Laboratory of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
Laboratory of Biopharmaceutics, Department of Pharmacology, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
J Pharm Sci. 2017 Sep;106(9):2642-2649. doi: 10.1016/j.xphs.2017.03.011. Epub 2017 Mar 18.
Overexpression and activation of P-glycoprotein (P-gp), which mediates efflux transport of various anticancer drugs in cancer cells, is associated with multidrug resistance (MDR). On the other hand, malignant cancer cells frequently undergo epithelial-to-mesenchymal transition (EMT), thereby acquiring high migratory mobility and invasive ability. Snail is a transcriptional factor that represses multiple other factors, and its overexpression is a trigger of EMT. Because both P-gp and Snail are involved in malignant evolution of cancer, in this work, we evaluated whether EMT induced by overexpression of Snail influences P-gp expression and activity. Snail-overexpressing cells showed downregulation of epithelial markers, E-cadherin, occludin, and claudin-1, and upregulation of mesenchymal markers, vimentin and ZEB1. Although Western blot analysis showed that P-gp expression levels were similar in Mock and Snail-overexpressing cells, the results of P-gp functional assays with P-gp substrates rhodamine123 and paclitaxel indicated that P-gp is activated in Snail-overexpressing cells. Indeed, Snail-overexpressing cells showed greater viability than Mock cells in the presence of paclitaxel. We observed caveolin-1 dephosphorylation and decreased growth factor receptor-bound protein 2 (GRB2) expression in Snail-overexpressing cells. These findings suggest a novel pathway leading to cancer MDR, in which Snail induces EMT concomitantly with a decrease in GRB2-mediated caveolin-1 phosphorylation, resulting in activation of P-gp.
P-糖蛋白（P-gp）的过度表达和激活介导了癌细胞中多种抗癌药物的外排转运，与多药耐药（MDR）有关。另一方面，恶性癌细胞经常经历上皮-间充质转化（EMT），从而获得高迁移性和侵袭能力。Snail 是一种转录因子，它抑制多个其他因子，其过表达是 EMT 的触发因素。由于 P-gp 和 Snail 都参与了癌症的恶性演变，在这项工作中，我们评估了由 Snail 过表达诱导的 EMT 是否影响 P-gp 的表达和活性。Snail 过表达细胞表现出上皮标志物 E-钙黏蛋白、occludin 和 claudin-1 的下调，以及间充质标志物 vimentin 和 ZEB1 的上调。尽管 Western blot 分析显示 Mock 和 Snail 过表达细胞中的 P-gp 表达水平相似，但 P-gp 功能测定用 P-gp 底物 rhodamine123 和紫杉醇的结果表明 P-gp 在 Snail 过表达细胞中被激活。事实上，在紫杉醇存在的情况下，Snail 过表达细胞的活力比 Mock 细胞更高。我们观察到 Snail 过表达细胞中 caveolin-1 的去磷酸化和生长因子受体结合蛋白 2（GRB2）表达的减少。这些发现表明了一种导致癌症 MDR 的新途径，其中 Snail 诱导 EMT 同时伴随着 GRB2 介导的 caveolin-1 磷酸化减少，导致 P-gp 的激活。
