Andreas J. Papoutsis

Resveratrol Prevents Epigenetic Silencing of BRCA-1 by the Aromatic Hydrocarbon Receptor in Human Breast Cancer Cells

The BRCA-1 protein is a tumor suppressor involved in repair of DNA damage. Epigenetic mechanisms contribute to its reduced expression in sporadic breast tumors. Through diet, humans are exposed to a complex mixture of xenobiotics and natural ligands of the aromatic hydrocarbon receptor (AhR), which contributes to the etiology of various types of cancers. The AhR binds xenobiotics, endogenous ligands, and many natural dietary bioactive compounds, including the phytoalexin resveratrol (Res). In estrogen receptor- alpha (ER alpha )-positive and BRCA-1 wild-type MCF-7 breast cancer cells, we investigated the influence of AhR activation with the agonist 2,3,7,8 tetrachlorobenzo(p)dioxin (TCDD) on epigenetic regulation of the BRCA-1 gene and the preventative effects of Res. We report that activation and recruitment of the AhR to the BRCA-1 promoter hampers 17 beta -estradiol (E2)-dependent stimulation of BRCA-1 transcription and protein levels. These inhibitory effects are paralleled by reduced occupancy of ER alpha , acetylated histone (AcH)-4, and AcH3K9. Conversely, the treatment with TCDD increases the association of mono-methylated-H3K9, DNA-methyltransferase-1 (DNMT1), and methyl-binding domain protein-2 with the BRCA-1 promoter and stimulates the accumulation of DNA strand breaks. The AhR-dependent repression of BRCA-1 expression is reversed by small interference for the AhR and DNMT1 or pretreatment with Res, which reduces TCDD-induced DNA strand breaks. These results support the hypothesis that epigenetic silencing of the BRCA-1 gene by the AhR is preventable with Res and provide the molecular basis for the development of dietary strategies based on natural AhR antagonists.

Gestational Exposure to the AhR Agonist 2,3,7, 8-Tetrachlorodibenzo-p-dioxin Induces BRCA-1 Promoter Hypermethylation and Reduces BRCA-1 Expression in Mammary Tissue of Rat Offspring: Preventive Effects of Resveratrol

Studies with murine models suggest that maternal exposure to aromatic hydrocarbon receptor (AhR) agonists may impair mammary gland differentiation and increase the susceptibility to mammary carcinogenesis in offspring. However, the molecular mechanisms responsible for these perturbations remain largely unknown. Previously, we reported that the AhR agonists 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) induced CpG methylation of the breast cancer‐1 (BRCA‐1) gene and reduced BRCA‐1 expression in breast cancer cell lines. Based on the information both the human and rat BRCA‐1 genes harbor xenobiotic responsive elements (XRE = 5′‐GCGTG‐3′), which are binding targets for the AhR, we extended our studies to the analysis of offspring of pregnant Sprague–Dawley rats treated during gestation with TCDD alone or in combination with the dietary AhR antagonist resveratrol (Res). We report that the in utero exposure to TCDD increased the number of terminal end buds (TEB) and reduced BRCA‐1 expression in mammary tissue of offspring. The treatment with TCDD induced occupancy of the BRCA‐1 promoter by DNA methyltransferase‐1 (DNMT‐1), CpG methylation of the BRCA‐1 promoter, and expression of cyclin D1 and cyclin‐dependent kinase‐4 (CDK4). These changes were partially overridden by pre‐exposure to Res, which stimulated the expression of the AhR repressor (AhRR) and its recruitment to the BRCA‐1 gene. These findings point to maternal exposure to AhR agonists as a risk factor for breast cancer in offspring through epigenetic inhibition of BRCA‐1 expression, whereas dietary antagonists of the AhR may exert protective effects.

BRCA-1 promoter hypermethylation and silencing induced by the aromatic hydrocarbon receptor-ligand TCDD are prevented by resveratrol in MCF-7 cells.

Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XREs=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XREs in the proximal BRCA-1 promoter and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-α-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17β-estradiol-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR; DNA methyl transferase (DNMT)1, DNMT3a and DNMT3b; methyl binding protein (MBD)2; and trimethylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses (1 μmol/L) the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation and the recruitment of the AhR, MBD2, H3K9me3 and DNMTs (1, 3a and 3b). Taken together, these observations provide mechanistic evidence for AhR agonists in the establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

Nutritional Targeting of Cyclooxygenase-2 for Colon Cancer Prevention

Factors related to diet and life style have been identified as primary determinants in about 80% of colorectal cancers. Non-steroidal anti-inflammatory drugs (NSAID) and selective cyclooxygenase-2 (COX-2) inhibitors (COXIB) reduce the relative risk of colon cancer. To overcome systemic COX inhibition associated with NSAID and COXIB, there is a growing interest in developing alternative colon cancer prevention strategies using diet-based approaches that target COX-2. The transition from aberrant crypt foci (ACF) to colon cancer is a multiyear process providing opportunities for nutritional targeting of genes influencing the course of this disease process at early stages of development. The activation of the proinflammatory gene COX-2 and PG production in the colonic mucosa are recognized risk factors in colon cancer. Many natural food components may impact colon cancer risk by interfering with ligand-activated receptors, signal transduction pathways, and transcription factors involved in stimulation of COX-2 expression. In this review, we highlight key upstream features of signaling pathways and transcriptional control of the COX-2 gene and discuss opportunities for dietary modulation of COX-2 expression in gastro-intestinal cancers with special emphasis on prevention of colorectal tumors. Review of the experimental evidence suggests that dietary strategies based on specific or cocktails of bioactive food components as well nutritional-pharmacological combinations targeted to regulation of COX-2 expression and activity may prove useful in the prevention of colon cancer. An integrated approach may offer the advantage of combined higher efficacies. Future studies should investigate the efficacy of combinations of bioactive food compounds on epigenetic regulation of the COX-2 gene and characterize potential synergies and amplification effects resulting from the concomitant use of bioactive food components and COX-2 inhibitors.

Genistein Prevents BRCA1 CpG Methylation and Proliferation in Human Breast Cancer Cells with Activated Aromatic Hydrocarbon Receptor

Abstract Background: Previous studies have suggested a causative role for agonists of the aromatic hydrocarbon receptor (AhR) in the etiology of breast cancer 1, early-onset (BRCA-1)–silenced breast tumors, for which prospects for treatment remain poor. Objectives: We investigated the regulation of BRCA1 by the soy isoflavone genistein (GEN) in human estrogen receptor α (ERα)–positive Michigan Cancer Foundation-7 (MCF-7) and ERα-negative sporadic University of Arizona Cell Culture-3199 (UACC-3199) breast cancer cells, respectively, with inducible and constitutively active AhR. Methods: In MCF-7 cells, we analyzed the dose- and time-dependent effects of GEN and (–)-epigallocatechin-3-gallate (EGCG) control, selected as prototype dietary DNA methyltransferase (DNMT) inhibitors, on BRCA-1 expression after AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in TCDD-washout experiments. We compared the effects of GEN and EGCG on BRCA1 cytosine-phosphate-guanine (CpG) methylation and cell proliferation. Controls for DNA methylation and proliferation were changes in expression of DNMT-1, cyclin D1, and p53, respectively. In UACC-3199 cells, we compared the effects of GEN and α-naphthoflavone (αNF; 7,8-benzoflavone), a synthetic flavone and AhR antagonist, on BRCA1 expression and CpG methylation, cyclin D1, and cell growth. Finally, we examined the effects of GEN and αNF on BRCA1, AhR-inducible cytochrome P450 (CYP)-1A1 (CYP1A1) and CYP1B1, and AhR mRNA expression. Results: In MCF-7 cells, GEN exerted dose- and time-dependent preventative effects against TCDD-dependent downregulation of BRCA-1. After TCDD washout, GEN rescued BRCA-1 protein expression while reducing DNMT-1 and cyclin D1. GEN and EGCG reduced BRCA1 CpG methylation and cell proliferation associated with increased p53. In UACC-3199 cells, GEN reduced BRCA1 and estrogen receptor-1 (ESR1) CpG methylation, cyclin D1, and cell growth while inducing BRCA-1 and CYP1A1. Conclusions: Results suggest preventative effects for GEN and EGCG against BRCA1 CpG methylation and downregulation in ERα-positive breast cancer cells with activated AhR. GEN and flavone antagonists of AhR may be useful for reactivation of BRCA1 and ERα via CpG demethylation in ERα-negative breast cancer cells harboring constitutively active AhR.

