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The aryl hydrocarbon receptor (AhR) is an environmentally sensitive transcription factor (TF) historically associated with carcinogenesis initiation via the activation of numerous carcinogens. Nowadays, the AhR has been attributed to multiple endogenous functions to maintain cellular homeostasis. Moreover, crosstalk, often reciprocal, has been found between the AhR and several other TFs, particularly estrogen receptors (ERs) and nuclear factor erythroid 2-related factor-2 (Nrf2). Adequate modulation of these signaling pathways seems to be an attractive strategy for cancer chemoprevention. Several naturally occurring and synthetically modified AhR or ER ligands and Nrf2 modulators have been described. Sulfur-containing derivatives of glucosinolates, such as indole-3-carbinol (I3C), and stilbene derivatives are particularly interesting in this context. I3C and its condensation product, 3,3'-diindolylmethane (DIM), are classic examples of blocking agents that increase drug-metabolizing enzyme activity through activation of the AhR. Still, they also affect multiple essential signaling pathways in preventing hormone-dependent cancer. Resveratrol is a competitive antagonist of several classic AhR ligands. Its analogs, with ortho-methoxy substituents, exert stronger antiproliferative and proapoptotic activity. In addition, they modulate AhR activity and estrogen metabolism. Their activity seems related to a number of methoxy groups introduced into the stilbene structure. This review summarizes the data on the chemopreventive potential of these classes of phytochemicals, in the context of AhR and its crosstalk modulation.

Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired  DNA adducts may induce mutations in critical genes, leading to the initiation of chemical carcinogenesis. Therefore,  detection, identification, and quantification of DNA adducts is essential for cancer risk assessment. Over the last 50 years, the major DNA adducts formed by different classes of environmental carcinogens were characterized. With the development of techniques such as 32P-postlabeling, their measurement was implemented into molecular epidemiology. Advances in liquid chromatography-tandem mass spectrometry (LC-MS ) made the measurement of adducts more precise  and allowed to gain knowledge about their identity and structures. Therefore,  opened the way to  DNA adductomics, the  "omics" approach investigating DNA adducts comprehensively, similarly to proteomics. This review presents the historical perspective of DNA adducts research and the emerging field of adductomics.

Pancreatic cancer is a disease in which deregulation of signaling pathways plays a key role, thus searching for their novel modulators is a promising therapeutic strategy. Hence, in this study, the effect of phytochemical combinations on the canonical and non-canonical activation of Nrf2 and its interaction with the NF-κB pathway was evaluated in extensively proliferating pancreatic cancer cell line, PSN-1, in comparison to non-cancerous MS1 cells. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cell survival were assessed in PSN-1 cells. The tumor burden was evaluated in mice carrying xenografts. PSN-1 cells were more sensitive to the tested compounds as compared to the MS1 cell line. Combination of xanthohumol and phenethyl isothiocyanate was more effective than single compounds at decreasing the canonical and non-canonical activation of Nrf2 in PSN-1 cancer cells. Decreased activation of NF-κB, and subsequent reduced cytosolic COX-2 and nuclear STAT3 level indicated their anti-inflammatory and pro-apoptotic activities. In vivo studies showed the partial response in groups treated with xanthohumol or the combination of xanthohumol and phenethyl isothiocyanate. Overall, these results suggest that the combination of xanthohumol and phenethyl isothiocyanate may be a promising therapeutic candidate against pancreatic cancer.

