Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA

Zhang, Wei; Li, Jibin; Allen, Samantha; Weiskircher, Erica A.; Huang, Yuehua; George, Rebecca A.; Fong, Ramon G.; Owen, Albert; Hidalgo, Ismael J.

doi:10.1124/dmd.108.023309

Cited by 22 publications

(14 citation statements)

References 25 publications

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“…One approach that could furnish unambiguous identification of transporter involvement is the use of RNA interference to knock down the expression of relevant transporters. This technique has already been used to selectively knock down the expression of BCRP, P-gp, and multidrug resistance-associated protein 2 in Caco-2 cells (Zhang et al, 2009), which proved valuable in elucidating the role of efflux transporters in the biliary efflux of ximelagatran (Darnell et al, 2010) and several statin drugs (Li et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

The Role of a Basolateral Transporter in Rosuvastatin Transport and Its Interplay with Apical Breast Cancer Resistance Protein in Polarized Cell Monolayer Systems

Li¹,

Wang²,

Zhang³

et al. 2012

Drug Metab Dispos

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ABSTRACT:Membrane transporters can play a clinically important role in drug absorption and disposition; Caco-2 and Madin-Darby canine kidney (MDCK) cells are the most widely used in vitro models for studying the functions of these transporters and associated drug interactions. Transport studies using these cell models are mostly focused on apical transporters, whereas basolateral drug transport processes are largely ignored. However, for some hydrophilic drugs, a basolateral uptake transporter may be required for drugs to enter cells before they can interact with apical efflux transporters. The objective of this study was to evaluate potential differences in drug transport across Caco-2 and MDCK basolateral membrane that could cause discrepancy in the identification of efflux transporter substrates and to elucidate the underlying factors that may cause such differences, using rosuvastatin as a model substrate. Bidirectional transport results in Caco-2 and breast cancer resistance protein-MDCK cells demonstrated the necessity of an uptake transporter at the basolateral membrane for rosuvastatin. Kinetic study revealed saturable and nonsaturable processes for rosuvastatin uptake across the Caco-2 basolateral membrane, with the saturable process encompassing >75% of overall rosuvastatin basolateral uptake at concentrations below the K m (4.2 M). Furthermore, rosuvastatin basolateral transport exhibited cis-inhibition and trans-stimulation phenomena, indicating a facilitated diffusion mechanism. This basolateral transporter appeared to be a prerequisite for rosuvastatin and perhaps for other hydrophilic substrates to interact with apical efflux transporters. Deficit of such a basolateral transporter in certain cell models may lead to false-negative results when screening drug interactions with apical efflux transporters.

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Section: Discussionmentioning

confidence: 99%

The Role of a Basolateral Transporter in Rosuvastatin Transport and Its Interplay with Apical Breast Cancer Resistance Protein in Polarized Cell Monolayer Systems

Li¹,

Wang²,

Zhang³

et al. 2012

Drug Metab Dispos

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“…The use of the post-transcriptional gene silencing by RNA interference to specifically knock down drug transporters is a rapidly growing field of research (Celius et al, 2004;Hua et al, 2005;Tian et al, 2005;Watanabe et al, 2005;Li et al, 2006;Yue et al, 2009;Zhang et al, 2009). Even though siRNA and shRNA have emerged as powerful tools to study gene function in a sequence-specific manner, off-target effects have been observed in several studies (Jackson et al,FIG.…”

Section: Discussionmentioning

confidence: 99%

“…Lentivirus plasmid vectors containing shRNA inserts targeting human P-gp (GenBank accession number NM_000927) and MRP2 (GenBank accession number NM_000392) genes were obtained from Sigma-Aldrich. The transduction procedure was described previously (Zhang et al, 2009). In brief, the cells were plated in 96-well tissue culture plates at a density of 5 ϫ 10 6 cell/well and transduced with 1 g of P-gp-or MRP2-targeting shRNA viral vector.…”

Section: Methodsmentioning

confidence: 99%

Investigation of the Involvement of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 in the Efflux of Ximelagatran and Its Metabolites by Using Short Hairpin RNA Knockdown in Caco-2 Cells

