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

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.

Glucosylceramide synthase blockade down-regulates P-glycoprotein and resensitizes multidrug-resistant breast cancer cells to anticancer drugs.

Overexpression of glucosylceramide synthase (GCS), a pivotal enzyme in glycolipid biosynthesis, contributes to cancer cell resistance to chemotherapy. We previously showed that transfection of doxorubicin-resistant MCF-7-AdrR cells with GCS antisense restored cell sensitivity to doxorubicin and greatly enhanced sensitivity to vinblastine and paclitaxel. In that study, doxorubicin promoted generation of ceramide in MCF-7-AdrR/GCS antisense cells; the present study implicates factors in addition to ceramide that augment sensitivity to chemotherapy. Although GCS antisense cells showed enhanced ceramide formation compared with MCF-7-AdrR when challenged with paclitaxel, GCS antisense cells also showed a 10-fold increase in levels of intracellular drug (paclitaxel and vinblastine). In addition, transfected cells had dramatically decreased expression (80%) of P-glycoprotein and a 4-fold decrease in the level of cellular gangliosides. Chemical inhibition of GCS produced the same effects as antisense transfection: exposure of MCF-7-AdrR cells to the GCS inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 5.0 micromol/L, 4 days) decreased ganglioside levels, restored sensitivity to vinblastine, enhanced vinblastine uptake 3-fold, and diminished expression of MDR1 by 58%, compared with untreated controls. A similar effect was shown in vinblastin-resistant KB-V0.01 cells; after 7 days with PPMP (10 micromol/L), MDR1 expression fell by 84% and P-glycoprotein protein levels decreased by 50%. MCF-7-AdrR cells treated with small interfering RNAs to specifically block GCS also showed a dramatic decrease in MDR1 expression. This work shows that limiting GCS activity down-regulates the expression of MDR1, a phenomenon that may drive the chemosensitization associated with blocking ceramide metabolism. The data suggest that lipids play a role in the expression of multidrug resistance.

Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy.

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.