Nonsteroidal Anti-Inflammatory Drugs Induced Endothelial Apoptosis by Perturbing Peroxisome Proliferator-Activated Receptor- Transcriptional Pathway

Recent studies have shown that use of nonsteroidal antiinflammatory drugs (NSAIDs) is associated with an increased risk of myocardial infarction. To explore whether NSAIDs may induce endothelial apoptosis and thereby enhance atherothrombosis, we treated human umbilical vein endothelial cells (HUVECs) with sulindac sulfide (SUL), indomethacin (IND), aspirin (ASA), or sodium salicylate (NaS), and we analyzed apoptosis. SUL and/or IND significantly increased annexin V-positive cells, cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3. ASA and NaS at 1 mM did not induce PARP cleavage or caspase-3 and at 5 mM, ASA but not NaS increased apoptosis. Because peroxisome proliferator-activated receptor -mediated 14-3-3 up-regulation was reported to play a crucial role in protecting against apoptosis, we determined whether NSAIDs suppress this transcriptional pathway. SUL, IND, and ASA (5 mM) suppressed PPAR and 14-3-3 proteins in a manner parallel to PARP cleavage. Neither ASA nor NaS at 1 mM interfered with PPAR or 14-3-3 expression. SUL inhibited PPAR promoter activity, which correlated with 14-3-3 promoter suppression. Suppression of 14-3-3 was associated with increased Bad translocation to mitochondria. Neither carbaprostacylin nor 4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy)propyloxyphenoxy acetic acid (L-165041) prevented HUVECs from SUL-induced apoptosis. Because of suppression of ectopic PPAR by sulindac, adenoviral PPAR transduction failed to restore 14-3-3 or prevent PPAR cleavage. Our findings suggest that NSAIDs, but not aspirin ( 1 mM) induce endothelial apoptosis via suppression of PPAR -mediated 14-3-3 expression. Nonsteroidal anti-inflammatory drugs (NSAIDs) make up a group of compounds of diverse chemical structures distinct from corticosteroids, but they possess common steroid-like anti-inflammatory actions (Simon and Mills, 1980). Recent reports reveal that NSAIDs such as sulindac and indomethacin prevent carcinogen-induced tumors in rats (Narisawa et al., 1981; Pollard and Luckert, 1983; Piazza et al., 1995) and reduce adenomas in Min mice, which spontaneously develop adenomatous polyposis (Boolbol et al., 1996; Jacoby et al., 1996). Epidemiological and controlled human clinical trials have confirmed that NSAID use reduces the risk of cancers and reduces adenomas in familial adenomatous polyposis (Kune et al., 1988; Giardiello et al., 1993; Steinbach et al., 2000). Although the mechanisms by which NSAIDs control cancer growth are not completely understood, several reports suggest that they induce cancer cell apoptosis (Shiff et al., 1995). NSAIDs induce apoptosis by tilting the balance between antiapoptotic and proapoptotic Bcl-2 family proteins toward the proapoptotic members, notably, Bax and Bad (Sheng et al., 1998; Zhang et al., 2000). Results from our laboratory suggest that the antitumor actions of sulindac and indomethacin are attributable to suppression of poly(ADP-ribose) polymerase (PPAR)) and PPAR -mediated 14-3-3 expression, resulting in reduction of cytosolic 14-3-3 proteins and consequently and increase in Bad translocation to mitochondria in which it induces apoptosis (Liou et al., 2007b). Aspirin has been reported to possess similar antitumor This work was supported by grants from National Health Research Institutes of Taiwan and National Institutes of Health Grant HL50675. J.-Y.L. and C.-C.W. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.049569. ABBREVIATIONS: NSAID, nonsteroidal anti-inflammatory drug; PPAR, peroxisome proliferator-activated receptor; COX, cyclooxygenase; HUVEC, human umbilical vein endothelial cell; cPGI2, carbaprostacyclin; L0165041, 4-(3-(2-propyl-3-hydroxy-4-acetyl)phenoxy)propyloxyphenoxy acetic acid; HSP60, 60-kDa heat shock protein; PPRE, peroxisome proliferator response element; Ad, adenovirus; GFP, green fluorescent protein; pNA, p-nitroaniline; CHO, aldehyde; MG-132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PG, prostaglandin; SUL, sulindac sulfide; IND, indomethacin; ASA, aspirin; NaS, sodium salicylate. 0026-895X/08/7405-1399–1406$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 5 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 49569/3394153 Mol Pharmacol 74:1399–1406, 2008 Printed in U.S.A. 1399 at A PE T Jornals on A uust 4, 2017 m oharm .aspeurnals.org D ow nladed from properties as NSAIDs. Aspirin was efficacious in preventing carcinogen-induced colon tumors in rats (Craven and DeRubertis, 1992; Reddy et al., 1993). Epidemiological studies show that aspirin use is associated with a significant reduction in cancers, notably colon cancers in humans (Kune et al., 1988; Thun et al., 1991). A randomized double-blind clinical trial has shown that aspirin reduces adenomatous polyposis in familial adenomatous polyposis patients (Baron et al., 2003). Aspirin and sodium salicylate at very high concentrations ( 10 mM) were reported to induce apoptosis of human leukemia and lymphoma cells (Bellosillo et al., 1998; Klampfer