Darnell

Karlsson²,

Owen³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Liver and bile secretion can be an important first-pass and clearance route for drug compounds and also the site of several drug-drug interactions. In the clinical program for ximelagatran development, an unexpected effect of erythromycin on the pharmacokinetics of the direct thrombin inhibitor ximelagatran and its metabolites was detected. This interaction was believed to be mediated by inhibition of drug transporters, which normally extrude the drug into the bile. Previous Caco-2 cell experiments indicated the involvement of an active efflux mechanism for ximelagatran, hydroxy-melagatran, and melagatran possibly mediated by P-glycoprotein (P-gp). However, the inhibitors used may not have been specific enough and the possibility that transporters other than P-gp were important in the Caco-2 cell assay cannot be excluded. In this study we used RNA interference, a post-transcriptional gene silencing mechanism in which mRNA is degraded in a sequence-specific manner, to specifically knock down P-gp or multidrug resistance-associated protein 2 (MRP2) transporters in Caco-2 cells. The data obtained from bidirectional transport studies in these cells indicate a clear involvement of P-gp but not of MRP2 in the transport of ximelagatran, hydroxy-melagatran, and melagatran across the apical cell membrane. The present study shows that short hairpin RNA Caco-2 cells are a valuable tool to investigate the contribution of specific transporters in the transcellular transport of drug molecules and to predict potential sites of pharmacokinetic interactions. The results also suggest that inhibition of hepatic P-gp is involved in the erythromycin-ximelagatran interaction seen in clinical studies.The direct thrombin inhibitor ximelagatran was envisaged to have a low potential for pharmacokinetic drug-drug interactions (DDIs) based on the preclinical information showing that ximelagatran, its intermediate metabolites, and melagatran are neither substrates nor inhibitors of the major cytochrome P450 isoenzymes (Bredberg et al., 2003). The preclinical results were supported by the results from three specific healthy volunteer studies showing no clinically significant DDIs between ximelagatran and diclofenac, diazepam, or nifedipine when coadministered simultaneously (Bredberg et al., 2003). The phase I DDI study showing a clear interaction with erythromycin, therefore, came as a surprise. Melagatran area under the curve increased by 82%, whereas ximelagatran plasma levels seemed to be essentially unchanged. Ximelagatran is bioconverted to melagatran via two intermediates, hydroxy-melagatran and ethyl-melagatran (Fig. 1), and an elevation in the plasma concentrations was seen for both intermediates, after coadministration with erythromycin (Eriksson et al., 2006).Several in vivo and in vitro studies have been performed to investigate the nature of the erythromycin-ximelagatran interaction (Eriksson et al., 2006; Sjö din et al., 2008). The pharmacokinetic profile in humans indicated that the elevation of ximelagatran and its m...

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“…However, there still remain some arguments that the inhibitors may affect the function of other proteins. Vector -based RNAi was used to establish a stable Caco -2 cell line with a persistent knockdown of effl ux transporters (Celius et al, 2004 ;Watanabe et al, 2005 ;Zhang et al, 2009, Darnell et al, 2010Graber -Maier et al, 2010 ). The effective sites of RNAi were selected using siRNA libraries and single siRNAs, and MDR1 stable knockdown Caco -2 cells were constructed using a tRNAval -shRNA expression vector (Watanabe et al, 2005 ).…”

Section: Applications In Identifying New Transportersmentioning

confidence: 99%

Applications of iRNA Technologies in Drug Transporters and Drug Metabolizing Enzymes

Liao

Xia

2012

ADME‐Enabling Technologies in Drug Design and Development

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Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA

Cited by 22 publications

References 25 publications

The Role of a Basolateral Transporter in Rosuvastatin Transport and Its Interplay with Apical Breast Cancer Resistance Protein in Polarized Cell Monolayer Systems

The Role of a Basolateral Transporter in Rosuvastatin Transport and Its Interplay with Apical Breast Cancer Resistance Protein in Polarized Cell Monolayer Systems

Investigation of the Involvement of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 in the Efflux of Ximelagatran and Its Metabolites by Using Short Hairpin RNA Knockdown in Caco-2 Cells

Applications of iRNA Technologies in Drug Transporters and Drug Metabolizing Enzymes

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