title: Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1.
abstract: Small-cell lung cancers (SCLCs) initially respond to chemotherapy, but are often resistant at recurrence. The non-steroidal anti-inflammatory drug indomethacin is an inhibitor of multidrug resistance protein 1 (MRP1) function. The doxorubicin-resistant MRP1-overexpressing human SCLC cell line GLC(4)-Adr was highly sensitive for indomethacin compared with the parental doxorubicin-sensitive line GLC(4). The purpose of this study was to analyse the relationship between hypersensitivity to indomethacin and MRP1 overexpression. The experimental design involved analysis of the effect of MRP1 downregulation on indomethacin-induced cell survival and apoptosis in GLC(4)-Adr and GLC(4), using siRNA. In addition the effect of indomethacin on glutathione levels and mitochondrial membrane potential was investigated. Small interfering RNAs directed against MRP1 reduced MRP1 mRNA levels twofold and reduced efflux pump function of MRP1, which was reflected by a 1.8-fold higher accumulation of MRP1 substrate carboxyfluorescein, in si-MRP1 versus si-Luciferase-transfected GLC(4)-Adr cells. Multidrug resistance protein 1 downregulation decreased initial high apoptosis levels 2-fold in GLC(4)-Adr after indomethacin treatment for 24 h, and increased cell survival (IC(50)) from 22.8+/-2.6 to 30.4+/-5.1 microM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC(4) or on glutathione levels in both lines. Although indomethacin (20 microM) for 2 h decreased glutathione levels by 31.5% in GLC(4)-Adr, complete depletion of cellular glutathione by L-buthionine (S,R)-sulphoximine only resulted in a small increase in indomethacin-induced apoptosis in GLC(4)-Adr, demonstrating that a reduced cellular glutathione level is not the primary cause of indomethacin-induced apoptosis. Indomethacin exposure decreased mitochondrial membrane potential in GLC(4)-Adr cells, suggesting activation of the mitochondrial apoptosis pathway. Indomethacin induces apoptosis in a doxorubicin-resistant SCLC cell line through an MRP1-dependent mechanism. This may have implications for the treatment of patients with MRP1-overexpressing tumours.
authors.affiliation: Department of Medical Oncology, University of Groningen and University Medical Center, PO Box 30.001, 9700 RB, Groningen, The Netherlands.
authors.forename: D J A
authors.initials: DJ
authors.lastname: de Groot
authors.forename: M
authors.initials: M
authors.lastname: van der Deen
authors.forename: T K P
authors.initials: TK
authors.lastname: Le
authors.forename: A
authors.initials: A
authors.lastname: Regeling
authors.forename: S
authors.initials: S
authors.lastname: de Jong
authors.forename: E G E
authors.initials: EG
authors.lastname: de Vries
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000378:metabolism; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005434:Flow Cytometry; D005978:Glutathione / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D034741:RNA, Small Interfering; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000903:Antibiotics, Antineoplastic; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004317:Doxorubicin; D005978:Glutathione; D007213:Indomethacin