title: Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes multidrug resistance by a novel mechanism involving the hijacking of lysosomal P-glycoprotein (Pgp).
abstract: Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2',7'-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia patric.jansson@sydney.edu.au.
authors.forename: Patric J
authors.initials: PJ
authors.lastname: Jansson
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamagishi
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Akanksha
authors.initials: A
authors.lastname: Arvind
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Nicole
authors.initials: N
authors.lastname: Seebacher
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Elaine
authors.initials: E
authors.lastname: Gutierrez
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Stacy
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Sanaz
authors.initials: S
authors.lastname: Maleki
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Danae
authors.initials: D
authors.lastname: Sharp
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia.
authors.forename: Sumit
authors.initials: S
authors.lastname: Sahni
authors.affiliation: From the Molecular Pharmacology and Pathology Program, Department of Pathology, University of Sydney, Sydney, New South Wales 2006, Australia d.richardson@med.usyd.edu.au.
authors.forename: Des R
authors.initials: DR
authors.lastname: Richardson
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D005260:Female; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D061986:MCF-7 Cells; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D034622:RNA Interference; D013882:Thiosemicarbazones / Q000378:metabolism / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology; D023041:Xenograft Model Antitumor Assays
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D013882:Thiosemicarbazones; C539263:di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone; D014747:Vinblastine; D004317:Doxorubicin
keywords: Chemical Biology; Chemoresistance; Chloroquine; Dp44mT; Drug Delivery; Drug Transport; Lysosome; Novel Drug Targets; P-glycoprotein