Background : Increasing evidence suggests that combinations of phytochemicals are more efficient than single components in the modulation of signaling pathways involved in cancer development. In this study, the impact of phenethyl isothiocyanate (PEITC), indole-3-carbinol (I3C), xanthohumol, (X), and resveratrol (RES) and their combinations on the activation and expression of Nrf2 and NF-κB in human hepatocytes and HCC cells were evaluated. Methods : THLE-2 and HepG2 cells were exposed to single phytochemicals and their combinations for 24 h. The activation of Nrf2 and NF-κB, expression of their target genes, and effect on cells survival were assessed. The tumor burden was evaluated in mice carrying xenografts. Results : All phytochemicals enhanced the activation and expression of Nrf2 and its target genes SOD and NQO1 in HepG2 cells. The increased expression of NQO1 (~90%) was associated with increased ROS generation. X + PEITC downregulated NF-κB activation reducing binding of its active subunits to DNA resulting in diminished COX-2 expression. In contrast to single phytochemicals, X + PEITC induced apoptosis. Moderate reduction of tumor burden in mice carrying xenografts following X and PEITC or their combination was observed. Conclusions : Since Nrf2 is overexpressed in HCC its reduced activation together with diminished level of NF-κB by X + PEITC may be considered as a strategy to support conventional HCC therapy.

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.

Our previous study demonstrated that new oleanolic acid oxime (OAO) derivatives and their conjugates with aspirin (ASP) inhibit NF-κB activation. Evidence exists that the downregulation of NF-κB negatively interferes with the Nrf2 signaling pathway. This study aimed to evaluate the effect of these compounds on Nrf2 activation and its cellular consequences in human hepatoma HepG2 cells and immortalized normal hepatocytes THLE-2. The results showed the enhanced activation and expression of Nrf2 as a result of treatment with OAO derivatives themselves and to less extent by their ASP conjugates, mainly in HepG2 cells. The association between cytotoxicity evaluated in our previous study and Nrf2 activation was observed. In this regard, compounds (18) with morpholide substituent at the C-17 position of OAO molecule and (12) with methyl ester substituent at the same position of OAO molecule to the most extent activated Nrf2 and subsequently cell cycle arrest at G2/M, leading to increased apoptosis and the number of resting HepG2 cells. The derivative of OAO (18) substituted with ASP (19) also affected Nrf2 activation and expression, but this effect was less pronounced in comparison with non-conjugated OAO. However, conjugation enhanced Nrf2 activation in normal THLE-2 cells. These results confirmed our earlier suggestion that OAO derivatives conjugated with ASP have the potential for application in the liver cancer chemoprevention. OAO themselves, particularly OAO substituted with morpholide, may be considered therapeutic agents, which may support conventional treatment strategy. Further studies are required to confirm this suggestion., (Copyright © 2020. Published by Elsevier B.V.)

Phytochemicals such as phenethyl isothiocyanate (PEITC), indole-3-carbinol (I3C), xanthohumol (XAN), and resveratrol (RES) have been shown to target signaling pathways that are involved in the proliferation and survival of different pancreatic cancer (PC) cell lines. While the activity of these compounds alone was extensively studied, their combinations were never assessed. Thus, the aim of this study was to evaluate and compare the effect of PEITC, I3C, XAN, and RES and their combinations on the expression and activation of NF-κB and Nrf2 in human PC cell line PANC-1. The combination of XAN and PEITC was more efficient than the single compounds in reducing the binding of NF-κB p65 subunits to DNA by 47-60% and expression of p65 gene by 28-48%. The combination of XAN and PEITC also enhanced the activation and expression of Nrf2 and subsequently the expression of GSTP, NQO1, and SOD genes which are controlled by this transcription factor. Modulation of the activity of NF-κB and Nrf2 by the combination of XAN and PEITC was found to lead to reduced proliferation of PANC-1 cells. These results suggest that the combination of XAN and PEITC might be considered as a novel strategy for the prophylaxis and/or treatment of PC., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pomegranate juice is a rich source of ellagitannins (ETs) believed to contribute to a wide range of pomegranate's health benefits. While a lot of experimental studies have been devoted to Alzheimer disease and hypoxic-ischemic brain injury, our knowledge of pomegranate's effects against Parkinson's disease (PD) is very limited. It is suggested that its neuroprotective effects are mediated by ETs-derived metabolites-urolithins. In this study, we examined the capability of pomegranate juice for protection against PD in a rat model of parkinsonism induced by rotenone. To evaluate its efficiency, assessment of postural instability, visualization of neurodegeneration, determination of oxidative damage to lipids and α-synuclein level, as well as markers of antioxidant defense status, inflammation, and apoptosis, were performed in the midbrain. We also check the presence of plausible active pomegranate ETs-derived metabolite, urolithin A, in the plasma and brain. Our results indicated that pomegranate juice treatment provided neuroprotection as evidenced by the postural stability improvement, enhancement of neuronal survival, its protection against oxidative damage and α-synuclein aggregation, the increase in mitochondrial aldehyde dehydrogenase activity, and maintenance of antiapoptotic Bcl-xL protein at the control level. In addition, we have provided evidence for the distribution of urolithin A to the brain.

