The Drug Salicylamide Is an Antagonist of the Aryl Hydrocarbon Receptor That Inhibits Signal Transduction Induced by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin
Total Page：16
File Type：pdf, Size：1020Kb
[CANCER RESEARCH 64, 429–434, January 1, 2004] The Drug Salicylamide Is an Antagonist of the Aryl Hydrocarbon Receptor That Inhibits Signal Transduction Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Christopher J. MacDonald, Henry P. Ciolino, and Grace Chao Yeh Cellular Defense and Carcinogenesis Section, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, Maryland ABSTRACT (a)pyrene, and heterocyclic amines such as 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine. These classes of xenobiotics are all 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environ- suspected human carcinogens. For example, TCDD, a widespread mental contaminant, that has been linked with a variety of deleterious environmental contaminant generated by a variety of industrial pro- effects on human health, including increased cancer rates and reproduc- tive anomalies. The detrimental effects of TCDD are mediated via the aryl cesses, has been linked to an increase in many types of human hydrocarbon receptor (AhR), a transcription factor that regulates the cancers, including hepatocellular cancer (3, 4). In animal models, expression of the carcinogen-activating enzymes cytochromes P-450 TCDD has been shown to cause acute toxicity (5), increased oxidative (CYP) 1A1, 1A2, and 1B1. In the present study, we examined the ability stress (6), impaired ovulation (7), and immunotoxicity (8). These of synthetic derivatives of salicylic acid to affect TCDD-stimulated AhR- effects in animal models are believed to closely reflect the deleterious mediated signal transduction in human hepatoma HepG2 cells. Salicyl- effects of TCDD in humans (9). TCDD has the highest affinity for the amide (SAL), an analgesic drug, caused a potent and long-lasting inhibi- AhR of any known compound, and is considered the prototypical AhR tion of TCDD-induced CYP enzyme activity. Acetylsalicylic acid (aspirin) ligand. Its interaction with the AhR is essential to its toxicity, as and the naturally occurring phytochemical salicylic acid had no effect on demonstrated in AhR-knockout mice, which are impervious to TCDD CYP activity. SAL inhibited the increase in CYP1A1, -1A2, and -1B1 exposure (10). mRNA levels that occurs on exposure to TCDD. TCDD-induced transcrip- tion of these genes was also inhibited by SAL, but not by aspirin or Because of the central role of the AhR in the toxicity of TCDD and salicylic acid, as demonstrated by luciferase reporter assays. The tran- genotoxicity of numerous xenobiotics, the development of inhibitors scription of the CYP1 family of genes is regulated by the interaction of of AhR-mediated signal transduction has been an important goal of TCDD-activated AhR with the xenobiotic-responsive element present in chemoprevention science. In the present study, we have examined the the promoter regions of these genes. As shown by electrophoretic mobility effects of the salicylates on the AhR and the metabolic pathway it shift assay, SAL completely blocked the binding of TCDD-activated AhR regulates. Salicylic acid was first identified in willow bark extracts as to the xenobiotic responsive element. Also, SAL substantially blocked the an active anti-inflammatory compound more than 200 years ago, and binding of TCDD to the cytosolic AhR. These results demonstrate that its synthetic derivative, acetylsalicylic acid (aspirin), has be used to SAL, a commonly used analgesic, is a potent inhibitor of AhR-mediated treat a number of human maladies for over a century. Recent studies signal transduction, and may be an effective agent in the prevention of have demonstrated that salicylate usage is associated with a reduction TCDD-associated disease. of incidence of several types of cancer (11–13), and animal studies have shown that salicylates are potent inhibitors of chemically in- INTRODUCTION duced carcinogenesis (14, 15). We have, therefore, examined the effects of salicylates on the AhR function in HepG2 cells, a well- The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed differentiated human liver cancer cell line that has been extensively cytosolic protein that is a member of the basic helix–loop–helix used as a model system to study the AhR (16–19). We demonstrated Per-Arnt-Sim super family of transcriptional regulatory proteins (1, that the synthetic salicylate, salicylamide (SAL), is a potent and 2). On binding the ligand, the “activated” AhR translocates to the long-lasting inhibitor of TCDD-induced signal transduction mediated nucleus, in which it forms a heterodimer with its partner, the aryl by the AhR. hydrocarbon nuclear translocator. Together, they form a transcription factor that binds to the xenobiotic-responsive element (XRE) present in the promoter region of a number of genes. The best characterized MATERIALS AND METHODS molecular response to AhR activation is the transcriptional induction Materials. Human hepatoma HepG2 