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

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.

Lung cancer is the tumour type with the highest incidence in the Western world. A total of 25% of lung cancers are of the small-cell lung cancer (SCLC) type. These tumours are well known for their initial sensitivity to chemotherapeutic agents and thereafter frequently recur at which time the tumour is drug resistant (Glisson, 2003). A common mechanism for drug resistance shared by chemotherapeutic drugs is the failure of the tumour cells to go into apoptosis. Interestingly, tumour cells have an independent pathway available, which can be used to induce apoptosis, namely, the death receptor ligand signalling pathway (Younes and Kadin, 2003). This has raised interest to exploit this pathway to circumvent drug resistance. Fas and Fas ligand (FasL) belong to the TNF family of death receptors and ligands (Trauth et al, 1989;Itoh et al, 1991;Suda et al, 1993). Fas expression is present in many tumours and tumour cell lines including in SCLC Muller et al, 1997;Niehans et al, 1997;Fulda et al, 1998;Kawasaki et al, 2000). After trimerisation of Fas on the cell membrane by extracellular FasL (Huang et al, 1996), Fas-associated Death Domain (FADD) and caspase-8 bind to the intracellular death domains of Fas and induce a death signal in the cell . This leads to the activation of a cascade of caspases and eventually to cell death. In addition, several antiapoptosis proteins regulate the Fas-mediated death pathway. Important antiapoptosis proteins are decoy receptor 3 (DcR3), Fas-associated phosphatase-1 (FAP-1), the long and short isoform of FLICE-inhibitory protein (FLIP 1 and FLIP S ) and the inhibitors of apoptosis family (IAPs) (Sato et al, 1995;Deveraux et al, 1997;Yanagisawa et al, 1997;Pitti et al, 1998;Li et al, 2000).
There is an alternative pathway for death receptor-induced apoptosis that involves the mitochondria (Scaffidi et al 1998(Scaffidi et al , 1999. This pathway is controlled by proapoptotic and antiapoptotic proteins from the Bcl-2 family. One of the key proapoptotic proteins in this pathway is Bid. When caspase-8 is activated in the initial phase of death receptor-induced apoptosis, it can cleave Bid. The p15 form of truncated Bid (tBid) translocates to the mitochondria where cytochrome c is released. Cytochrome c activates caspase-9, which activates downstream effector caspases resulting in apoptosis (Luo et al, 1998).
In several tumour cell lines, including SCLC cell lines, Fas membrane expression is upregulated after exposure to chemotherapeutic agents (Friesen et al, 1999). This can enhance sensitivity to apoptosis-inducing anti-Fas antibody. Therefore, induction of Fas-mediated apoptosis together with chemotherapy may be an option to overcome drug resistance. At the moment, the major problem of FasL or stimulating anti-Fas antibody is the liver toxicity observed in mice (Ogasawara et al, 1993). However, several attempts are ongoing to circumvent liver toxicity.
Another option to modulate drug resistance is the inhibition of expression of antiapoptotic members of the Bcl-2 family of apoptosis with nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs act by cyclooxygenase (COX) inhibition but can also affect death receptor-mediated apoptosis pathways (Bagrij et al, 2001) and induce apoptosis by downregulation of Bcl-2 family members (Liu et al, 1998, Crosby andDuBois, 2003). In SCLC cell lines, Bcl-2 family members have been described as important factors in chemotherapeutic drug resistance and therefore downregulation of Bcl-2 family members with an NSAID can be an interesting modality to circumvent drug resistance (Sartorius and Krammer, 2002). Human lung adenocarcinoma cells, exposed to NSAIDs showed an effective reduction of the antiapoptosis Bcl-2 family member Mcl-1 (Lin et al, 2001).
In this study, we investigated the possibility of utilising the Fasmediated apoptosis route and indomethacin to modulate doxorubicin resistance in an acquired doxorubicin resistant SCLC cell line.

