Abstract
The uptake and efflux of doxorubicin (Dox) were investigated in a human bladder cancer cell line (UM-UC-6) and in a multi-drug resistant (mdr) subline (UM-UC-6Dox). Unlike previous reports, the initial uptake kinetics of Dox, and its accumulation and retention to steady-state were modelled mathematically. Cells were incubated with Dox and the amount of Dox in the cellular and medium phases was measured by a specific HPLC method. When monitored for 1 min from 0.02 μM to 25 μM Dox, the uptake was very rapid but was significantly faster in the resistant cell line. The initial rate of uptake at t=0 followed Michaelis-Menten kinetics yielding Vmax values (the maximal rate of uptake) of 15.0 ± 1.7 and 12.9 ± 1.2 nmol/106/min and Km (rate at Vmax/2) of 25.2 ± 4.7 and 16.4 ± 2.9 μM for UM-UC-6 and UM-UC-6Dox, respectively. There was no metabolism of Dox by keto-reduction or reductive hydrolysis. At 1.0 μM the uptake of Dox to steady-state was biexponential but there was no difference in total cellular Dox concentration between the two cell lines at equilibrium. A 3 compartment sequential closed model was fitted yielding significantly different values for the intercompartmental and hybrid rate constants, indicating altered intracellular distribution in resistant cells. Verapamil (10 μM), trifluoperazine (10 μM) or Tween 80 (0.005%) had no effect on the uptake or efflux of Dox. The UM-UC-6Dox line appeared to show atypical mdr characteristics since net drug accumulation was not lowered and classic P-glycoprotein inhibitors were not effective. The primary mechanism of Dox resistance is not enhanced metabolism or lowered intracellular concentrations.
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