[CANCER RESEARCH 60, 3338 –3342, July 1, 2000] Advances in Brief Exisulind Induction of Apoptosis Involves Guanosine 3,5-Cyclic Monophosphate Phosphodiesterase Inhibition, Protein Kinase G Activation, and Attenuated -Catenin W. Joseph Thompson, 1 Gary A. Piazza, Han Li, Li Liu, John Fetter, Bing Zhu, Gerhard Sperl, Dennis Ahnen, and Rifat Pamukcu Cell Pathways, Inc., Horsham, Pennsylvania 19044 [W. J. T., G. A. P., H. L., L. L., J. F., G. S., R. P.]; Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688 [W. J. T., B. Z.]; and University of Colorado and the Denver Veterans Affairs Medical Center, Denver, Colorado 80220 [D. A.] Abstract Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition. SW480 colon tumor cells contain guanosine 3,5-monophosphate (cGMP) phosphodiesterase (PDE) isoforms of the PDE5 and PDE2 gene families that are inhibited by exisulind and new synthetic analogues. The analogues maintain rank order of potency for PDE inhibition, apoptosis induction, and growth inhibition. A novel mechanism for exisulind to induce apoptosis is studied involving sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction not found with selective PDE5 or most other PDE inhibitors. Accumulated -catenin, shown to be a substrate for PKG, is decreased by exisulind, suggesting a mechanism to explain apoptosis induction in neoplastic cells harboring adenomatous polyposis coli gene mutations. Introduction Chemotherapeutic and chemopreventive agents such as sulindac and similar NSAIDs 2 induce apoptosis (1, 2). Exisulind, the oxidative me- tabolite of sulindac, induces apoptosis and inhibits growth of tumor cell lines of diverse origins (3–5), suggesting that an important survival pathway is modified by the drug. Exisulind is not an NSAID because it lacks the hallmark cyclooxygenase inhibitory activities of NSAIDs (6). The mechanism of exisulind-induced apoptosis is independent of p53, Bcl-2, and cell cycle arrest (4, 5). Exisulind inhibits tumor growth in rodent models of colon, mammary, prostate, bladder, and lung carcino- genesis (3, 6 – 8). We report here studies indicating that exisulind induces apoptosis in colon tumor cells by inhibiting cGMP PDE, causing a persistent increase in cellular cGMP, and inducing PKG. This approach has led to the development of a novel new class of chemopreventive and antineoplastic drugs that lack NSAID-induced gastrointestinal, renal, and hematological toxicities. However, exisulind did show dose-limiting tox- icity of mild to moderate hepatic enzyme elevations in some FAP patients that was reversible on dose reduction. Cyclic nucleotide PDEs consist of 10 gene families, each having one or more isoforms. These enzymes are being used as pharmaceutical targets for new drugs designed to manipulate cellular processes modu- lated by cAMP and/or cGMP (9 –12). PDE inhibitors influence many pathologies, but their use as anticancer agents has not been developed (13, 14). The majority of PDE isozyme inhibitors are not proapoptotic in epithelial-derived tumor cells, although inhibitors of PDE1 and PDE4 isoforms induce apoptosis in lymphoid cells (14, 15). We found that like exisulind, nonselective PDE5 inhibitors MY5445 and dipyridamole in- duced apoptosis in HT29, SW480, and T84 human colon tumor cell lines used for these studies. Therefore, the hypothesis that exisulind may induce apoptosis via cGMP PDE inhibition was tested. Materials and Methods Cell Growth. SW480 and HT29 cells were grown in RPMI 1640, 2 mM glutamine, 1% antibiotic/antimycotic solution, and 5% FBS in 150-cm 2 flasks or dishes. SW480 cells were also grown in Corning 850-cm 2 roller bottles with the addition of 25 mM HEPES for the fast protein liquid chromatography profile. T84 cells were grown in 47% ATCC Ham’s F-12 media, 47% Sigma DMEM, 1% antibiotic/antimycotic solution, 8.4 mM sodium bicarbonate, and 5% serum, pH 7.25. Cell lines were grown using serum from Sigma in 5% CO 2 at 37°C. Cells were harvested at 70 –100% confluence with either Trypsin/ EDTA (Life Technologies, Inc.) or Pancreatin (Life Technologies, Inc.) and either used fresh or were frozen on liquid nitrogen and stored at -70°C. Protein Purification. SW480 cells were grown in roller bottles at 0.5 rpm. Approximately 600 million cells were manually homogenized in 5 mM Tris- acetate, 5 mM magnesium acetate, 0.1 mM EDTA, 0.8% Triton X-100, 10 M benzamidine, 10 M N--p-tosyl-L-lysine chloromethyl ketone (TLCK), 2000 units/ml aprotinin, 2 M leupeptin, and 2 M pepstatin A (pH 7.5). After ultracentrifugation at 100,000  g at 4°C for 1 h, supernatants were diluted 5-fold with the buffer minus Triton and loaded at 1 ml/min onto an 18-ml DEAE Trisacryl M column (BioSepra) using Pharmacia AKTA/fast protein liquid chro- matography. The column was washed with 8 mM TRIS-acetate, 5 mM magnesium acetate, and 0.1 mM EDTA (pH 7.5). Enzymes were eluted with a gradient of 0 –1 M sodium acetate at a flow rate of 1 ml/min into 1.5-ml fractions. Apoptosis and Cell Growth Inhibition. DNA fragmentation in SW480 cells at 10,000 cells/well in 96-well plates was measured using a double antibody ELISA kit (Boehringer Mannheim) that detects DNA/histone com- plexes. After 24 h, cells were dosed and grown for an additional 48 h. Growth inhibition was determined by plating cells at 1000 cells/well in 96-well plates. Cells were dosed after 24 h and incubated for 6 days. Cells were fixed with 10% trichloroacetic acid at 4°C for 1 h, rinsed five times with deionized H 2 O, and incubated for 10 min with 0.4% sulforhodamine B in 1% acetic acid. Plates were rinsed four times with 1% acetic acid, dried 30 min, and solubilized in 10 mM Tris. Absorbance was determined at 540 nM using a Molecular Devices Spectra Max 340 plate reader. cGMP and cAMP RIA. cGMP and cAMP levels were measured by RIA. Approximately 5  10 6 cells were used for each assay. After drug treatment, cells were washed with cold PBS. Cyclic nucleotides were extracted with 0.2 N HCl/50% methanol and dried. The dried samples were reconstituted in water and acetylated before RIA with anti-cGMP and anti-cAMP antibodies. The results were expressed in fmol of cGMP/cAMP generated per mg protein of the cells. PKG Activation. SW480 cells were treated with compounds for 48 h, and PKG activity was measured using a substrate of cloned GST fusion protein of a fragment of human PDE5 bound to GSH-Sepharose affinity beads. The PDE5 fragment contains its phosphorylation site (Ser-92) and cGMP binding domains (residue 35–530, relative to bovine PDE5). Cell lysate (100 g), substrate (20 g), 0.25 M protein kinase inhibitor, 4.5 mM magnesium, and [- 32 P]ATP (10 Ci; 190 M) with or without added cGMP (8 M) were Received 2/3/00; accepted 5/18/00. 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 To whom requests for reprints should be addressed, at Cell Pathways, Inc., 702 Electronic Drive, Horsham, PA 19044. 2 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; cGMP PDE, guanosine 3',5'-cyclic monophosphate phosphodiesterase; PKG, protein kinase G; FAP, familial adenomatous polyposis; GST, glutathione S-transferase; APC, adenomatous polyposis coli. 3338 Research. on August 18, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from