Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer.
In our present study, a P-glycoprotein-EGFP (P-gp-EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P-glycoprotein (P-gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P-gp-EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12-48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P-gp-EGFP. Significant daunorubicin accumulation occurred in transfected cells when P-gp-EGFP was localized predominantly within the ER, and accumulation remained high when P-gp-EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P-gp-EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P-gp-EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P-gp-EGFP and retention of P-gp-EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P-gp-EGFP can be used to define the dynamics of P-gp traffic in a transient expression system, and demonstrates that localization of P-gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P-gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells.
Fu
Dong
D
The Faculty of Pharmacy, The University of Sydney, Australia.
Bebawy
Mary
M
Kable
Eleanor P W
EP
Roufogalis
Basil D
BD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003630:Daunorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000502:physiology*; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D000935:Antifungal Agents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D008985:Monensin; D003630:Daunorubicin