Nuclear expression of VDR and AHR is mutually exclusive in glandular cells in endometriosis.

The vitamin D receptor (VDR) and aryl hydrocarbon receptor (AHR) are two nuclear receptors that exert their effects by binding with ligands and forming a molecular complex. These complexes translocate to the nucleus and activate the expression of a series of genes which have a response element to VDR or AHR. Both receptors have been identified in the pathogenesis of endometriosis, a common disease characterized by the formation of endometrium-like tissue in ectopic zones. Despite numerous therapies, there is no definitive cure for endometriosis at the pharmacological level. Our study aims to describe the location and the expression of VDR and AHR at the protein level. For this purpose, an evaluation was performed using tissue from the three normal phases of the endometrium (proliferative, early, and late secretory) and in endometriosis by immunohistochemistry, using anti-VDR and anti-AHR antibodies. We demonstrate that in the nuclei of glandular cells in endometriosis, the expression of VDR and AHR is mutually exclusive-when the expression of one receptor is high, the other one is low-suggesting a possible target in the treatment of endometriosis. We also identify a significant change in the expression of glandular cytoplasmic AHR between the proliferative and late secretory endometrium.

Genetic and epigenetic changes in the eutopic endometrium of women with endometriosis: association with decreased endometrial αvß3 integrin expression.

About 40% of women with infertility and 70% of women with pelvic pain suffer from endometriosis. The pregnancy rate in women undergoing IVF with low endometrial integrin αvß3 (LEI) expression is significantly lower compared to the women with high endometrial integrin αvß3 (HEI). Mid-secretory eutopic endometrial biopsies were obtained from healthy controls (C; n=3), and women with HEI (n=4) and LEI (n=4) and endometriosis. Changes in gene expression were assessed using human gene arrays and DNA methylation data were derived using 385 K Two-Array Promoter Arrays. Transcriptional analysis revealed that LEI and C groups clustered separately with 396 differentially expressed genes (DEGs) (P<0.01: 275 up and 121 down) demonstrating that transcriptional and epigenetic changes are distinct in the LEI eutopic endometrium compared to the C and HEI group. In contrast, HEI vs C and HEI vs LEI comparisons only identified 83 and 45 DEGs, respectively. The methylation promoter array identified 1304 differentially methylated regions in the LEI vs C comparison. The overlap of gene and methylation array data identified 14 epigenetically dysregulated genes and quantitative RT-PCR analysis validated the transcriptomic findings. The analysis also revealed that aryl hydrocarbon receptor (AHR) was hypomethylated and significantly overexpressed in LEI samples compared to C. Further analysis validated that AHR transcript and protein expression are significantly (P<0.05) increased in LEI women compared to C. The increase in AHR, together with the altered methylation status of the 14 additional genes, may provide a diagnostic tool to identify the subset of women who have endometriosis-associated infertility.

R-sulforaphane modulates the expression profile of AhR, ERα, Nrf2, NQO1, and GSTP in human breast cell lines.

Our previous study showed remarkable differences in the effect of R-sulforaphane (R-SFN) on the expression of CYPs 19, 1A1, 1A2, and 1B1 in ER(+) MCF7, ER( -) MDA-MB-231, and non-tumorigenic immortalized MCF10A (8). This study aimed to evaluate the effect of R-SFN on phase II enzymes induction and expression of AhR, Nrf2, and ERα in the same breast cell lines. The results showed increased expression of GSTP as a result of treatment with R-SFN in breast cancer cells. An increased NQO1 transcript and protein levels were found in all breast cells, with the most significant increase in MCF7 cells. Similarly, the enhancement of Nrf2 expression was noticed in all tested cells. AhR gene transcript and protein were decreased in MCF7 cells. In MDA-MB-231, increased AhR mRNA was not confirmed at the protein level. No differences were found in the expression of ERα. Overall, the results of the present study extended our earlier suggestions on the possible interference of R-SFN with estrogens homeostasis in breast cancer cells differing in ERα status, as well as in non-tumorigenic immortalized breast epithelial cells. While some of R-SFN effects might be beneficial and useful in breast cancer prevention, the others, particularly GSTP induction, may lead to adverse effects.

MiR-124a Mediates the Impairment of Intestinal Epithelial Integrity by Targeting Aryl Hydrocarbon Receptor in Crohn's Disease.

