Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines

Abstract

The LLC-PK1:MDR1, LLC-PK1 and Caco-2 cell lines were used to investigate whether rhodamine-123 or doxorubicin would be the preferred substrate to study P-glycoprotein (P-gp) functionality in vitro. Both rhodamine-123 and doxorubicin showed highly polarised transport in the Caco-2 cell line and the LLC-PK1:MDR1 cell line, indicating that P-gp is actively transporting these drugs. However, for rhodamine-123 polarised transport was also seen in the monolayers of the wild-type LLC-PK1 cell line, indicating the presence of another active transporter for this compound. Polarised transport of doxorubicin in the Caco-2 and the LLC-PK1:MDR1 cell lines could be inhibited by the P-gp inhibitors SDZ-PSC 833 (PSC 833), cyclosporin A (CsA), verapamil and quinine, but not by the inhibitors for the organic cation carrier systems cimetidine and tetraethylammonium (TEA). Polarised transport of rhodamine-123 in the Caco-2 cell line could only be inhibited by P-gp inhibitors. In the LLC-PK1:MDR1 and LLC-PK1 cell lines transport was also inhibited by inhibitors for the organic cation transport systems. In conclusion, rhodamine-123 is a substrate for both P-gp and the organic cation carrier systems in the kidney cell line. This indicates that rhodamine-123 is not selective enough to study P-gp functionality in cell systems were organic cation carrier systems are also present. Doxorubicin appears to be a more selective P-gp substrate and therefore more useful in studying P-gp functionality in vitro.

Introduction

P-glycoprotein (P-gp) is an energy-dependent drug efflux pump and belongs to the family of ATP binding cassette transport proteins (Higgins, 1992). It is highly expressed on different types of tumour cells and on the apical surfaces of several epithelial (e.g., intestine) and endothelial cells (e.g., human brain capillary blood vessels that represent the blood–brain barrier) (Cordon-Cardo et al., 1990). P-gp transports different classes of substrates, like chemotherapeutic drugs (e.g., anthracyclines, taxanes and vinca alkaloids), steroid hormones, HIV protease inhibitors and fluorescent dyes (Chaudhary & Roninson, 1991, Ueda et al., 1992, Kim et al., 1998). Although these substrates differ considerably in chemical structures, the general feature of P-gp substrates is their relatively hydrophobic, amphiphilic nature (Gottesman and Pastan, 1993). Furthermore, Seelig (1998) argued that P-gp recognises substrates by recognition elements formed by two (type I unit) or three electron donor groups (type II unit) with a fixed spatial separation. Another phenomenon which may explain the variety of P-gp substrates is that two or even three substrate binding sites on P-gp were found with different substrate affinities (Shapiro et al., 1999).

Two model compounds, rhodamine-123 and doxorubicin, are frequently used to study P-gp activity. Doxorubicin is a chemotherapeutic agent that is not only transported by P-gp, but also induces its expression in tumour cells (Beck et al., 1988, Safa et al., 1994, Zhang et al., 1995).

Rhodamine-123 is a fluorescent dye and has often been used to study the functionality of P-gp (Nare et al., 1994). In vivo, microdialysis studies in a P-gp knockout mice model (Schinkel et al., 1995a, Schinkel et al., 1995b) showed an increased uptake of rhodamine-123 into the brain in mdr1a (−/−) mice, which lack P-gp at the level of the blood–brain barrier, while in mdr1a (+/+) mice the uptake was a factor 3–4 times lower (De Lange et al., 1998). However, although rhodamine-123 is often used to study the functionality of P-gp, it seems not to be a specific P-gp substrate. Masereeuw et al. (1997) showed that in the isolated perfused rat kidney rhodamine-123 was preferentially secreted by a transporter of the organic cation carrier systems and not by P-gp. Furthermore, the CHO-K1 cell line, which does not display reactivity with the monoclonal antibody C219 raised against P-gp, shows an active rhodamine-123 efflux, which cannot be related to P-gp (Pétriz et al., 1997). In addition, it has been suggested that in adriamycin resistant human leukaemia K562 cells rhodamine-123 is not solely excreted by P-gp (Denis-Gay et al., 1995).

This could mean, that the role of other transporter(s) in the efflux from cells of rhodamine-123 is (are) underestimated and that rhodamine-123 is not a good substrate to investigate P-gp functionality. Therefore, it was of interest to investigate whether rhodamine-123 or doxorubicin would be the preferred substrate to study P-gp functionality in vitro. For this study a normal LLC-PK1 cell line and a LLC-PK1 cell line transfected with the human MDR1 gene (encoding for P-gp) was used (Schinkel et al., 1995a, Schinkel et al., 1995b). In addition, a Caco-2 cell line was used, which exhibits an age-dependent expression of P-gp (Hosoya et al., 1996). Rhodamine-123 and doxorubicin were used as substrates to study drug transport in all three cell lines. Furthermore, PSC 833 and CsA were used as inhibitors of P-gp and verapamil and quinine were used as non-selective inhibitors for both P-gp and the organic cation carrier systems, while cimetidine and TEA were used as inhibitors of the organic cation carrier systems.