An aryl hydrocarbon receptor agonist suppresses the growth of human umbilical vein endothelial cells in vitro: Potent effect with polyunsaturated fatty acids.

Human umbilical vein endothelial cells (HUVECs) are a pivotal component of the hematopoietic microenvironment linked to the modulation of the immune response, inflammation and carcinogenesis. HUVEC expresses the aryl hydrocarbon receptor (AHR), which regulates gene expression by binding to the xenobiotic-responsive element. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent agonist for AHR signalling. Treatment with TCDD (0.1-100 nmol/L) was found to suppress the proliferation and to stimulate the death of HUVEC. TCDD's effects were abolished by culturing with CH223191, an inhibitor of AHR signalling. Mechanistically, TCDD treatment increased the protein levels of cell growth suppressors, including p53, Rb, p21 and regucalcin, and caspase-3 implicated in apoptotic cell death, and decreased the levels of Stat3, mitogen-activated protein kinase (MAPK/Erk1/2) and phospho-MAPK/Erk1/2. Treatment with polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid, eicosapentaenoic acid and arachidonic acid, suppressed the proliferation and stimulated the death of HUVEC in vitro, and decreased the levels of Stat3, MAPK/Erk1/2 and phospho-MAPK/Erk1/2 and increased caspase-3. Notably, the effects of TCDD in suppressing proliferation and stimulating death of HUVEC were modulated by coculturing with PUFAs. These effects were reversed by treatment with CH223191, an inhibitor of AHR. Treatment with both TCDD and PUFAs collaboratively enhanced the levels of AHR, CYP1A1, p53, p21, Rb and regucalcin. Moreover, TCDD suppressed migration with wound healing of HUVEC. Notably, the combination of TCDD and PUFAs revealed potent suppressive effects on angiogenesis of HUVEC, potentially related to disorders of the stromal microenvironment.

The role of AHR-inducible cytochrome P450s in metabolism of polyunsaturated fatty acids.

The environmental pollutant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) is the prototype of a large number of non-genotoxic carcinogens, dietary phytochemicals and endogenous metabolites that act via binding the aryl hydrocarbon receptor (AHR). The TCDD-liganded AHR massively upregulates CYP1A1, CYP1A2 and CYP1B1 in many mammalian organs. We demonstrated that TCDD treatment markedly increases the levels of several epoxides and diol metabolites of the epoxides of both ω-6 and ω-3 polyunsaturated fatty acids (PUFA) in the liver and lungs of mice, in an aryl hydrocarbon receptor-dependent fashion, and most likely via the activities of the CYP1 family members. ω-6 Epoxides are known to stimulate tumor growth, angiogenesis, and metastasis in mice. Interestingly, ω-3 epoxides have the opposite effect on these parameters. TCDD and other AHR agonists may, therefore, impact angiogenesis, growth and metastasis of tumors in either a positive or negative way, depending on the relative levels of ω -6 epoxides and ω-3 epoxides generated in the host and/or tumor cells. This is of potential relevance to carcinogenesis by AHR agonists in the human, since the human population is exposed to widely varying ω-6: ω-3 PUFA ratios in the diet.

SIN3A, generally regarded as a transcriptional repressor, is required for induction of gene transcription by the aryl hydrocarbon receptor.

CYP1A1 bioactivates several procarcinogens and detoxifies several xenobiotic compounds. Transcription of CYP1A1 is highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the aryl hydrocarbon receptor. We recently described an RNAi high throughput screening performed in the Hepa-1 mouse hepatoma cell line, which revealed that SIN3A is necessary for the induction of CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) enzymatic activity by TCDD. In the current studies, we sought to provide insight into the role of SIN3A in this process, particularly because studies on SIN3A have usually focused on its repressive activity on transcription. We report that ectopic expression of human SIN3A in Hepa-1 cells enhanced EROD induction by TCDD and efficiently rescued TCDD induction of EROD activity in cells treated with an siRNA to mouse SIN3A, thus validating a role for SIN3A in CYP1A1 induction. We demonstrate that SIN3A is required for TCDD induction of the CYP1A1 protein in Hepa-1 cells but not for expression of the aryl hydrocarbon receptor protein. In addition, siRNAs for SIN3A decreased TCDD-mediated induction of CYP1A1 mRNA and EROD activity in human hepatoma cell line Hep3B. We establish that TCDD treatment of Hepa-1 cells rapidly increases the degree of SIN3A binding to both the proximal promoter and enhancer of the Cyp1a1 gene and demonstrate that increased binding to the promoter also occurs in human Hep3B, HepG2, and MCF-7 cells. These studies establish that SIN3A physically interacts with the CYP1A1 gene and extends the transcriptional role of SIN3A to a gene that is very rapidly and dramatically induced.

