Role of 14-3-3 sigma in over-expression of P-gp by rifampin and paclitaxel stimulation through interaction with PXR.

Abstract

In this study, we presented the role of 14-3-3σ to activate CK2-Hsp90β-PXR-MDR1 pathway on rifampin and paclitaxel treated LS174T cells and in vivo LS174T cell-xenografted nude mouse model. Following several in vitro and in vivo experiments, rifampin and paclitaxel were found to be stimulated the CK2-Hsp90β-PXR-MDR1 pathway. Of the proteins in this pathway, Pregnane X receptor (PXR) is a representative transcription factor of multidrug resistance protein 1 (MDR1). We constructed FLAG-PXR-LS174T stable cell lines and discovered 22 proteins that interacted with PXR on rifampin treatment. Among them, Hsp90β and 14-3-3σ were isolated for further study. Both the proteins were found to be localized in cytoplasm on rifampin treatment by using confocal microscopy. On the other hand, PXR was found to be localized in nucleus after rifampin and paclitaxel treatment by using cell fractionation assay. In Western blot analysis, rifampin did not influence the expression of 14-3-3σ protein. Transient transfection of 14-3-3σ into LS174T cells induced overexpression of PXR; however, P-glycoprotein (P-gp) was not changed significantly. P-gp overexpression was induced only when 14-3-3σ transfected LS174T cells were treated with rifampin and paclitaxel, whereas 14-3-3σ inhibition by nonpeptidic inhibitor, BV02 and 14-3-3σ siRNA reduced rifampin induced PXR and P-gp expression. Cell survival rates were much higher at 14-3-3σ-LS174T stable cell lines than LS174T cells following paclitaxel and vincristine treatment. This data indicates that 14-3-3σ contributes to P-gp overexpression through interaction with PXR with rifampin and paclitaxel treatment.