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 GLC4-Adr and its parental doxorubicin-sensitive line GLC4 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 GLC4-Adr. In addition, GLC4-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 GLC4-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC4-Adr but not in GLC4 cells. Surprisingly, in GLC4-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 GLC4-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 GLC4. 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.

Figure 1

Basic mRNA and protein expression levels of proapoptotic proteins in GLC₄ and GLC₄-Adr in the Fas-mediated apoptosis pathway were determined at the mRNA level (A) and protein level (B). Representative examples of three independent experiments are shown.

Figure 2

Basic mRNA and protein expression levels of antiapoptotic proteins in GLC₄ and GLC₄-Adr in the Fas-mediated apoptosis pathway were determined at the mRNA level (A) and protein level (B). Representative examples of three independent experiments are shown.

Figure 3

Fas localisation in GLC₄ and GLC₄-Adr determined with the mouse monoclonal CH11 anti-Fas antibody (Upstate Biotechnology) using confocal laser microscopy.

Figure 4

Fas-mediated apoptosis induction. Apoptosis induction in GLC₄ and GLC₄-Adr was determined after exposure to medium (stripes), cycloheximide (white), anti-Fas antibody 1 μg ml⁻¹ (grey) or both (black) using the apoptosis assay. Data represent the mean±s.d. of three independent experiments (^*P<0.05).

Figure 5

Fas-mediated caspase cleavage. PARP and caspase-8 cleavage were determined after exposing GLC₄ and GLC₄-Adr to anti-Fas antibody for 24 h and cycloheximide for 24 and 2 h preincubation. Representative example of three independent experiments.

Figure 6

Indomethacin-mediated apoptosis. Apoptosis induction in GLC₄ and GLC₄-Adr was determined after exposure to different concentrations of indomethacin for 48 h.

Figure 7

(A) Indomethacin-induced activation of the death receptor apoptosis pathway. Caspase-8, Bid and PARP cleavage were determined after exposing GLC₄ and GLC₄-Adr to 0, 25, 50 and 100 μM indomethacin for 16 and 24 h. Representative example of three experiments are shown. (B) Expression of Bcl-2, Bc1-X_S/L and Mcl-1 after 24 h of 25 and 50 μM indomethacin exposure.

Figure 8

Effects of modulators on caspase-3 activation in GLC₄-Adr after 48 h of exposure to. (1) medium, (2) indomethacin (25 μM), (3) doxorubicin (3 μM), (4) anti-Fas antibody (1 μg ml⁻¹), (5) anti-Fas antibody and doxorubicin (3 μM), (6) indomethacin (25 μM) and doxorubicin (3 μM). Exposure to the anti-Fas antibody was only for the last 24 h. Data represent the mean±s.d. of three independent experiments (^*P<0.05).

Figure 9

Inhibition of indomethacin-induced apoptosis. Apoptosis induction in GLG₄-Adr after exposure to 50 μM indomethacin (grey) or 0 μM indomethacin (black) in combination with the caspase-8 inhbitor zIETD-fmk, the caspase-9 inhibitor zLEHD or the broad-spectrum caspase inhibitor zVAD-fmk for 24 h.