New hydroxystilbenoid derivatives endowed with neuroprotective activity and devoid of interference with estrogen and aryl hydrocarbon receptor-mediated transcription

We have synthesized a series of new (E) stilbenoid derivatives containing hydroxy groups at ring positions identical or similar to those of trans-resveratrol and bearing one or two bulky electron donating groups ortho to 4′-OH and we have evaluated their neuroprotective activity using glutamate-challenged HT22 hippocampal neurons to model oxidative stress-induced neuronal cell death. The most active derivatives, 5-{(E)-2-[3,5-bis(1-ethylpropyl)-4-hydroxyphenyl]ethenyl}-1,3-benzenediol (2), 5-[(E)-2-(3,5-di-tert-butyl-4-hydroxyphenylethenyl)]-1,3-benzenediol (4) and 5-{(1E,3E)-4-[3,5-bis(1-ethylpropyl)-4-hydroxyphenyl]-1,3-butadienyl}-1,3-benzenediol (6), had EC₅₀ values of 30, 45 and 12 nM, respectively, and were ca. 100 to 400-fold more potent than resveratrol. Derivatives 2, 4 and 6 lacked cytotoxic activity against HT22 cells and estrogen receptor agonist or antagonist activity in estrogen response element-dependent gene expression and in estrogen-dependent proliferation of MCF-7 human breast cancer cells. In addition, they were incapable of interfering with aryl hydrocarbon receptor-mediated xenobiotic response element-dependent gene expression. Derivatives 2, 4 and 6 might assist in the development of lead candidates against oxidative stress-driven neurodegenerative diseases that will not increase endocrine cancer risk nor affect drug activation and detoxification mechanisms.     

Analysis of the complex relationship between nuclear export and aryl hydrocarbon receptor-mediated gene regulation

The aryl hydrocarbon receptor (AHR) contains signals for both nuclear import and nuclear export (NES). The purpose of the studies in this report was to determine the relationship between the nuclear export of the AHR and AHR-mediated gene regulation. Blockage of nuclear export in HepG2 cells with leptomycin B (LMB) resulted in increased levels of AHR-AHR nuclear translocator (ARNT) complex in the nucleus and correlative reductions in agonist-stimulated AHR degradation. However, LMB exposure inhibited agonist-mediated induction of numerous AHR-responsive reporter genes by 75 to 89% and also inhibited induction of endogenous CYP1A1. LMB did not transform the AHR to a ligand binding species or affect activation by TCDD (2, 3,7,8-tetrachlorodibenzo-p-dioxin). Mutagenesis of leucines 66 and 71 of the putative AHR NES resulted in a protein with reduced function in dimerization to ARNT and binding to DNA, while alanine substitution at leucine 69 (AHR(A69)) resulted in an AHR that bound with ARNT and associated with DNA. AHR(A69) protein injected directly into the nuclei of E36 cells remained nuclear following 6 h of agonist stimulation. In transient-transfection assays, AHR(A69) accumulated within the nucleus was not degraded efficiently following agonist exposure. Finally, AHR(A69) supported induction of AHR-responsive reporter genes in an agonist-dependent manner. These findings show that it is possible to generate an AHR protein defective in nuclear export that is functional in agonist-mediated gene induction. This implies that the negative effect of LMB on agonist-mediated gene induction is independent of the nuclear export of the AHR.     

Differential ligand-dependent activation and a role for Y322 in aryl hydrocarbon receptor-mediated regulation of gene expression

The aryl hydrocarbon receptor (AHR) mediates the toxic effects of halogenated aromatic hydrocarbons (HAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF). Non-traditional activators, including omeprazole (Omp), are thought to regulate AHR action through phosphorylation rather than binding to the receptor. In this study, we examined the ability of these compounds to induce AHR-dependent regulation of cytochrome P450 1A1 (CYP1A1) and CYP1B1 in T-47D human breast cancer cells. The role of Y322, a residue implicated in Omp-dependent activation of AHR was also investigated. All four compounds induced CYP1A1 and CYP1B1 mRNA expression, with Omp differing from the HAHs. Chromatin immunoprecipitation assays revealed ligand- and gene-selectivity in the recruitment patterns of AHR coactivators. We also found that residue Y322 of human AHR was important for maximum activation of AHR by 2,3,7,8-TCDD and 2,3,4,7,8-PeCDF, but required for 2,3,7,8-TCDF and Omp in an AHR-deficient MCF-7 human breast cancer cell line. In summary, this study provides evidence for context- and ligand-selective differences in coactivator recruitment in AHR-regulated gene expression and reveal an important role of Y322 in AHR activation.     