Health Benefits of Traditional culinary and Medicinal Mediterranean Plants

Publisher Summary This chapter focuses on the health benefits of traditional culinary and medicinal plants of the Mediterranean region. The Mediterranean diet is mentioned to be rich in fruits and vegetables, monounsaturated fatty acids and olive oil and the diet has a negative association with incidence of metabolic syndrome that contributes to an increased risk for cardiovascular disease. Mediterranean plants are believed to prevent or cure a wide range of ailments based on their bioactive components that can exert anti-oxidant, anti-inflammatory, anti-carcinogenic or anti-diabetic activities. A table is presented that gives a list of Mediterranean plants and their health benefits as well as brief accounts of the medicinal use of the plants and includes rosemary, licorice, chamomile and olive oil among others. The consumption of olive oil has a strong correlation with reduced hypertension, cancers of the prostate, breast and colon. It highlights the mechanism of action of the bioactive components and the molecular targets derived from these plants.

Abstract 4318: Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Sporadic breast cancers, which represent the vast majority (∼90%) of breast tumor cases, do not have mutations in the tumor suppressor gene, BRCA-1 but have absent or markedly reduced levels of BRCA-1 similar to those observed in hereditary BRCA-1 tumors. Therefore, understanding the non-mutational mechanisms that contribute to repression of BRCA-1 has important implications for the prevention of both hereditary and sporadic breast tumors. Agonists of the aromatic hydrocarbon receptor (AhR) are ubiquitous in the environment and include dietary compounds, metabolites of fatty acids, industrial pollution, and photoproducts generated in the skin from ultraviolet radiation. In cultured estrogen-receptor (ER)α-positive MCF-7 breast cancer cells, we found that activation of the AhR with the prototype ligand 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) repressed by 50% estradiol-induced BRCA-1 expression. Conversely, the post-treatment with the soy isoflavone genistein and tea (-)-epigallocatechin-3-gallate (EGCG) at physiological doses (1 μM) rescued within 24 to 48 h BRCA-1 expression to levels ∼2.0 to 3.0-fold higher than those measured in cells treated with estradiol alone. Genistein and EGCG reversed BRCA-1 promoter hypermethylation in AhR-activated MCF-7 cells. In ERα-negative and AhR-overexpressing UACC-3199 breast cancer cells, genistein (1 μM) reactivated BRCA-1 expression. The post-treatment of AhR-activated MCF-7 cells with genistein and EGCG antagonized the recruitment of DNA-methyl transferase-1 (DNMT-1) enzyme to the BRCA-1 promoter. These results suggest the involvement of epigenetic mechanisms in the repression of BRCA-1 and reversal effects of genistein and EGCG against breast tumorigenesis associated with activation of the AhR. Citation Format: Ornella I. Selmin, Andreas J. Papoutsis, Donato F. Romagnolo. Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4318.

Abstract 1115: Epigenetic regulation of BRCA-1 by xenobiotic and dietary ligands of the aryl hydrocarbon receptor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Mutations of BRCA-1 account for only 5-10% of breast cancer cases, whereas sporadic breast tumors represent the remaining 90-95%. In sporadic breast cancers there is silencing of BRCA-1 in the absence of mutations. Factors contributing to this etiology may include environmental ligands of the aryl hydrocarbon receptor (AhR) such as dioxins (ex.2,3,7,8 tetrachlorodibenzene(p)dioxin, TCDD) and polycyclic aryl hydrocarbons (PAHs). Dietary ligands of the AhR with potential protective effects include the phytoalexin resveratrol found in grapes. In this study, we investigated the mechanisms responsible for repression of BRCA-1 expression by the activated AhR. The results of DNA binding studies in MCF-7 breast cancer cells documented that TCDD-induced the association of the AhR to xenobiotic responsive elements (XRE) harbored in the BRCA-1 promoter region. The cotreatment with resveratrol reduced AhR recruitment in a dose-dependent fashion, prevented the TCDD-dependent repression of BRCA-1 transcription and restored BRCA-1 protein expression. The treatment with TCDD induced the recruitment of histone deacetylase-1, DNA methyltransferase-1, methyl-binding protein-2, and reduced the association of acetylated histone-4 and acetylated histone-K9. These effects were abrogated by cotreatment with resveratrol. We propose that dietary antagonists of the AhR such as resveratrol may be useful in the prevention of epigenetic BRCA-1 silencing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1115.