Wanda Baer-Dubowska

The effect of dietary polyphenols on the epigenetic regulation of gene expression in MCF7 breast cancer cells

The CpG island methylator phenotype is characterized by DNA hypermethylation in the promoters of several suppressor genes associated with the inactivation of various pathways involved in tumorigenesis. DNA methylation is catalyzed by specific DNA methyltransferases (DNMTs). Dietary phytochemicals particularly catechol-containing polyphenols were shown to inhibit these enzymes and reactivate epigenetically silenced genes. The aim of this study was to evaluate the effect of a wide range of dietary phytochemicals on the activity and expression of DNMTs in human breast cancer MCF7 cell line and their effect on DNA and histone H3 methylation. All phytochemicals inhibited the DNA methyltransferase activity with betanin being the weakest while rosmarinic and ellagic acids were the most potent modulators (up to 88% inhibition). While decitabine led to a partial demethylation and reactivation of the genes, none of the tested phytochemicals affected the methylation pattern or the expression of RASSF1A, GSTP1 or HIN1 in MCF7 cells. The global methylation of histone H3 was not affected by any of the tested phytochemicals or decitabine. The results of our study may suggest that non-nucleoside agents are not likely to be effective epigenetic modulators, in our experimental model at least. However, a long-term exposure to these chemicals in diet might potentially lead to an effect, which can be sufficient for cancer chemoprevention.

Epigenetic diagnostics of cancer — the application of DNA methylation markers

In recent years it has become apparent that epigenetic events are potentially equally responsible for cancer initiation and progression as genetic abnormalities. DNA methylation is the main epigenetic modification in humans. Two DNA methylation lesions coexist in human neoplasms: hypermethylation of promoter regions of specific genes within a context of genomic hypomethylation. Aberrant methylation is found at early stages of carcinogenesis and distinct types of cancer exhibit specific patterns of methylation changes. Tumor specific DNA is readily obtainable from different clinical samples and methylation status analysis often permits sensitive disease detection. Methylation markers may also serve for prognostic and predictive purposes as they often reflect the metastatic potential and sensitivity to therapy. As current findings show a great potential of recently characterised methylation markers, more studies in the field are needed in the future. Large clinical studies of newly developed markers are especially needed. The review describes the diagnostic potential of DNA methylation markers.

Pterostilbene is equally potent as resveratrol in inhibiting 12‐O‐tetradecanoylphorbol‐13‐acetate activated NFκB, AP‐1, COX‐2, and iNOS in mouse epidermis

Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kappaB (NF-kappaB) induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. The aim of the present study was to further explore the effect of resveratrol on TPA-induced signaling pathways in mouse epidermis and to compare with its dimethylether, pterostilbene. Resveratrol and pterostilbene significantly reduced activator protein 1 (AP-1) and NF-kappaB activation. In the case of AP-1, the binding of c-Jun subunit was particularly affected, while only slight effect on c-Fos binding to TPA-responsive element (AP-1 binding consensus sequence) (TRE) site was observed. Both stilbenes inhibited the activation of NF-kappaB by blocking the translocation of p65 to the nucleus and increasing the retention of IkappaBa in the cytosol. The latter might be related to decreased activity of IkappaB kinase and/or proteasome 20S. Reduced activation of transcription factors decreased the expression and activity of COX-2 and inducible nitric oxide synthase (iNOS). In most assays, pterostilbene was either equally or significantly more potent than resveratrol. Pterostilbene might show higher biological activity due to its possible better bioavailability, since substitution of hydroxy with methoxy group increases lipophilicity.

Influences of chronic venlafaxine, olanzapine and nicotine on the hippocampal and cortical concentrations of brain-derived neurotrophic factor (BDNF)

Brain-derived neurotrophic factor (BDNF) is a key neurotrophic factor in the brain. It plays an important role in the etiopathogenesis and pharmacotherapy of mental disorders, such as depression or schizophrenia. In recent years, studies have shown that cognitive processes, which are impaired in the course of mental disorders, significantly change BDNF levels in the brain. Administered to rats at a dose of 20 mg/kg (b.d. for 5 weeks), venlafaxine (VEN) increases BDNF levels in the hippocampus and cortex, compared to controls. Administered at a dose of 0.5 mg/kg (b.d. for 5 weeks), olanzapine (OLA) significantly increases BDNF levels in both the cortex and the hippocampus. Similarly, nicotine (NIC) administered at a dose of 0.2 mg/kg (b.d. for 5 weeks) increases BDNF concentrations in both the hippocampus and the cortex. Combined administration of NIC with VEN or OLA does not increase BDNF levels in the hippocampus or the cortex. Based on our study, it can be claimed that BDNF mediates behavioral responses only to drugs used individually and participates in the antidepressant and procognitive effects of the study compounds. BDNF also initiates plastic changes and modulation of synaptic activity in rat brains.