Potential role of epigenetic mechanisms in the regulation of drug metabolism and transport.

This is a report of a symposium on the potential role of epigenetic mechanisms in the control of drug disposition sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2013 meeting in Boston, MA, April 21, 2013. Epigenetics is a rapidly evolving area, and recent studies have revealed that expression of drug-metabolizing enzymes and transporters is regulated by epigenetic factors, including histone modification, DNA methylation, and noncoding RNAs. The symposium speakers provided an overview of genetic and epigenetic mechanisms underlying variable drug metabolism and drug response, as well as the implications for personalized medicine. Considerable insight into the epigenetic mechanisms in differential regulation of the dioxin-inducible drug and carcinogen-metabolizing enzymes CYP1A1 and 1B1 was provided. The role of noncoding microRNAs in the control of drug metabolism and disposition through targeting of cytochrome P450 (P450) enzymes and ATP-binding cassette membrane transporters was discussed. In addition, potential effects of xenobiotics on chromatin interactions and epigenomics, as well as the possible role of long noncoding RNAs in regulation of P450s during liver maturation were presented.

2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A(2) to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved.

HIF-1 expression is associated with CCL2 chemokine expression in airway inflammatory cells: implications in allergic airway inflammation.

BACKGROUND: The pathogenesis of allergic airway inflammation in asthmatic patients is complex and characterized by cellular infiltrates and activity of many cytokines and chemokines. Both the transcription factor hypoxia inducible factor-1 (HIF-1) and chemokine CCL2 have been shown to play pivotal roles in allergic airway inflammation. The interrelationship between these two factors is not known. We hypothesized that the expression of HIF-1 and CCL2 may be correlated and that the expression of CCL2 may be under the regulation of HIF-1. Several lines of evidence are presented to support this hypothesis. METHODS: The effects of treating wild-type OVA (ovalbumin)-sensitized/challenged mice with ethyl-3,4-dihydroxybenzoate (EDHB), which upregulate HIF, on CCL2 expression, were determined. Mice conditionally knocked out for HIF-1ß was examined for their ability to mount an allergic inflammatory response and CCL2 expression in the lung after intratracheal exposure to ovalbumin. The association of HIF-1α and CCL2 levels was also measured in endobronchial biopsies and bronchial fluid of asthma patients after challenge. RESULTS: We show that both HIF-1α and CCL2 were upregulated during an OVA (ovalbumin)-induced allergic response in mice. The levels of HIF-1α and CCL2 were significantly increased following treatment with a pharmacological agent which upregulates HIF-1α, ethyl-3,4-dihydroxybenzoate (EDHB). In contrast, the expression levels of HIF-1α and CCL2 were decreased in the lungs of mice that have been conditionally knocked out for ARNT (HIF-1ß) following sensitization with OVA when compared to levels in wild type mice. In asthma patients, the levels of HIF-1α and CCL2 increased after challenge with the allergen. CONCLUSIONS: These data suggest that CCL2 expression is regulated, in part, by HIF-1 in the lung. These findings also demonstrate that both CCL2 and HIF-1 are implicated in the pathogenesis of allergic airway inflammation.

Human CYP2S1 metabolizes cyclooxygenase- and lipoxygenase-derived eicosanoids.

CYP2S1 is a recently described dioxin-inducible cytochrome P450. We previously demonstrated that human CYP2S1 oxidizes a number of carcinogens but only via the peroxide shunt. In this article, we investigated whether human CYP2S1 can metabolize cyclooxygenase- and lipoxygenase-derived lipid peroxides in a NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G(2) (PGG(2)) (K(m) = 0.267 ± 0.072 µM) into several products including 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin H(2) (PGH(2)) (K(m) = 11.7 ± 2.8 µM) into malondialdehyde, 12-HHT, and thromboxane A(2) (TXA(2)). The turnover to 12-HHT by human CYP2S1 (1.59 ± 0.04 min(-1)) is 40-fold higher than that of TXA(2) (0.04 min(-1)). In addition to PGG(2) and PGH(2) metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic acids (5S-, 12S-, and 15S-) and 13S-hydroperoxyoctadecadienoic acid into 5-oxo-eicosatetraenoic acid (turnover = 16.7 ± 0.3 min(-1)), 12-oxo-eicosatetraenoic acid 1 (11.5 ± 0.9 min(-1)), 15-oxo-eicosatetraenoic acid (16.9 ± 0.8 min(-1)), and 13-octadecadienoic acid (20.2 ± 0.9 min(-1)), respectively. Other cytochromes P450 such as CYP1A1, 1A2, 1B1, and 3A4 underwent similar conversions but at slower rates. The fatty acid hydroperoxides were also converted by human CYP2S1 to several epoxyalcohols. Our data indicate that fatty acid endoperoxides and hydroperoxides represent endogenous substrates of CYP2S1 and suggest that the enzyme CYP2S1 may play an important role in the inflammatory process because some of the products that CYP2S1 produces play important roles in inflammation.

Hypoxia inducible factor promotes murine allergic airway inflammation and is increased in asthma and rhinitis.

BACKGROUND: New therapies are necessary to address inadequate asthma control in many patients. This study sets out to investigate whether hypoxia-inducible factor (HIF) is essential for development of allergic airway inflammation (AAI) and therefore a potential novel target for asthma treatment. METHODS: Mice conditionally knocked out for HIF-1ß were examined for their ability to mount an allergic inflammatory response in the lung after intratracheal exposure to ovalbumin. The effects of treating wild-type mice with either ethyl-3,4-dihydroxybenzoate (EDHB) or 2-methoxyestradiol (2ME), which upregulate and downregulate HIF, respectively, were determined. HIF-1α levels were also measured in endobronchial biopsies and bronchial fluid of patients with asthma and nasal fluid of patients with rhinitis after challenge. RESULTS: Deletion of HIF-1ß resulted in diminished AAI and diminished production of ovalbumin-specific IgE and IgG(1) . EDHB enhanced the inflammatory response, which was muted upon simultaneous inhibition of vascular endothelial growth factor (VEGF). EDHB and 2ME antagonized each other with regard to their effects on airway inflammation and mucus production. The levels of HIF-1α and VEGF increased in lung tissue and bronchial fluid of patients with asthma and in the nasal fluid of patients with rhinitis after challenge. CONCLUSIONS: Our results support the notion that HIF is directly involved in the development of AAI. Most importantly, we demonstrate for the first time that HIF-1α is increased after challenge in patients with asthma and rhinitis. Therefore, we propose that HIF may be a potential therapeutic target for asthma and possibly for other inflammatory diseases.

Role of epigenetic mechanisms in differential regulation of the dioxin-inducible human CYP1A1 and CYP1B1 genes.

The aryl hydrocarbon receptor (AhR) mediates induction of CYP1A1 and CYP1B1 by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (dioxin) via binding to xenobiotic-responsive elements (XREs) in their enhancer regions. CYP1A1 and CYPIB1 were both inducible by dioxin in human MCF-7 cells. However, only CYP1A1 was inducible in human HepG2 cells. Further experiments focused on providing an explanation for this last observation. Dioxin induced the recruitment of AHR and the transcriptional coactivators p300 and p300/cAMP response element-binding protein binding protein-associated factor (PCAF) to the CYP1B1 enhancer in HepG2 cells but failed to induce recruitment of RNA polymerase II (polII) or the TATA binding protein (TBP) and acetylations of histones 3 and 4 or methylation of histone 3 at the promoter. Because p300 was required for dioxin induction of the aforementioned histone modifications at the CYP1B1 promoter and for induction of CYP1B1 transcription (in MCF-7 cells), the recruitments of p300 and AhR, although necessary, are not sufficient for eliciting the above responses to dioxin. Cytosine residues within CpG dinucleotides at the enhancer, including those within the XREs, were partially methylated, whereas those at the promoter were fully methylated. Treatment of HepG2 cells with 5-aza-2'-deoxycytidine led to partial demethylation of the promoter, restored polII and TBP binding, and CYP1B1 inducibility. Thus, the deficiency of CYP1B1 induction in HepG2 cells is ascribable to cytosine methylation at the promoter, which prevents recruitment of TBP and polII. It is noteworthy that our data indicate that stable recruitment of p300 and PCAF to the CYP1B1 gene does not require their tethering to the promoter and to the enhancer.