Background: "Orphan" cytochromes are a new group of P450 cytochromes without a fully recognized biological role. The expression of these CYPs in tumors is higher than that in normal tissues, which makes them attractive as chemopreventive and/or therapeutic targets. In this study, we compared the effect of synthetic methoxy stilbenes and resveratrol on the expression of two orphan cytochromes, CYP2S1 and CYP2W1, in breast cancer cells., Methods: Breast cancer cells, lines MCF7 and MDA-MB-231, were treated for 72 h with tested compounds. The expression of CYP2S1 and CYP2W1 was evaluated at the transcript and protein levels by RT-PCR and Western blot, respectively., Results: The constitutive expression of both isoforms was confirmed at the mRNA and protein levels. CYP2S1 and CYP2W1 showed higher expression in MDA-MB-231 cells. In MCF7 cells treated with stilbenes, the expression of both CYPs was increased at the mRNA level, whereas at the protein level this effect was confirmed for CYP2S1 alone. In contrast, in estrogen receptor-negative MDA-MB-231 cells treated with stilbenes, the expression of both CYPs decreased, but mostly at the transcript level., Conclusions: The results of the present study confirmed the constitutive expression of CYP2S1 and CYP2W1 in breast cancer cells, although their relatively low level of expression suggests that they may be less involved in the transformation of therapeutic agents in these types of tumors. Stilbenes, particularly 3MS and 4MS, can modulate the expression of "orphan" CYPs more efficiently than resveratrol., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to evaluate the effect of new oleanolic acid oxime (OAO) derivatives and their conjugates with aspirin (ASP) on the expression and activation of NF-κB in human hepatoma HepG2 cells. OAO derivatives showed a stronger cytotoxic effect against HepG2 cells compared with their conjugates with aspirin. Moreover, conjugation of OAO with ASP led to enhanced downregulation of NF-κB expression and activation. Among the hybrids with ASP, compounds: 19, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid morpholide and 13, 3-(2-acetoxy)benzoyloxyiminoolean-12-en-28-oic acid methyl ester, differing, respectively, in morpholide and methyl ester groups at the C-17 position of oleanolic acid (OA) molecule were the most efficient. COX-2 transcript and protein levels were also diminished after treatment with these compounds. The results of this study indicate that the new derivatives of OAO and particularly their conjugates with ASP, downregulate the expression of COX-2 in HepG2 cells by modulating the NF-κB signaling pathway and suggest their potential application in the prevention of liver inflammation and cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Naturally occurring oleanolic acid (OA) possesses a hepatoprotective activity and ability to inhibit proliferation of human hepatocellular carcinoma cells. Both properties might be related to its anti-inflammatory activity. Its low bioavailability justifies the search for more hydrophilic OA derivatives. The aim of this study was the design and synthesis of four novel OA oxime derivatives conjugated with succinic acid at the C-3 position of oleanane skeleton structure and evaluation of their effect on NF-κB and STATs expression and activation in HepG2 cells. The expression of NF-κB and cyclooxygenase-2 (COX-2), STAT5A/B and STAT3 with its target genes: BAX, BCL-XL and MYC was evaluated after 24 h treatment with tested compounds. The comparison of the levels of cytosolic and nuclear NF-κB subunits p50, p65 and STATs proteins was used as the measure of their activation. The results pointed out the 3-succinyloxyiminoolean-12-en-28-oic acid morpholide (SMAM) as the most potent modulator of NF-κB and STAT3. SMAM significantly reduced the expression and activation of NF-κB as well as its nuclear protein level of p65 subunit. This compound also reduced the expression and activation of STAT3 and STAT5A/B. Combined effect of SMAM on these transcription factors resulted in reduced expression of COX-2, MYC and anti-apoptotic BCL-XL genes. Simultaneously, the increased expression of pro-apoptotic BAX gene was observed. In the cells treated with 3-succinyloxyiminoolean-12-en-28-oic acid (SMAA) the increased expression of BAX was also found. The effects of 3-succinyloxyiminoolean-12-en-28-oic acid benzyl ester (SMAEB) and 3-succinyloxyiminoolean-12-en-28-oic acid methyl ester (SMAEM) were moderate and ambiguous in relation to the tested factors. Moreover, the coordinated action of SMAM on NF-κB and STAT3 confirms their close association in HepG2 cells. We conclude that SMAM efficiently downregulates the key elements of signaling pathways involved in inflammatory driven HCC. Thus, may be considered as a potential chemopreventive or therapeutic agent in this type of cancer., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 IC 50 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.