Cell lines
GLC 4 was derived from a pleural effusion in our laboratory and kept in culture in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS) (both from Life Technologies, Breda, The Netherlands). GLC 4 -Adr obtained resistance to doxorubicin, but also to a wide range of other chemotherapeutic agents, by stepwise increasing concentrations of doxorubicin (Zijlstra et al, 1987;de Jong et al, 1990;Meijer et al, 1991;Versantvoort et al, 1995). GLC 4 -Adr is 190.6716.2 times more resistant to doxorubicin than its parental cell line. The doxorubicin resistance in GLC 4 -Adr is due to a downregulation of the activity of DNA-topoisomerase II (TOPO II) and amplification and overexpression of the MRP-1 gene GLC 4 -Adr was exposed to 1.2 mM doxorubicin twice weekly. GLC 4 -Adr was cultured without doxorubicin for 20 days prior to experiments. Cells were incubated at 371C in a humidified atmosphere with 5% CO 2 . Cells from exponentially growing cultures were used for all experiments.

SDS gel electrophoresis and Western blot
Proteins for Western blot analysis were extracted by lysing cells with sample buffer containing 0.125 M Tris-HCl, 2% SDS, 10% glycerol and 0.001% bromophenol blue. Samples were boiled for 5 min. An amount of 10 mg protein was run on 10% SDS polyacrylamide gels at 200 V and transblotted onto polyvinylidene difluoride membranes (PVDF) (Millipore, Bedford, UK) with a semi-dry blot system. Equal protein loading was confirmed by Ponceau red staining of membranes and Coomassie blue staining of the gels. Membranes were activated in methanol for 5 min and washed three times with H 2 O and once with TBS without Tween 20. Membranes were then blocked for 1 h in TBS supplemented with 5% skim milk and probed with the primary antibody for 1 h. Membranes were washed three times with TBS and incubated with the horseradish peroxidase-bound secondary antibody for 1 h at room temperature. Membranes were washed three times with TBS and bands were visualised with chemoluminescence POD or Lumilight þ (Roche Diagnostics, Basel, Switzerland). All experiments were performed three times.

Confocal laser microscopy
The intracellular localisation of Fas in the cell lines was determined with confocal laser microscopy. Cells were washed cells once with RPM3 1640 medium 10% FCS. Glass slides were coated with 0.1% poly-L-lysine and dried at room temperature. A volume of 50 ml of 4 Â 10 5 cells/ml were put on glass slides and left to adhere to the slides for 1 h. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS: 6.4 mM Na 2 HPO 4 , 15 mM KH 2 PO 4 ; 0.14 mM NaCl; 2.7 mM KC1; pH 7.2) supplemented with 3.3 mM CaCl 2 for 15 min. Cells were washed twice with PBS and incubated for 1 h with the CHl 1 anti-Fas antibody. After incubation with the primary antibody, cells were washed twice and incubated with a FITC coupled rabbit anti-mouse antibody for 30 min and washed twice before they were analysed on a Leica confocal laser microscope.

Flow cytometry
To determine Fas membrane expression cells were harvested from the culture medium by centrifugation at 110 g for 5 min and washed twice with cold PBS supplemented with 2% FCS and 0.1% sodium azide. Cells were then incubated for 1 h with the PElabelled anti-human Fas DX2 antibody, which was diluted 1 : 10 in cold PBS supplemented with 2% FCS and 0.1% sodium azide for 1 h on ice in the dark and washed twice with cold PBS. Analysis was performed on a Coulter Elite Flow cytometer (Becton Dickinson, Mount View, CA, USA) with Winlist and Winlist 32 software (Verity Software House, Inc., Topsham, ME, USA). Fas membrane expression was determined as mean fluorescence intensity (MFI). To study also the effect of indomethacin on Fas membrane expression, cells were incubated for 24 h with indomethacin. These experiments were performed three times.

Mutation screening of Fas
DNA was extracted from the cell lines using a standard laboratory technique. The Fas gene was screened for mutations by denaturing gradient gel electrophoresis of the extracted DNA. The entire coding region, including all splice site junctions, was amplified in 10 amplicons using primers and conditions as described earlier (Gronbaek et al, 1998). The amplicons were electrophoresed in a 9% polyacrylamide denaturing gradient gel containing 5% glycerol and 20 -60% urea-formamide (100% urea-formamide ¼ 7 M urea and 40% deionised formamide). The gels were stained with ethidium bromide and photographed under an UV transilluminator.

