Violetta Krajka-Kuźniak

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.

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.

Beetroot juice protects against N-nitrosodiethylamine-induced liver injury in rats

Red beetroot, a common ingredient of diet, is a rich source of a specific class of antioxidants, betalains. Our previous studies have shown the protective role of beetroot juice against carcinogen induced oxidative stress in rats. The aim of this study was to examine the effect of long term feeding (28 days) with beetroot juice on phase I and phase II enzymes, DNA damage and liver injury induced by hepatocarcinogenic N-nitrosodiethylamine (NDEA). Long term feeding with beetroot juice decreased the activities of enzymatic markers of cytochrome P450, CYP1A1/1A2 and CYP2E1. NDEA treatment also reduced the activities of these enzymes, but increased the activity of CYP2B. Moreover, combined treatment with beetroot juice and NDEA enhanced significantly CYP2B only. Modulation of P450 enzyme activities was accompanied by changes in the relevant proteins levels. Increased level and activity of NQO1 was the most significant change among phase II enzymes. Beetroot juice reduced the DNA damage increased as the result of NDEA treatment, as well as the biomarkers of liver injury. Collectively, these results confirm the protective effect of beetroot juice against oxidative damage shown in our previous studies and indicate that metabolic alterations induced by beetroot feeding may protect against liver damage.

The activation of the Nrf2/ARE pathway in HepG2 hepatoma cells by phytochemicals and subsequent modulation of phase II and antioxidant enzyme expression.

Previous studies have shown that naturally occurring phytochemicals, indole-3-carbinol, phenethyl isothiocyanate, protocatechuic acid, and tannic acid increased the activity and protein level of hepatic phase II enzymes in animal models. In order to further explore the mechanism of this activity, we investigated the effect of these compounds on the activation of nuclear factor erythroid-2-related factor 2 (Nrf2)-regulated transcription in human hepatocellular carcinoma HepG2 cells. Treatment with all the tested compounds resulted in the translocation from the cytosol and nuclear accumulation of active phosphorylated Nrf2. Furthermore, phenethyl isothiocyanate and indole-3-carbinol increased the transcript and protein levels of GSTA, GSTP, GSTM, GSTT, and NQO1. On the other hand, protocatechuic and tannic acids enhanced only the expression of GSTA, GSTM, and GSTT. The expression of genes encoding antioxidant enzymes CAT, SOD, GR, and GPx was increased after the treatment with all the tested phytochemicals. These results indicate that isothiocyanates/indoles and protocatechuic and tannic acids induce phase II and antioxidant gene expression in HepG2 cells through the Nrf2-Keap1-ARE signaling pathway. Moreover, the results of this study confirmed that the degradation products of glucosinolates are more effective inducers of phase II and antioxidant enzymes than protocatechuic and tannic acids.

Evaluation of the Effect of Beetroot Juice on DMBA-induced Damage in Liver and Mammary Gland of Female Sprague–Dawley Rats

Red beetroot contains a specific class of antioxidants collectively named betalains, which have been shown to have anticarcinogenic and anti‐inflamatory potential. We investigated the effect of beetroot juice on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12‐dimethylbenz[a]anthracene (DMBA). In the liver, pretreatment with beetroot juice significantly decreased levels and activities of the majority of tested biochemical parameters, elevated by DMBA. Feeding with beetroot juice decreased the activities of CYP1A1 and 1A2 and increased phase II enzymes. The activities of all enzymes tested were enhanced in the animals treated with DMBA alone and in combination with beetroot juice. The most significant changes in the level of the enzymes tested were observed for NAD(P)H:quinone oxidoreductase‐1. In mammary gland, beetroot juice induced the level of glutathione S‐transferase pi, enzyme involved in active metabolites of DMBA detoxification. The final effects of beetroot juice are tissue specific and depend on the class of carcinogen. Copyright

Modulation of CYP1A1, CYP1A2 and CYP1B1 Expression by Cabbage Juices and Indoles in Human Breast Cell Lines

Epidemiological studies have shown that consumption of cabbage and sauerkraut is connected with significant reduction of breast cancer incidences. Estrogens are considered a major breast cancer risk factor and their metabolism by P450 enzymes substantially contributes to carcinogenic activity. The aim of this study was to investigate the effect of cabbage and sauerkraut juices of different origin on the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) in breast cell lines MCF7, MDA-MB-231, and MCF10A. The effects of cabbage juices were compared with that exerted by indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM). The treatment with cabbage juices or indoles for 72 h affected the expression of CYP1 family genes in cell-type dependent manner. Their induction was found in all cell lines, but the ratio of CYP1A1 to CYP1B1 was 1.22- to 10.6-fold in favor to CYP1A1 in MCF7 and MCF10A cells. Increased levels of CYP1A2 in comparison with CYP1B1 were also observed in MCF7 cells. In contrast, in MDA-MB-231 cells CYP1B1 was preferentially induced. Since the cell lines investigated differ in invasion capacity, these results support epidemiological observations and partly explain the mechanism of the chemopreventive activity of white cabbage products.

