Actin disruption inhibits endosomal traffic of P-glycoprotein-EGFP and resistance to daunorubicin accumulation.
Intracellular traffic of human P-glycoprotein (P-gp), a membrane transporter responsible for multidrug resistance in cancer chemotherapy, was investigated using a P-gp and enhanced green fluorescent fusion protein (P-gp-EGFP) in human breast cancer MCF-7 cells. The stably expressed P-gp-EGFP from a clonal cell population was functional as a drug efflux pump, as demonstrated by the inhibition of daunorubicin accumulation and the conferring of resistance of the cells to colchicine and daunorubicin. Colocalization experiments demonstrated that a small fraction of the total P-gp-EGFP expressed was localized intracellularly and was present in early endosome and lysosome compartments. P-gp-EGFP traffic was shown to occur via early endosome transport to the plasma membrane. Subsequent movement of P-gp-EGFP away from the plasma membrane occurred by endocytosis to the early endosome and lysosome. The component of the cytoskeleton responsible for P-gp-EGFP traffic was demonstrated to be actin rather than microtubules. In functional studies it was shown that in parallel with the interruption of the traffic of P-gp-EGFP, cellular accumulation of the P-gp substrate daunorubicin was increased after cells were treated with actin inhibitors, and cell proliferation was inhibited to a greater extent than in the presence of daunorubicin alone. The actin dependence of P-gp traffic and the parallel changes in cytotoxic drug accumulation demonstrated in this study delineates the pathways of P-gp traffic and may provide a new approach to overcoming multidrug resistance in cancer chemotherapy.
Fu
Dong
D
Faculty of Pharmacy, The University of Sydney, Sydney, NSW 2006, Australia.
Roufogalis
Basil D
BD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism* / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003078:Colchicine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003630:Daunorubicin / Q000378:metabolism* / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D000903:Antibiotics, Antineoplastic; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D053778:Thiazolidines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D003513:Cycloheximide; C037068:latrunculin B; D003078:Colchicine; D003630:Daunorubicin