Apoptosis assay
Cells (1.5 Â l0 4 per well) were cultured in 96-well plates and optionally preincubated with 1 mg ml À1 cycloheximide for 2 h. Apoptosis was induced by adding the anti-Fas antibody 7C11 (1 mg ml À1 ) for 24 h. To determine whether indomethacin induces apoptosis, cells were incubated with different concentrations of indomethacin. To investigate whether apoptosis induction with indomethacin is Fas-mediated, cells were optionally preincubated with 2 mg ml À1 NOK-1 and incubated with indomethacin for 24 h thereafter. Apoptosis was defined as the appearance of apoptotic bodies and/or chromatin condensation, using a fluorescence microscope. Results were expressed as the percentage of apoptotic cells in a culture by counting at least 200 cells per well. All apoptosis assays were performed three times in two-fold.

Inhibition of indomethacin-induced apoptosis
At 1 h prior to indomethacin exposure, cells were incubated with 20 mM broad-spectrum caspase inhibitor zVAD-fmk, caspase-8 inhibitor zIETD-fmk or caspase-9 inhibitor zLEHD-fmk (all from Calbiochem, Breda, The Netherlands). Cells were exposed to the combination of indomethacin and caspase inhibitor for 24 h after which acridine orange was added and the percentage apoptotic cells was calculated. Results are expressed as the percentage of apoptotic cells in a culture by counting at least 200 cells per well. All apoptosis assays were performed three times in two-fold.

Caspase-3 activation assay
The cleavage assay was carried out in six-well plates according to Thornberry et al (2000). Activity of caspase-3 was assayed according to the manufacturer's instructions using the fluorescence peptide substrate Ac-DEVD-AFC (Biomol Tebu-bio, Heerhugowaard, The Netherlands). Fluorescence from free 7-amino-4trifluoromethyl coumarin (AFC) was monitored in a FL600 Fluorimeter Bio-tek plate reader (Beun de Ronde, Abcoude, The Netherlands) using 380 nm excitation and 508 nm emission wavelengths. Relative caspase-3 activity was calculated by the fluorescence of a sample of treated cells by a sample of untreated cells. Protein from all samples was isolated to confirm apoptosis with PARP cleavage on Western blot. Experiments were performed three times.

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
The cell lines were cultured in HAM/F12 and DMEM medium (1 : 1) (Life Technologies) supplemented with 20% FCS. The effect of doxorubicin and indomethacin on survival was tested MTT assay as described previously (Timmer-Bosscha et al, 1989). Cells were incubated for 4 days at 371C and 5% CO 2 in a humidified environment with a range of indomethacin concentrations and 10 and 2000 nM doxorubicin for GLC 4 and GLC 4 -Adr respectively. The modulating effect of indomethacin (10 and 20 mM) and MK-571 (50 mM) on cell survival by doxorubicin were also tested in the MTT assay using continuous incubation. After a 4-day culture period, MTT (5 mg ml À1 in PBS) was added and formazan crystal production was measured as described previously. Controls consisted of media without cells (background extinction) and cells incubated with medium instead of chemotherapeutic agents. Experiments were performed three times in quadruplicate.

Statistics
All experiments were performed at least three times on different occasions. Analysis included double-sided nonpaired t-test. A P-value o0.05 was considered significant.

