Study On The Impact Of ABCC2(1249G>A)Polymorphism On The Efflux Of Sorafenib And The Molecular Mechanismp

Objective:Multidrug resistance-associated protein 2(MRP2),encoded by the ABCC2 gene,is an efflux transporter of several endogenous substrates and xenobiotics.Here we aims to investigated the impact of ABCC2(1249G>A)polymorphism on the efflux of anti-tumor drug sorafenib and conduct a preliminary study on the molecular mechanism.It is conducive to provide some reference on the individualized treatment of the clinical application of sorafenib.Methods:1 Biological engineering technology was used to construct the lentiviral vectors carrying human ABCC2-1249G or ABCC2-1249A,then the recombined lentiviral vectors were packaged and transfected to HEK293 cells.After a certain time of screen,HEK293 cells overexpressing ABCC2-1249G and ABCC2-1249A were selected and cultivated.2 cck-8 kit was applied the evaluate and compare the sensibility of ABCC2-1249G and ABCC2-1249A on the toxicity of sorafenib.3 Liquid-liquid extraction was used to extract sorafenib in cells and high performance liquid chromatography(HPLC)was used to the quantitative determination of sorafenib.4 The MRP2 was isolated by ProteoExtract(?)Native Membrane Protein Extraction Kit and then applied to estimate the ATPase activity.Results:Recombinant HEK293 cells expressing ABCC2-1249G and ABCC2-1249A were constructed,the RT-PCR and western-blot showed comparable expression level in two different recombinant cells.The determination of the inhibition concentration of 50%cell growth(IC50)of sorafenib confirmed that the recombinant cells overexpressing ABCC2-1249A displayed a significant increase in resistance to sorafenib compared with ABCC2 1249G recombinant cells and control group cells.Accumulation and efflux assay showed that Sorafenib accumulation was decreased in ABCC2-1249A cells in comparison to ABCC2-1249G cells at comparable expression level.The ATPase activity of isolated ABCC2-1249A membrane proteins stimulated by sorafenib was proved to be higher than that of isolated ABCC2-1249G membrane proteins.Conclusion:As a kind of ATP dependent transporters,MRP-2 participates in the transmembrane transport of sorafenib.And the ABCC2 polymorphism 1249G>A increases the ATPase activity of MRP2,leading to greater efflux of sorafenib.