title: Effect of ABCG2 on cytotoxicity of platinum drugs: interference of EGFP.
abstract: ATP-binding drug efflux transporters decrease intracellular concentrations of cytotoxic drugs, causing multidrug resistance in cancer. In this study, we examined possible interactions of ABCG2 transporter with platinum cytotoxic drugs. We demonstrate here an interference of platinum drugs with enhanced green fluorescence protein (EGFP) in the cellular models, where EGFP was employed as a reporter gene. Cytotoxicity of cisplatin (CIP), carboplatin (CAP) and oxaliplatin (OXP) was significantly lowered in MDCKII cells transfected with ABCG2 transporter and EGFP reporter. The IC(50) values in MDCKII-ABCG2 were 25.7, 164 and 165 microM for CIP, CAP and OXP, respectively, whereas IC(50) for the same cytostatics in MDCKII cells were as follows: 15.4, 133 and 50.3 microM. Addition of fumitremorgin C (FTC), a potent ABCG2 inhibitor, significantly suppressed the resistance of MDCKII-ABCG2 to OXP, suggesting that OXP interacts with ABCG2. However, FTC did not change the sensitivity of the cells to CIP and CAP. We assume that EGFP rather than ABCG2 causes the diminished toxicity of the platinum cytostatics in the transfected cells. This hypothesis was confirmed in human Hep2 cells expressing EGFP: using MTT test, IC(50) of 30.0, 247 and 27.9 microM were obtained for CIP, CAP and OXP, respectively, while 12.3, 106 and 20.5 microM were observed in the parent Hep2 cells. Employing neutral red cytotoxicity assay, similar data were obtained (IC(50) 7.73, 685 and 112 microM for CIP, CAP, and OXP, respectively, in the Hep2-EGFP cells and 1.65, 79.4 and 24.5 microM in the parent Hep2 cells). Caspase-3/7 assay revealed lower susceptibility of EGFP expressing Hep2 cells to apoptosis induced by CIP when compared to the parent cell line. We therefore conclude that EGFP in transfected cells interferes with cytotoxicity of platinum drugs by hindering the drug induced apoptosis and could cause misinterpretation of results obtained in cytotoxicity studies.
authors.affiliation: Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Ceckova
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Vackova
authors.forename: Hana
authors.initials: H
authors.lastname: Radilova
authors.forename: Antonin
authors.initials: A
authors.lastname: Libra
authors.forename: Martin
authors.initials: M
authors.lastname: Buncek
authors.forename: Frantisek
authors.initials: F
authors.lastname: Staud
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D016190:Carboplatin / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity; D002478:Cells, Cultured; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D004285:Dogs; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D049452:Green Fluorescent Proteins / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism*; D009944:Organoplatinum Compounds / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity*; D000077150:Oxaliplatin; D014162:Transfection / Q000379:methods
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D009363:Neoplasm Proteins; D009944:Organoplatinum Compounds; C485184:enhanced green fluorescent protein; D000077150:Oxaliplatin; D049452:Green Fluorescent Proteins; D016190:Carboplatin; D002945:Cisplatin