Purpose
This study investigates the subcellular pharmacokinetics of drug efflux in cancer cells and explores the role of the multivesicular body (MVB) in facilitating efflux of doxorubicin, a widely used DNA-targeting anticancer agent, from the nucleus.
Methods
Human erythroleukemic K562 cells were pulsed with doxorubicin and then chased in drug-free media to allow for efflux. Microscopy and biochemical techniques were used to visualize the subcellular localization of the drug and measure drug content and distribution during the efflux period. To explore the role of the MVB in doxorubicin efflux, K562 cells were transfected with dominant negative mutant forms of VPS4a–GFP chimeras.
Results
Although the intracellular concentration of drug exceeds the extracellular concentration, nuclear efflux of doxorubicin occurs in living cells at a faster rate than doxorubicin unbinding from isolated nuclei into drug-free buffer. In cells expressing dominant negative VPS4a, doxorubicin accumulates in VPS4a-positive vesicles and drug sequestration is inhibited, directly implicating the MVB pathway in the egress route of doxorubicin in this cell type.
Conclusions
Cellular membranes are a component of the doxorubicin efflux mechanism in K562 cells. Dominant-negative GFP chimeric mutants can be used to elucidate the role of specific membrane trafficking pathways in subcellular drug transport routes.
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Abbreviations
- MDR:
-
multidrug resistance
- MVB:
-
multivesicular body
- VPS4a:
-
vacuolar protein sorting 4a
- GFP:
-
green fluorescent protein
- HEPES:
-
4-2-hydroxyethyl-1-piperazineethanesulfonic acid
- EGTA:
-
ethyleneglycol-bis-(β-aminoethylether)-N, N, N′, N′-tetraacetic acid
- EDTA:
-
ethylenediamine-tetraacetic acid
- ER:
-
endoplasmic reticulum
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Acknowledgments
We thank Wesley Sundquist for the GFP–VPS4a EQ and KQ constructs. This work was supported by a grant from the National Institutes of Health (CA104686, G.R.R.). V.Y.C. was supported by a Pre-Doctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation and a Pharmacological Sciences Training Grant from the National Institute of General Medical Sciences (GM07767). Contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS.
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Chen, V.Y., Posada, M.M., Blazer, L.L. et al. The Role of the VPS4A-Exosome Pathway in the Intrinsic Egress Route of a DNA-Binding Anticancer Drug. Pharm Res 23, 1687–1695 (2006). https://doi.org/10.1007/s11095-006-9043-0
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DOI: https://doi.org/10.1007/s11095-006-9043-0