Differences between the Fas-mediated apoptosis pathway of GLC 4 and GLC 4 -Adr
The genes encoding the proapoptotic proteins FasL, Fas, FADD, and caspase-8 were all expressed at the mRNA level in GLC 4 and GLC 4 -Adr ( Figure 1A). The proteins FasL, Fas, FADD, caspase-8, Bid, caspase-9, caspase-3 and PARP were also present in both cell lines ( Figure 1B). GLC 4 contained less Bid and caspase-8 compared to GLC 4 -Adr, while there were no differences in the protein expression of FasL, Fas, FADD, Bax, caspase-9, caspase-10, caspase-3, and PARP between the two lines ( Figure 1B). Mutation analysis of the entire Fas gene revealed no aberrant patterns in both cell lines.
Antiapoptosis genes were present in GLC 4 and GLC 4 -Adr. RT -PCR analysis revealed a higher expression of FLIP 1 , FLIP S and DcR3 in GLC 4 -Adr compared to GLC 4 (Figure 2A). Western blot analysis showed no differences in expression of the apoptosis inhibitors FAP-1, FLIP, Bcl-2, Bcl-X L and XIAP between the cell lines. The nonspecific anti-XIAP and Bc1-X L , immunoreactive molecule as indicated (*) served as an internal loading control (Deveraux et al, 1999, Delhalle et al, 2002 (Figure 2B). There were also no differences in COX-2 expression ( Figure 2B).
Confocal laser microscopy revealed that in both cell lines Fas was present in the cytoplasm and at the cell membrane ( Figure 3). However, as determined by flow cytometry GLC 4 -Adr contained 3.1-fold more Fas on the cell membrane than GLC 4 . MFI were on average 15.4 in GLC 4 and 47.5 in GLC 4 -Adr.

Anti-Fas antibody induces apoptosis
Functionality of the Fas pathway was tested by exposure to anti-Fas antibody (24 h) and a combination of anti-Fas antibody (24 h) and cycloheximide (2 h preincubation). The anti-Fas antibody alone hardly induced apoptosis. Cotreatment with cycloheximide largely increased apoptosis in GLC 4 -Adr (40%) but had almost no effect in GLC 4 (8%) (Figure 4). Caspase-8 activation was used as intracellular determinants for activation of the Fas pathway and PARP cleavage as a marker for apoptosis. Surprisingly, no effect of anti-Fas antibody alone on caspase-8 cleavage was found. Intermediate cleavage products of caspase-8 (p41/p43) were detected after exposure to cycloheximide alone or in combination with anti-Fas antibody in both cell lines ( Figure 5). Active caspase-8 (pi8 product) was especially observed in GLC 4 -Adr but only after cotreatment with cycloheximide and anti-Fas antibody. These results indicate the presence of intracellular inhibitors) of the Fasmediated apoptosis pathway, presumably at the level of caspase-8.

Indomethacin induces apoptosis in GLC 4 -Adr but not in GLC 4
Indomethacin alone had hardly any effect on apoptosis induction in GLC 4 but already induced apoptosis (28%) in GLC 4 -Adr at 25 mM ( Figure 6). GLC 4 and GLC 4 -Adr were exposed to 0, 25, 50 and 100 mM indomethacin for 16 and 24 h in order to study the apoptosis-inducing effect of indomethacin in more detail. Cleavage of caspase-8, Bid and PARP was investigated with Western blotting. Caspase-8, Bid, caspase-9 and PARP activation in GLC 4 -Adr occurred 16 h after the addition of 50 mM indomethacin. In GLC 4 -Adr 100 mM indomethacin induced clearly detectable levels of activated caspase-8 (p 18) as well as massive cleavage of full-length caspase-8 and PARP. Indomethacin more effectively induced caspase-8 activation than the combination of anti-Fas antibody and cycloheximide in GLC 4 -Adr. However, even at these high indomethacin concentrations, no activation of caspase-8, Bid and PARP was observed in GLC 4 ( Figure 7A). To further investigate the mechanism by which indomethacin induces apoptosis, GLC 4 and GLC 4 -Adr cells were exposed to 25 and 50 mM indomethacin and protein expression levels of antiapoptotic Bcl-2 family members were analysed. No changes in Bcl-2, Bcl-X S/L or Mcl-1 expression were observed in both cell lines ( Figure 7B).
To determine more quantitatively the effect of various drug combinations on apoptosis in GLC 4 -Adr, caspase-3 activation was measured with the DEVD-AFC cleavage assay. Doxorubicin alone showed minimal caspase-3 activation. Doxorubicin in combination with anti-Fas antibody had a slightly additive effect on caspase-3 activation. The combination of doxorubicin with indomethacin was, however, the most effective combination to induce caspase-3 activation (Figure 8).

