Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

Abstract

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC 4 -Adr and its parental doxorubicinsensitive line GLC 4 were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC 4 -Adr. In addition, GLC 4 -Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC 4 -Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC 4 -Adr but not in GLC 4 cells. Surprisingly, in GLC 4 -Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC 4 -Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC 4 . Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.

Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1

“…We previously reported that exposure of the MRP1-overexpressing doxorubicin-resistant SCLC cell line GLC 4 -Adr to indomethacin resulted in caspase-8-and caspase-9-dependent apoptosis induction, suggesting the involvement of the extrinsic and intrinsic apoptosis pathways. In contrast, exposure of the parental cell line GLC 4 to indomethacin did not induce apoptosis (De Groot et al, 2005). Indomethacin as well as MK571 increase doxorubicin sensitivity in these GLC 4 cell lines.…”

“…Compared with the parental cell line GLC 4 , the doxorubicinresistant sub-line GLC 4 -Adr strongly overexpresses MRP1 and is highly sensitive to indomethacin (De Groot et al, 2005). The role of MRP1 in indomethacin sensitivity was investigated using an MRP1 siRNA approach.…”

“…Previously, we have demonstrated that indomethacin-induced apoptosis in GLC 4 -Adr was caused by caspase-8 as well as caspase-9 activation (De Groot et al, 2005). However, the role of mitochondria in indomethacin-induced apoptosis in GLC 4 -Adr was not established.…”

Abundant Fas expression by gastrointestinal stromal tumours may serve as a therapeutic target for MegaFasL

“…Fas membrane expression was determined in GIST cells by flow cytometry as described previously (De Groot et al, 2005). Phycoerythrin (PE)-conjugated mouse anti-Fas monoclonal antibody (clone DX-2, 1 : 10; BD Pharmingen, Alphen aan de Rijn, The Netherlands) was used.…”

“…Western blotting was performed as described previously (De Groot et al, 2005). Primary antibodies were goat anti-Fas (polyclonal, 1 : 400; Santa Cruz, Heerhugowaard, The Netherlands), mouse anti-FasL and mouse anti-caspase 3 (clone 33 and clone 19, respectively, 1 : 1000; BD Transduction Laboratories, Alphen aan den Rijn, The Netherlands), rabbit anti-cleaved caspase 3 and rabbit anti-caspase 6 (1 : 1000; Cell Signalling Technology, Bioké, Leiden, The Netherlands), mouse anti-caspase 8 (clone 1C12, 1 : 500; Cell Signalling), and mouse anti-actin (clone C4, 1 : 20 000; ICN Biomedicals, Zoetermeer, The Netherlands).…”