Indomethacin induces apoptosis via a MRPI-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRPI

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 (MRPI) function. The doxorubicin-resistant MRPI-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 MRPI overexpression. The experimental design involved analysis of the effect of MRPI 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 MRPI reduced MRPI mRNA levels twofold and reduced efflux pump function of MRPI, which was reflected by a 1.8-fold higher accumulation of MRPI substrate carboxyfluorescein, in si-MRPI 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 (IC50) from 22.8 +/- 2.6 to 30.4 +/- 5.1 mM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC4 or on glutathione levels in both lines. Although indomethacin (20 mM) 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 MRPI-dependent mechanism. This may have implications for the treatment of patients with MRPI-overexpressing tumours.