The effect of initiating doses of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene on the expression of PAH activating enzymes and its modulation by plant phenols

Polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) are environmental pollutants, which undergo metabolic activation to exert their carcinogenic effects. Our earlier studies showed that naturally occurring plant phenols, protocatechuic, chlorogenic, tannic acids and resveratrol, besides inhibiting B[a]P and DMBA binding to DNA, modulate the activity of the enzymes involved in PAHs activation. The aim of the present study was further examination of the effect of these compounds on the expression and activities of CYP1A1/1A2, CYP1B1, CYP2B, and phase 2 enzymes in female BALB/C mouse epidermis treated with an initiating dose of B[a]P or DMBA. Application of a single 400 nmol dose of B[a]P alone significantly (by 119-127%) increased the activities of ethoxy- (EROD) and methoxy- (MROD) resorufin dealkylases and to lesser extent penthoxyresorufin depentylase (PROD) (by 32%). Western blot analysis with CYP1A1/1A2, CYP1B1 and CYP2B-specific antibodies showed the increase of CYP1A1/1A2 and CYP2B levels in B[a]P-treated animals. Phase 2 enzymes, gluthatione S-transferase and NAD(P)H:quinone oxidoreductase-1 (NQO1) were also significantly increased. In contrast to B[a]P, application of the initiating dose of DMBA (10 nmol) on mouse skin did not change the activities or protein levels of cytochrome P450, however increased the activities of NQO1 and GST. Pretreatment of mice with phenolic compounds one hour before B[a]P application significantly decreased the activities of all alkoxyresorufin dealkylases in comparison with the group of mice treated only with B[a]P. The sole exception was tannic acid which did not affect the PROD activity. This polyphenol, however, decreased the protein level of CYP1A1/1A2 and CYP1B1 isozymes enhanced by B[a]P. All phenolics, particularly resveratrol, significantly (by 129-174%) increased the activity of NQO1 in comparison with B[a]P-treated animals. On the other hand, pretreatment with phenolic compounds significantly diminished NQO1 activity in comparison with DMBA-treated group. These results indicate that the reduction of B[a]P-DNA adducts observed in our earlier studies may result from the decreased B[a]P activation by investigated plant phenols. In case of DMBA-DNA adducts, the scavenging or masking the binding sites to be occupied by DMBA reactive metabolites is more probable. Moreover, the lack of cytochrome P450 induction by the initiating dose of DMBA suggests that the constitutive expression of P450, particularly CYP1B1 is sufficient for DMBA activation and subsequent DNA adducts formation.

Xanthohumol induces phase II enzymes via Nrf2 in human hepatocytes in vitro

The aim of this study was to investigate whether xanthohumol may exert chemoprotective activity through the modulation of the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway in immortalized normal THLE-2 hepatocytes and a hepatocellular carcinoma HepG2 cell line. Cells were incubated in the presence of xanthohumol and the activation of Nrf2 and expression of genes controlled by this transcription factor were evaluated. Additionally, p53 level was assessed. Xanthohumol increased the expression and led to the activation of Nrf2 in both cell lines. However, in contrast to normal cells the expression of genes controlled by this transcription factor was not affected in HepG2 cells, except for GSTA and GSTP. Xanthohumol, beside the induction of GSTs and HO-1, significantly elevated NQO1 expression in concert with p53 level in normal hepatocytes. The activation of Nrf2 pathway and subsequently phase II enzymes in concert with p53 induction in normal hepatocytes may account for the molecular mechanism of the chemopreventive activity of xanthohumol. On the other hand its cytotoxicity towards HCC cells shown in this study indicates that it may also be considered as potentially chemotherapeutic.

Phloretamide, an apple phenolic compound, activates the Nrf2/ARE pathway in human hepatocytes.

The aim of this study was to evaluate the effect of phloretamide (PA), an apple constituent, on the activation of the Nrf2 transcription factor and the expression of its target genes: glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) in normal human THLE-2 hepatocytes and the hepatoma HepG2 cell line. PA did not show significant cytotoxicity towards THLE-2 cells but such an effect was observed in HepG2 cells (IC(50) ∼200μM). The treatment of cells with PA resulted in the translocation of Nrf2 from cytosol to nucleus in both cell lines, but increased the level of its transcript and protein only in THLE-2 cells. In this cell line an increased level of GSTA, GSTP, GSTT, NQO1 mRNA was also observed. Increased expression of GSTs was confirmed by enhancement of their protein levels. The increase in p53 protein content observed in THLE-2 may be associated with its stabilization induced by the enhancement of NQO1 level. PA did not affect Nrf2, GSTs, NQO1 or HO-1 expression in HepG2 cells. These results suggest that PA has rather chemopreventive than chemiotherapeutic potential and acts similarly as apple dihydrochalcones through the induction of detoxification/antioxidative enzymes.

Chokeberry (Aronia melanocarpa) juice modulates 7,12-dimethylbenz[a]anthracene induced hepatic but not mammary gland phase I and II enzymes in female rats

Chokeberry is a rich source of procyanidins known to have several types of biological activity including anticarcinogenic potential in experimental models. In this study we examined the effect of chokeberry juice on the hepatic and mammary gland carcinogen metabolizing enzyme expression altered by the polycyclic aromatic hydrocarbon, 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley rats were gavaged with chokeberry juice (8 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. Pretreatment with chokeberry juice reduced the activity of CYP1A1 and increased that of CYP2B involved in metabolic activation/detoxication of DMBA in rat liver, as well as expression and activity of phase II enzymes. Chokeberry juice had no effect on these parameters in the mammary gland and DMBA induced DNA damage in rat blood cells. These results together with our earlier observations indicate that metabolic alterations induced by chokeberry feeding are tissue specific and depend on the class of carcinogen.