Division of Gastroenterology and Hepatology, University Hospital of Basel, CH-4031, Basel, Switzerland.
Mol Pharm. 2010 Oct 4;7(5):1618-28. doi: 10.1021/mp100040f. Epub 2010 Aug 11.
P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) play an important role in transport of a wide variety of endogenous compounds, drugs and toxins. Transport of some drugs, for example the tyrosine kinase inhibitor imatinib, is influenced by both P-gp and BCRP. Establishing an intestinal Caco-2 cell culture model with specific knock-downs of P-gp and BCRP and double knock-down of both proteins, we aimed to elucidate the impact of each transporter on transport of imatinib. Stable single and double knock-downs of P-gp and BCRP were obtained by RNA interference (RNAi). Transporter expression was measured on RNA and protein level using real-time RT-PCR and Western blot, respectively. Functional activity was quantified by transport of specific substrates across Caco-2 cells. MDR1 and BCRP mRNA expression was reduced to 75% and 90% compared to wild-type control in single MDR1- and BCRP-knock-down clones, respectively. In double knock-down clones, MDR1 expression decreased to 95% and BCRP expression to 80%. Functional activity of P-gp and BCRP was diminished as transport of the P-gp-specific substrate (3)H-digoxin and the BCRP-specific substrate (14)C-PhIP was augmented in the opposite direction, when the respective transporter was knocked down. Similar effects were observed by chemical inhibition of the respective transporter. Bidirectional transport studies with (14)C-imatinib revealed an abrogation of asymmetric transport when P-gp was knocked down, either in single or double knock-down clones compared to wild-type cells. This was not observed in single BCRP-knock-down clones. In conclusion, this newly established cell system with single and concomitant knock-down of P-gp and BCRP can be used to quantify the specific partial impact of the transporters on transport of substrates that are transported by both proteins. For imatinib transport, the contribution of P-gp seems to be more important compared to BCRP in this Caco-2 cell system.
P-糖蛋白（P-gp/MDR1/ABCB1）和乳腺癌耐药蛋白（BCRP/ABCG2）在多种内源性化合物、药物和毒素的转运中发挥重要作用。一些药物的转运，例如酪氨酸激酶抑制剂伊马替尼，受到 P-gp 和 BCRP 的影响。通过 RNA 干扰（RNAi）建立具有特定 P-gp 和 BCRP 敲低以及两种蛋白双重敲低的肠 Caco-2 细胞培养模型，我们旨在阐明每种转运蛋白对伊马替尼转运的影响。通过 RNA 干扰（RNAi）获得 P-gp 和 BCRP 的稳定单和双敲低。使用实时 RT-PCR 和 Western blot 分别在 RNA 和蛋白质水平上测量转运体表达。通过特定底物穿过 Caco-2 细胞的转运来定量功能活性。与野生型对照相比，单 MDR1 和 BCRP 敲低克隆中 MDR1 和 BCRP 的 mRNA 表达分别降低到 75%和 90%。在双敲低克隆中，MDR1 表达降低到 95%，BCRP 表达降低到 80%。当敲低相应的转运体时，P-gp 特异性底物（3）H-地高辛和 BCRP 特异性底物（14）C-PhIP 的转运增加，P-gp 和 BCRP 的功能活性减弱。用相应转运体的化学抑制剂进行的双向转运研究也观察到了类似的效果。用（14）C-伊马替尼进行的双向转运研究表明，与野生型细胞相比，当 P-gp 敲低时，无论是在单或双敲低克隆中，不对称转运都被阻断。在单 BCRP 敲低克隆中未观察到这种情况。总之，这个新建立的具有 P-gp 和 BCRP 单和同时敲低的细胞系统可用于定量测量转运蛋白对由两种蛋白转运的底物的特定部分影响。在这个 Caco-2 细胞系统中，与 BCRP 相比，伊马替尼的转运似乎更依赖于 P-gp。
