Attenuation of Pancreatic Cancer In Vitro and In Vivo via Modulation of Nrf2 and NF-κB Signaling Pathways by Natural Compounds.

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.

Comparison of the Impact of Xanthohumol and Phenethyl Isothiocyanate and Their Combination on Nrf2 and NF-κB Pathways in HepG2 Cells In Vitro and Tumor Burden In Vivo.

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.

R-sulforaphane modulates the expression profile of AhR, ERα, Nrf2, NQO1, and GSTP in human breast cell lines.

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.

Activation of the Nrf2 response by oleanolic acid oxime morpholide (3-hydroxyiminoolean-12-en-28-oic acid morpholide) is associated with its ability to induce apoptosis and inhibit proliferation in HepG2 hepatoma cells.

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.

Combination of xanthohumol and phenethyl isothiocyanate inhibits NF-κB and activates Nrf2 in pancreatic cancer cells.

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.

Oleanolic acid oxime derivatives and their conjugates with aspirin modulate the NF-κB-mediated transcription in HepG2 hepatoma cells.

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.

Expression of CYP2S1 and CYP2W1 in breast cancer epithelial cells and modulation of their expression by synthetic methoxy stilbenes.

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.

Morpholide derivative of the novel oleanolic oxime and succinic acid conjugate diminish the expression and activity of NF-κB and STATs in human hepatocellular carcinoma cells.

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.

Neuroprotective Effects of Pomegranate Juice against Parkinson's Disease and Presence of Ellagitannins-Derived Metabolite-Urolithin A-In the Brain.

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.

Evaluation of the effect of the new methoxy-stilbenes on expression of receptors and enzymes involved in estrogen synthesis in cancer breast cells.

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 IC50 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.

Resveratrol and its methoxy derivatives modulate the expression of estrogen metabolism enzymes in breast epithelial cells by AhR down-regulation.

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.

Evaluation of Safety and Antioxidant Activity of Yellow Tea (Camellia sinensis) Extract for Application in Food.

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.

Evaluation of Safety of Iron-Fortified Soybean Sprouts, a Potential Component of Functional Food, in Rat.

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.

INFLUENCE OF CLOUDY APPLE JUICE ON N-NITROSODIETHYLAMINE- INDUCED LIVER INJURY AND PHASES I AND II BIOTRANSFORMATION ENZYMES IN RAT LIVER.

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.

Cabbage Juices and Indoles Modulate the Expression Profile of AhR, ERα, and Nrf2 in Human Breast Cell Lines.

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.

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

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.

Modulating potential of L-sulforaphane in the expression of cytochrome p450 to identify potential targets for breast cancer chemoprevention and therapy using breast cell lines.

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.

The effect of resveratrol and its methylthio-derivatives on the Nrf2-ARE pathway in mouse epidermis and HaCaT keratinocytes.

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.