The effects of tannic acid on cytochrome P450 and phase II enzymes in mouse liver and kidney

Tannic acid, a naturally occurring plant polyphenol, was shown to decrease the mutagenicity and/or carcinogenicity of several amines derivatives and polycyclic aromatic hydrocarbons in rodents. The aim of this study was to evaluate the effect of tannic acid on the activities of murine cytochrome P450 and phase II enzymes. The activities of ethoxyresorufin-O-deethylase (EROD), methoxyresorufin-O-demethylase (MROD), p-nitrophenol hydroxylase (PNPH), glutathione S-transferase (GST), UDP-glucuronosyltransferase (UDPGT) and NAD(P)H:quinone oxidoreductase (NQO1) were measured in the liver and kidney microsomes of female Swiss mice treated intraperitoneally (i.p.) with tannic acid in the dose range of 20-80 mg/kg. At the highest dose, tannic acid decreased the activities of EROD and MROD by 25-28% in mouse liver, while the activity of both hepatic and renal PNPH was reduced by approximately 50% as result of treatment. Moreover, Western blot analysis with CYP2E1 specific antibody showed a significant decrease in the levels of hepatic CYP2E1 in tannic acid treated animals. This polyphenol affected also the phase II enzymes in both tissues examined. The activity of GST was elevated in kidneys, but reduced in livers of the animals treated with tannic acid. The most striking effect was the inhibition of hepatic NQO1. The effect was dose dependent and almost 90% inhibition was observed after the treatment with tannic acid at the dose of 60 and 80 mg/kg. The same treatment caused the approximately 60% inhibition of renal NQO1. These results indicate that tannic acid, beside of scavenging active metabolites of chemical carcinogens, can change their metabolism by modulating the enzymes involved in xenobiotics activation and/or detoxification pathways.

Alteration in Phase I and II Enzyme Activities and Polycyclic Aromatic Hydrocarbons-DNA Adduct Formation by Plant Phenolics in Mouse Epidermis

Several naturally occurring plant phenols were shown to inhibit the mutagenicity and/or tumorigenicity of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs). In this study, the effect of the topical application of three structurally diverse phenolic acids and trihydroxystilbene, resveratrol, on epidermal aryl hydrocarbon hydroxylase (AHH), phase II enzymes, as well as the binding of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) to epidermal DNA were compared. The single, topical application of 8 and 16 μmol of protocatechuic or chlorogenic acid increased the activity of AHH by 10-30%, whereas resveratrol in a dose of 16 μmol almost completely (99%) inhibited the enzyme activity. Phenolic acids also increased the activities of phase II enzymes. Resveratrol did not affect the glutathione S-transferase activity but induced UDP glucuronosyltransferase (by ~100-150%) and to a lesser extent NAD(P)H:quinone oxidoreductase. In a dose of 16 μmol all phenolic acids afforded 40-50% inhibition of covalent benzo[a]pyrene-diol-epoxide (B[a]PDE) binding to DNA. Resveratrol had no effect on B[a]PDE adduct formation but reduced the levels of all the major DMBA adducts. Phenolic acids, particularly tannic acid, mostly affected the formation of syn- and anti-DMBADE dAdo adducts. These results indicate that both the modulation of carcinogen activating enzymes and the prevention of their ultimate metabolites binding to DNA by naturally occurring phenolics are involved in the antitumorigenic activity of these compounds. For phenolic acids, however, their interactions with reactive PAH metabolites and/or blocking of a specific binding site in a genome seem more important. Derivatives of stilbene, such as resveratrol, affect DNA adduct formation and thus the initiation of tumorigenesis through the interaction with the Ah receptor rather than the scavenging active metabolites.

Frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT in laryngeal squamous cell carcinomas and adjacent normal mucosa

Laryngeal cancers are the most frequent cancers of the head and neck region. While recent observations indicate the occurrence of an epigenetic field defect in head and neck cancer patients, a detailed exploration of the characteristic changes in the DNA methylation profile in laryngeal cancer patients was lacking. The aim of this study was to assess the methylation frequency of seven genes in a group of patients with primary laryngeal squamous cell carcinoma. Along tumor sections, matching samples of normal mucosa from epiglottis and trachea were analyzed. Gene methylation was assessed using the methylation-specific polymerase chain reaction. We found frequent gene hypermethylation in both the tumor and normal mucosa samples. The methylation of MGMT in tumor cells was associated with lymph node involvement. We report that laryngeal squamous cell carcinomas are characterized by frequent hypermethylation of DAPK, RARbeta, MGMT, RASSF1A and FHIT. Moreover, evidence is shown for the occurrence of a large epigenetically changed field of epithelial cells in laryngeal cancer patients. Our findings indicate the high prevalence of epigenetic abnormalities in laryngeal tumors.

