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Clinical Cancer Research Vol. 8, 354-360, February 2002
© 2002 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

Sulindac Enhances Tumor Necrosis Factor-{alpha}-mediated Apoptosis of Lung Cancer Cell Lines by Inhibition of Nuclear Factor-{kappa}B1

Kevin S. Berman, Udit N. Verma, Gwyndolen Harburg, John D. Minna, Melanie H. Cobb and Richard B. Gaynor2

Departments of Pharmacology [K. S. B., M. H. C.], Internal Medicine [U. N. V., J. D. M., R. B. G.], Simmons Cancer Center [U. N. V., G. H., R. B. G.], and Hamon Center for Therapeutic Oncology [J. D. M.], The University of Texas Southwestern Medical Center, Dallas, Texas 75390


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Programmed cell death (apoptosis) is induced by certain anticancer therapies, and resistance to apoptosis is a major mechanism by which tumors evade these therapies. The transcription factor nuclear factor (NF)-{kappa}B, which is frequently activated by treatment of cancer cells with different chemotherapeutic agents, promotes cell survival, whereas its inhibition leads to enhanced apoptosis. Recently, sulindac and other nonsteroidal anti-inflammatory drugs have been shown to inhibit tumor necrosis factor (TNF)-{alpha}-mediated NF-{kappa}B activation. Here, we demonstrate that treatment of the non-small cell lung carcinoma cells NCI-H157 and NCI-H1299 with sulindac greatly enhances TNF-{alpha}-mediated apoptosis. We further show that sulindac inhibits TNF-{alpha}-mediated activation of NF-{kappa}B DNA binding and nuclear translocation of NF-{kappa}B. These results suggest that sulindac and other nonsteroidal anti-inflammatory drug inhibitors of NF-{kappa}B activation may serve as useful agents in cancer chemotherapy.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Many cancer chemotherapeutic agents ultimately act by causing the cell to undergo apoptosis (1) . Paradoxically, many chemotherapeutic agents that promote apoptosis also activate the transcription factor NF-{kappa}B3 (2, 3, 4, 5) . This transcription factor induces genes that promote cell survival and block apoptosis (6 , 7) . Furthermore, NF-{kappa}B also up-regulates genes important for tumor proliferation and metastasis (5) . NF-{kappa}B may be one of several factors inducing tumor growth, as suggested by the fact that some tumors have constitutively high levels of NF-{kappa}B in their nuclei and that NF-{kappa}B is important for cell survival (8, 9, 10) . These results suggest the hypothesis that blocking NF-{kappa}B activation may augment cancer chemotherapy.

Because of the importance of NF-{kappa}B in disease states as well as in the stress response, much effort has been directed toward defining the pathway that leads to its activation. NF-{kappa}B is normally sequestered in the cytoplasm in its inactive form, bound by a family of inhibitory proteins known as I{kappa}B. IKKß can phosphorylate I{kappa}B, leading to its degradation and the translocation of NF-{kappa}B to the nucleus, where it can bind DNA and regulate transcription (11) . Aspirin and sodium salicylate can inhibit the activation of NF-{kappa}B by binding to and inhibiting the activity of IKKß, whereas other NSAIDs such as indomethacin are deficient in this activity (12) . Sulindac is an NSAID structurally related to indomethacin but differs from indomethacin in its ability to bind IKKß and inhibit NF-{kappa}B activation in response to TNF-{alpha} (13) . Sulindac is predominantly metabolized in the colon to sulindac sulfide and sulindac sulfone. Similar to sulindac, the sulfide and sulfone compounds can inhibit IKKß. However, only sulindac sulfide is able to block prostaglandin synthesis by inhibiting COX-2 (14) . Sulindac, and to a lesser extent aspirin, have been shown to cause regression of colonic polyps and block their progression to cancer (15) . Sulindac and its metabolites are more potent inhibitors of NF-{kappa}B activation than aspirin and have been shown to inhibit proliferation and induce apoptosis in colon adenocarcinoma cells (13 , 16) . The mechanism by which sulindac and its metabolites cause apoptosis, however, remains controversial.

