The role of the VPS4A-exosome pathway in the intrinsic egress route of a DNA-binding anticancer drug.
OBJECTIVE
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.
Chen
Vivien Y
VY
Department of Pharmaceutical Sciences, University of Michigan College of Pharmacy, 428 Church St., Ann Arbor, Michigan 48109, USA.
Posada
Maria M
MM
Blazer
Levi L
LL
Zhao
Tong
T
Rosania
Gus R
GR
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000502:physiology*; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D002467:Cell Nucleus; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004317:Doxorubicin / Q000493:pharmacokinetics; D056827:Endosomal Sorting Complexes Required for Transport; D006801:Humans; D020014:K562 Cells; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases; D033921:Vesicular Transport Proteins
D000903:Antibiotics, Antineoplastic; D000970:Antineoplastic Agents; D056827:Endosomal Sorting Complexes Required for Transport; D012097:Repressor Proteins; D033921:Vesicular Transport Proteins; D004317:Doxorubicin; D004247:DNA; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human