Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligand(s) that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl-3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including: CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an ~ 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.The aryl hydrocarbon receptor (AHR) 1 is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors, a class of proteins that are considered environmental sensors. Prior to activation, the AHR is located in the cytoplasm of the cell, complexed with two heat-shock protein 90 molecules, X-associated

The present studies were performed to elucidate the mechanism of cytotoxicity of the aminoflavone analog (5-amino-2,3-fluorophenyl)-6,8-difluoro-7-methyl-4H-1-benzopyran-4-one (AF; NSC 686288), a novel flavone with potent in vitro and in vivo antiproliferative activity against a number of human tumor cell lines and with a unique pattern of antiproliferative activity in the National Cancer Institute tumor cell line screen. AF was extensively metabolized by cytochrome P450 (P450) 1A1 and 1A2 to several metabolites, one of which was identified by mass spectrometry as a potentially reactive hydroxylamine. Radiolabeled AF was converted by rat and human microsomes, by recombinant CYP1A1 and CYP1A2, and by sensitive human tumor cell lines to species that covalently bound macromolecules. Treatment of sensitive human MCF7 cells with AF resulted in increased CYP1A1 mRNA and CYP1A1/1A2 protein followed by covalent binding of an AF metabolite to DNA, phosphorylation and stabilization of p53, and increased expression of the p53 transcriptional target p21. Covalent binding of the AF metabolite was increased by pretreatment with the CYP1A inducer 3-methylcholanthrene and decreased by coincubation with the CYP1A inhibitor ␣-naphthoflavone. In contrast, induction of CYP1A1 and covalent binding of the AF metabolite did not occur in AF-resistant M14-MEL cells. These observations suggest that AF is uniquely able to induce its own metabolic activation via CYP1A1/1A2 in duction to cytotoxic DNA-damaging species directly in tumor cells. AF, and possibly other agents, may offer a treatment strategy for tumors responsive to CYP1A1/1A2 induction, such as breast, ovarian, and renal cancers. AF (Fig.

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The aryl hydrocarbon receptor (AHR) is regarded as an important homeostatic transcriptional regulator within physiological and pathophysiological processes, including xenobiotic metabolism, endocrine function, immunity, and cancer. Agonist activation of the AHR is considered deleterious based on toxicological evidence obtained with environmental pollutants, which mediate toxic effects through AHR. However, a multitude of plant-derived constituents, e.g., polyphenols that exhibit beneficial properties, have also been described as ligands for the AHR. It is conceivable that some of the positive aspects of such compounds can be attributed to suppression of AHR activity through antagonism. Therefore, we conducted a dioxin response element reporter-based screen to assess the AHR activity associated with a range of flavonoid compounds. Our screen identified two flavonoids (5-methoxyflavone and 7,4Ј-dimethoxyisoflavone) with previously unidentified AHR agonist potential. In addition, we have identified and characterized 6,2Ј,4Ј-trimethoxyflavone (TMF) as an AHR ligand that possesses the characteristics of an antagonist having the capacity to compete with agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo[a]pyrene, thus effectively inhibiting AHR-mediated transactivation of a heterologous reporter and endogenous targets, e.g., CYP1A1, independent of cell lineage or species. Furthermore, TMF displays superior action by virtue of having no partial agonist activity, in contrast to other documented antagonists, e.g., ␣-napthoflavone, which are partial weak agonists. TMF also exhibits no species or promoter dependence with regard to AHR antagonism. TMF therefore represents an improved tool allowing for more precise dissection of AHR function in the absence of any conflicting agonist activity.

There is increasing evidence that the aryl hydrocarbon receptor (AHR) plays a role in tumor progression through numerous mechanisms. We have previously shown that, in certain cancer cell lines that are typically non-responsive to cytokine-mediated IL6 induction, activation of the AHR with the agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin derepresses the IL6 promoter and allows for synergistic induction following IL1β treatment. The mechanism by which this occurs involves liganded AHR binding upstream from the transcription start site and dismissing HDAC-containing corepressor complexes, giving rise to a promoter structure that is more amenable to NF-κB activation. This fact, combined with observations of multiple endogenously-produced chemicals activating the AHR, led us to study its role in basal expression among high cytokine-producing cancer cell lines. The current study provides evidence that several head and neck squamous cell carcinoma cell lines have a level of constitutively bound AHR at the IL6 promoter, allowing for higher basal and readily inducible IL6 transcription. Treatment of these cell lines with an AHR antagonist led to dismissal of the AHR from the IL6 promoter and recruitment of corepressor complexes, thus diminishing cytokine expression. Head and neck squamous cell carcinoma is typically a high cytokine-producing tumor type, with IL6 expression levels correlating with disease aggressiveness. For this reason, AHR antagonist treatment could represent a novel adjuvant therapy for patients, lowering pro-growth and anti-apoptotic signaling with minimal systemic side effects.

We have characterized previously a class of aryl hydrocarbon receptor (AHR) ligand termed selective AHR modulators (SAhRMs). SAhRMs exhibit anti-inflammatory properties, including suppression of cytokine-mediated acute phase genes (e.g., Saa1), through dissociation of non-dioxin-response element (DRE) AHR activity from DRE-dependent xenobiotic gene expression. The partial AHR agonist ␣-naphthoflavone (␣NF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that ␣NF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of ␣NF derivatives identifies 3Ј,4Ј-dimethoxy-␣NF (DiMNF) as a candidate SAhRM. Competitive ligand binding validates DiMNF as an AHR ligand, and DRE-dependent reporter assays with quantitative mRNA analysis of AHR target genes reveal minimal agonist activity associated with AHR binding. Consistent with loss of agonist activity, DiMNF fails to promote AHR binding to DRE probes as determined through electromobility shift assay. Importantly, mRNA analysis indicates that DiMNF retains the suppressive capacity of ␣NF regarding cytokine-mediated SAA1 expression in Huh7 cells. Interestingly, predictive docking modeling suggests that DiMNF adopts a unique orientation within the AHR ligand binding pocket relative to ␣NF and may facilitate the rational design of additional SAhRMs. Microarray studies with a non-DRE binding but otherwise functional AHR mutant identified complement factor C3 as a potential SAhRM target. We confirmed this observation in Huh7 cells using 10 M DiMNF, which significantly repressed C3 mRNA and protein. These data expand the classes of AHR ligands exerting DRE-independent anti-inflammatory SAhRM activity, suggesting SAhRMs may have application in the amelioration of inflammatory disorders.