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Reversal agent inhibition of the multidrug resistance pump in human leukemic lymphoblasts
1Department of Medical Biology, University of Tennessee Medical Center, Knoxville 37920.
Abstract:
Multidrug resistant cancer cells of the MDR-1 phenotype utilize an ATP-dependent pump to excrete toxic drugs. Rhodamine 123 (R123) is a fluorescent substrate of the MDR pump. An assay for the ATP-dependent initial efflux of R123 from CEM/VLB100 human leukemic lymphoblasts has been developed. The MDR-1 cells were treated with a reversal agent and preloaded with 40.0 nM R123 in buffer at 30 degrees C that contained sodium azide and 2-deoxyglucose. The cells were rinsed with cold buffer and resuspended in L-glutamine/glucose solution at 23 degrees C. The cell suspension was passed through a filter and R123 in the filtrate was detected at 2-s intervals by fluorescence. Efflux of R123 was inhibited by the reversal agents amiodarone, cyclosporin A, Ro11-2933 (DMDP), quinidine, and the optical isomers of propranolol. The MDR pump is stereospecific for the (R)-diastereomer quinidine; however, the (S)-diastereomer quinine is a relatively weak inhibitor of the pump. Cyclosporin A was the most potent inhibitor tested against the efflux of R123 by the MDR pump.
Insights
Multidrug resistant cancer cells use an ATP-dependent pump to expel drugs. This study developed an assay to measure drug efflux, finding Cyclosporin A most effectively inhibited this process in MDR-1 cells.
Area of Science:
- Biochemistry
- Pharmacology
- Cell Biology
Background:
- Multidrug resistant (MDR) cancer cells possess ATP-dependent pumps that facilitate drug extrusion.
- Rhodamine 123 (R123) serves as a fluorescent substrate for these MDR pumps, enabling their study.
Purpose of the Study:
- To develop and validate an assay for measuring the ATP-dependent efflux of R123 from MDR-1 phenotype cancer cells.
- To evaluate the inhibitory effects of various reversal agents on R123 efflux mediated by the MDR pump.
Main Methods:
- CEM/VLB100 human leukemic lymphoblasts were preloaded with R123.
- Cells were treated with reversal agents and incubated under specific temperature and buffer conditions.
- R123 efflux was measured by detecting fluorescence in filtrate over time using a developed assay.
Main Results:
- The assay successfully measured ATP-dependent R123 efflux from MDR-1 cells.
- Several agents, including amiodarone, cyclosporin A, DMDP, quinidine, and propranolol isomers, inhibited R123 efflux.
- Cyclosporin A demonstrated the highest potency in inhibiting R123 efflux, and the MDR pump showed stereospecificity for quinidine.
Conclusions:
- A functional assay for MDR-mediated drug efflux has been established.
- Various compounds can inhibit MDR pump activity, with Cyclosporin A being a potent inhibitor.
- The MDR pump exhibits stereospecificity, highlighting potential for targeted therapeutic strategies.