Pharmocoepigenetics: a new approach to predicting individual drug responses and targeting new drugs

Epigenetics is the study of heritable changes in genes and gene expression that do not involve DNA nucleotide sequences. Epigenetic modifications include DNA methylation, several forms of histone modifications, and microRNA expression. Because of its dynamic nature, epigenetics provides a link between the genome and the environment and fills the gap between DNA and proteins. Advances in epigenetics and epigenomics (the study of epigenetics on a genome-wide basis) have influenced pharmacology, leading to the development of a new specialty, pharmacoepigenetics, the study of the epigenetic basis for variations in drug response. Many genes encoding enzymes, drug transporters, nuclear receptors, and drug targets are under epigenetic control. This review describes the known epigenetic regulation of drug-metabolizing enzymes and other proteins that might affect drug response and compounds that modify the epigenetic status.

The negative regulators of Wnt pathway—DACH1, DKK1, and WIF1 are methylated in oral and oropharyngeal cancer and WIF1 methylation predicts shorter survival

The deregulation of Wnt signaling has recently emerged as one of the drivers of head and neck cancers. This is frequently related to the methylation of several antagonists of this pathway. This study aimed at the assessment of the profile of methylation of Wnt pathway antagonists and the determination of the prognostic value of the methylation of selected genes in oral carcinomas. The methylation of DACH1, DKK1, LKB1, PPP2R2B, RUNX3, SFRP2, and WIF-1 was analyzed in 16 oral squamous cell carcinoma cell lines using the methylation-specific polymerase chain reaction. The methylation of selected genes was further analyzed in tumor sections from 43 primary oral carcinoma patients. The analysis of oral carcinoma cell lines showed very frequent methylation of SFRP2 and WIF-1 and also a less frequent methylation of DACH1 and DKK1. On the other hand, RUNX3 was methylated only in one cell line, while LKB1 and PPP2R2B were not methylated in any of the cell lines. The biallelic methylation of DKK1 correlated with the low level of expression of this gene. Further evaluation of the methylation of DACH1, DKK1, and WIF1 in a clinical patient group confirmed the frequent methylation of WIF1 and intermediate or low frequency of methylation of DACH1 or DKK1, respectively. Importantly, the methylation of WIF-1 correlated with shorter survival in oral cancer patients. Overall, the methylation of the antagonists of Wnt pathway is frequently detected in oral squamous cell carcinomas. The methylation of WIF1 may be considered a prognostic marker in oral cancers.

Effect of tannic acid on benzo[a]pyrene‐DNA adduct formation in mouse epidermis: Comparison with synthetic gallic acid esters

Tannic acid, a naturally occurring plant phenol, was shown to inhibit the mutagenicity and/or tumorigenicity of several polycyclic aromatic hydrocarbons in mouse skin. In this study the effect of topical application of tannic acid on epidermal aryl hydrocarbon hydroxylase, glutathione S-transferase, and binding of benzo[a]pyrene (B[a]P) to epidermal DNA was compared with the activity of synthetic gallic acid esters. Single topical application of 8 mumol octyl and dodecyl gallate had no effect on the induction of aryl hydrocarbon hydroxylase, whereas propyl gallate and tannic acid increased the enzyme activity by nearly 200%. Application of the phenolics one hour before 0.2 mumol of B[a]P enhanced the enzyme activity, but the observed differences were not significant in comparison with a B[a]P-treated group of mice. Application of dodecyl and octyl gallates to mouse skin resulted in three- and twofold increases, respectively, in the activity of glutathione S-transferase. Combined treatment with dodecyl gallate and B[a]P also resulted in significant enhancement of this enzyme activity. Application of the same dose of tannic acid to mouse skin one hour before the application of 0.2 or 1 mumol of B[a]P afforded 60% inhibition of covalent benzo[a]pyrene-diol-epoxide binding to epidermal DNA. Gallic acid esters with the exception of dodecyl gallate were less effective inhibitors of benzo[a]pyrene-diol-epoxide binding, especially when the higher dose of B[a]P was used. These results indicate that the antitumorigenic activity of tannic acid involves the interaction of the ultimate carcinogen with DNA rather than an altered metabolism. The linkage between gallic acid and glucose in natural plant phenols is also more effective at inhibiting B[a]P binding to epidermal DNA than the linkage with the alkyl group in synthetic gallates.