Growing evidence suggested that microRNAs (miRNAs) contributed to the progression of Crohn's disease (CD), but the exact physiological functions of many miRNAs in CD patients still remain illusive. In this study, we explore the potent pathogenicity of miRNAs in CD. Expressions of miRNAs and aryl hydrocarbon receptor (AHR) protein were determined in the colitic colon of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis mice and CD patients. Colitis was induced in wild-type (WT), miR-124a overexpression (miR-124a-Nju), and AHR knockout (AHR-/-) mice. Intestinal barrier function was evaluated in colitis mice and Caco2 monolayers. There was a negative relationship between miR-124a and AHR protein in inflamed colons from CD patients. MiR-124a-Nju and AHR-/- mice treated with TNBS had more severe intestinal inflammation than WT mice. Both miR-124a-Nju mice and AHR-/- mice underwent evident intestinal barrier destruction, and anti-miR-124a administration could reverse this dysfunction in miR-124a-Nju mice but not in AHR-/- mice. In vitro studies revealed that miR-124a mimics downregulated the expression of AHR and tight junction proteins and induced hyperpermeability by increasing miR-124a expression, which was abrogated by miR-124a inhibitor and AHR antagonist FICZ. This study suggests that miR-124a can induce intestinal inflammation and cause intestinal barrier dysfunction by supressing AHR.

IDO, TDO, and AHR overexpression is associated with poor outcome in diffuse large B-cell lymphoma patients in the rituximab era.

Although Indoleamine 2,3-dioxygenase (IDO), tryptophan-2,3-dioxygenase (TDO), and aryl hydrocarbon receptor (AHR) are involved in cancer immune escape, their prognostic impact on diffuse large B-cell lymphoma (DLBCL) is unknown.To examine the prognostic impact of IDO, TDO, and AHR on patients with DLBCL.This was a retrospective study on treatment-naïve patients with newly diagnosed DLBCL at the Henan Province People's Hospital between 01/2012 and 06/2015. Patients with inflammatory reactive lymph nodes were included as controls. All cases were reviewed by 2 pathologists. IDO, TDO, and AHR positivity was determined through immunochemistry. Survival was examined using the Kaplan-Meier method and multivariable Cox analyses.The positive expression of TDO (50.0% vs 16.7%, P = .005) and AHR (60.0% vs 8.3%, P < .001) were higher in DLBCL than in inflammatory control. The overall survival of IDO, TDO, and AHR positive expression in DLBCL patients was 34.6, 26.7, and 32.2 months, respectively, which is significantly shorter than that of the corresponding negative patients (49.0 months, P = .04; 58.2 months, P < .001; 58.0 months, P < .001; respectively). The multivariable analysis showed that TDO expression and Ann-Arbor stage were independently associated with PFS (TDO: HR = 8.347, 95%CI: 2.992-23.289, P < .001; stage: HR = 2.729, 95%CI: 1.571-4.739, P < .001) and OS (TDO: HR = 9.953, 95%CI: 3.228-30.686, P < .001; stage: HR = 2.681, 95%CI: 1.524-4.719, P = .001) in DLBCL patients.Overexpression of IDO, TDO, and AHR is associated with poor survival of patients with DLBCL and could be involved in the immune escape of cancer cells. Further studies are necessary to determine whether these proteins can be targeted by treatment regimens.

Altered Gene expression of ABC transporters, nuclear receptors and oxidative stress signaling in zebrafish embryos exposed to CdTe quantum dots.

Adenosine triphosphate-binding cassette (ABC) transporters, including P-glycoprotein (Pgp) and multi-resistance associated proteins (Mrps), have been considered important participants in the self-protection of zebrafish embryos against environmental pollutants, but their possible involvement in the efflux and detoxification of quantum dots (QDs), as well as their regulation mechanism are currently unclear. In this work, gene expression alterations of ABC transporters, nuclear receptors, and oxidative stress signaling in zebrafish embryos after the treatment of mercaptopropionic acid (MPA)CdTe QDs and MPA-CdSCdTe QDs were investigated. It was observed that both QDs caused concentration-dependent delayed hatching effects and the subsequent induction of transporters like mrp1&2 in zebrafish embryos, indicating the protective role of corresponding proteins against CdTe QDs. Accompanying these alterations, expressions of nuclear receptors including the pregnane X receptor (pxr), aryl hydrocarbon receptor (ahr) 1b, and peroxisome proliferator-activated receptor (ppar)-ß were induced by QDs in a concentration- and time-dependent manner. Moreover, elevated oxidative stress, reflected by the reduction of glutathione (GSH) level and superoxide dismutase (SOD) activities, as well as the dramatic induction of nuclear factor E2 related factor (nrf) 2, was also found. More importantly, alterations of pxr and nrf2 were more pronounced than that of mrps, and these receptors exhibited an excellent correlation with delayed hatching rate in the same embryos (R2 > 0.8). Results from this analysis demonstrated that the induction of mrp1 and mrp2 could be important components for the detoxification of QDs in zebrafish embryos. These transporters could be modulated by nuclear receptors and oxidative stress signaling. In addition, up-regulation of pxr and nrf2 could be developed as toxic biomarkers of CdTe QDs.

