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Doxorubicin cellular pharmacokinetics and DNA breakage in a multi-drug resistant B16 melanoma cell line
R Supino1, M Mariani, G Capranico
1Division of Experimental Oncology B, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan.
Abstract:
Mechanisms of anthracycline resistance have been investigated in a B16 murine melanoma cell subline selected by continuous in vitro exposure to increasing concentrations of doxorubicin (DX). Altered drug pharmacokinetics were observed in resistant B16 cells as compared to the sensitive counterpart. In fact, cellular DX uptake - as determined by a fluorescence method - was lower in resistant than in sensitive cells. Furthermore, drug efflux rate was shown to be higher in resistant than in sensitive cells; treatment of cells with the metabolic inhibitor sodium azide decreased drug efflux rate in resistant but not in sensitive cells, suggesting the presence of an energy-dependent drug extrusion mechanism in the resistant B16 cells. However, since drug-induced cell killing did not correlate with cellular DX contents in sensitive and resistant cells, drug resistance of B16 subline could not be completely explained by the observed differences in drug pharmacokinetics. Since drug-induced DNA breaks have been related to drug cytotoxicity, DNA cleavage was also measured by alkaline elution methods. The number of DNA breaks produced by DX was decreased in resistant cells as compared to sensitive cells at the same cellular drug accumulation. The results are consistent with the view that anthracycline resistance may be multifactorial and probably arises following multiple biochemical changes.
Insights
This study investigated doxorubicin (DX) resistance in melanoma cells. Resistant cells showed altered drug uptake and efflux, but resistance was multifactorial, involving reduced DNA damage.
Area of Science:
- Biochemistry
- Pharmacology
- Cancer Biology
Background:
- Anthracyclines, like doxorubicin (DX), are vital chemotherapy agents.
- Understanding drug resistance mechanisms is crucial for improving cancer treatment efficacy.
- Melanoma cell lines provide a model for studying drug resistance.
Purpose of the Study:
- To investigate the mechanisms of anthracycline resistance in a B16 murine melanoma cell subline selected for doxorubicin (DX) resistance.
- To elucidate the roles of altered drug pharmacokinetics and DNA damage in conferring DX resistance.
Main Methods:
- Cellular doxorubicin (DX) uptake and efflux were measured using fluorescence assays.
- The effect of metabolic inhibitors (sodium azide) on drug efflux was assessed.
- DNA damage induced by DX was quantified using alkaline elution techniques.
Main Results:
- Resistant B16 melanoma cells exhibited lower DX uptake and higher DX efflux compared to sensitive cells.
- An energy-dependent drug extrusion mechanism was suggested in resistant cells.
- DX-induced DNA breaks were reduced in resistant cells, even at equivalent drug accumulation levels.
- Drug resistance could not be fully explained by pharmacokinetic alterations alone.
Conclusions:
- Anthracycline resistance in B16 melanoma cells is multifactorial.
- Altered drug pharmacokinetics (uptake and efflux) contribute to resistance.
- Reduced DNA damage in resistant cells is a significant factor in anthracycline resistance.
- Multiple biochemical changes likely underlie the development of drug resistance.