TNF-{alpha} is an inflammatory cytokine originally identified as a protein that can kill cells in vitro and induce hemorrhagic necrosis in transplantable tumors (17) . Although TNF-{alpha} stimulates apoptosis by binding the receptor TNF-R1, it also activates NF-{kappa}B. It has been proposed that inhibition of NF-{kappa}B could potentiate the cytotoxicity of TNF-{alpha}. Studies supporting this idea include cell lines deficient in NF-{kappa}B or through the use of adenovirus-delivered or -transfected dominant-negative I{kappa}B mutants (an inhibitor of NF-{kappa}B function), which renders cells more susceptible to the apoptotic effects of TNF-{alpha} (2 , 18 , 19) . These results suggest that TNF-{alpha} and other cancer chemotherapy agents that induce apoptosis and stimulate NF-{kappa}B activity may be more effective if given in combination with agents that could inhibit NF-{kappa}B. Although these results have potential clinical implications, there have been no studies reported in lung or other common cancers of combining sulindac treatment with agents such as TNF-{alpha}. Because TNF-{alpha} is a strong inducer of apoptosis when NF-{kappa}B is inhibited and because sulindac potently inhibits NF-{kappa}B activity, we examined the effects of TNF-{alpha} when combined with sulindac on inducing apoptosis in lung cancer cell lines.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Viability Assays.
The NSCLC cell lines H157 (squamous cell) and H1299 (large cell) were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 5% fetal bovine serum at 37°C in 5% CO2. MTT assays were used to measure the number of viable cells. In this assay, this membrane permeant dye is reduced by mitochondrial reductases in living cells, and spectrophotometric measurement allows quantitation of cell viability. Equal numbers of cells were cultured in 24-well plates. Cells treated with NSAIDs were preincubated for 2 h with either 1 mM sulindac (Sigma Chemical Co.) diluted from a 100 mM stock in Tris-HCl (pH 8.0) or with 25 µM indomethacin diluted from a 25 mM stock in Tris (pH 8.0). Recombinant purified human TNF-{alpha} (Ref. 20 ; kindly provided by Steve Sprang, University of Texas-Southwestern, Dallas, TX) was diluted in PBS with BSA at 1 mg/ml.

At the indicated times after adding TNF-{alpha}, 200 µl of MTT (Sigma Chemical Co.; 5 mg/ml in PBS) was added to the culture medium. Cells were then incubated for 3 h at 37°C. After the incubation period, 2 ml of 10% Triton X-100 plus 0.1 N HCl in isopropanol were added to the culture medium to dissolve formazan crystals. The plates were mixed for 5–10 min on a gyratory shaker, and the absorbance was measured at a wavelength of 595 nm on a plate reader (Molecular Devices, Inc). Z-VAD-FMK (Calbiochem) was added to indicated plates 30 min before the sulindac preincubation.

Terminal Deoxynucleotidyl Transferase-mediated FITC-dUTP Nick End Labeling TUNEL Assay for Detection of Apoptotic Cells.
H1299 cells were grown in RPMI 1640, as described above for MTT assay, in 100-mm Petri dishes. Confluent cells (80–90%) were exposed to: (a) vehicle only; (b) 1 mM sulindac; (c) 25 µM indomethacin; (d) 20 ng/ml TNF-{alpha}; (e) sulindac and TNF-{alpha}; and (f) indomethacin and TNF-{alpha}. Thirty-six h after exposure to these agents, cells were harvested by trypsinization and washed two times in PBS. FITC-conjugated dUTP was used to label the cells with DNA nicking, using Apoptosis Detection System kit from Promega Corp. (Madison, WI). Guidelines suggested by the manufacturer were followed for staining of cells. Briefly, after washing with PBS, cells were fixed with 1% methanol-free formaldehyde for 20 min on ice. After fixation, cells were washed two times in PBS and stored overnight in 70% ethanol at -20°C to permeabilize. Next, cells were washed of ethanol, equilibrated with equilibrating buffer supplied with the kit, and incubated at 37°C with FITC-conjugated nucleotide mix and TdT enzyme in equilibration buffer for 60 min. Negative controls for each treatment group were incubated as above, except without TdT. After incubation, reaction was terminated by the addition of 1 ml of 20 mM EDTA to each tube. Cells were washed with 1 ml of PBS two times containing 0.1% Triton X-100 and 5 mg/ml BSA. After a final wash, cells were resuspended in 0.5 ml of freshly diluted propidium iodide at a concentration of 5 µg/ml in PBS and incubated at room temperature for 30 min. Flow cytometric analysis was performed on FACScan (Becton-Dickinson, San Diego, CA), acquiring data for FL1 (FITC) and FL3 (propidium iodide) in addition to forward and side scatter.

Caspase-3 Assay.
Caspase-3 activity was measured with the colorimetric caspase-3 substrate I assay (Calbiochem). Cells were treated with NSAIDs and TNF-{alpha} as in the MTT assays, and at the indicated times, cells were washed once in cold PBS and lysed in a buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM DTT, and 0.1 mM EDTA. Cell lysates were incubated with the assay buffer containing 50 mM HEPES (pH 7.4), 100 mM NaCl, 10 mM DTT, 0.1 mM EDTA, and 10% glycerol so that the final 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate concentration was 0.1%. The caspase-3 substrate was added, and the absorbance was measured at a wavelength of 405 nm on a plate reader (Molecular Devices, Inc.) at 20-min intervals for 2 h.

EMSA.
EMSA was performed as described previously (12) . Briefly, cells treated with NSAIDs were preincubated for 2 h with PBS, 3 mM sulindac, or 25 µM indomethacin and then treated with TNF-{alpha} for 20 min at a final concentration of 10 ng/ml. Cells were washed with cold PBS and harvested into a buffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 50 mM NaF, 1 mM sodium orthovanadate, 0.1% NP40, and 0.5 mM ß-glycerophosphate supplemented with leupeptin and aprotinin (50 µg/ml each). Cells were incubated on ice for 10 min, vortexing every 2 min. Lysates were clarified at 14,000 rpm, and the supernatant was collected (cytosolic fraction). Pellets were resuspended in a buffer containing 20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT. After a 20-min incubation on ice, insoluble material was sedimented, and the supernatant was collected (nuclear fraction). The nuclear fractions were then dialyzed for 1 h at 4°C in a buffer containing 20 mM HEPES (pH 7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 1 mM PMSF. For EMSA, 7.5 µg of nuclear protein were incubated on ice for 15 min with poly(deoxyinosinic-deoxycytidylic acid) at 1 mg/ml and 20,000 cpm of labeled probe in binding buffer containing 10 mM HEPES (pH 7.9), 0.5 mM DTT, 0.1 mM EDTA, 50 mM KCl, 15% glycerol, and 5 mM MgCl2. Reactions were resolved on 4% acrylamide gels and autoradiographed.

NF-{kappa}B Immunofluorescence Assay.
H157 cells were grown on coverslips to 60–80% confluence and incubated either with PBS, sulindac (3 mM), or indomethacin (25 µM) for 2 h before TNF-{alpha} (10 ng/ml) treatment for 20 min. Coverslips were then washed with PBS and fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were washed with PBS and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Cells were then incubated for 20 min in a moisture chamber with 3% normal donkey serum. Coverslips were incubated for 1 h at room temperature with a NF-{kappa}B p65 rabbit polyclonal antibody (sc-372; Santa Cruz Biotechnology) and a lamin-B goat polyclonal antibody (sc-6217; Santa Cruz Biotechnology). Cells were washed three times with PBS and then incubated with donkey antirabbit FITC-tagged and donkey antigoat rhodamine red-X tagged secondary antibody for 1 h. Cells were then washed three times in PBS (30 min total), and coverslips were mounted and analyzed with confocal microscopy.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Sulindac Enhances TNF-{alpha}-induced Cytotoxicity.
The purpose of these studies was to determine whether pharmacological suppression of NF-{kappa}B activation by sulindac potentiated cell killing by TNF-{alpha} (13) . To evaluate the effects of sulindac and TNF-{alpha} on cell proliferation and cell death, we used the NSCLC cell lines H1299 and H157. The majority of NSCLCs in general are refractory to currently available chemotherapy agents; thus, these cells provide a demanding test of this concept. H1299 is derived from a large cell lung carcinoma, and H157 is derived from a squamous cell lung carcinoma (21) .

TNF-{alpha} by itself had virtually no effect on the growth of the two cell lines. Cells exposed to TNF-{alpha} continued to proliferate at a rate similar to untreated cells (Fig. 1)Citation . Sulindac (1 mM) alone caused only a 30% reduction in the number of viable cells for either of the NSCLC cell lines over the course of 1–2 days. This minor effect on viability is similar to that of colon carcinoma cell lines exposed to sulindac (15) . The combination of TNF-{alpha} (1.25–20 ng/ml) and sulindac (1 mM) resulted in a dramatic reduction in the number of viable cells over the same time period (Fig. 1)Citation . Within 1–2 days, the majority of cells of both types died, and the number of viable cells was <10% of the original number. Doses of TNF-{alpha} as low as 1.25 ng/ml in combination with sulindac (1 mM) resulted in death of nearly all of the cells. Thus, low concentrations of TNF-{alpha} result in large amounts of cell death in the presence of sulindac. By contrast, the NSAID indomethacin at 25 µM, a concentration that nearly completely inhibits COX-2 activity, had no effect on cell growth either alone or when combined with TNF-{alpha} (Fig. 1)Citation . The rates of proliferation of NSCLC cell lines H358 and H661 were not affected by treatment with TNF-{alpha} and/or sulindac (data not shown).



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  		Fig. 1. Sulindac enhances TNF-{alpha}-mediated cell death. H1299 (left) and H157(right) cell viability as measured by the MTT assay is shown. Cells were treated with the indicated amounts of TNF-{alpha} after a 2-h incubation with either sulindac (1 mM) or indomethacin (25 µM). Bars, SE.

 
Sulindac and TNF-{alpha} Synergistically Induce Cell Death by a Caspase-mediated Apoptotic Pathway.
TNF-{alpha} causes cell death by activating caspases that trigger a well-characterized death pathway (22) . To ascertain that the observed reduction in cell viability was attributable to apoptosis, terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick end labeling and flow cytometry was performed to detect apoptotic cells. Sulindac and TNF-{alpha} caused a significantly greater amount of apoptotic cells than either sulindac or TNF-{alpha} treatments alone (Fig. 2)Citation . Indomethacin (25 µM) alone and with TNF-{alpha} did not result in levels of apoptosis above baseline (not shown). These results were consistent with the cell viability assays (Fig. 1)Citation .



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  		Fig. 2. Flow cytometric analysis of H1299 cells undergoing apoptosis. H1299 cells were exposed to sulindac (1 mM), indomethacin (25 µM), or in combination with TNF-{alpha} (20 ng/ml) for 36 h. At the end of incubation, cells were harvested and subjected to TdT-mediated dUTP nick-end labeling assay using the Apoptosis Detection System, Fluorescein kit from Promega as described in "Materials and Methods." Results analyzed with CellQuest software (Becton-Dickinson) are shown for control, sulindac, TNF-{alpha}, and sulindac + TNF-{alpha} groups. The percentage of cells undergoing apoptosis as indicated by FITC-dUTP labeling in different groups is given above the bars in each panel. The number of apoptotic cells treated with indomethacin alone or in combination with TNF-{alpha} was similar to control (data not shown).

 
To confirm the role of caspases in the apoptosis, we measured caspase-3 activity. Although TNF-{alpha} treatment alone resulted in basal levels of apoptosis, treatment of cells with TNF-{alpha} and sulindac resulted in a dramatic increase in caspase-3 activity, with a 35-fold increase over basal levels (Fig. 3A)Citation . Sulindac alone resulted in levels of caspase-3 activity that were only 3-fold above basal levels. Indomethacin, alone or in combination with TNF-{alpha}, did not result in levels of caspase-3 activity that were above baseline (Fig. 3A)Citation . Treatment of cells with the caspase inhibitor Z-VAD-FMK (25 µM) significantly inhibited cell death in the presence of sulindac and TNF-{alpha} (Fig. 3B)Citation . These data support the idea that TNF-{alpha} and sulindac induce cell death through a caspase-mediated apoptotic pathway.



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  		Fig. 3. Sulindac and TNF-{alpha} induce apoptosis through a caspase-mediated pathway. A, caspase-3 activity of H1299 cells was measured 24 h after being treated with or without TNF-{alpha} (1.25–20 ng/ml) in the presence or absence of a 2-h preincubation with sulindac (1 mM) or indomethacin (25 µM). Bars, SE. B, numbers of H1299 cells after TNF-{alpha} and sulindac treatment with and without the caspase inhibitor Z-VAD-FMK. H1299 cells were treated with sulindac (1 mM) and TNF-{alpha} (10 ng/ml) as above, with and without a 30-min pretreatment with 25 µM Z-VAD-FMK. MTT assays were performed 30 h after the addition of TNF-{alpha}, and the number of viable cells was compared with that of untreated cells. Bars, SE.

 
Sulindac Inhibits NF-{kappa}B DNA Binding and the Translocation of NF-{kappa}B to the Nucleus.
One explanation for the synergy in apoptosis observed with TNF-{alpha} and sulindac treatment is that sulindac inhibits the nuclear translocation of NF-{kappa}B and thus NF-{kappa}B activation of cellular genes. To test this idea, gel retardation analysis was performed with nuclear extracts prepared from H1299 cells and H157 cells. Nuclear extracts were incubated with oligonucleotides corresponding to NF-{kappa}B binding sites. Stimulation of NF-{kappa}B binding in response to TNF-{alpha} was inhibited by pretreatment of cells with sulindac but not by indomethacin (Fig. 4A)Citation . The specificity of this binding has been tested previously in H1299 and H157 cells by supershift assays and competitor assays with unlabeled oligonucleotides (23 , 24) . Confocal microscopy analysis with TNF-{alpha}-treated H157 cells also demonstrated the inhibition of NF-{kappa}B nuclear translocation by treatment with sulindac but not by indomethacin (Fig. 4B)Citation . Lamin-B (red), which localizes to the nuclear membrane is shown. Sulindac, but not indomethacin, blocked the nuclear translocation of the NF-{kappa}B p65 component (green). These results indicate that sulindac inhibits NF-{kappa}B nuclear translocation.



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  		Fig. 4. Sulindac inhibits nuclear translocation of NF-{kappa}B. A, H157 (top) or H1299 (bottom) cells were either untreated or treated with sulindac (Sul; 3 mM) or indomethacin (Indo; 25 µM) before TNF-{alpha} treatment (10 ng/ml) for 20 min. Untreated (Unt) cells were not treated with an NSAID or TNF-{alpha}. Nuclear extracts were prepared and analyzed by EMSA for NF-{kappa}B binding activity. Duplicate gels using SP1 oligo were run to ascertain that lanes were loaded equally (not shown). B, H157 cells were treated as above, and immunofluorescence was used to track NF-{kappa}B p65 localization (green), with lamin B as a marker of the nuclear membrane (red).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
TNF-{alpha} either alone or in combination with sulindac was tested for its ability to induce apoptosis of lung cancer cells. We found that TNF-{alpha} or sulindac alone had little effect on apoptosis, whereas the combination of these agents dramatically increased apoptosis of lung cancer cells. In addition, sulindac inhibited the nuclear translocation and DNA binding of NF-{kappa}B. These effects were not seen with the NSAID indomethacin, which does not have NF-{kappa}B inhibitory activity. Taken together, these results suggest additional preclinical studies are warranted to determine the potential efficacy of combining sulindac (or its metabolites) or similar NF-{kappa}B inhibitory agents with standard chemotherapy agents. Obviously, both preclinical and Phase I clinical trials will be required to monitor toxicity of such combination approaches.

Many current anticancer agents are directed at inducing apoptosis of malignant cells, and thus resistance to apoptosis is a major problem in achieving effective cell killing of cancer cells. Paradoxically, many of the agents that induce apoptosis also activate pathways that promote antiapoptotic signals, thereby mediating the resistance of the tumor to the drug (4 , 5) . One mechanism resulting in resistance to apoptosis after chemotherapy is the activation of the transcription factor NF-{kappa}B (2 , 4 , 5 , 19 , 25) . The NF-{kappa}B family of transcription factors is activated by many stress stimuli (26) and promotes the transcription of genes that allow resistance to apoptosis. When activated, NF-{kappa}B increases the expression of several antiapoptotic genes, such as the cellular inhibitors of apoptosis c-IAP1 and c-IAP2 (27) . Once induced by NF-{kappa}B, these genes are capable of suppressing the activation of the caspase cascade that results in cell death. This mechanism is important for the survival of cancer cells and suggests a role for NF-{kappa}B in tumor growth (10) . Furthermore, several genes important for angiogenesis and metastasis are also up-regulated by NF-{kappa}B (28) . Thus, activation of the NF-{kappa}B pathway by chemotherapy may in part explain resistance in solid tumors.

TNF-{alpha} is an inflammatory cytokine that can induce apoptosis but also potently activates the NF-{kappa}B pathway. Because sulindac inhibits NF-{kappa}B activation via its inhibition of the kinase IKKß, which is a critical step in activating this pathway, we tested how cells would respond to TNF-{alpha} in the presence of sulindac. Our data demonstrate that TNF-{alpha}, over a wide range of concentrations, in combination with sulindac results in synergistic cytotoxicity in two histologically distinct NSCLC cell lines. Furthermore, the mechanism by which these agents induce cell death is by apoptosis, as shown by the increased caspase activity seen with this treatment. Notably, treatment of the NSCLC cell lines H358 and H661 with TNF-{alpha} and/or sulindac did not result in increased levels of cell death as compared with untreated cells (data not shown). The lack of response was likely attributable to defects in TNF-{alpha} binding and NF-{kappa}B activation. Thus, the susceptibility of cells to TNF-{alpha} and sulindac will likely differ among various neoplasms.

The dramatic effects of TNF-{alpha} and sulindac suggest that sulindac inhibition of NF-{kappa}B may serve as an effective therapy in cancer. However, the concentration of sulindac used in our studies (1–3 mM) is greater than the serum concentration achieved in patients by conventional administration (29) . Sulindac sulfide and sulindac sulfone are much more potent inhibitors of the NF-{kappa}B pathway than sulindac and reach comparable serum concentrations (13 , 29) . Although we have not yet tested combination therapy of sulindac sulfide and sulindac sulfone with TNF-{alpha}, their ability to even more potently block NF-{kappa}B may make them better agents than sulindac. Because sulindac induces the regression of neoplastic colonic polyps, others recently have utilized sulindac sulfide and sulindac sulfone in combination with standard chemotherapy. They have found that the sulfide and sulfone metabolites of sulindac do indeed synergize with paclitaxel and cisplatin to enhance cytotoxicity (30) . In the future, the development of other agents that potently block the NF-{kappa}B pathway may prove important in cancer chemotherapy.

Our proposed model of sulindac inhibition of NF-{kappa}B activation helps to explain the greater cytotoxicity observed with these combination therapies. Many commonly used therapeutic agents activate NF-{kappa}B, perhaps as a result of the stress associated with their interference in a variety of cellular processes. The anthracycline antibiotics daunorubicin and doxorubicin that cross-link DNA activate NF-{kappa}B, and their cytotoxicity is greatly enhanced by inhibition of NF-{kappa}B (2) . Paclitaxel, camptothecin, CPT-11, and cisplatin are other therapeutic agents known to activate NF-{kappa}B in addition to their effects on causing apoptosis of cancer cells (28) . Combination therapy with these agents and sulindac may prove to be more effective than chemotherapy alone against certain forms of cancer.