Indomethacin induces caspase-8 and caspase-9 activation independently from Fas
The nature of the indomethacin-induced caspase-8 activation was further investigated. Apoptosis induction in GLC 4 -Adr could not be prevented by preincubation with the anti-FasL NOK-1 antibody (data not shown). This means that indomethacin-induced apoptosis is not caused by autocrine or paracrine Fas/FasL interactions. In addition, indomethacin at a concentration of 50 mM for 24 h did not affect Fas membrane expression (results not shown).

Modulation of chemotherapy-induced growth inhibition by indomethacin
To investigate growth inhibition after exposure to indomethacin and doxorubicin the MTT assay was used. GLC 4 -Adr cells are 190.6716.2 times more resistant to doxorubicin as compared to GLC 4 . Two relatively nontoxic concentrations of indomethacin (10 and 20 mM) that induced some caspase activation in GLC 4 -Adr, were used in combination with doxorubicin in an MTT assay. A dose of 10 and 20 mM of indomethacin induced, respectively, 1 and 2% growth inhibition in GLC 4 , and respectively 15 and 17% in GLC 4 -Adr. In the presence of 20 mM indomethacin there was no effect on doxorubicin sensitivity in GLC 4 , while a 2.7-fold increase in doxorubicin sensitivity was observed in GLC 4 -Adr (Table 1). Indomethacin, like doxorubicin, is also a substrate for the MRPl drug efflux pump, which is overexpressed in GLC 4 -Adr. We observed a similar increase in doxorubicin sensitivity comparing the effect of indomethacin with the effect of MK-571, a wellestablished inhibitor of MRPl, in the MTT assay (Table 1).

