Abstract
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.
Highlights
High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer
Comparing sequences of GCS in human, rat, and mouse by gene alignment analysis, the homology values in target regions of antisense GCS (asGCS) ODN-1 and ODN-7 were 100% and 85%, respectively, and the homology values for asGCS ODN-5, ODN-9, ODN-10, and ODN-11 were 90%
All asGCS ODNs inhibited the growth of MCF-7-AdrR cells, albeit variably; the EC50 values ranged from 0.3 M to 2.2 M
Summary
High glucosylceramide synthase (GCS) activity is one factor contributing to multidrug resistance (MDR) in breast cancer. We demonstrate that antisense GCS (asGCS) ODN-7 blocked cellular GCS expression and selectively increased the cytotoxicity of anticancer agents. Oligonucleotides blocking glucosylceramide synthase expression selectively reverse drug resistance in cancer cells. The loss of ceramide generation can cause cellular resistance to apoptosis in response to ionizing radiation, tumor necrosis factor-␣, and doxorubicin [12,13,14]. We found that GCS expression is upregulated in metastatic breast cancer and in multidrug-resistant cancer cell lines (our unpublished data). Overall, these studies indicate that enhanced expression of GCS contributes to poor chemotherapy response
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