EVects of cyclosporin at various concentrations on dexamethasone intracellular uptake in multidrug resistant cells

Background—The multidrug resistance phenomenon results from the expression of P-glycoprotein (P-gp), a drug-eZux pump. Corticosteroids are substrates for P-gp, whose function can be inhibited by cyclosporin. This study evaluates the ability of cyclosporin to modulate dexamethasone uptake in multidrug resistant cells. Methods—The K 562 cell line, which does not express P-gp and a P-gp expressing clone, K562/ADM, were used. Cells were incubated with H3-dexamethasone in the absence or presence of cyclosporin at various concentrations. Then, cells were washed, lysed, and radioactivity was measured. Results—The uptake of dexamethasone alone was higher in sensitive than in resistant cells. Addition of cyclosporin induced a dose dependent increase of dexamethasone uptake in resistant cells, whereas the drug did not influence dexamethasone uptake in parental cells. Conclusion—Cyclosporin, at therapeutic concentrations induces a moderate, but significant increase in dexamethasone accumulation in multidrug resistant cells. Thus, cyclosporin might increase the intestinal absorption of corticosteroids or their accumulation in mononuclear cells, or both, thereby increasing their therapeutic eYcacy. (Ann Rheum Dis 2000;59:146–148) Primary or acquired resistance of cancer to cytotoxic drugs is part of a larger phenomenon described as multidrug resistance (MDR). 2 MDR results from the expression of a 170 kDa membrane glycoprotein (P-gp), which acts as a drug-eZux pump. 2 P-gp or its encoding gene (mdr 1) has been detected in many human cancers, in either untreated or previously treated tumours. Reversal of drug resistance has been obtained by diVerent agents, which usually act as competitive inhibitors. These so called resistance modifying agents include verapamil, quinine derived compounds and cyclosporin. Several observations suggest that some corticosteroids could be physiological substrates for P-gp. These compounds have been proposed to interfere with the MDR system by either modulating mdr 1 gene expression or acting as competitive inhibitors for P-gp drug transport function. The potential interactions between corticosteroids and the MDR system raise interesting questions, including its potential role in some resistances to corticosteroids. The observation that corticosteroids are substrates for P-gp, and that cyclosporin inhibits P-gp function suggest that cyclosporin might diminish the cellular eZux and increase the intracellular accumulation of corticosteroids in patients treated with both drugs, such as some rheumatoid arthritis (RA) patients. By the use of high concentrations of cyclosporin, we have previously shown that cyclosporin increases dexamethasone intracellular accumulation in cell lines expressing P-gp. Others have shown that cyclosporin and other immunosuppressants potentiate a dexamethasone mediated transcriptional response in LMCAT cells by inhibiting eZux of this corticosteroid. Such inhibition might or might not be mediated through P-gp blockade. However, the concentrations of cyclosporin used were much higher than clinically relevant concentrations. This study evaluates the eVects of clinically relevant concentrations of cyclosporin on dexamethasone intracellular uptake in multidrug resistant cells. Methods We used a human leukaemic resistant cell line (K562/ADM) derived from the parental K562 cell line by continuous exposure to increasing concentrations of doxorubicin. These clones were kindly provided by Dr Tsuoro (Cancer Chemotherapy Center, Tokyo, Japan). The MDR phenotype is not expressed by K562 but is overexpressed in K562/ADM. The expression of P-gp protein in K562 and K562/ADM cells was checked by cytofluorimetry using MRK16, a specific mouse monoclonal antibody (Immunotech, Marseille, France). Cells were grown in suspension in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 2 mM L-glutamine and 10 % fetal bovine serum at 37°C in a humified atmosphere (5% carbon dioxide and 95% air). K 562 and K562/ADM cells were seeded in 96 well flat bottom microtitre plates (2.10 cells per well) and were incubated for four hours at 37°C in culture medium with 50 μg/ml dexamethasone (99.92% dexamethasone and 0.08% 1, 2, 4,6, 7-(H)-dexamethasone, Amersham; Les Ulis; France), in the absence or in the presence of cyclosporin A at various concentrations (0.05, 0.15, 0.6, 1 and 10 μmol/l). Each determination was made in triplicate. The dexamethasone concentration was high, at least with respect to pharmacological serum concentrations. We used such a concentration Ann Rheum Dis 2000;59:146–148 146 Department of Rheumatology, Dijon University Hospital, France