cells were from American Type Cul- of CYP1A1, CYP1A2, and CYP1B1, the genes that encode the 1A1, ture Collection (Manassas, VA). RPMI 1640, TRIzol, and LipofectAMINE 1A2, and 1B1 enzyme isoforms, respectively, of cytochrome P-450 were from Life Technologies, Inc. (Grand Island, NY). Glutamine and fetal (CYP). These enzymes catalyze the epoxidation of certain classes of bovine serum were from BioFluids (Rockville, MD). Polydeoxyinosinic-de- xenobiotics, resulting in the generation of highly reactive electrophilic oxycytidylic acid, protease inhibitors, ethoxyresorufin, acetylsalicylic acid metabolites. These metabolites can covalently bind specific residues (aspirin), salicylic acid and SAL were from Sigma (St. Louis, MO). [32P]dATP 32 of DNA, causing DNA adduct formation that may result in mutation and [ P]dCTP were from DuPont-NEN (Boston, MA). TCDD was from the 3 and subsequent cellular transformation. The AhR and the genes that it Midwest Research Institute (Kansas City, MO). [ H]TCDD was from Chem- syn Science Labs, Lanexa, KS. The Omniscript kit was from Qiagen (Valencia, regulates are, therefore, central in mediating the genotoxicity of many CA). Tris-borate gels, Tris-borate running buffer, and high-density sample environmental contaminants. buffer were from Invitrogen (Carlsbad, CA). Ligands of the AhR include several classes of environmental car- Cell Culture. HepG2 human hepatoma cells were grown with RPMI 1640 cinogens, including polyhalogenated biphenyls such as 2,3,7,8-tetra- supplemented with 2 mM glutamine and 10% fetal bovine serum in a 5% CO2 chlorodibenzo-p-dioxin (TCDD), aryl hydrocarbons such as benzo- humidified incubator at 37°C. For all of the treatment compounds, DMSO was used as the vehicle and did not exceed 0.1%. Received 4/10/03; revised 9/23/03; accepted 9/29/03. Carcinogen Activation Capacity in HepG2 Cells. The ability of SAL to The costs of publication of this article were defrayed in part by the payment of page affect CYP enzyme activity was evaluated in intact cells by measurement of charges. This article must therefore be hereby marked advertisement in accordance with ethoxyresorufin-O-deethylase (EROD) activity. In 24-well plates, HepG2 cells 18 U.S.C. Section 1734 solely to indicate this fact. were incubated with increasing concentrations of salicylates in the presence of Requests for reprints: Grace Chao Yeh, Basic Research Laboratory, Building 538/ Room 141, National Cancer Institute-Frederick, Frederick, MD 21702-1201. Phone: 1nM TCDD for 24 h, for dose-response experiments. For time course exper- (301) 846-5369; Fax: (301) 846-6395; Email: [email protected]. iments, cells were incubated with increasing concentrations of SAL, or with 429 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. SAL INHIBITS AhR FUNCTION AND CYP1 EXPRESSION Ligand-Binding Assay. AhR ligand binding was measured according to the methods of Raha et al. (26). Cytosolic protein (1.5 mg/500 l) was incubated with 20 nM [3H]TCDD in the presence of DMSO (5 l/500 l), 5 mM SAL, or 10 M TCDD (unlabeled) for2hat4°C. Samples were centri- fuged in linear gradients of 5–30% sucrose (v/v) in 12 ml of Beckman Quick-Seal rotor tubes at 65,000 rpm for2hat4°C in a Beckman VTI-65–1 rotor. Fractions (ϳ500 l each) of the gradients were collected, and radioac- tivity was determined by scintillation counting. Statistical Analysis. Statistical analyses were performed with STATVIEW Statistical Analysis Software (SAS Institute, San Francisco, CA). Differences between group mean values were determined by a one-factor ANOVA, fol- lowed by Fisher’s protected least significant difference post hoc analysis for pairwise comparison of means. A two-sided P value of Ͻ0.05 was considered significant. CYP mRNA levels in Figs. 3 and 4 are shown on a linear scale. Fig. 1. Structures of salicylic acid, aspirin, and salicylamide (SAL). RESULTS other test compounds at concentrations that inhibit 50% (IC50), in the presence Effect of Salicylates on TCDD-Induced CYP Enzyme Activity. of1nM TCDD for various times from 6 to 144 h. Medium was decanted, and In this study, we examined three salicylates the structures of which cells were washed once with PBS. Medium containing 5 M ethoxyresorufin are illustrated in Fig. 1. The cellular capacity for enzymatic acti- was added. Increasing fluorescence as a result of the conversion of vation of procarcinogens was measured by EROD assay. Treatment ethoxyresorufin to resorufin by CYP enzymes was measured using a CytoFluor of HepG2 cells with 1 nM TCDD resulted in an increase in multiwell plate reader (Applied Biosystems, Foster City, CA), with an exci- EROD-specific activity from nondetectable levels to 17 pmol/min/ tation of 530 nm and emission at 590 nm. The reaction was allowed to run for 30 min. well in controls (Fig. 2A). The addition of SAL caused a concen- Reverse Transcription-PCR. In 6-well plates, cells were treated with various concentrations of SAL alone, or with 100 pM TCDD, for 8 h. Cells were washed twice with PBS, and total RNA was isolated with TRIzol reagent per manufacturer’s instructions.