Ceramide and glucosylceramide upregulate expression of the multidrug resistance gene MDR1 in cancer cells

Abstract

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 microg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 microg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1-(14)C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 mug/ml [1-(14)C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.

Fig. 1

Influence of C8-ceramide and C8-GC on MDR1 expression in human breast cancer cells. Cells (500,000) were seeded into 6-cm dishes and the following day supplemented with (A) C8-ceramide (5 µg/ml medium) or (B) C8-GC (10 µg/ml medium). Controls received ethanol vehicle (0.1% final concentration). After 72 h, total RNA was extracted and analyzed by qPCR. Fold increases in MDR1 were calculated using actual number of gene copies per unit β-actin expression. Results are the average of triplicate experiments, and experiments were repeated several times.

Fig. 2

Conversion of C8-ceramide to C8-GC and uptake of G8-GC when supplemented to breast cancer cells. (A) C8-ceramide conversion. Cells were grown in the absence and presence of C8-ceramide (5 µg/ml medium) for 72 h. (B, C) Uptake. Cells were grown in the absence and presence of C8-GC (10 µg/ml medium) for 48 h. Total lipids were extracted from washed cells and analyzed by TLC (200 µg total lipid/lane). Lipids were resolved using a solvent system containing chloroform/methanol/ammonium hydroxide (70:35:4, v/v), and visualization was by sulfuric acid char. Commercial standards (Std) used were GC, glucosylceramide (brain), C8-GC, phosphatidylethanolamine (PE), phosphatidylcholine (PC). In (A) the oval denotes C8-GC synthesized from C8-ceramide supplement. In (B, C) the ovaldenotes C8-GC that was found intracellularly after cells were incubated with C8-GC supplement.

Fig. 3

The influence of chronic exposure to C8-ceramide on MDR1 mRNA, P-gp levels, and cell morphology in MDA-MB-231 cells. (A) MDR1 mRNA levels by RT-PCR in MDA-MB-231 cells and in high passage C8-ceramide cells (MDA-MB-231/C8cer, passage 22). Samples were subjected to RT-PCR analysis (0.5 µg RNA/tube) and products were resolved on 1% agarose gels. β-actin was employed as housekeeping gene. (B) P-gp levels by Western blot in MDA-MB-231 and in MDA-MB-231/C8cer cells at passages 12 and 22. Aliquots (100 µg cell protein) were electrophoresed for Western blot analysis of P-gp (C219 antibody). KB-Ch^R-8-5 (colchicine-resistant human epidermoid carcinoma) cell protein (50 µg) was used as a positive control for P-gp. For comparative purposes, 0.25 µg protein from MCF-7-AdrR cells was sufficient to detect P-gp in this highly drug-resistant cancer cell line (data not shown). (C) Cellular morphology. MDA-MB-231 cells, after first being exposed to 2.5 µg/ml C8-ceramide were grown continuously in the presence of 5 µg/ml C8-ceramide. MDA-MB-231/C8cer cells shown on right are from passage 19. Photomicrographs at 200× magnification.

Fig. 4

Doxorubicin and paclitaxel sensitivity in MDA-MB-231 (■) and MDA-MB-231/C8cer cells (●). MDA-MB-231/C8cer cells (passage 17) were used in the chemosensitivity assays, (A) doxorubicin and (B) paclitaxel. C8-ceramide was not in the medium during the experiment. Cell viability was conducted as described in Materials and methods. Data are the mean ± S.D. of six replicates. Experiments were repeated giving similar results.

Fig. 5

Rhodamine uptake and efflux in MDA-MB-231 and MDA-MB-231/C8cer cells. The experiments were conducted as detailed in Materials and methods. Cyclosporine A was present during rhodamine uptake. F.U., fluorescence unites. Uptake refers to intracellular F.U. after 60 min rhodamine exposure. Efflux refers to intracellular F.U. remaining after 60 min reincubation.

Fig. 6

Metabolism of and influence of C8-ceramide on GC formation in breast cancer cells. Cells in 6-well plates were grown in medium containing [³H]galactose (10 µCi/ml 5% FBS medium) in the absence and presence of C8-ceramide (5 µg/ml) for 24 h. Cellular total lipid extracts were analyzed by TLC. Zonal analysis (incremental scraping) was done by LSC only on the GC area of the chromatogram. Results are the mean ± S.D. from triplicate cultures.

Fig. 7

Metabolism of [¹⁴C]C6-ceramide by breast cancer cell lines. Cells in 6-well plates were grown with [¹⁴C]C6-ceramide (500,000 cpm/ml, 5 µg/ml) for 24 h, after which total lipids were extracted and analyzed by TLC for radiolabeled free C6-ceramide, C6-GC, C6-SPM and C6-LacCer by LSC. For MDA-MB-231/C8cer cells (see right panel), the C8-ceramide was removed from the medium at seeding (6-well plates) and was also absent during the [¹⁴C]C6-ceramide exposure period. Results are the mean±S.D. from triplicate cultures.

Fig. 8

Influence of GCS inhibition on ceramide-induced MDR1 upregulation in MDA-MB-435 cells. (A) Influence of ethylenedioxy-P4 (Etdioxy-P4) on cellular GC synthesis. Monolayer cultures (6-cm dishes) were incubated with [¹⁴C]C6-ceramide mixed with unlabeled C6-ceramide (84,500 cpm/ml, 5 µg/ml) for 24 h in the absence or presence of ethylenedioxy-P4. [¹⁴C]CG-GC was quantitated in the total lipid extract by TLC and LSC. (B) Influence of GCS inhibitor, ethylenedioxy-P4, on MDR1 expression. Cells in 10-cm dishes were exposed to the agents indicated for 24 h. Total RNA was extracted and MDR1 mRNA was measured by qPCR. C6-ceramide, 5.0 µg/ml; ethylenedioxy-P4, 0.2 µM. Fold increase was calculated as in Fig. 1.