title: Uptake of compounds that selectively kill multidrug-resistant cells: the copper transporter SLC31A1 (CTR1) increases cellular accumulation of the thiosemicarbazone NSC73306.
abstract: Acquired drug resistance in cancer continues to be a challenge in cancer therapy, in part due to overexpression of the drug efflux transporter P-glycoprotein (P-gp, MDR1, ABCB1). NSC73306 is a thiosemicarbazone compound that displays greater toxicity against cells expressing functional P-gp than against other cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (CTR1, SLC31A1). Overexpression of P-gp sensitizes LLC-PK1 cells to NSC73306. Cisplatin (IC50 = 77 μM), cyclosporin A (IC50 = 500 μM), and verapamil (IC50 = 700 μM) inhibited cellular accumulation of [(3)H]NSC73306. Cellular hypertoxicity of NSC73306 to P-gp-expressing cells was inhibited by cisplatin in a dose-dependent manner. Cells transiently expressing the cisplatin uptake transporter CTR1 (SLC31A1) showed increased [(3)H]NSC73306 accumulation. In contrast, CTR1 knockdown decreased [(3)H]NSC73306 accumulation. The presence of NSC73306 reduced CTR1 levels, similar to the negative feedback of CTR1 levels by copper or cisplatin. Surprisingly, although cisplatin is a substrate of CTR1, we found that CTR1 protein was overexpressed in high-level cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. We confirmed that the CTR1 protein was functional, as uptake of NSC73306 was increased in KB-CP20 cells compared to their drug-sensitive parental cells, and downregulation of CTR1 in KB-CP20 cells reduced [(3)H]NSC73306 accumulation. These results suggest that NSC73306 is a transport substrate of CTR1.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: King Leung
authors.initials: KL
authors.lastname: Fung
authors.forename: Abisola K
authors.initials: AK
authors.lastname: Tepede
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Pluchino
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Pouliot
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Pixley
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Hall
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D027682:Cation Transport Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002945:Cisplatin / Q000008:administration & dosage / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D000079923:Copper Transporter 1; D016572:Cyclosporine / Q000737:chemistry; D018432:Drug Resistance, Multiple / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000493:pharmacokinetics*; D020128:Inhibitory Concentration 50; D018374:LLC-PK1 Cells; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D013552:Swine; D013882:Thiosemicarbazones / Q000737:chemistry; D014700:Verapamil / Q000008:administration & dosage
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D007211:Indoles; C511311:NSC73306; D034741:RNA, Small Interfering; C490079:SLC31A1 protein, human; D013882:Thiosemicarbazones; D003300:Copper; D016572:Cyclosporine; D014700:Verapamil; D002945:Cisplatin