P-glycoprotein mediates drug resistance via a novel mechanism involving lysosomal sequestration.
Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.
From the Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, Blackburn Building (D06), University of Sydney, Sydney, New South Wales 2006, Australia.
Tetsuo
T
Yamagishi
Sumit
S
Sahni
Danae M
DM
Sharp
Akanksha
A
Arvind
Patric J
PJ
Jansson
Des R
DR
Richardson
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; C497811:LAMP2 protein, human; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D009363:Neoplasm Proteins
Chemical Biology; Chemoresistance; Chloroquine; Doxorubicin; Drug Resistance; Drug Transport; Lysosomes; Multidrug Resistance; P-glycoprotein