Curcumin and its derivatives inhibit 2,3,7,8,-tetrachloro-dibenzo-p-dioxin-induced expression of drug metabolizing enzymes through aryl hydrocarbon receptor-mediated pathway

Abstract

Certain dioxins, including 2,3,7,8,-tetrachloro-dibenzo-p-dioxin (TCDD), are exogenous ligands for an aryl hydrocarbon receptor (AhR) and induces various drug-metabolizing enzymes. In this study, we examined the effect of curcumin on expression of drug-metabolizing enzymes through the AhR and NF-E2 related factor 2 (Nrf2) pathways. Curcumin dose-dependently inhibited TCDD-induced expression of phase I enzyme cytochrome P450 1A1 (CYP1A1) and phase II enzymes NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO-1) but not tert-butyl hydroquinone-induced NQO1 and HO-1, suggesting that curcumin inhibited only AhR pathway, but not Nrf2 one directly. Furthermore, we used 14 curcumin derivatives and obtained the correlation between hydrophobicity of the compounds and suppressive effect against AhR transformation. Results from the quantitative structure active correlative analysis indicated that methoxy groups and β-diketone structure possessing keto-enol tautomerism in curcumin were necessary to inhibit AhR transformation, and the addition of methyl and methoxy group(s) to the curcumin increased the inhibition effect.     

Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer

The mammalian CYP1A1, CYP1A2, and CYP1B1 genes (encoding cytochromes P450 1A1, 1A2, and 1B1, respectively) are regulated by the aromatic hydrocarbon receptor (AHR). The CYP1 enzymes are responsible for both metabolically activating and detoxifying numerous polycyclic aromatic hydrocarbons (PAHs) and aromatic amines present in combustion products. Many substrates for CYP1 enzymes are AHR ligands. Differences in AHR affinity between inbred mouse strains reflect variations in CYP1 inducibility and clearly have been shown to be associated with differences in risk of toxicity or cancer caused by PAHs and arylamines. Variability in the human AHR affinity exists, but differences in human risk of toxicity or cancer related to AHR activation remain unproven. Mouse lines having one or another of the Cyp1 genes disrupted have shown paradoxical effects; in the test tube or in cell culture these enzymes show metabolic activation of PAHs or arylamines, whereas in the intact animal these enzymes are sometimes more important in the role of detoxification than metabolic potentiation. Intact animal data contradict pharmaceutical company policies that routinely test drugs under development; if a candidate drug shows CYP1 inducibility, further testing is generally discontinued for fear of possible toxic or carcinogenic effects. In the future, use of "humanized" mouse lines, containing a human AHR or CYP1 allele in place of the orthologous mouse gene, is one likely approach to show that the AHR and the CYP1 enzymes in human behave similarly to that in mouse.     

Transcriptional activation of c-fos protooncogene by 17beta-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition.

17Beta-estradiol (E2) induced c-fos protooncogene mRNA levels in MCF-7 human breast cancer cells, and maximal induction was observed within 1 h after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibited the E2-induced response within 2 h. The molecular mechanism of this response was further investigated using pFC2-CAT, a construct containing a -1400 to +41 sequence from the human c-fos protooncogene linked to a bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In MCF-7 cells transiently transfected with pFC2-CAT, 10 nM E2 induced an 8.5-fold increase of CAT activity, and cotreatment with 10 nM TCDD decreased this response by more than 45%. Alpha-Naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist, blocked the inhibitory effects of TCDD; moreover, the inhibitory response was not observed in variant Ah-nonresponsive MCF-7 cells, suggesting that the AhR complex was required for estrogen receptor cross-talk. The E2-responsive sequence (-1220 to -1155) in the c-fos gene promoter contains two putative core pentanucleotide dioxin-responsive elements (DREs) at -1206 to -1202 and -1163 to -1159. In transient transfection assays using wild-type and core DRE mutant constructs, the downstream core DRE (at -1163 to -1159) was identified as a functional inhibitory DRE. The results of photo-induced cross-linking, gel mobility shift, and in vitro DNA footprinting assays showed that the AhR complex interacted with the core DRE that also overlapped the E2-responsive GC-rich site (-1168 to -1161), suggesting that the mechanism for AhR-mediated inhibitory effects may be due to quenching or masking at the Sp1-binding site.     

The aryl hydrocarbon receptor, more than a xenobiotic-interacting protein

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.     

Identification of new aryl hydrocarbon receptor (AhR) antagonists using a zebrafish model

A new series of 1,3-diketone, heterocyclic and α,β-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC₅₀ of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion.