Background: To provide insight for the development of more effective clinical agents, the authors attempted to elucidate the mechanisms of action of multidrug resistance (MDR) modulators. Previously, the authors found that MDR modulators blocked the conversion of ceramide to glucosylceramide in MDR cells, thereby enhancing cytotoxicity. Because ceramide is a critical component of the apoptosis signaling cascade, the current study examined the impact of therapy using agents that elicit ceramide formation combined with agents that block ceramide glycosylation.
Methods: Doxorubicin-resistant human breast carcinoma cells (MCF-7-AdrR) were treated with either doxorubicin, tamoxifen, cyclosporine A, or the cyclosporine A analog SDZ PSC 833 (PSC 833) or with combinations thereof, and ceramide and glucosylceramide metabolisms were measured by cell radiolabeling. Cell viability was quantitated spectrophotometrically and apoptosis was evaluated analyzing DNA integrity by gel electrophoresis.
Results: Whereas cyclosporine A blocked the generation of glucosylceramide in MCF-7-AdrR cells, a chemical cousin, PSC 833, elicited a 3-fold increase in glucosylceramide and a 5-fold increase in ceramide levels at 24 hours. The PSC 833 response was time-dependent(as early as 30 minutes) and dose-dependent (as low as 0.1 microM). The appearance of ceramide foreran the generation of glucosylceramide. Sphingomyelin levels were not decreased in response to PSC 833; however, Fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Adding tamoxifen, which blocks ceramide glycosylation, to the PSC 833 regimen boosted ceramide levels 11-fold over controls and caused DNA fragmentation. A 3-component regimen comprised of tamoxifen, doxorubicin, and PSC 833 increased ceramide levels 26-fold and brought cell viability to zero.
Conclusions: These results demonstrate that MDR modulators can be used separately, in combination, or in conjunction with chemotherapy at clinically relevant concentrations to manipulate cellular ceramide levels and restore sensitivity in the drug resistant setting. As such, this represents a new direction in the treatment of cancer.