Aryl Hydrocarbon Receptor-Dependent inductions of omega-3 and omega-6 polyunsaturated fatty acid metabolism act inversely on tumor progression.

The Western diet contains a high ratio of omega-6 (ω6) to omega-3 (ω3) polyunsaturated fatty acids (PUFA). The prototypical aryl hydrocarbon receptor (AHR) ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), induces CYP1 family enzymes, which can metabolize PUFA to epoxides. Mice fed ω3-rich or ω6-rich diets were treated with TCDD and injected subcutaneously with AHR-competent Hepa1-GFP hepatoma cells or AHR-deficient LLC lung cancer cells. TCDD reduced the growth rates of the resulting tumors in ω3-fed mice and inhibited their metastasis to the liver and/or lung, but had the opposite effects in mice fed ω6 PUFA. These responses were likely attributable to the corresponding PUFA epoxides generated in tumor cells and/or host, since many depended upon co-administration of a soluble epoxide hydrolase (EPHX2) inhibitor in males, and/or were associated with increases in epoxide levels in tumors and sites of metastasis. Equivalent effects occurred in females in the absence of EPHX2 inhibition, probably because this sex expressed reduced levels of EPHX2. The responses elicited by TCDD were associated with effects on tumor vascularity, tumor cell proliferation and/or apoptosis. Thus environmental AHR agonists, and potentially also endogenous, nutritional, and microbiome-derived agonists, may reduce or enhance cancer progression depending on the composition of dietary PUFA, particularly in females.

Functional characterization of human cytochrome P450 2S1 using a synthetic gene-expressed protein in Escherichia coli.

Human cytochrome P450 2S1 was recently identified and shown to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin and hypoxia. It is highly expressed in epithelial cells of tissues that are exposed to the environment and in many tumors of epithelial origin. The biological function of CYP2S1 has not yet been determined, although its possible role in carcinogen metabolism has been suggested. In this report, we investigated its ability to metabolize carcinogens. To obtain a large quantity of active enzyme for substrate screening, we overexpressed CYP2S1 in Escherichia coli (200 nM culture), using a synthetic gene approach. High-level expression allowed us to achieve purification of CYP2S1 with high specific content and purity (16 nmol/mg). Despite high-level expression, we found that CYP2S1 was not readily reduced by cytochrome P450 reductase, and thus no activity was found using NADPH. However, the oxidative activity of CYP2S1 was supported by cumene hydroperoxide or H(2)O(2), such that CYP2S1 oxidized many important environmental carcinogens, including benzo[a]pyrene, 9,10-dihydro-benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene-7,8-dihydrodiol, aflatoxin B1, naphthalene, and styrene, with high turnover. Most substrates tested were converted to detoxification products, except in the case of benzo[a]pyrene-7,8-dihydrodiol, which was converted into the very potent carcinogenic metabolite 7,8-dihydrodiol-trans-9,10-epoxide at a relatively efficient rate (K(m) = 12.4 +/- 2 microM, turnover = 2.3 min(-1)). This metabolite formation was also supported both in vitro and in vivo by fatty acid hydroperoxides described in the accompanying report (p. 1044). Together, these data indicate that CYP2S1 contributes to the metabolism of environmental carcinogens via an NADPH independent activity.

Fatty acid hydroperoxides support cytochrome P450 2S1-mediated bioactivation of benzo[a]pyrene-7,8-dihydrodiol.

