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Idarubicin DNA intercalation is reduced by MRP1 and not Pgp
M E Smeets1, R A Raymakers, G Vierwinden
1Division of Hematology and Central Hematology Laboratory, University Hospital Nijmegen, The Netherlands.
Abstract:
Currently available data regarding the substrate specificity of the multi-drug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-associated protein (MRP1) for idarubicin are inconclusive. A multiparameter flow cytometry method was developed which allows simultaneous quantitative measurement of total cellular fluorescence and the amount of anthracyclines intercalated into the DNA. Anthracycline DNA intercalation was measured by fluorescence resonance energy transfer (FRET) between Hoechst 33342 and anthracyclines. Daunorubicin and idarubicin accumulation were studied and compared in established cell lines expressing Pgp and MRP1. The data demonstrate that daunorubicin DNA intercalation is affected by both Pgp and MRP1 whereas idarubicin DNA intercalation is affected only by MRP1. MRP1 and Pgp function could be blocked completely by 5 microM PAK 104P, while higher concentrations of verapamil, PSC 833 and cyclosporin A were necessary to attain complete blocking of MRP1 compared to Pgp. Daunorubicin DNA intercalation correlates better with cell survival and is more sensitive at physiological MDR expression as observed in hematopoietic progenitors than daunorubicin levels measured by total cellular fluorescence. In conclusion, idarubicin DNA intercalation is reduced by MRP1 but not by Pgp. PAK-104P is an effective modulator for both Pgp and MRP1 and may further improve idarubicin efficacy.
Insights
Multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 affect daunorubicin DNA intercalation, but only MRP1 impacts idarubicin. PAK-104P effectively modulates both Pgp and MRP1, potentially enhancing idarubicin efficacy.
Area of Science:
- Pharmacology
- Molecular Biology
- Cancer Research
Background:
- Multidrug resistance (MDR) mechanisms, including P-glycoprotein (Pgp) and MDR-associated protein 1 (MRP1), reduce cancer chemotherapy effectiveness.
- Substrate specificity of Pgp and MRP1 for the anthracycline idarubicin remains unclear, impacting treatment strategies.
Purpose of the Study:
- To investigate and compare the effects of Pgp and MRP1 on the DNA intercalation of daunorubicin and idarubicin.
- To evaluate the efficacy of modulators in blocking Pgp and MRP1 activity and their impact on anthracycline DNA intercalation.
Main Methods:
- Development of a multiparameter flow cytometry assay to simultaneously measure total cellular fluorescence and DNA-intercalated anthracyclines.
- Utilized fluorescence resonance energy transfer (FRET) for quantitative measurement of anthracycline DNA intercalation.
- Compared daunorubicin and idarubicin accumulation and DNA intercalation in cell lines expressing Pgp and/or MRP1, with and without modulators (PAK 104P, verapamil, PSC 833, cyclosporin A).
Main Results:
- Daunorubicin DNA intercalation was significantly affected by both Pgp and MRP1.
- Idarubicin DNA intercalation was reduced by MRP1 but not by Pgp.
- PAK-104P completely blocked both Pgp and MRP1 at 5 μM, while other modulators required higher concentrations.
- Daunorubicin DNA intercalation correlated better with cell survival than total cellular fluorescence, especially in hematopoietic progenitors.
Conclusions:
- Idarubicin's DNA intercalation is selectively reduced by MRP1, not Pgp.
- PAK-104P demonstrates potent dual inhibition of Pgp and MRP1, suggesting its potential to improve idarubicin's clinical efficacy.
- The developed FRET-based flow cytometry method provides a sensitive tool for assessing anthracycline DNA intercalation and MDR modulation.