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.

The article presents an evaluation of the safety of yellow tea (Camellia sinensis) extract consumption and its antioxidant activity in an animal model. Wistar rats were exposed through diet to 2, 6, and 10 g yellow tea extract/kg feed for 90 days. No signs of toxicity and no differences in mean body weight gain in the treated and control rats were recorded throughout the experiment. No statistically significant differences in hematology findings and clinical chemistry parameters were observed between controls and treated groups. Microscopic examination of tissue sections revealed no pathology attributable to yellow tea extract intake. Lipid peroxidation level in the liver was slightly increased in medium-dose males and high-dose females and decreased in two female groups receiving 2 and 6 g/kg of the extract tested. Content of carbonyl groups in protein, as well as the basal level of DNA damage, was not changed. In a majority of rats, the activity of antioxidant enzymes was increased except superoxide dismutase in high-dose groups, glutathione peroxidase in high-dose females, glutathione reductase in low- and mid-dose groups, and glutathione S-transferase in mid-dose females and high-dose males. It could be concluded that rats tolerated well dietary treatment with yellow tea extract up to 0.8 g/kg b.w./day for 90 days. Results showed that yellow tea extract at the doses tested did not demonstrate adverse effects and improved the antioxidant status in the liver of rats.

Ferritin-iron is currently considered as one of the most promising iron forms to prevent iron deficiency anaemia. We found that the cultivation of soybean seeds in a solution of ferrous sulfate results in material with extremely high iron content - 560.6 mg Fe/100 g of dry matter, while ferritin iron content was 420.5 mg/100 g dry matter. To assess the potential adverse effects of a preparation containing such a high concentration of iron, male and female Wistar rats were exposed via diet to 10, 30, 60 g soybean sprouts powder/kg feed for 90 days. There were no differences in final body weight and mean food consumption between controls and rats administered sprouts. No statistically significant differences in haematology and clinical chemistry parameters were found between controls and treated rats. Microscopic examination of 22 tissues did not reveal any pathology due to soybean sprouts intake. Long term administration of the test material did not cause oxidative damage to DNA and protein in the liver as evidenced by the unchanged basal levels of DNA damage as well as carbonyl groups content. Lipid peroxidation was slightly increased only in females. The activity of several antioxidant enzymes: superoxide dismutase, glutathione peroxidase and glutathione S-transferase was increased, which substantially enhanced the antioxidant status in the liver from the rats treated with soybean sprouts. Hence, the material tested can be recommended as a component of food supplements for individuals with iron deficiency anaemia and inflammatory bowel diseases.

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.

