Kinetics of daunorubicin transport in Ehrlich ascites tumor cells with different expression of P-glycoprotein.

The classical multidrug resistance (MDR) phenotype is characterized by a decrease in the intracellular drug concentration in resistant cells as compared to sensitive cells. P-glycoprotein (P-gp) is thought to be responsible for an active efflux of lipophilic drugs. Four Ehrlich ascites tumor cell lines selected in vivo for resistance to daunorubicin (DNR) and their sensitive counterpart were investigated. The resistant sublines EHR2/0.1, EHR2/0.2, EHR2/0.4, and EHR2/0.8 were developed by treatment of tumor bearing mice with DNR 0.1, 0.2, 0.4, and 0.8 mg/kg x 4 weekly, respectively. One passage from EHR2/0.1, EHR2/0.2, and EHR2/0.4 and two passages from EHR2/0.8 were investigated. Western blot analysis showed significantly different amounts of P-gp (a 6-fold variation). Efflux of DNR in a drug free medium was investigated with and without presence of verapamil (VER). Efflux from sensitive and resistant cells was described by mono- and bi-exponential kinetics, respectively. In all cases but one, a correlation between resistance, expression of P-gp, P-gp mediated efflux capacity and effect of VER was established. In passage No. 12 of EHR2/0.8, however, a high expression of P-gp was found in spite of a low degree of resistance and a low efflux capacity. In this subline the effect of VER did not correlate to the expression of P-gp. Active efflux seemed to be saturable and was suggested to constitute the major route of efflux in MDR cells. A dose-response relationship was established for the effect of VER on efflux. In conclusion, the results support that P-gp acts as a drug efflux pump. No simple correlation, however, could be established between P-gp and drug transport in all the investigated cell lines. Other factors which might influence transmembranous transportation of DNR are suggested. The active efflux capacity of the cell lines seemed to determine the degree of resistance and the sensitivity to circumvention by VER.