Role of 14-3-3 sigma in over-expression of P-gp by rifampin and paclitaxel stimulation through interaction with PXR
Role of 14-3-3 sigma in over-expression of P-gp by rifampin and paclitaxel stimulation through interaction with PXR
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DOI：
10.1016/j.cellsig.2017.01.001
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发表时间：
2017-02-01
影响因子：
4.8
通讯作者：
Shin, Jae-Gook
中科院分区：
文献类型：
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作者：
Kim, So Won;Hasanuzzaman, Md.;Shin, Jae-Gook
在本研究中，我们展示了 14-3-3 sigma 在利福平和紫杉醇处理的 LS174T 细胞和体内 LS174T 细胞异种移植裸鼠模型上激活 CK2-Hsp90 beta-PXR-MDR1 通路的作用。经过多次体外和体内实验，发现利福平和紫杉醇可刺激 CK2-Hsp90 beta-PXR-MOR1 通路。在该途径的蛋白质中，孕烷X受体（PXR）是多药耐药蛋白1（MDR1）的代表性转录因子。我们构建了 FLAG-PXR-LS174T 稳定细胞系，并发现了 22 种在利福平治疗时与 PXR 相互作用的蛋白质。其中，分离出Hsp90 beta和14-3-3 sigma以供进一步研究。使用共聚焦显微镜发现，在利福平处理后，这两种蛋白质都定位于细胞质中。另一方面，通过细胞分级分析发现，利福平和紫杉醇处理后，PXR定位于细胞核。在Western blot分析中，利福平不影响14-3-3 sigma蛋白的表达。将 14-3-3 sigma 瞬时转染至 LS174T 细胞可诱导 PXR 过度表达；然而，P-糖蛋白（P-gp）没有显着变化。仅当用利福平和紫杉醇处理 14-3-3 sigma 转染的 LS174T 细胞时，才会诱导 P-gp 过表达，而非肽抑制剂 BV02 和 14-3-3 sigma siRNA 的 14-3-3 sigma 抑制会降低利福平诱导的 PXR 和 P-gp 表达。紫杉醇和长春新碱治疗后，143-3 sigma-LS174T 稳定细胞系的细胞存活率比 LS174T 细胞高得多。该数据表明，14-3-3 sigma 通过与利福平和紫杉醇治疗下的 PXR 相互作用，导致 P-gp 过度表达。 (C) 2017 Elsevier Inc. 保留所有权利。
In this study, we presented the role of 14-3-3 sigma to activate CK2-Hsp90 beta-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-Hsp90 beta-PXR-MOR1 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, Hsp90 beta and 14-3-3 sigma 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 14-3-3 sigma protein. Transient transfection of 14-3-3 sigma into LS174T cells induced overexpression of PXR; however, P-glycoprotein (P-gp) was not changed significantly. P-gp overexpression was induced only when 14-3-3 sigma transfected LS174T cells were treated with rifampin and paclitaxel, whereas 14-3-3 sigma inhibition by nonpeptidic inhibitor, BV02 and 14-3-3 sigma siRNA reduced rifampin induced PXR and P-gp expression. Cell survival rates were much higher at 143-3 sigma-LS174T stable cell lines than LS174T cells following paclitaxel and vincristine treatment. This data indicates that 14-3-3 sigma contributes to P-gp overexpression through interaction with PXR with rifampin and paclitaxel treatment. (C) 2017 Elsevier Inc. All rights reserved.