title: Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells.
abstract: In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.
authors.affiliation: Department of Molecular Toxicology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands. Cnubben@voeding.tno.nl
authors.forename: N H
authors.initials: NH
authors.lastname: Cnubben
authors.forename: A J
authors.initials: AJ
authors.lastname: Rommens
authors.forename: M J
authors.initials: MJ
authors.lastname: Oudshoorn
authors.forename: P J
authors.initials: PJ
authors.lastname: Van Bladeren
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology; D018528:ATP-Binding Cassette Transporters / Q000502:physiology; D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism*; D001692:Biological Transport; D001711:Biotransformation; D001943:Breast Neoplasms / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D005260:Female; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase / Q000502:physiology*; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013852:Thiotepa / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D018906:Antineoplastic Agents, Alkylating; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; D007527:Isoenzymes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013852:Thiotepa; C496556:GSTP1 protein, human