The Uptake and Efflux of Doxorubicin by a Sensitive Human Bladder Cancer Cell Line and Its DoxorubicinResistant Subline

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 microM to 25 microM 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/10(6)/min and Km (rate at Vmax/2) of 25.2 +/- 4.7 and 16.4 +/- 2.9 microM for UM-UC-6 and UM-UC-6Dox, respectively. There was no metabolism of Dox by keto-reduction or reductive hydrolysis. At 1.0 microM 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 microM), trifluoperazine (10 microM) 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.

Establishment and characterization of a bladder cancer cell line with enhanced doxorubicin resistance by mevalonate pathway activation

“…As measured by a specific reporter gene system, the resistant daughter cell line showed a higher response to intracellular doxorubicin, excluding drug efflux as the major resistance mechanism. This is unexpected, as this was a dominant mechanism in previously established cell lines [10][11][12][13][14][15]. In accordance with other reports, however, the resistance was associated with a diminished apoptotic response to doxorubicin.…”

“…Michel (Department of Urology, Mannheim Medical Center, Mannheim, Germany). The cells were cultured in high-glucose (4.5 g/l) Dulbecco'sm o d i f i e dE a g l e 'sm e d i u m (DMEM) (Gibco-Invitrogen, Mannheim, Germany) supplemented with 10 % foetal bovine serum (FBS) (Biochrom AG, Berlin, Germany), penicillin (100 U/ml) und streptomycin (100 μg/ml, Biochrom AG) at 37°C with 5 % CO 2 .T h e clonally related cell lines UM-UC-6 and UM-UC-6dox [10,11] were a generous gift from Dr. H.B. Grossman (MD Anderson Cancer Center, Houston, USA) and were cultured identically except that minimal essential medium (MEM) (Gibco-Invitrogen) was used instead of DMEM.…”

“…In two previously published doxorubicin-resistant cell lines, RT112/RT112-D21 [12] and UM-UC6/UM-UC-6dox [10,11], the mechanism underlying resistance was identified as classical multidrug resistance by overexpressed ABC efflux transporters. To investigate whether the same mechanism was responsible in the BFTC-905-DOXO-II daughter cell line, we employed the previously described doxorubicin-responsive reporter plasmid pE9ns 2 GFP, which harbours the GFP reporter gene under the control of a synthetic promoter that is strongly activated by intracellular doxorubicin [24,25].…”

“…Their molecular analysis yielded an enhanced drug efflux as a major chemoresistance mechanism, either alone [10][11][12] or in association with a partial or complete loss of the therapeutic target, DNA topoisomerase II [13,14], or with a concomitant activation of antiapoptotic signalling pathways [15]. The analysis of clinical tumours, while corroborating the clinical significance of increased expression of drug efflux pumps like ABCB1 (MDR-1) or ABCC1 (MRP), suggested nevertheless that this is unlikely to represent the only resistance mechanism [16][17][18][19].…”

MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells

“…However, other mechanisms of drug resistance also occurs such as those involving the DNA topoisomerase II gene, MDR-associated protein expression, and glutathione (Naito et al, 1995;Kim et al, 1995;Ahn et al, 1994). We previously established and characterized the doxorubicin-resistant subline UM-UC-6dox (Shinohara et al, 1993;Usansky et al, 1991). UM-UC-6dox is 6.7-fold more resistant to doxorubicin than its parental human bladder cancer cell line UM-UC-6, as calculated by the IC 50 ratio, and exhibits an atypical drug resistance pattern.…”