Expression, Localization, and Functional Characteristics of Breast Cancer Resistance Protein in Caco-2 Cells

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ABSTRACT:This report describes a newly identified potential of grapefruit juice (GFJ) in mediating pharmacokinetic drug interactions due to its capability to inhibit esterase. The study demonstrates that GFJ inhibits purified porcine esterase activity toward p-nitrophenyl acetate and the prodrugs lovastatin and enalapril. In rat and human hepatic or gut S9 fractions and rat gut lumen, GFJ inhibited the hydrolysis of enalapril and lovastatin, which are known to be metabolized principally by esterases, lovastatin being metabolized also by CYP3A. In Caco-2 cells, with minimal CYP3A activity, permeability of these prodrugs was increased in the presence of GFJ. In rats, oral coadministration of GFJ or an esterase inhibitor, bis-(p-nitrophenylphosphate), with the prodrugs led to respective increases in plasma area under the curve by 70% or 57% for enalaprilat and 279% or 141% for lovastatin acid. In addition, portal vein-cannulated rats pretreated with GFJ at ؊15 and ؊2 h before lovastatin administration (10 mg/kg p.o.) as a solution, 1) in water and 2) in GFJ, showed, respectively, a 49% increase (CYP3A-inhibited) and a 116% increase (both CYP3A and gut esteraseinhibited) in the portal plasma exposure to the active acid, compared with a non-GFJ pretreatment group. Overall, along with the CYP3A inactivation by GFJ, the decreased esterase activity also played a significant role in increasing the metabolic stability and permeability of esters leading to enhancement of exposure to the active drugs in rats. These new esterase inhibition findings indicate that the potential of drug interaction between ester prodrugs and GFJ should also be considered in the clinic.Since the first report of the grapefruit juice (GFJ) effect on the oral bioavailability of felodipine (Bailey et al., 1989(Bailey et al., , 1991, the effect of grapefruit juice ingestion on oral pharmacokinetics has been reported for approximately 40 drugs (Saito et al., 2005), generally related to CYP3A inhibition. These drugs differ in their chemical and pharmacological properties but are, in common, extensively metabolized by CYP3A. The mechanism of action thus was postulated to be competitive and mechanism-based inhibition of CYP3A4/5 (hereafter referred to as CYP3A) in the small intestine by GFJ (Schmiedlin-Ren et al., 1997;He et al., 1998). Although some recent reports point to the inhibitory effects of grapefruit juice on the function of P-glycoprotein (Zhou et al., 2004) and OATP (Dresser et al., 2002), the contribution to the bioavailability of drugs that are substrates of P-glycoprotein and OATP has not been well established. It is interesting to note that the magnitude of GFJ effect varied greatly, and we noted that the magnitude of GFJ effect was not proportional to the extent of CYP3A-mediated intestinal metabolism. As an example, cyclosporine, which is extensively metabolized in human intestine by CYP3A, led to only a weak interaction with GFJ (Ducharme et al., 1995), the interaction unlikely to be of clinical significance. However, lovastatin, which...

Section: Discussionmentioning

confidence: 99%

“…Caco-2 cell cultures were prepared as described (Xia et al, 2005), and monolayers with the transepithelial electrical resistance values greater than 250 ohm ⅐ cm 2 were used. The single-directional transport studies were performed at 37°C in air.…”

Section: Methodsmentioning

confidence: 99%

Esterase Inhibition Attribute of Grapefruit Juice Leading to a New Drug Interaction

Callery²,

Gan³

et al. 2007

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“…Inhibition of ABCG2 activity using specific inhibitors was shown to influence the pharmacokinetic properties of substrates of ABCG2 in mice (Allen et al, 2002) and humans (Kruijtzer et al, 2002;van Herwaarden and Schinkel, 2006). ABCG2 is also present in Caco-2 cells, which are widely used for permeability screening and drug-transporter interaction studies focusing on ABCB1 [P-glycoprotein (P-gp)] (Xia et al, 2005). Altogether, the central modulating effect of the transporter on the pharmacokinetic parameters of certain drugs indicates that it is important to test the interaction of drug candidates with ABCG2 at the early phase of drug development.…”

mentioning

confidence: 99%

ABCG2 (Breast Cancer Resistance Protein/Mitoxantrone Resistance-Associated Protein) ATPase Assay: A Useful Tool to Detect Drug-Transporter Interactions

Glavinas¹,

Kis²,

Ákos³

et al. 2007

ABSTRACT:The ATPase assay using membrane preparations from recombinant baculovirus-infected Spodoptera frugiperda ovarian (Sf9) cells is widely used to detect the interaction of compounds with different ATP-binding cassette transporters. However, Sf9 membrane preparations containing the wild-type ABCG2 transporter show an elevated baseline vanadate-sensitive ATPase activity, which cannot be further stimulated by substrates of ABCG2. Therefore, this assay system cannot be used for the detection of ABCG2 substrates. To overcome this difficulty we 1) purified membranes from a selected human cell line expressing wild-type ABCG2, and 2) inhibited the baseline ATPase activity with different inhibitors. In our modified assay, ABCG2 substrates were able to stimulate the baseline ATPase activity of ABCG2 expressed in membranes of human cells. Furthermore, using the specific ABCG2 inhibitors Ko143 or Ko134 allowed us to suppress the baseline vanadate-sensitive ATPase activity. Substrates of ABCG2 could stimulate this suppressed baseline ATPase, resulting in a better signal-to-background ratio and a robust assay to detect substrates of the ABCG2 transporter. The ATPase assay and the direct vesicular transport measurements for estrone-3-sulfate were in good accordance.

“…In this study, permeability assays were performed as described previously . Both the BCRP (Xia et al, 2005) or PhA (Robey et al, 2004), were used in bidirectional permeability assays to determine BCRP function. On predetermined days postseeding, monolayer integrity was monitored using transepithelial electrical resistance (TEER).…”

Section: Methodsmentioning

confidence: 99%

“…BCRP is a 655-amino acid polypeptide (72 kDa) and contains six putative transmembrane domains and four potential N-glycosylation sites (Priebsch et al, 2006). BCRP is similar to half the duplicated P-gp or multidrug resistance protein 1 (MRP1) molecule and functions as a homodimer bridged by disulfide bonds (Doyle et al, 1998;Kage et al, 2002;Xia et al, 2005) or potentially as a multimer. Like P-gp, BCRP is an efflux transporter that can alter the pharmacokinetics of coadministered drugs or result in toxicity when inhibited.…”

mentioning

confidence: 99%

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

Zhang¹,

Li²,

Allen³

et al. 2009

ABSTRACT:A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based short hairpin RNA (shRNA). Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1), and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp in particular play a significant role in pharmacokinetics because of their expression at several key interfaces. Overexpression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. The shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative polymerase chain reaction (qPCR), Western blotting, immunofluorescence microscopy, and bidirectional transport of probe substrates, estrone-3-sulfate (E3S), and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates.