Peroxisome Proliferator-Activated Receptor- Is a Target of Nonsteroidal Anti-Inflammatory Drugs Mediating Cyclooxygenase-Independent Inhibition of Lung Cancer Cell Growth

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of different cancer cell types, suggesting a broad role for their cyclooxygenase (COX) targets and eicosanoid products in tumor cell growth. Sulindac sulfide, a COX inhibitor, inhibited the growth of non–small-cell lung cancers (NSCLC) both in soft agar and as xenografts in nude mice. Importantly, the concentration of sulindac sulfide required to inhibit NSCLC cell growth greatly exceeded the concentration required to inhibit prostaglandin (PG) E2 synthesis in NSCLC cells, suggesting that NSAID inhibition of cell growth is mediated by additional targets distinct from COX. Both sulindac sulfide and ciglitazone, a defined peroxisome proliferator-activated receptor(PPAR ) agonist, stimulated a promoter construct containing a PPAR response element linked to luciferase and potently inhibited NSCLC cell growth at similar concentrations, indicating a role for PPAR as a target of NSAID action in these cells. Overexpression of PPAR in NSCLC cells strongly inhibited the transformed growth properties of the cells, providing a molecular confirmation of the results obtained with the PPAR agonists. Increased expression of PPAR , as well as ciglitazone and sulindac sulfide induced expression of E-cadherin, which has been linked to increased differentiation of NSCLC. Despite the fact that SCLC cell lines expressed little or no cytosolic phospholipase A2, COX-1, or COX-2, sulindac sulfide and PPAR agonists also inhibited the transformed growth of these lung cancer cells. We propose that PPAR serves as a target for NSAIDs that accounts for COX-independent inhibition of lung cancer cell growth. Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of compounds that block eicosanoid production through the inhibition of cyclooxygenase (COX) activity (Smith et al., 1994). In addition to their general use as inhibitors of inflammation, pain, and fever, NSAIDs have an emerging utility as chemotherapeutics for the prevention and treatment of human cancer (Marnett, 1992; Duperron and Castonguay, 1997). The observed chemoprevention of colon cancer by the NSAID sulindac (Rao et al., 1995) and epidemiological studies indicating that NSAIDs decrease the risk for developing lung cancer (Schreinemachers and Everson, 1994) are consistent with an emerging role for eicosanoid biosynthetic pathways in human cancer development. A large number of studies have now demonstrated that NSAIDs may exert some of their cellular actions through COX-independent mechanisms (reviewed in Tegeder et al., 2001). Among these potential targets of NSAIDs is the peroxisome proliferator-activated receptor (PPAR) family of nuclear receptors that function as ligand-dependent transcription factors (Spiegelman, 1997). Three isoforms have been described, PPAR , , and , all of which bind to specific DNA sequences as heterodimers with the retinoic acid X-receptors (DiRenzo et al., 1997). PPAR has been shown to be activated by the synthetic antidiabetic thiazolidinediones, such as ciglitazone and troglitazone (Lehmann et al., 1995), as well as by prostaglandin D and J derivatives, which may function as endogenous activators (Forman et al., 1995). Whereas the function of PPAR in the setting of human cancer is controversial, recent findings indicate that loss of PPAR expression is associated with colon tumorigenesis, and activation of PPAR leads to inhibition of anchorageindependent growth of colon cancer cell lines (Brockman et Supported by National Institutes of Health grants CA58157, DK19928, and DK39902. ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; COX, cyclooxygenase; PPAR, peroxisome proliferator-activated receptor; SCLC, small-cell lung cancer; NSCLC, non–small-cell lung cancer; cPLA2, cytosolic phospholipase A2; PPAR-RE, peroxisome proliferator-activated receptor–response element; TTBS, Tris-buffered saline-Tween 20; NFB, nuclear factor B; PG, prostaglandin; APC, adenomatous polyposis coli; -gal, -galactosidase. 0026-895X/02/6205-1207–1214$7.00 MOLECULAR PHARMACOLOGY Vol. 62, No. 5 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1844/1021256 Mol Pharmacol 62:1207–1214, 2002 Printed in U.S.A. 1207 at A PE T Jornals on Sptem er 9, 2017 m oharm .aspeurnals.org D ow nladed from al., 1998), suggesting that this gene may function as a tumor suppressor. Lung cancer is a heterogeneous disease that is generally categorized into small-cell lung cancer (SCLC) and non– small-cell lung cancer (NSCLC). As a group, the NSCLCs constitute the bulk of lung cancers and are subdivided into squamous, adenocarcinoma, and large-cell carcinoma phenotypes. Gain-of-function mutations in K-Ras are observed in approximately 30% of adenocarcinomas and just under 10% of other NSCLC types (Giaccone, 1996). These mutations seem to be virtually absent in SCLC (Mitsudomi et al., 1991). We and others have previously reported that a subset of NSCLC cell lines expressing oncogenic forms of Ras exhibit high levels of prostaglandin production, whereas SCLC cell lines produce little or no prostaglandins (Heasley et al., 1997). High levels of prostaglandin production by NSCLC cells are correlated with increased expression of both cytosolic phospholipase A2 (cPLA2) and COX-2 (Heasley et al., 1997). Moreover, expression of gain-of-function Ras was both necessary and sufficient to mediate increased transcription of these enzymes (Van Putten et al., 2001). Based on the restricted expression of cPLA2 and COX-2 and synthesis of prostaglandins by lung cancer cells noted in our studies and in the literature, a selective action of NSAIDs on various lung cancer cells would be predicted. In fact, preliminary studies in our laboratory revealed a widespread inhibitory action of NSAIDs on NSCLC and SCLC cell lines. In this study, we have examined the role of PPAR as a potential target of NSAIDs mediating growth inhibition of diverse lung cancer cells. In light of multiple potential effects of both NSAIDs and PPAR activators, we employed both pharmacological and molecular approaches to assess the role of this pathway as a target of NSAIDs mediating the inhibition of transformed growth of NSCLC and SCLC cells. Materials and Methods Materials. Antibodies to PPAR , cPLA2, COX-1, COX-2, and E-cadherin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Sulindac sulfide, NS-398, ciglitazone, and WY 14,463 were purchased from Biomol (Plymouth Meeting, PA). The PPAR -Gal4 expression plasmid was a generous gift of Dr. Jeffrey Flier (Beth Israel Hospital, Boston, MA). Expression plasmids encoding PPAR and constructs encoding a consensus PPAR-response element ligated to a luciferase reporter (PPAR-RE) were the gift of Carl Clay (Wake Forest University Baptist Medical Center, WinstonSalem, NC. Cell Culture and Transfection. Non–small-cell lung cancer cell lines (H2122, A549, H460) and small-cell lung cancer cell lines (H345, SHP-77) were obtained from the University of Colorado Health Sciences Center Cancer Center Tissue Culture Core. H2122, A549, H460, and SHP-77 cells were maintained in RPMI containing 10% fetal bovine serum and H345 cells were grown in HITES medium (RPMI medium containing 10 nM hydrocortisone, 5 g/ml insulin, 10 g/ml transferrin, 10 nM 17 -estradiol, 30 nM sodium selenite, and 0.1% bovine serum albumin). Cells were transfected by electroporation as described previously (Heasley et al., 1997). Two million cells were electroporated in 0.4-cm electroporation cuvettes (Bio-Rad, Hercules, CA) using a geneZAPPER (IBI, Madison, WI). After electroporation, cells were incubated in standard media for 48 h. Cells were then harvested and firefly luciferase and -galactosidase activity determined as described previously (Heasley et al., 1997). Results are expressed as luciferase units normalized to milliunits of -galactosidase. For stable transfections, the PPAR 1 cDNA (Gurnell et al., 2000) was inserted into the pLNCX2 retroviral expression vector (BD Biosciences Clontech, Palo Alto, CA) and transfected into 293T cells along with vectors encoding gag, pol, and env proteins to make recombinant virus, as described previously (Van Putten et al., 2001). Medium from the 293T cells was used to transfect the ecotropic retroviral-producing GP E-86 cell line, then medium from the infected GP E-86 cells was used to transfect the amphotropic retroviral-producing packaging cell line, PA317. Medium from the LNCX2-PPAR PA317 packaging cell line was used to stably transfect H2122 cell lines, as described above. Polybrene (8 g/ml) was added to the retrovirus-containing medium collected from the packaging cells and filtered before two sequential 24-h incubations with subconfluent layers of cells. The infected cells were replated, selected for G418 resistance, and expanded. Clones were screened for expression of PPAR by immunoblotting with a specific anti-PPAR antibody. Control cell lines (pLNCX2) were selected by infecting cells with a virus lacking a cDNA insert. Growth Assay and Tumor Cell Growth in Athymic Mice. For determination of anchorage-independent growth, single-cell suspensions of the indicated NSCLC or SCLC lines were prepared and aliquots containing 10,000 cells were suspended in 1.5 ml of RPMI 1640 medium containing 10% fetal bovine serum and 0.3% Nobel agar and layered over a base prepared in 35-mm dishes of RPMI 1640 medium, 10% fetal bovine serum, and 0.5% agarose supplemented with the various inhibitors at twice the indicated concentration. For H345 cells, HITES medium (RPMI 1640 medium with the following additives per liter: 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM -estradiol, 10 mM HEPES, and 2 mM L-glutamine) was used. The dishes were incubated for 3 to 4 weeks at 37°C in a humidified CO2 incubator. Live colonies were stained for 5 to 20 h at 37°C with nitro blue tetrazolium chloride (1 mg/ ml), visualized under a microscope, and counted. For determination of growth under standard conditions, cells were plated in 96-well plates. After 24 h, various concentrations of inhibitors were added. Cells were assayed for live cells 72 h later by the CellTiter 96 AQueous One Solution Cell Proliferation Assay ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay; Promega, Madison, WI). Results are given as percentage of live cells. For studies of tumor growth in vivo, athymic mice were inoculated subcutaneously in the flanks with the indicated tumor cells (10 cells/flank). Seven days after inoculation, mice were treated daily with sulindac sulfide (5 mg/kg) or vehicle administered intraperitoneally. Seven animals were used per treatment and tumor volumes were measured every 3 days. Immunoblot Analyses. Cells were collected in phosphate-buffered saline and, after centrifugation (5 min, 1,000g), were lysed in mitogen-activated protein kinase lysis buffer (Heasley et al., 1996). Nuclei and cell debris were removed by microcentrifugation (5 min, 10,000g) and portions containing 100 to 200 g of protein were mixed with SDS sample buffer and submitted to SDS-PAGE on 7.5% 1208 Wick et al. at A PE T Jornals on Sptem er 9, 2017 m oharm .aspeurnals.org D ow nladed from acrylamide gels. The resolved polypeptides were transferred electrophoretically to nitrocellulose (MSI, Westboro, MA) and the filters were blocked extensively in Tris-buffered saline containing 0.1% Tween 20 (TTBS) and 3% nonfat dry milk. After an incubation (16–24 h) with the indicated antibodies in TTBS/3% milk, the filters were washed with four changes of TTBS and bound antibodies were visualized with horseradish peroxidase-coupled secondary reagents and enhanced chemiluminescence according to the manufacturer’s specifications.