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.

Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells.

High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. Enforced GCS overexpression has been shown to disrupt ceramide-induced apoptosis and to confer resistance to doxorubicin. To examine whether GCS is a target for cancer therapy, we have designed and tested the effects of antisense oligodeoxyribonucleotides (ODNs) to GCS on gene expression and chemosensitivity in multidrug-resistant cancer cells. Here, we demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Pretreatment with asGCS ODN-7 increased doxorubicin sensitivity by 17-fold in MCF-7-AdrR (doxorubicin-resistant) breast cancer cells and by 10-fold in A2780-AD (doxorubicin-resistant) ovarian cancer cells. In MCF-7 drug-sensitive breast cancer cells, asGCS ODN-7 only increased doxorubicin sensitivity by 3-fold, and it did not influence doxorubicin cytotoxicity in normal human mammary epithelial cells. asGCS ODN-7 was shown to be more efficient in reversing drug resistance than either the GCS chemical inhibitor d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol or the P-glycoprotein blocking agents verapamil and cyclosporin A. Experiments defining drug transport and lipid metabolism parameters showed that asGCS ODN-7 overcomes drug resistance mainly by enhancing drug uptake and ceramide-induced apoptosis. This study demonstrates that a 20-mer asGCS oligonucleotide effectively reverses MDR in human cancer cells.