DISCUSSION
This is the first study that illustrates the effective circumvention of doxorubicin resistance by indomethacin-induced activation of the death receptor apoptosis pathway in a doxorubicin-resistant SCLC cell line independent of Fas. Fas-mediated apoptosis could only be induced in GLC 4 -Adr but not in GLC 4 in the presence of the protein synthesis inhibitor cycloheximide, demonstrating that the death receptor-mediated apoptosis pathway is functional in the chemotherapy resistant cell line when a cellular block is removed. Interestingly, indomethacin induced apoptosis in GLC 4 -Adr but not in GLC 4 in the absence of cycloheximide. No marked intensity differences were observed for pro-and antiapoptotic proteins involved in the mitochondrial apoptosis pathway. In contrast, several proapoptotic proteins important for the death receptor apoptosis pathway were higher expressed in the doxorubicin-resistant cell line GLC 4 -Adr compared to GLC 4 . For instance, the Fas-membrane expression was 3.1-fold higher in GLC 4 -Adr compared to the parental cell line. The higher Fas membrane expression may be due to the repetitive incubation with doxorubicin. It may also serve to facilitate a growth advantage to GLC 4 -Adr as was demonstrated in several Fas-positive tumour cell lines (Owen-Schaub et al, 1994;Siegel et al, 2000). The fact that the difference in Fas membrane Inhibition of indomethacin-induced apoptosis. Apoptosis induction in GLG 4 -Adr after exposure to 50 mM indomethacin (grey) or 0 mM 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. Indomethacin circumvents doxorubicin resistance DJA de Groot et al expression does not correlate with the Fas expression in total cell lysates may be due to a different distribution of Fas, cytoplasmic and on the cell membrane, as was described in prostate carcinoma cell lines and neuroblastoma cell lines Usla et al, 1997;Bennett et al, 1998;Sodeman et al, 2000). The expression levels of the proapoptotic proteins caspase-8 and Bid were also elevated in GLC 4 -Adr compared to GLC 4 . Bid has been described to transport and recycle mitochondrial membrane phospholipids (Esposti et al, 2001). Since doxorubicin has toxic properties towards mitochondrial membranes, increased expression of Bid in GLC 4 -Adr may be an additional resistance mechanism. The sensitivity of GLC 4 -Adr to Fas-mediated apoptosis, in the presence of cycloheximide, as compared to GLC 4 can therefore be due to the higher Fas-membrane levels as well as elevated expression levels of caspase-8 and Bid or a combination of these factors. Anti-Fas antibody alone induced minimal caspase-3 activation which was only slightly increased by combining it with doxorubicin in GLC 4 -Adr. Owing to the limited modulatory effects of doxorubicin on Fas-mediated apoptosis an alternative was sought.
The NSAID indomethacin has been identified as an apoptosisinducing agent in different in vivo models and among the several mechanisms involved it can induce caspase-3-mediated apoptosis (Fujii et al, 2000;Kim et al, 2000;Sanchez-Alcazar et al, 2003). The apoptosis-inducing effect of indomethacin in GLC 4 -Adr is, however, not based on Fas/FasL interaction. Indomethacin did not affect Fas membrane expression and apoptosis is not decreased when cells are pretreated with an inhibiting anti-FasL antibody prior and during indomethacin exposure. Indomethacin alone induced extensive apoptosis in GLC 4 -Adr with activation of caspase-8, caspase-9 and PARP cleavage even at low doses. This did not occur in GLC 4 . The apoptosis-inducing effect of indomethacin will therefore most likely be due to a Fas receptorindependent effect on the death receptor-apoptosis pathway. However, we cannot exclude the involvement of other death receptors. Inhibition of either caspase-8 or caspase-9 by zIETDfmk and zLEHD-fink, respectively, decreased indomethacininduced apoptosis. Therefore, indomethacin-mediated apoptosis induction in the GLC 4 cell lines depends on a functional mitochondrial apoptosis pathway, which is probably absent in GLC 4 due to the decreased Bid expression. Indomethacin, however, did not decrease expression of Bcl-2, Bcl-X S/L or Mcl-1 in GLC 4 or GLC 4 -Adr which is in contrast to results described for lung adenocarcinoma cell lines (Lin et al, 2001). The feet that indomethacin can activate caspase-8, Bid and caspase-9 in GLC 4 -Adr makes it a good alternative for agonistic anti-Fas antibody. Indomethacin added to doxorubicin largely increased doxorubicin effects on caspase-3 activation and cytotoxicity in GLC 4 -Adr cells. Indomethacin, like doxorubicin, is a substrate for the MRP1 drug efflux pump, which is overexpressed in GLG 4 -Adr (Versantvoort et al, 1995;Draper et al, 1997;Touhey et al, 2002). Therefore, a subsequent increase in cellular doxorubicin concentration by indomethacin may have partly played a role. Other mechanisms by which indomethacin might induce apoptosis are increased glutathione extrusion mediated by MRP1 (Trompier et al, 2004) or increased ATP consumption by MRP1 ATPase activity in analogy to the observed verapamil-induced ATP consumption in P-glycoprotein-overexpressing cells (Broxterman et al, 1989).
The role of indomethacin in modulation of doxorubicin toxicity, however, cannot completely be explained by the MRP1 inhibitory effect. MK-571 is a far more effective inhibitor of MRP1-mediated drug efflux than indomethacin (Bagrij et al, 2001). Despite the similar fold of doxorubicin sensitisation with either drug, this suggests that the observed effect of indomethacin on doxorubicin sensitivity is due to an increase in drug accumulation as well as MRP1-dependent or independent caspase activation in GLQ-Adr cells.
Interestingly, the NSAIDs have recently caught much attention in the treatment of tumours in combination with chemotherapy to potentiate their effect (Gupta and DuBois, 2001). The first clinical report on a combination of celecoxib, an NSAID and selective cyclooxygenase-2 inhibitor, with chemotherapy appeared (Altorki et al, 2003). It showed an enhanced response to preoperative paclitaxel and carboplatin in early-stage non-small-cell lung cancer. This approach in SCLC may also be of interest not only because of Cox-2 inhibition but also because of the effect observed by us on the alternative apoptotic route compared to the route used by chemotherapy. The observed extensive potentiation of doxorubicin-induced inhibition of cell survival at achievable clinical doses indomethacin (Gupta and DuBois, 2001), deserves testing in the clinic. The potential effect of these concentrations of indomethacin on other chemotherapeutic drugs requires further testing in preclinical models.
Overall, it can be concluded that indomethacin increases the cytotoxic activity of doxorubicin in a doxonibicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas.