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Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line
L A Hazlehurst1, N E Foley, M C Gleason-Guzman
1Department of Biochemistry, Pharmacology, and Internal Medicine, H. Lee Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612, USA.
Abstract:
Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.
Insights
A novel drug efflux pump causes low-level mitoxantrone resistance in myeloma cells. This resistance, reversed by fumitremorgin C, involves reduced intracellular drug accumulation and altered distribution.
Area of Science:
- Pharmacology
- Molecular Biology
- Cancer Research
Background:
- Acquired drug resistance in cancer is a complex phenomenon.
- Multiple resistance mechanisms can emerge concurrently or sequentially.
- Understanding these mechanisms is crucial for effective cancer therapy.
Purpose of the Study:
- To investigate the mechanisms of mitoxantrone resistance in human myeloma cells.
- To characterize a novel drug transport mechanism that emerges during selection.
- To evaluate the potential of fumitremorgin C in overcoming drug resistance.
Main Methods:
- Selection of human myeloma cell line 8226 with mitoxantrone.
- Assessment of drug resistance levels and intracellular drug concentrations.
- Confocal microscopy to determine drug distribution.
- Evaluation of chemosensitizing agent fumitremorgin C.
- Analysis of topoisomerase II alpha and beta expression.
Main Results:
- A novel, ATP-dependent efflux pump conferred 10-fold mitoxantrone resistance (8226/MR4 cell line).
- This cell line also showed cross-resistance to BBR 3390 with altered drug distribution.
- Fumitremorgin C reversed resistance in the novel transporter line but not in P-glycoprotein-positive cells.
- Higher mitoxantrone resistance (8226/MR20) correlated with reduced topoisomerase II expression, not further efflux reduction.
Conclusions:
- Low-level mitoxantrone resistance is mediated by a novel drug efflux pump.
- This transporter is distinct from P-glycoprotein and multidrug resistance-associated protein.
- Fumitremorgin C can be used to study this novel transporter and potentially overcome resistance.
- Increased resistance involves reduced drug accumulation, altered distribution, and topoisomerase II activity changes.