In the accompanying report (p. 1031), we showed that a novel dioxin-inducible cytochrome P450, CYP2S1, efficiently metabolizes benzo[a]pyrene-trans-7,8-dihydrodiol (BaP-7,8-diol) into the highly mutagenic and carcinogenic benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BaP-diol-t-epoxide), using cumene hydroperoxide in lieu of NADPH/O(2). Lipid hydroperoxide-supported P450 oxidation has been reported in several cases. However, it has not yet been described for the bioactivation of BaP-7,8-diol. In this report, we demonstrate that CYP2S1 can use various fatty acid hydroperoxides to support epoxidation of BaP-7,8-diol at a much higher rate than with cumene hydroperoxide. Kinetic analyses with several fatty acid hydroperoxides revealed that 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13-HpODE) was the most potent oxidant tested (K(m), 3.4 +/- 0.8 microM; turnover, 4.51 +/- 0.13 min(-1)), followed by 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 2.8 +/- 0.7 microM; turnover, 3.7 +/- 0.1 min(-1)), 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (K(m), 2.7 +/- 0.8 microM; turnover, 3.69 +/- 0.09 min(-1)), and 15S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (K(m), 11.6 +/- 0.3 microM; turnover, 0.578 +/- 0.030 min(-1)). The antioxidant butylated hydroxyanisole inhibited CYP2S1-catalyzed epoxidation by 100%, suggesting that epoxidation proceeds by a free radical mechanism. Other cytochromes P450, including CYP1A1, CYP1B1, CYP1A2, and CYP3A4, were also able to epoxidize BaP-7,8-diol using various fatty acid hydroperoxides, although at slower rates than CYP2S1. The cytotoxicity of BaP-7,8-diol significantly increased in mammalian cells overexpressing CYP2S1, and BaP-diol-t-epoxide formation in these cells also increased in the presence of 13-HpODE. Together, these results suggest that fatty acid hydroperoxides can serve as physiological cofactors in supporting in vivo CYP2S1-catalyzed oxidation of BaP-7,8-diol, and that fatty acid hydroperoxides and CYP2S1 may play important roles in benzo[a]pyrene-induced carcinogenesis.

The aryl hydrocarbon receptor nuclear translocator (Arnt) is required for tumor initiation by benzo[a]pyrene.

Benzo[a]pyrene (B[a]P) is a ligand for the aryl hydrocarbon receptor (Ahr). After binding ligand, Ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) protein, and the dimer upregulates the transcription of Cyp1a1, Cyp1b1 and other enzymes involved in the metabolic activation of B[a]P. Arnt null mice die in utero. Mice in which Arnt deletion occurs constitutively in the epidermis die perinatally. In the current study, mice were developed in which the Arnt gene could be deleted specifically in adult skin epidermis. This deletion had no overt pathological effect. Homozygosity for a null reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase allele was introduced into the above mouse strain to render it more susceptible to tumor initiation by B[a]P. Deletion of Arnt in the epidermis of this strain completely prevented the induction of skin tumors in a tumor initiation-promotion protocol in which a single topical application of B[a]P acted as the tumor-initiating event, and tumor promotion was provided by repeated topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA). In contrast, deletion of Arnt did not prevent the induction of skin tumors in a protocol also using TPA as the promoter but using as the initiator N-methyl-N'-nitro-N-nitrosoguanidine, whose activity is unlikely to be affected by the activity of Ahr, Arnt or their target genes. These observations demonstrate that Arnt is required for tumor initiation by B[a]P in this system.

A CRISPR/Cas9 Whole-Genome Screen Identifies Genes Required for Aryl Hydrocarbon Receptor-Dependent Induction of Functional CYP1A1.

Environmental pollutants including halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, including benzo[a]pyrene, exert their deleterious effects through the activation of the aryl hydrocarbon receptor (AHR) and by the resulting transcription of genes not yet fully identified. Ligand-bound AHR translocates from cytoplasm to nucleus, where it dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. The AHR/ARNT dimer binds to enhancer regions of responsive genes to activate transcription. AHR also mediates carcinogenesis caused by PAHs, likely via CYP1A1, CYP1A2, and CYP1B1, which are massively induced by activated AHR in many tissues and generate carcinogenic electrophilic derivatives of PAHs. In the current study, we have used the mouse GeCKOv2 genome-wide CRISPR/Cas9 library to identify novel genes in the AHR pathway by taking advantage of a B[a]P selection assay that we previously used to identify core AHR pathway genes in Hepa-1c1c7 murine hepatoma cells. Besides Ahr, Arnt, and Cyp1a1, we report the identification of multiple additional putative AHR pathway genes including several that we validated. These include cytochrome P450 reductase (Por), which mediates redox regeneration of cytochromes P450, and 5 genes of the heme biosynthesis pathway: delta-aminolevulinate synthase 1 (Alas1), porphobilinogen deaminase (Hmbs), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), and ferrochelatase (Fech): heme being an essential prosthetic group of cytochrome P450 proteins. Notably, several of these genes were identified by GeCKO screening, despite not being identifiable by reverse genetics approaches. This indicates the power of high-sensitivity genome-wide genetic screening for identifying genes in the AHR pathway.