The aryl hydrocarbon receptor regulates nucleolar activity and protein synthesis in MYC-expressing cells.

MYC enhances protein synthesis by regulating genes involved in ribosome biogenesis and protein translation. Here, we show that MYC-induced protein translation is mediated by the transcription factor aryl hydrocarbon receptor (AHR), which is induced by MYC in colonic cells. AHR promotes protein synthesis by activating the transcription of genes required for ribosome biogenesis and protein translation, including OGFOD1 and NOLC1. Using surface sensing of translation (SUnSET) to measure global protein translation, we found that silencing AHR or its targets diminishes protein synthesis. Therefore, targeting AHR or its downstream pathways could provide a novel approach to limit biomass production in MYC-driven tumors.

Effects of simvastatin on nuclear receptors, drug metabolizing enzymes and transporters expression in Human Umbilical Vein Endothelial Cells.

BACKGROUND: Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS: HUVEC were exposed to TCDD (10nM), oltipraz (100µM) and simvastatin (1µM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS: Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS: This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.

Malassezia pachydermatis up-regulates AhR related CYP1A1 gene and epidermal barrier markers in human keratinocytes.

Cytochrome P450 CYP1A1 and CYP1B1 enzymes are regulated by the aryl hydrocarbon receptor (AhR), a transcription factor activated by a variety of ligands among which Malassezia metabolites. In this study, we analyzed the modulation of CYP1A1, CYP1B1, and AhR in human keratinocytes infected with different strains of Malassezia pachydermatis, as well as the upregulation of some genes involved in the epidermal homeostasis. We demonstrated that all the strains induced AhR activation and its nuclear translocation in HaCaT cells infected for 24 h, compared to untreated cells. The expression of CYP1A1 and CYP1B1, prototypical markers of the AhR signaling pathway, were upregulated with the level of CYP1A1 mRNA approximately 100-fold greater than that for CYP1B1. Filaggrin, involucrin, and TGaseI, proteins involved in epidermal differentiation, were all modulated by Malassezia pachydermatis strains, with the strongest induction observed for filaggrin. By contrast, quinone oxidoreductase 1 (NQO1), which is part of the antioxidant defense system involved in detoxification, was not modulated in our experimental model. In conclusions, our findings suggest that Malassezia pachydermatis infection of human keratinocytes induces activation of the AhR, and increases the expression of its responsive genes and markers of epidermal differentiation, paving the way for occurrence/exacerbation of pathological skin conditions.

Significance of A-to-I RNA editing of transcripts modulating pharmacokinetics and pharmacodynamics.

RNA editing is a post-transcriptional process that alters the nucleotide sequence of RNA transcripts to generate transcriptome diversity. Among the various types of RNA editing, adenosine-to-inosine (A-to-I) RNA editing is the most frequent type of RNA editing in mammals. Adenosine deaminases acting on RNA (ADAR) enzymes, ADAR1 and ADAR2, convert adenosines in double-stranded RNA structures into inosines by hydrolytic deamination. Inosine forms a base pair with cytidine as if it were guanosine; therefore, the conversion may affect the amino acid sequence, splicing, microRNA targeting, and miRNA maturation. It became apparent that disrupted RNA editing or abnormal ADAR expression is associated with several diseases including cancer, neurological disorders, metabolic diseases, viral infections, and autoimmune disorders. The biological significance of RNA editing in pharmacokinetics/pharmacodynamics (PK/PD)-related genes is starting to be demonstrated. The authors conducted pioneering studies to reveal that RNA editing modulates drug metabolism potencies in the human liver, as well as the response of cancer cells to chemotherapy agents. Awareness of the importance of RNA editing in drug therapy is growing. This review summarizes the current knowledge on the RNA editing that affects the expression and function of drug response-related genes. Continuing studies on the RNA editing that regulates pharmacokinetics/pharmacodynamics would provide new beneficial information for personalized medicine.