Cloudy apple juice (CAJ) is a rich source of nutrients as well as non-nutrient components including high quantity of polyphenols, particularly oligomeric procyanidins, which are considered as potential chemopreventive agents that protect against the action of chemical carcinogens. The aim of this study was to examine the effect of CAJ alone or in combination with hepatocarcinogenic N-nitrosodiethylamine (NDEA) on liver damage biomarkers, including DNA damage, and the phase I and II enzymes in rat. The forced feeding with CAJ alone for 28 days, has slightly reduced the activities of phase I enzymes, MROD (CYP1A2 biomarker) and PNPH (CYP2El biomarker), while phase II enzymes, glutathione S-transferase (GST) and, Nad(p)h: quinone oxidoreductase-1 (NQO1), were elevated. Combined treatment of rats with CAJ and NDEA significantly reduced the levels of hepatic ALT and SDH (by ~100%) as compared to values from NDEA-treated animals. CAJ pretreatment further increased the PROD (CYP2B biomarker) and NQO1 activities increased by NDEA administration. Modulation of enzymes activities was accompanied by the changes in the proteins levels. These results indicate that CAJ may protect liver against damage induced by NDEA. Moreover, a significant decrease of SDH activity by CAJ may confirm its potential anti-diabetic activity.

The L-sulforaphane (SFN) component of broccoli sprout showed anticancer activity in several preclinical studies including breast cancer. Estrogens are considered major risk factors in breast carcinogenesis. The aim of this study was to evaluate the effect of SFN on the expression of cytochrome P450 involved in the estrogen metabolism in breast cancer cell lines MCF7 and MDA-MB-231 and in non-tumorigenic MCF10A cell line. The expression of CYP19, CYP1A1, 1A2, 1B1 was determined at the transcript and protein levels. There were found some remarkable differences in the effect of SFN at a dose of 5 µmol/L on CYP19 expression: in ER(+) MCF7 significant reduction, in ER(-) MDA-MB-231 an increased expression and unchanged expression in MCF10A cell line. The effect of SFN on CYPs (1A1, 1A2, 1B1) involved in estrogen catabolism was to a lesser extent dependent on breast cell line. The slightly reduced CYP1A1 protein level was observed in all cell lines tested. An increased level of CYP1A2 and decreased level of CYP1B1 expression were found in MCF10A. These results indicate that the naturally occurring L isomer of SFN may affect the expression of P450s involved in estrogen metabolism. This effect may contribute to the anticancer activity of SFN in breast tissue., (Copyright © 2014 John Wiley & Sons, Ltd.)

Our previous studies showed the diversified effect of cabbage juices and indoles on the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) in breast epithelial cells differing in ER status, i.e., in tumorigenic-MCF7, MDA-MB-231 and non-tumorigenic-MCF10A cells. The aim of the present study was to further investigate the mechanism of chemopreventive action of cabbage juice and its active components by evaluating their effect on the expression of AhR, ERα, and Nrf2 using the same treatment regimen. The mRNA level of AhR and ERα was changed in a cell type-dependent manner and in general correlated with previously observed modulation of CYP expression. However, in most cases the alterations in mRNA were not accompanied by the changes in the level of relevant proteins. Marked differences were also found in the effect of cabbage juices and indoles; although both cabbage juices and indoles increased most of the NQO1 transcript levels in all tested lines, indoles also enhanced GSTP transcription in MCF7 and MDA-MB-231. Overall, the results of this study partly explain the mechanism behind the chemopreventive activity of white cabbage products and indicate that modulation of the expression of specific transcription factors may play an important role in this process.

Epidermal growth factor receptor (EGFR) interacting with Stat3 is considered to be an attractive therapeutic target. In the current study, we investigated the effect of resveratrol and its two 4'-methylthio-trans-stilbene derivatives (3-M-4'-MTS; S2) (3,5-DM-4″-MTS; S5) on EGFR and Stat3 activation in human immortalized HaCaT keratinocytes and epidermoid carcinoma A431 cells. In the HaCaT cells both derivatives, similarly as resveratrol, decreased the total level of the EGFR receptor. In the A431 cells, resveratrol in the higher dose significantly (p < 0.05) reduced Y1173 and Y1068 EGFR residue phosphorylation, while S2 affected only the phosphorylation of the Y1068 residue. In this cell line, resveratrol in both tested doses and the S2 derivative in the lower concentration significantly diminished Stat3 binding capacity to the DNA consensus site. The effect of the tested compounds on Stat3 activation in HaCaT cells was only slightly affected. These results indicate that methylthiostilbenes are not more potent modulators of the EGFR/Stat3 complex than resveratrol and that introducing an additional methoxy group makes them less effective.

