhibitors was without effect. The different outcome may be attributable to cell type-dependence or the fact that the myeloid leukemia cells used by Plo et al (23) highly overexpress Pgp as a result of drug selection or transfection with the mdr-1 gene, whereas the human neuroblastoma cell lines used in our study were neither selected nor transfected.…”

“…Third, when closely associated, sphingolipids could directly modulate ABC transporter efflux function. In this context, evidence has been obtained for gangliosides GD 3 and GM 3 as Pgp regulators through the modulation of Pgp phosphorylation in acute myeloid leukemia cells (23). Finally, gangliosides have been shown to be upregulated in MDR human ovarian and hepatic tumor cells, the latter overexpressing Pgp (34,35).…”

“…When closely associated, sphingolipids could directly modulate Pgp or MRP1 efflux function. In this context, evidence has been obtained for gangliosides GD 3 and GM 3 as Pgp regulators through the modulation of Pgp phosphorylation in acute myeloid leukemia cells (23).…”

Accumulation of lactosylceramide and overexpression of a PSC833-resistant P-glycoprotein in multidrug-resistant human sarcoma cells

“…In GARF cells, the overexpression of Pgp was accompanied by an increased LacCer level. Plo et al have shown that treatment with PDMP results in increased rhodamine 123 retention and potent chemosensitization of Pgp-expressing leukemic cells (27). In the above study, PDMP increased ceramide levels but reduced ganglioside levels.…”