Evaluation of the effect of the new methoxy-stilbenes on expression of receptors and enzymes involved in estrogen synthesis in cancer breast cells.

Our previous study showed that the new synthetic methoxy-stilbenes, 3,4,2'-trimethoxy-trans-stilbene (3MS), 3,4,2',4'-tetramethoxy-trans-stilbene (4MS), and 3,4,2',4',6'-pentamethoxy-trans-stilbene (5MS), modulate the constitutive expression of enzymes and receptors involved in estrogen metabolism in breast immortalized epithelial MCF10 cells. In this study, we evaluated the effect of 3MS, 4MS, and 5MS in comparison to resveratrol activity in MCF7 estrogen-dependent and MDA-MB-231 estrogen-independent breast cancer cell lines. 3MS similarly to resveratrol reduced the expression of estrogen receptor α in MCF7 cells. However, in these cells, 5MS reduced the most CYP19, the gene encoding aromatase, at mRNA transcript level. In contrast, in the MDA-MB-231 cells, the most efficient inhibitor of CYP19 expression was 3MS, reducing the level of its protein by ~ 25%. This stilbene also inhibited the aromatase activity in a recombinant protein system with IC50 value ~ 85 µM. Treatment with the methoxy-stilbenes reduced the level of estradiol in culture medium. The most significant reduction was exerted by 3MS. None of the tested stilbenes including resveratrol changed significantly the expression of AhR, although CYP1A1 protein level was slightly reduced in MDA-MB-231 cells, while CYP1B1 expression was increased in these cells as a result of treatment with 3MS, but only at the transcript level. Overall, these results show weak or moderate effect of the new methoxy-stilbenes on the expression of key proteins involved in estrogens metabolism in cancer breast cells. However, the reduced CYP19 expression and activity upon 3MS treatment in metastatic MDA-MB-231 cells require the further studies.

Resveratrol and its methoxy derivatives modulate the expression of estrogen metabolism enzymes in breast epithelial cells by AhR down-regulation.

Our earlier studies have shown that compared to resveratrol, its analogs with ortho-methoxy substituents exert stronger antiproliferative and proapoptotic activity. Since estrogens are considered the major risk factors of breast carcinogenesis, the aim of this study was to evaluate the effect of 3,4,2'-trimethoxy (3MS), 3,4,2',4'-tetramethoxy (4MS), and 3,4,2',4',6'-pentamethoxy (5MS) trans-stilbenes on the constitutive expression of the enzymes involved in estrogen metabolism, as well as receptors: AhR and HER2 in breast epithelial cell line MCF10A. The results showed different effect of resveratrol and its methoxy derivatives on the expression of genes encoding key enzymes of estrogen synthesis and catabolism. Resveratrol at the doses of 1 and 5 µmol/L increased the level of CYP19 transcript and protein level, while 5MS reduced mRNA transcript of both CYP19 and STS genes. Resveratrol and all its derivatives reduced also SULT1E1 mRNA transcript level. The reduced expression of AhR, CYP1A1, and 1B1 was also found as a result of treatment with these compounds. The most significant changes were found in the case of AhR. The most potent inhibitor of CYP1A1 and 1B1 genes expression was 5MS, which reduced the levels of mRNA transcript and protein of both CYPs from 31 to 89% of the initial levels. These results indicate that methoxy derivatives of resveratrol might be efficient modulators of estrogen metabolism. Moreover, the number of methoxy groups introduced to stilbene structure may play a certain role in this effect.

Protective effects of levamisole, acetylsalicylic acid, and α-tocopherol against dioxin toxicity measured as the expression of AhR and COX-2 in a chicken embryo model.