Background: Resveratrol is a natural stilbene derivative whose chemopreventive activity has been well established. Our previous studies have shown that modification of the stilbene backbone with the methylthio group may influence selectivity and inhibitory potency toward P450 isozymes. The aim of this study was to further investigate the mechanism of their potential chemopreventive activity by evaluating the effect of two 4'-methylthio-trans-stilbene derivatives possessing one (3-M-4'-MTS; S2) and two (3,5-DM-4'-MTS; S5) additional methoxy groups on constitutive nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation in immortalized human HaCaT keratinocytes., Methods: The synthesis of MTS was performed as described earlier. Translocation of NF-κB and AP-1 was evaluated by Western blot analysis. Binding of p65 (NF-κB) and c-Jun and c-Fos subunits (AP-1) to consensus oligonucleotide was assessed by ELISA. Real-time PCR and Western blot were used to evaluate COX-2 and iNOS expression., Results: We found differential modulation of signaling pathways depending on the stilbene structure after 24h of cells treatment. The S2 compound, in contrast to S5 and resveratrol, significantly reduced NF-κB activation by blocking the translocation of the p65 subunit to the nucleus, and decreasing IκB kinase activity. All compounds, but particularly S5, increased c-Jun binding to the AP-1 consensus sequence, while c-Fos binding was not affected., Conclusions: We conclude that methylthiostilbenes differently modulate constitutive signal transduction pathways in HaCaT cells. These observations should be taken into account in designing new stilbene derivatives with potential chemopreventive activity., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Apples abundant in phenolic compounds show a variety of biological activities that may contribute to health beneficial effects against cardiovascular diseases, diabetes, obesity and cancer. We investigated the effect of cloudy apple juice (CAJ) on the hepatic and mammary gland carcinogen metabolizing enzymes, DNA damage and liver injury, altered by 7,12-dimethylbenz[a]anthracene (DMBA). Sprague-Dawley female rats were gavaged with CAJ (10 ml/kg b.w.) for 28 consecutive days. DMBA was administered i.p. on the 27th and the 28th days. In the liver, feeding with CAJ 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 CAJ. The most significant changes in the level of the hepatic enzymes tested were observed for GST alpha and NQO1. In mammary gland CAJ induced an increase in the level of GST mu and GST pi, while DMBA and CAJ combined administration elevated GST pi only. This may be beneficial as GST pi is involved in the DMBA detoxification. Additionally, pretreatment with CAJ reduced the level of most of the blood biochemical liver and kidney markers elevated as a result of DMBA treatment. These findings indicate that CAJ may interfere with enzyme system involved in carcinogen metabolism. However, this effect seems to be dependent on tissue and carcinogen and is moderately effective in the case of DMBA. Moreover, CAJ can also provide some protection against the liver and kidney damage.

Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4'-MTS (S2), 3,5-DM-4'-MTS (S5) and 3,4,5-TM-4'-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH.

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 © 2013 John Wiley & Sons, Ltd.)

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20 μm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequences were increased only in the THLE-2 cells and were accompanied by the phosphorylation of serine/threonine kinase (AKT), c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). BET also significantly increased the mRNA and protein levels of GSTP, GSTT, GSTM and NQO1 in these cells. Conversely, besides the translocation of Nrf2 from the cytosol to the nucleus, BET did not modulate any of the other parameters measured in the HepG2 cells. BET did not change the methylation of GSTP1 in these cells either. These results indicate that BET through the activation of Nrf2 and subsequent induction of the expression of genes controlled by this factor may exert its hepatoprotective and anticarcinogenic effects. Moreover, the activation of mitogen-activated protein kinases may be responsible for the activation of Nrf2 in the THLE-2 cells.