2,3,7,8­tetrachlorodibenzo­p­dioxin suppresses the growth of human colorectal cancer cells in vitro: Implication of the aryl hydrocarbon receptor signaling.

Human colorectal cancer is the third most common cancer disease with a 5­year survival rate of 55% in USA in 2016. The investigation to identify novel biomarker factors with molecular classification may provide notable clinical information to prolong the survival of patients with colorectal cancer. The aryl hydrocarbon receptor (AHR) binds the AHR nuclear translocator in the cytoplasm of various types of cells, including liver cells, and then binds to the xenobiotic responsive element on various genes. AHR was initially discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD). The present study was undertaken to determine whether TCDD, an agonist of AHR signaling, impacts the growth of RKO human colorectal cancer cells in vitro. Treatment with TCDD (0.1­100 nM) revealed suppressive effects on colony formation and proliferation of RKO cells, and stimulated death of these cells with subconfluence. These effects of TCDD were abolished by pretreatment with CH223191, an inhibitor of AHR signaling. Western blot analysis demonstrated that TCDD treatment decreased AHR levels and elevated cytochrome P450 family 1 subfamily A member 1 (CYP1A1) levels, indicating a stimulation of AHR signaling. TCDD treatment caused an increase in nuclear factor­κB p65 and ß­catenin levels, although it did not have an effect on Ras levels. Notably, TCDD treatment increased the levels of p53, retinoblastoma, p21 and regucalcin, which are depressors of carcinogenesis. Additionally, action of TCDD on cell proliferation and death were not revealed in regucalcin­overexpressing RKO cells, and regucalcin overexpression depressed AHR signaling associated with CYP1A1 expression. Thus, AHR signaling suppresses the growth of colorectal cancer cells, indicating a role as a significant targeting molecule for colorectal cancer.

Prolonged survival of renal cancer patients is concomitant with a higher regucalcin gene expression in tumor tissues: Overexpression of regucalcin suppresses the growth of human renal cell carcinoma cells in vitro.

Renal cell carcinoma (RCC), which is a type of cancer found in the kidney tubule, is among the 10 most frequently occurring human cancers. Regucalcin plays a potential role as a regulator of transcriptional activity, and its downregulated expression or activity may contribute to the promotion of human cancers. In this study, we investigated the involvement of regucalcin in human RCC. Regucalcin expression was compared in 23 normal and 29 tumor samples of kidney cortex tissues of patients with clear cell RCC obtained through the Gene Expression Omnibus (GEO) database (GSE36895). Regucalcin expression was downregulated in the tumor tissues. The prolonged survival of patients with clear cell RCC was demonstrated to be associated with a higher regucalcin gene expression in the TCGA dataset. The overexpression of regucalcin suppressed the colony formation, proliferation and the death of human clear cell RCC A498 cells in vitro. Mechanistically, the overexpression of regucalcin induced the G1 and G2/M phase cell cycle arrest of A498 cells through the suppression of multiple signaling components, including Ras, PI3 kinase, Akt and mitogen­activated protein (MAP) kinase. Importantly, the overexpression of regucalcin led to an elevation in the levels of the tumor suppressors, p53, Rb and the cell cycle inhibitor, p21. The levels of the transcription factors, c­fos, c­jun, nuclear factor­κB p65, ß­catenin and signal transducer and activator of transcription 3, were suppressed by regucalcin overexpression. On the whole, the findings of this study suggest that regucalcin plays a suppressive role in the promotion of human RCC. The overexpression of regucalcin by gene delivery systems may thus prove to be a novel therapeutic strategy for RCC.

CXCR4 and CXCL12 down-regulation: a novel mechanism for the chemoprotection of 3,3'-diindolylmethane for breast and ovarian cancers.

Cruciferous vegetables are thought to protect against numerous types of cancer. 3,3'-Diindolylmethane (DIM) is an acid-catalyzed product generated during the consumption of cruciferous vegetables and appears to be chemoprotective for breast cancer. The interaction between the chemokine receptor, CXCR4, and its unique ligand, CXCL12, is known to mediate the progression and metastasis of breast and other cancers. Organs to which these cancers metastasize secrete CXCL12, which binds to CXCR4 expressed on the surface of primary cancer cells. This process subsequently stimulates the invasive properties of the cancer cells and attracts them to the preferred organ sites of metastases. We have found that DIM down-regulates both CXCR4 and CXCL12 in MCF-7 and MDA-MB-231 breast cancer cells as well as in BG-1 ovarian cancer cells at the transcriptional level and in an estrogen-independent manner. We demonstrate that the potential of MDA-MB-231 and BG-1 cells for chemotaxis and invasion towards CXCL12, but not towards IL-6 or fetal bovine serum, respectively, is inhibited by DIM. Furthermore, we show that DIM down-regulates CXCR4 under hypoxia and CXCL12 under estradiol-inducing conditions. Our data suggest that one mechanism whereby DIM protects against breast, ovarian, and possibly other cancers is through the repression of CXCR4 and/or CXCL12, thereby lowering the invasive and metastatic potential of these cells.