et al., 1999; Pique et al., 2000). However, it remains unknown whether aspirin at therapeutic concentrations ( 1 mM) causes cell damage. Furthermore, mechanism by which high, suprapharmacological concentrations of aspirin and salicylate ( 5 mM) induce apoptosis has not been elucidated. It is well recognized that selective cyclooxygenase (COX)-2 inhibitors are associated with increased rick of myocardial infarction (Mukherjee et al., 2001; Fitzgerald, 2004). Recent reports indicate that all classes of NSAIDs are also associated with risk of myocardial infarction (Chan et al., 2006). The reasons for the adverse cardiovascular complications of NSAIDs are not entirely clear. Because NSAIDs induce cancer cell apoptosis, we postulated that their cardiovascular toxicity may be due to endothelial cell apoptosis. In this report, we evaluated the effect of sulindac and indomethacin as well as aspirin and sodium salicylate on PARP cleavage and/or other apoptotic markers in human umbilical vein endothelial cells (HUVECs), and we determined changes in PPAR and 14-3-3 expressions. The results show that sulindac and indomethacin induced apoptosis and suppressed PPAR and 14-3-3 expressions in a correlative manner. Aspirin and sodium salicylate at 1 mM had no effect, but aspirin at 5 mM exerted a similar apoptotic action as NSAIDs. Materials and Methods Pharmacological Reagents. Sulindac sulfide and indomethacin were purchased from Calbiochem (San Diego, CA), and aspirin and sodium salicylate were from Sigma-Aldrich (St. Louis, MO). All reagents except sodium salicylate were dissolved in ethanol. Sodium salicylate was dissolved in filtered distilled water. Carbaprostacylin (cPGI2; 50 M) and L-165041 (50 M) were obtained from Cayman Chemical (Ann Arbor, MI). They were dissolved in ethanol. The final concentrations of ethanol vehicle were 0.1%. Cell Culture. HUVECs were collected from fresh umbilical veins and cultured as described previously (Xu et al., 1999). In all experiments, only cultures up to five passages were used before experiments. HUVECs were washed and cultured in serum-free medium containing the testing drugs for 24 h. Because it was difficult to transfect HUVECs with conventional liposome-based carriers, we used ECV304 cells for selected transfection experiments. ECV304 cells are immortalized cells derived from HUVECs. They retain cobble stone-like morphology and stain positively for von Willebrand factor. Like HUVECs, they possess enzymes for prostacyclin synthesis. ECV304 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. Preparation of Mitochondrial Fraction. HUVECs treated with sulindac sulfide for 4 h were washed three times with phosphatebuffered saline and harvested by centrifugation. Mitochondrial fractions were prepared by a mitochondria isolation kit (Sigma-Aldrich) as described previously (Liou et al., 2006). The mitochondrial and cytosolic fractions were purified by two-step gradient centrifugation and stored at 20°C. Mitochondrial and cytosolic Bad protein levels were analyzed by Western blotting. The 60-kDa heat shock protein (HSP60) was used as a mitochondrial marker. Western Blot Analysis. HUVECs were lysed with radioimmunoprecipitation assay buffer containing protease inhibitors. Western blots were performed as described previously (Liou et al., 2006). For cell apoptosis evaluation, 20 g of lysate protein was applied to each lane. A rabbit PARP antibody (1:1000 dilution) (Cell Signaling Technology Inc., Danvers, MA) was used to detect full-length PARP (116 kDa) and cleaved PARP (carboxyl-terminal catalytic fragment, 89 kDa). For 14-3-3 analysis, 20 g of lysate protein was applied to each lane. 14-3-3 was detected with a rabbit polyclonal antibody (1:2000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For PPAR protein analysis, 30 g of cell lysate proteins was loaded to each lane. PPAR was detected with a rabbit polyclonal antibody against PPAR (1:500 dilution) (Cayman Chemical). Donkey antirabbit IgG conjugated with horseradish peroxidase was purchased from Santa Cruz Biotechnology, Inc. Protein bands were visualized by enhanced chemiluminescence (Pierce Chemical, Rockford, IL). Plasmid Constructs and Luciferase Reporter Assay. The human 14-3-3 promoter ( 1625 to 24) was subcloned into pGL3 luciferase reporter as described previously (Liou et al., 2006). PPRE reporter conjugated to luciferase was kindly provided by Drs. K. W. Kinzler and B. Vogelstein (Johns Hopkins University, Baltimore, MD; He et al., 1999). To achieve high transfection efficiency, the endothelial-like ECV304 cells were treated with a mixture containing reporter constructs and FuGENE 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) for 24 h, washed, and replaced with fresh medium containing the testing drugs for an additional 24 h. Cells were lysed, and the luciferase activity was measured using a kit from Promega (Madison, WI). The emitted light was determined in a luminometer. Protein concentrations of cell lysates were determined by a protein assay kit (Bio-Rad, Hercules, CA). Luciferase activity was expressed as relative light units per microgram of