Donato F. Romagnolo

Flavonoids and cancer prevention: a review of the evidence.

The objective of this work is to review data from epidemiological and preclinical studies addressing the potential benefits of diets based on flavonoids for cancer prevention. Flavonoids are subdivided into subclasses including flavonols, flavones, flavanones, flavan-3-ols, anthocyanidins, and isoflavones. Epidemiological studies suggest dietary intake of flavonoids may reduce the risk of tumors of the breast, colon, lung, prostate, and pancreas. However, some studies have reported inconclusive or even harmful associations. A major challenge in the interpretation of epidemiological studies is that most of the data originate from case-control studies and retrospective acquisition of flavonoid intake. Differences in agricultural, sociodemographics, and lifestyle factors contribute to the heterogeneity in the intake of flavonoids among populations residing in the United States, Europe, and Asia. Dose and timing of exposure may influence the anticancer response to flavonoid-rich diets. A limited number of intervention trials of flavonoids have documented cancer preventative effects. Proposed anticancer mechanisms for flavonoids are inhibition of proliferation, inflammation, invasion, metastasis, and activation of apoptosis. Prospective studies with larger sample sizes are needed to develop biomarkers of flavonoid intake and effect. Mechanistic studies are needed to ascertain how flavonoid-rich diets influence gene regulation for cancer prevention.

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.

Rosmarinic Acid Antagonizes Activator Protein-1–Dependent Activation of Cyclooxygenase-2 Expression in Human Cancer and Nonmalignant Cell Lines

One mechanism through which bioactive food components may exert anticancer effects is by reducing the expression of the proinflammatory gene cyclooxygenase-2 (COX-2), which has been regarded as a risk factor in tumor development. Rosmarinic acid (RA) is a phenolic derivative of caffeic acid present in rosemary (Rosmarinus officinalis). Previous research documented that RA may exert antiinflammatory effects. However, the mechanisms of action of RA on COX-2 expression have not been investigated. Here, we report that in colon cancer HT-29 cells, RA (5, 10, and 20 micromol/L) reduced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 promoter activity (P < 0.05) and protein levels (P < 0.05). In addition, the cotreatment with RA reduced (5 micromol/L, P < 0.05; 10 and 20 micromol/L, P < 0.01) TPA-induced transcription from a control activator protein-1 (AP-1) promoter-luciferase construct and repressed binding of the AP-1 factors c-Jun (10 micromol/L; P < 0.01) and c-Fos (10 micromol/L; P < 0.05) to COX-2 promoter oligonucleotides harboring a cAMP-response element (CRE). The anti-AP1 effects of RA were also examined in a nonmalignant breast epithelial cell line (MCF10A) in which RA antagonized the stimulatory effects of TPA on COX-2 protein expression (5 micromol/L, P < 0.05; 10 and 20 micromol/L, P < 0.01), the recruitment of c-Jun and c-Fos (10 micromol/L; P < 0.01) to the COX-2/CRE oligonucleotides, and activation of the extracellular signal-regulated protein kinase-1/2 (ERK1/2) (10 micromol/L; P < 0.01), a member of the mitogen-activated protein kinase pathway. Additionally, RA antagonized ERK1/2 activation in colon HT-29 and breast MCF-7 cancer cells (10 micromol/L; P < 0.01). Thus, we propose that RA may be an effective preventative agent against COX-2 activation by AP-1-inducing agents in both cancer and nonmalignant mammary epithelial cells.

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.

Inhibition of BRCA-1 expression by benzo[a]pyrene and its diol epoxide.

The objective of this study was to investigate whether polycyclic aromatic hydrocarbons (PAHs) contribute to the etiology of sporadic breast cancer by altering the expression of BRCA‐1. Acute exposure to the PAH benzo[a]pyrene (B[a]P) inhibited in a time‐ and dose‐dependent fashion cell proliferation and levels of BRCA‐1 mRNA and protein in estrogen receptor (ER)–positive breast MCF‐7 and ovarian BG‐1 cancer cells. Moreover, the acute exposure to B[a]P abrogated estrogen induction of BRCA‐1 in MCF‐7 cells. The loss of BRCA‐1 expression was prevented by the aromatic hydrocarbon receptor (AhR) antagonist α‐naphthoflavone, suggesting participation of the AhR pathway. BRCA‐1 exon 1a transcripts were downregulated by B[a]P faster than exon 1b mRNA was. Long‐term exposure to B[a]P (40 nM for 15 mo) lowered BRCA‐1 mRNA levels in subclones of MCF‐7 and BG‐1 cells, whereas expression of BRCA‐1 in these clones was reverted to normal levels by washing out of B[a]P. The mechanisms of BRCA‐1 repression by B[a]P were further investigated by examining the effects of the halogenated aryl hydrocarbon 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and the B[a]P metabolite 7r,8t‐dihydroxy‐9t,10t‐epoxy‐7,8,9,10‐tetrahydrobenzo[a]pyrene (BPDE). While TCDD did not influence basal BRCA‐1 mRNA and protein levels at any of the doses (from 10 nM to 1 μM) tested in this study, treatment with 50 nM BPDE drastically reduced BRCA‐1 mRNA levels, indicating that metabolism of B[a]P to BPDE may contribute to downregulation of BRCA‐1. Conversely, ER‐negative breast MDA‐MB‐231 and HBL‐100 cancer cells were refractory to treatment with B[a]P or TCDD and expressed constant levels of BRCA‐1 mRNA and protein. We conclude that B[a]P may be a risk factor in the etiology of sporadic breast cancer. Mol. Carcinog. 26:100–118, 1999.

