This project will determine the influence of ceramide, ceramide metabolites, and glucosylceramide synthase (GCS) on expression of the multidrug-resistant phenotype, using cancer cells in vitro, and tumor xenographs in vivo, as model systems. GCS catalyzes the formation of glucosylceramide (GC), the building block of higher glycolipids (cerebrosides, gangliosides). Glycolipids such as GC are also substrates for ABC transporter proteins, of which the MDR1 protein, P-glycoprotein (P-gp) is a member. Overexpression of MDR1/P-gp in cells represents a major impediment to effective treatment of many infectious and malignant diseases. Our objective is to improve treatment of disease. If lipids are involved in expression of the MDR1 phenotype, then blocking lipid metabolism at a specific juncture may be a means of limiting multidrug resistance. Many agents - chemotherapy drugs, nitrous oxide, gamma-irradiation - promote an increase in cellular ceramide, which elicits apoptosis. However, high levels of ceramide can also trigger overexpression of GCS due to increased availability of the enzymes lipoidal substrate, leading to elevated levels of GC. Because of the relationship between chemotherapy drugs and ceramide generation, and because high levels of GC are found in multidrug-resistant cancer cells and tumors, we hypothesize that lipids enhance expression of the multidrug-resistant phenotype. This enhancement would dull cellular responses to antibiotics, antitumor agents, and HIV protease inhibitors.
The aims of this proposal are: 1. to determine the influence of GCS on MDR1/P-gp expression;2. to determine the influence of GCS modulation by PPMP, a GCS inhibitor, on MDR1/P-gp expression and response to doxorubicin in an in vitro multidrug-resistant breast tumor xenograft model;3. to determine the influence of lipids (ceramide, glycolipids) on expression of the multidrug-resistant phenotype, 4. to determine the influence of chemotherapy drugs on GCS and MDR1 expression. We have ordered the Aims to facilitate testing of proof of principle.
Aim 1 will demonstrate the power of targeting GCS as an approach to limiting MDR1 expression, using in vitro models.
Aim 2, using animal models, will evaluate the power of targeting GCS as an approach to treating drug resistance, in vivo.
Aims 3 and 4 focus on mechanisms, and their outcomes will not detract from the possible therapeutic value of the study. This is the first study to investigate the influence of lipids on the multidrug-resistant phenotype. The results could lead to improved treatment of HIV/AIDS, malignant disorders, and bacterial and parasitic infectious diseases, as treatment of these diseases is classically hindered by drug resistance.
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