Cytochromes P450 (P450) are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms' genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1) that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues.

Purpose: The aim of this study was to evaluate the effect of white cabbage and sauerkraut juices of different origin and indole-3-carbinol (I3C) and diindolylmethane (DIM) on expression of CYP19 gene encoding aromatase, the key enzyme of estrogen synthesis., Methods: Human breast cell lines (MCF7, MDA-MB-231 and MCF10A) were examined to compare the action of cabbage juices versus their active components (I3C, DIM). Real-time PCR and Western blot were used in order to analyse CYP19 mRNA and protein, respectively., Results: Remarkable differences in the effect on CYP19 transcript and protein level were found between the cabbage juices (in 2.5-25 mL/L concentrations) and indoles (in 2.5-50 μM doses) in the three cell lines. While cabbage juices at the lower doses diminished the aromatase expression in nontumorigenic/immortalized MCF10A breast cells (0.25-0.86-fold change, P < 0.05), I3C and DIM were more efficient in decreasing the aromatase expression in estrogen-dependant MCF7 breast cancer cells (0.24-0.82-fold change, P < 0.05). Inhibition of aromatase by juice obtained from cabbage grown on industrial farm was correlated with the induction of apoptosis (1.7-1.8-fold change, P < 0.01) in MCF10A cells. In estrogen-independent MDA-MB-231 cells, up-regulation of CYP19 expression by I3C and DIM (1.5-2.0-fold change, P < 0.05) was observed. Similarly, in MCF7 cells juices increased aromatase expression (1.1-2.2-fold change, P < 0.05)., Conclusion: These results, particularly that obtained in nontumorigenic/immortalized MCF10A cells, suggest that chemopreventive activity of cabbage against breast cancer observed in epidemiological studies may be partly explained by inhibition of the aromatase expression.

Introduction: Cytochrome P450 (CYP) families 1 - 3, besides oxidizing environmental and dietary chemicals, leading to their elimination, catalyze the bioactivation of exogenous as well as endogenous carcinogens. Phytochemicals, particularly those which are active food components, were shown to be able to affect specific CYP expression and/or activity in animal models and in human in vitro systems. Human intervention studies involving healthy volunteers were also performed. This review describes human CYP modulation by naturally occurring phytochemicals which can not only affect carcinogen metabolism in humans, but also change the drug response., Areas Covered: The authors present an overview of carcinogens metabolizing human CYP modulation in different model systems as well as studies on human dietary intervention. Furthermore, the authors provide examples of the phytochemicals that affect CYP expression and activity., Expert Opinion: CYP, which are involved in carcinogen activation, can metabolize a range of substrates and inducing CYP by one substrate may also increase the metabolism of another. The ultimate proof of the efficacy of CYP modulation strategy for chemoprevention may be provided by clinical trials involving risk populations, which are difficult to perform. The new human-like models are highly desired for the study of modulation of carcinogen-metabolizing CYP.

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.