Polychlorinated dibenzo-p-dioxins and dibenzofurans (dioxins) are classed as persistent organic pollutants and have adverse effects on multiple functions within the body. Dioxins are known carcinogens, immunotoxins, and teratogens. Dioxins are transformed in vivo, and interactions between the products and the aryl hydrocarbon receptor (AhR) lead to the formation of proinflammatory and toxic metabolites. The aim of this study was to determine whether α-tocopherol (vitamin E), acetylsalicylic acid (ASA), and levamisole can decrease the amount of damage caused by dioxins. Fertile Hubbard Flex commercial line chicken eggs were injected with solutions containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or containing TCDD and the test compounds. The chicken embryos and organs were analyzed after 7 and 13 days. The levels at which AhR and cyclooxygenase-2 (COX-2) proteins (which are induced during inflammation) were expressed were evaluated by performing immunohistochemical analyses on embryos treated with TCDD alone or with TCDD and the test compounds. TCDD caused developmental disorders and increased AhR and COX-2 expression in the chicken embryo tissues. Vitamin E, levamisole, ASA, and ASA plus vitamin E inhibited AhR and COX-2 expression in embryos after 7 days and decreased AhR and COX-2 expression in embryos after 13 days. ASA, levamisole, and ASA plus vitamin E weakened the immune response and prevented multiple organ changes. Vitamin E was not fully protective against developmental changes in the embryos.

Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes.

Induction of expression of aryl hydrocarbon receptor-dependent genes in human HepaRG cell line modified by shRNA and treated with ß-naphthoflavone.

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. In this study, the effects of administration of ß-naphthoflavone (BNF), a potent AhR ligand, on the expression of AhR-dependent genes were examined by microarray and qPCR analysis in both, differentiated and undifferentiated HepaRG cell lines. To prove that BNF-induced changes of investigated genes were indeed AhR-dependent, we knock down the expression of AhR by stable transfection of HepaRG cells with shRNA. Regardless of genetical identity, our results clearly demonstrate different expression profiles of AhR-dependent genes between differentiated and undifferentiated HepaRG cells. Genes involved in metabolism of xenobiotics constitute only minute fraction of all genes regulated by AhR in HepaRG cells. Participation of AhR in induction of expression of genes associated with regulation of apoptosis or involved in cell proliferation as well as AhR-dependent inhibition of genes connected to cell adhesion could support suggestion of involvement of AhR not only in initiation but also in progression of carcinogenesis. Among the AhR-dependent genes known to be involved in metabolism of xenobiotics, cytochromes P4501A1 and 1B1 belong to the most inducible by BNF. On the contrary, expression of GSTA1 and GSTA2 was significantly inhibited after BNF treatment of HepaRG cells. Among the AhR-dependent genes that are not involved in metabolism of xenobiotics SERPINB2, STC2, ARL4C, and TIPARP belong to the most inducible by BNF. Our results imply involvement of Ah receptor in regulation of CYP19A1, the gene-encoding aromatase, and an enzyme responsible for a key step in the biosynthesis of estrogens.

Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes≥2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction.

Aryl hydrocarbon receptor-microRNA-212/132 axis in human breast cancer suppresses metastasis by targeting SOX4.

BACKGROUND: MicroRNAs (miRNAs) are a class of short non-coding RNAs that pave a new avenue for understanding immune responses and cancer progression. Although the miRNAs are involved in breast cancer development, their axis with the transcription factors that show therapeutic potential in breast cancer is largely unknown. Previous studies showed anti-metastatic roles of agonist-activated aryl hydrocarbon receptor (Ahr) in various breast cancer cell lines. Recently, we demonstrated that agonist-activated Ahr induced a highly conserved miRNA cluster, named miR-212/132, in murine cellular immune compartment. Therefore, current study was performed to examine if this miRNA cluster mediates the anti-metastatic properties of Ahr agonists. METHODS: The expression of miR-212/132 cluster and coding genes were examined by real-time PCR, and the protein levels were detected by western blot. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3'-diindolylmethane (DIM) were used to activate Ahr in MDA-MB-231 and T47D breast cancer cells. Chromatin immunoprecipitation (ChIP) assay was used to identify the binding site(s) for Ahr on miR-212/132 promoter. For prediction of potentially target gene of the miRNA cluster, bioinformatics analysis was carried out, and to test targeting, luciferase activity was quantified. Besides, biological effects of Ahr-miR-212/132 axis were examined in vitro by cell migration, expansion and invasion, and examined in vivo by orthotopic model of spontaneous metastasis. RESULTS: The miR-212/132 cluster was transcriptionally activated in MDA-MB-231 and T47D cells by TCDD and DIM, and this activation was regulated by Ahr. A reciprocal correlation was identified between Ahr agonists-induced miR-212/132 and the pro-metastatic SRY-related HMG-box4 (SOX4), and a new specific binding sites for miR-212/132 were identified on the untranslated region (3'UTR) of SOX4. Interestingly, miR-212/132 over-expression showed direct anti-migration, anti-expansion and anti-invasion properties, and an inhibition of the miRNA cluster mitigated the anti-invasive properties of TCDD and DIM. Further in vivo studies demonstrated that the Ahr-miR-212/132-SOX4 module was induced by Ahr activation. CONCLUSION: Taken together, the findings provide the first evidences of the synergistic anti-metastatic properties of miR-212/132 cluster through suppression of SOX4. Also, current study suggest a new miRNA-based mechanism elucidating the anti-metastatic properties of Ahr agonists, suggesting possibility of using miR-212/132 to control metastasis in breast cancer patients.

Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis.

The novel Aryl hydrocarbon receptor inhibitor biseugenol inhibits gastric tumor growth and peritoneal dissemination.

Biseugenol (Eug) is known to antiproliferative of cancer cells; however, to date, the antiperitoneal dissemination effects have not been studied in any mouse cancer model. In this study, Aryl hydrocarbon receptor (AhR) expression was associated with lymph node and distant metastasis in patients with gastric cancer and was correlated with clinicolpathological pattern. We evaluated the antiperitoneal dissemination potential of knockdown AhR and Biseugenol in cancer mouse model and assessed mesenchymal characteristics. Our results demonstrate that tumor growth, peritoneal dissemination and peritoneum or organ metastasis implanted MKN45 cells were significantly decreased in shAhR and Biseugenol-treated mice and that endoplasmic reticulum (ER) stress was caused. Biseugenol-exposure tumors showed acquired epithelial features such as phosphorylation of E-cadherin, cytokeratin-18 and loss mesenchymal signature Snail, but not vimentin regulation. Snail expression, through AhR activation, is an epithelial-to-mesenchymal transition (EMT) determinant. Moreover, Biseugenol enhanced Calpain-10 (Calp-10) and AhR interaction results in Snail downregulation. The effect of shCalpain-10 in cancer cells was associated with inactivation of AhR/Snail promoter binding activity. Inhibition of Calpain-10 in gastric cancer cells by short hairpin RNA or pharmacological inhibitor was found to effectively reduced growth ability and vessel density in vivo. Importantly, knockdown of AhR completed abrogated peritoneal dissemination. Herein, Biseugenol targeting ER stress provokes Calpain-10 activity, sequentially induces reversal of EMT and apoptosis via AhR may involve the paralleling processes. Taken together, these data suggest that Calpain-10 activation and AhR inhibition by Biseugenol impedes both gastric tumor growth and peritoneal dissemination by inducing ER stress and inhibiting EMT.

Effects of individual polychlorinated naphthalene (PCN) components of Halowax 1051 and two defined, artificial PCN mixtures on AHR and CYP1A1 protein expression, steroid secretion and expression of enzymes involved in steroidogenesis (CYP17, 17ß-HSD and CYP19) in porcine ovarian follicles.

In this study we tried to answer a question which component of Halowax 1051 is responsible for, observed in previously published study, androgenic effects of the mixture, and whether it is possible to draw conclusions about the action of mixtures by examining the effect of an indicator congener. Ovarian follicles were incubated with individual congeners of an artificial mixture for 6-24h. At the end of the incubation period, media were collected for determination of progesterone (P4), androstenedione (A4), testosterone (T) and estradiol (E2) levels by enzyme immunoassay, and follicles were retained for an examination of aryl hydrocarbon receptor (AHR), cytochrome p450 enzymes (CYP1A1, CYP17, CYP19), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) protein expression by Western blotting. CN73 in dose 50pg/ml after 6h had no effect and decreased AHR expression after 24h, while at dose 400pg/ml increased AHR protein expression after 6h of exposure which remained elevated after 24h. CN74 and CN75 at both concentrations tested (25 and 50pg/ml) stimulated AHR protein expression after 6h and decreased it after 24h of exposure. Individual congeners induced a rapid increase in CYP1A1 protein expression, with a rank order of efficacy of CN73>CN74=CN75. All congeners increased P4/A4 and T/E2 secretion ratios in association with a decrease in the A4/T ratio, pointing to androgenic and anti-estrogenic properties of PCNs in ovarian follicles. The most potent congener in this context was CN73. The effects of mixtures were comparable to those of CN74 and CN75, and were not as strong as those observed for CN73. Collectively, these data suggest antagonistic actions of single congeners in a mixture, indicating that the actions of a mixture cannot be predicted based on the actions of individual congeners.