There are at least four reported mechanisms of action attributed to sulindac and its metabolites that can lead to apoptosis. Although all three forms of sulindac inhibit NF-{kappa}B, the sulfide metabolite also can block COX-2 and inhibit PPAR{delta} (31) . Sulindac sulfone can inhibit phosphodiesterase (32) . Although some have attributed the apoptosis of sulindac to COX-2 inhibition, others have shown that cells that do not express COX-2 undergo apoptosis and that levels of prostaglandins are not necessarily affected by sulindac (33) . Furthermore, sulindac and its sulfone metabolite do not efficiently block COX-2 activity, so their ability to cause apoptosis is likely independent of COX-2 inhibition. The ability of sulindac and the sulfone metabolite to block PPAR{delta} has not been reported. The ability of sulindac sulfone to inhibit phosphodiesterase is currently being investigated. The inhibition of the NF-{kappa}B pathway may lead to the increased apoptosis observed in carcinoma cell lines in response to treatment with TNF-{alpha} in combination with sulindac, sulindac sulfide, and sulindac sulfone. Inhibition of NF-{kappa}B is a common mechanism by which sulindac and its metabolites can cause apoptosis, as they all are capable of inducing cell death at concentrations at which they also block IKKß activity.

The use of NSAIDs in cancer therapy has attracted much attention since the discovery that sulindac and aspirin can cause regression of adenomatous polyps in the colon. However, the diverse actions of these agents have generated much controversy over the mechanisms responsible for their anticancer effects (see above). Inhibition of NF-{kappa}B both in cell culture and mouse models of cancer promotes apoptosis, and thus NSAID blockade of NF-{kappa}B is an attractive explanation for their anticancer effects. Although the role of blocking COX-2 in cancer may not necessarily be to induce apoptosis, it may be important in blocking tumor growth by inhibiting angiogenesis (34) .

Sulindac or one of its metabolites that can block COX-2 as well as NF-{kappa}B may be the NSAID that is most useful in cancer therapy. In particular, the metabolite sulindac sulfide shows promise because it is the most potent inhibitor of IKKß and blocks both COX-2 and PPAR{delta} function. Cancer therapies that activate the NF-{kappa}B pathway induce much more apoptosis in cancer cells when the NF-{kappa}B pathway is blocked (2) . As a result, future studies will focus on chemotherapy combinations using sulindac with therapies known to activate the NF-{kappa}B pathway. Because chemotherapeutic drugs commonly used for many different forms of cancer activate NF-{kappa}B, treatment with sulindac may improve current chemotherapy regiments for the treatment of solid tumors.


   ACKNOWLEDGMENTS
 
We thank members of the Cobb, Minna, and Gaynor labs for assistance and guidance. We thank Hal Berman for intellectual insight and Alejandra Herrera for assistance in preparing the figures.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants P50 CA70907 (Lung Cancer Specialized Program of Research Excellence; to J. D. M.), CA74128 (to R. B. G.), and GM53032 (to M. H. C.) from the NIH and a grant from the Cancer Research Foundation of North Texas. K. S. B. was supported in part by the Medical Scientist Training Program and the Perot Family Foundation. Back

2 To whom requests for reprints should be addressed, Division of Hematology-Oncology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8594. Phone: (214) 648-4996; Fax: (214) 648-4152; E-mail: gaynor{at}utsw.swmed.edu Back

3 The abbreviations used are: NF-{kappa}B, nuclear factor-{kappa}B; TNF, tumor necrosis factor; NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; NSCLC, non-small cell lung cancer; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium bromide; Z-VAD-FMK, z-Val-Ala-Asp-fluoromethyl ketone; EMSA, electrophoretic mobility shift assay; PMSF, phenylmethylsulfonyl fluoride; PPAR{delta}, peroxisome proliferator-activated receptor {delta}. Back

Received 7/30/01; revised 11/ 9/01; accepted 11/21/01.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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