This study investigated the effect of raw cabbage and sauerkraut juices on the activity and expression of CYP1A1, 1A2, 1B1 and 2B in Wistar rat liver and kidney. The results were compared with those of two commercially available products of glucosinolates degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Significant differences in the effect of the cabbage juices as well as I3C and PEITC between the liver and kidney were found. In the liver, both juices decreased the activities of enzymatic markers of CYP1A1 and CYP1A2 after 10 days of the experiment, while in the kidney an enhancement of the activity of these enzymes was observed on days 4 and 10. The increased activity of these enzymes and CYP1A1/1A2 protein level in the liver was found after 30 days of treatment with sauerkraut juice. A similar effect was observed as a result of PEITC treatment. I3C increased the expression and activity of hepatic CYPs at all time points investigated. In conclusion, the present study demonstrates that raw cabbage and sauerkraut juices may affect CYPs involved in the activation of carcinogens/xenobiotics and in this way exert anticarcinogenic activity. The final effects, however, depend on the time-frame of exposure and the type of tissue., (Copyright © 2011 John Wiley & Sons, Ltd.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The effect of raw cabbage and sauerkraut juices on the expression and activity of phase II enzymes, glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in the rat liver and kidney was compared with that of two commercially available products of glucosinolate degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Male Wistar rats were treated by oral administration with cabbage juices, I3C or PEITC for 4, 10 and 30 d. The results showed that juices, particularly sauerkraut juice as with I3C and PEITC, significantly increased GST and NQO1 activities in the rat liver. The only exception was the 30 d time point of feeding with raw cabbage juice. Cabbage juices, I3C and PEITC affected the hepatic GST μ to the greatest extent and GST α to a lesser extent. The results of the present study also showed that the treatment of rats with juices and compounds tested caused the translocation of the NF-E2-related transcription factor (Nrf2) active subunit from the cytosol to the nucleus, providing an argument for the involvement of this transcription factor in the induction of GST and NQO1. In contrast to the liver, cabbage juices affected only the renal GST θ, while treatment with I3C and PEITC significantly increased the activity of NQO1. Thus, the results of the present study indicate that induction of the key detoxifying enzymes by cabbage juices, particularly sauerkraut, may be responsible for their chemopreventive activity demonstrated by epidemiological studies and in animal models. However, the final effects might be organ or tissue dependent.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Chokeberry is a rich source of polyphenols, which may counteract the action of chemical carcinogens. The aim of this study was to examine the effect of chokeberry juice alone or in combination with N-nitrosodiethylamine (NDEA) on phase I and phase II enzymes and DNA damage in rat liver. The forced feeding with chokeberry juice alone decreased the activities of enzymatic markers of cytochrome P450, CYP1A1 and 1A2. NDEA treatment also decreased the activity of CYP2E1 but enhanced the activity of CYP2B. Pretreatment with chokeberry juice further reduced the activity of these enzymes. Modulation of P450 enzyme activities was accompanied by the changes in the relevant proteins levels. Phase II enzymes were increased in all groups of animals tested. Chokeberry juice augmented DNA damage and aggravated the effect of NDEA. These results indicate that chokeberry may protect against liver damage; however, in combination with chemical carcinogens it might enhance their effect.

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.

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.

A simple plant phenolic acid, protocatechuic acid and a polyphenol, tannic acid are potential chemopreventive agents which inhibited the chemically induced carcinogenesis in many experimental models. We previously demonstrated that those compounds modulate the activity of xenobiotic detoxifying enzymes, including GST in mouse liver, kidney and epidermis. Intraperitoneal (i.p.) treatment with protocatechuic acid in the dose of 80mg/kg for three consecutive days increased the GST activity in liver and kidney. In case of tannic acid the same effect was observed in kidney after i.p. administration of the single dose of 80mg/kg. Topical application of phenolic acids resulted in enhancement of epidermal GST activity. The focus of this study was to further investigate the effects of these phenolic acids on the protein levels of GST isozymes in the same tissues using the treatment protocols used in our previous studies. The results confirmed the expression of GST alfa, mu, pi and theta in mouse liver, kidney and epidermis. Treatment with protocatechuic acid resulted in an increase of the expression of GST class mu in liver, but did not affect this isoform in skin and kidney. This compound inhibited the level of kidney GST pi by 35%. Tannic acid decreased the expression of GST mu, alpha and theta in liver. Application of the equimolar doses of both phenolic acids on mouse skin resulted in reduced level of the GST alpha protein. The results of our study indicate that, although moderate, the effect of protocatechuic acid and tannic acid on GST subunits in mice may play certain role in biological activity of these compounds. Of special importance could be the increased expression of GST mu in liver which is involved in detoxification of many carcinogens including polycyclic aromatic hydrocarbons., (Copyright © 2007 Elsevier B.V. All rights reserved.)