Cyclooxygenase-2 Promoter Activation by the Aromatic Hydrocarbon Receptor in Breast Cancer MCF-7 Cells: Repressive Effects of Conjugated Linoleic Acid

Abstract The role of the aromatic hydrocarbon receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA > c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA > c9,t11-CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that the AhR may be a suitable target for prophylactic strategies that target COX-2 expression.

Deoxycholate, an Endogenous Tumor Promoter and DNA Damaging Agent, Modulates BRCA-1 Expression in Apoptosis-Sensitive Epithelial Cells: Loss of BRCA-1 Expression in Colonic Adenocarcinomas

Deoxycholate, a bile salt present at high levels in the colonic lumen of individuals on a high-fat diet, is a promoter of colon cancer. Deoxycholate also causes DNA damage. BRCA-1 functions in repair of DNA and in induction of apoptosis. We show that, when cultured cells of colonic origin are exposed to deoxycholate at different concentrations, BRCA-1 expression is induced at a low noncytotoxic concentration (10 μM) but is strongly inhibited at higher cytotoxic concentrations (>100 μM). Indication of phosphorylation of BRCA-1 by deoxycholate (100 μM) at a lower dose was seen by Western blot analysis, whereas, at a higher dose, deoxycholate (200 and 300 μM) caused a complete loss of BRCA-1 expression. We show that BRCA-1 is substantially lower in colon adenocarcinomas from five patients compared with associated non-neoplastic colon tissue from the same patients, suggesting that the loss of BRCA-1 expression contributes to the malignant phenotype. In the non-neoplastic colon tissue, BRCA-1 was localized to the nongoblet cells. Our results imply that reduced expression of BRCA-1 may be associated with carcinoma of the colon.

Expression of Cytosolic NADP+-Dependent Isocitrate Dehydrogenase in Bovine Mammary Epithelium: Modulation by Regulators of Differentiation and Metabolic Effectors

The cytosolic NADP+-dependent isocitrate dehydrogenase (IDH1) catalyzes the conversion of isocitrate to α-ketoglutarate in the cytosol, and generates NADPH as a primary source of reducing equivalents for de novo fatty acid synthesis in bovine mammary gland. The enzymatic activity of IDH1 increases dramatically in early lactation in bovine mammary tissue. We hypothesized that the expression of IDH1 in bovine is modulated by regulators of mammary epithelial differentiation. To test this hypothesis, we first examined the changes in IDH1 expression in late pregnancy (20 days) and at various stages (14, 90, 120, and 240 days) of lactation in bovine mammary tissue. IDH1 mRNA levels increased by 2.3-fold after parturition compared to late pregnancy and remained elevated thereafter. Next, we examined the effects of extracellular matrix and lactogenic hormones on the expression of IDH1 in cultured BME-UV bovine mammary epithelial cells. We found that expression of IDH1 mRNA increased in parallel with β-casein expression induced by extracellular matrix. Fetal calf serum and insulin repressed, whereas prolactin stimulated the expression of IDH1 mRNA in a dose-dependent fashion. The inhibitory effects of insulin on IDH1 mRNA levels were antagonized by cotreatment with prolactin. In contrast, treatment with prolactin in the presence of extracellular matrix further increased IDH1 mRNA and protein accumulation. Prolactin-induced IDH1 expression was inhibited by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126, and Janus tyrosine kinase 2 (Jak2) inhibitor AG490, suggesting that both MAPK and Jak2 contribute to regulation of IDH1 expression by prolactin. Finally, we report that treatment of BME-UV cells with α-ketoglutarate and palmitic acid reduced IDH1 transcript levels. Taken together, our data suggest that the expression of IDH1 in bovine mammary epithelium is modulated by regulators of differentiation including extracellular matrix and lactogenic hormones as well as metabolic effectors.

Epigenetics of breast cancer: Modifying role of environmental and bioactive food compounds.

SCOPE Reduced expression of tumor suppressor genes (TSG) increases the susceptibility to breast cancer. However, only a small percentage of breast tumors is related to family history and mutational inactivation of TSG. Epigenetics refers to non-mutational events that alter gene expression. Endocrine disruptors found in foods and drinking water may disrupt epigenetically hormonal regulation and increase breast cancer risk. This review centers on the working hypothesis that agonists of the aromatic hydrocarbon receptor (AHR), bisphenol A (BPA), and arsenic compounds, induce in TSG epigenetic signatures that mirror those often seen in sporadic breast tumors. Conversely, it is hypothesized that bioactive food components that target epigenetic mechanisms protect against sporadic breast cancer induced by these disruptors. METHODS AND RESULTS This review highlights (i) overlaps between epigenetic signatures placed in TSG by AHR-ligands, BPA, and arsenic with epigenetic alterations associated with sporadic breast tumorigenesis; and (ii) potential opportunities for the prevention of sporadic breast cancer with food components that target the epigenetic machinery. CONCLUSIONS Characterizing the overlap between epigenetic signatures elicited in TSG by endocrine disruptors with those observed in sporadic breast tumors may afford new strategies for breast cancer prevention with specific bioactive food components or diet.