Effects of atorvastatin on functional activity and expression of membrane transporters

Abstract

ATP Binding Cassette (ABC) proteins are a family of membrane cell transporters that promote the cellular efflux of several compounds. ABCB1, BCRP and MRP2 are ABC transporters that have been associated with the cellular efflux of drugs, mainly in the liver and bowel. While solute-linked carriers (SLC) transporters, such as OATP1B1, OATP2B1 and OCT1, are involved on drug uptake. These membrane transporters regulate the intracellular homeostasis of several drugs, modifying their disposition and efficacy. Statins are potent HMGCoA reductase inhibitors broadly used for hypercholesterolemia therapy. Atorvastatin is a substrate for ABCB1 and other drug transporters. Therefore, it is relevant to study the mechanisms involved in regulation of gene expression of membrane transporters by statins, in order to clarify the role of these transporters in lowering-cholesterol response. In this study, the effects of atorvastatin on mRNA and protein expression of the efflux (ABCB1, BCRP and MRP2) and uptake (OATP1B1, OATP2B1 and OCT1) transporters will be investigated in HepG2 and Caco-2 cells, and lymphocytes of hypercholesterolemic individuals. Total RNA and proteins will be extracted from the cultured cells incubated with different concentrations (0 to 20 uM) of atorvastatin. Protein and mRNA expression will be evaluated by Western blot and flow cytomety, and real time PCR, respectively. ABCB1 functional activity will be measured by flow cytometry and ATPase activity by colorimetric assay. ABCB1 mRNA stability will be analyzed by Northern blot and its relation with the ABCB1 C3435T polymorphism will be also investigated. The effects of atorvastatin on activating ABCB1 transcription factors (NF-B, AP-1 e SP1) will be evaluated by electrophoresis mobility shift assay (EMSA). The results of this study will provide new information regarding the modulation of the membrane transporters expression by atorvastatin and elucidate the role of these proteins on response to lowering-cholesterol drugs. (AU)