The mibefradil derivative NNC55-0396, a specific T-type calcium channel antagonist, exhibits less CYP3A4 inhibition than mibefradil.

A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca(2+) channel inhibitor without L-type Ca(2+) channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s--CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1--and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC(50) = 33 +/- 3 nM, K(i) = 23 +/- 0.5 nM) and Ro40-5966 (IC(50) = 30 +/- 7.8 nM, K(i) = 21 +/- 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC(50) = 300 +/- 30 nM, K(i) = 210 +/- 6 nM). More dramatically, mibefradil (IC(50) = 566 +/- 71 nM, K(i) = 202 +/- 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6beta-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC(50) = 11 +/- 1.1 microM, K(i) = 3.9 +/- 0.4 microM). Loss of testosterone 6beta-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (K(I) = 3.87 microM, K(inact) = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (K(I) = 83 nM, K(inact) = 0.048/min). In contrast, NNC55-0396 (IC(50) = 29 +/- 1.2 nM, K(i) = 2.8 +/- 0.3 nM) and Ro40-5966 (IC(50) = 46 +/- 11 nM, K(i) = 4.5 +/- 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC(50) = 129 +/- 21 nM, K(i) = 12.7 +/- 0.9 nM). Our results suggest that NNC55-0396 could be a more favorable T-type Ca(2+) antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.

2,3,7,8­Tetrachlorodibenzo­p­dioxin suppresses the growth of human liver cancer HepG2 cells in vitro: Involvement of cell signaling factors.

The aryl hydrocarbon receptor (AHR) is transcriptionally active in the form of a heterodimer with the AHR nuclear translocator, which then binds to the xenobiotic responsive element. AHR was originally discovered via its ligand, the polychlorinated hydrocarbon, 2,3,7,8­tetrachlorodibenzo­p­dioxin (TCDD). In this study, we investigated whether TCDD regulates the growth of human liver cancer HepG2 cells in vitro. TCDD (0.1­100 nM) was found to exert suppressive effects on the colony formation and proliferation of HepG2 cells, and stimulatory effects on the death of HepG2 cells when the cells reached subconfluence. The effects of TCDD on the HepG2 cells were abolished by culture with CH223191, an inhibitor of AHR signaling. The effects of TCDD were dependent on the concentration of serum, which contains various signaling factors. The effects of TCDD were not potentiated by culture with tumor necrosis factor­α, which activates the signaling of nuclear factor­κB (NF­κB). The results of western blot analysis revealed that TCDD increased the protein levels of p53, Rb, p21, and regucalcin, which are suppressors of the growth of tumor cells. Moreover, TCDD enhanced the NF­κB p65, ß­catenin, signal transducer and activator of transcription 3 (STAT3), Ras and Akt levels. Thus, the findings of this study indicate that TCDD may suppress liver cancer cell growth through various signaling pathways, mediated by AHR and its­related co­factors. Of note, the effects of TCDD were found to be potentiated by gemcitabine, which induces nuclear DNA damage in cancer cells, suggesting that their combined use may have potential as a suppressor of tumor cell growth.

ChIP-re-ChIP: Co-occupancy Analysis by Sequential Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) exploits the specific interactions between DNA and DNA-associated proteins. It can be used to examine a wide range of experimental parameters. A number of proteins bound at the same genomic location can identify a multi-protein chromatin complex where several proteins work together to regulate gene transcription or chromatin configuration. In many instances, this can be achieved using sequential ChIP; or simply, ChIP-re-ChIP. Whether it is for the examination of specific transcriptional or epigenetic regulators, or for the identification of cistromes, the ability to perform a sequential ChIP adds a higher level of power and definition to these analyses. In this chapter, we describe a simple and reliable method for the sequential ChIP assay.