title: Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.
abstract: P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Katayama
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Khyati
authors.initials: K
authors.lastname: Kapoor
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Ohnuma
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Atish
authors.initials: A
authors.lastname: Patel
authors.affiliation: Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892, USA.
authors.forename: William
authors.initials: W
authors.lastname: Swaim
authors.affiliation: Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, NIH, Bethesda, MD 20892, USA.
authors.forename: Indu S
authors.initials: IS
authors.lastname: Ambudkar
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: ambudkar@helix.nih.gov.
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D006801:Humans; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D061988:Proteasome Inhibitors / Q000494:pharmacology; D059748:Proteolysis
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000935:Antifungal Agents; D018942:Macrolides; D061988:Proteasome Inhibitors; C040929:bafilomycin A1
keywords: Degradation; Endosome; Half-life; Lysosome; P-glycoprotein; Proteasome