Influx of daunorubicin in multidrug resistant Ehrlich ascites tumour cells: correlation to expression of P-glycoprotein and efflux. Influence of verapamil

Abstract

Classic multidrug resistance is characterized by a decrease in the intracellular concentration of drugs in resistant cells as compared to sensitive cells. This is correlated with the presence of P-glycoprotein in the membrane. P-glycoprotein is responsible for an active efflux of drug. In this study we investigated the correlation between P-glycoprotein and influx of daunorubicin. Four Ehrlich ascites tumour cell lines selected in vivo for resistance to daunorubicin were investigated. The sublines EHR2/0.1, EHR2/0.2, passage no. 12 of EHR2/0.8, EHR2/0.4, and passage no. 72 of EHR2/0.8 were 6-, 6-, 5-, 33-, and 35-fold resistant to daunorubicin, respectively. All sublines overexpressed P-glycoprotein as determined with Western blot. Influx was measured over 40 sec. In glucose-enriched medium influx was significantly decreased in all but one of the resistant sublines. A correlation between P-glycoprotein, degrees of resistance, and influx was demonstrated in four sublines. Comparing influx experiments with efflux experiments (Nielsen et al., Biochem Pharmacol 1994, 47, 2125-2135) we found a linear relationship between influx and efflux in the resistant sublines (r = 0.97). Verapamil (5.5 microM, 11.0 microM) increased influx significantly in all resistant sublines, whereas the drug had no effect on sensitive cells. Verapamil (3.3 microM) increased influx in the EHR2/0.8 (passage no. 72) subline to the level of sensitive cells. Comparing this result with efflux experiments, verapamil was found to increase influx preferentially. Depletion of energy (medium without glucose including Na(+)-azide) increased influx in all resistant sublines. In EHR2/0.4 and EHR2/0.8 (passage no. 72) the influx, however, was still significantly decreased after depletion of energy. In these cells further addition of verapamil increased influx to the level of EHR2. These data were consistent with the hypothesis that P-glycoprotein effluxes drug directly from the plasma membrane.