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The aim was to assess the inhibitory effect of a series of naturally occurring trans -resveratrol analogues on cytochromes P450, namely CYP1A2 and CYP2E1, in vitro in order to analyse any structure–activity relationships. 3,5-Dimethoxy-4′-hydroxy- trans -stilbene (pterostilbene), 3,4′,5-trimethoxy- trans -stilbene (TMS), 3,4′-dihydroxy-5-methoxy- trans -stilbene (3,4′-DH-5-MS) and 3,5-dihydroxy-4′-methoxy- trans -stilbene (3,5-DH-4′-MS) inhibited the activity of CYP1A2, with K i   =  0.39, 0.79, 0.94 and 1.04  µM, respectively. Piceatannol (3,3′,4,5′-tetrahydroxy- trans -stilbene) was the least potent inhibitor of CYP1A2 with a K i   =  9.67  µM. Piceatannol and TMS in the concentration range 1–100  µM did not inhibit CYP2E1 activity. The activity of this enzyme likewise was not significantly influenced by pterostilbene and 3,5-DH-4′-MS with IC 50   >  100  µM, whereas 3,4′-DH-5-MS appeared to be a moderately potent, competitive inhibitor of CYP2E1 ( K i   =  42.6  µM). Structure–activity relationship analysis leads to the conclusion that the substitution of hydroxy groups of resveratrol with methoxy groups increases the inhibition of CYP1A2, yet the number and position of methylation are not essential. However, the 4′-hydroxy group in trans -resveratrol and its analogues may play an important role in the interaction with a binding site of CYP2E1. [ABSTRACT FROM AUTHOR]

1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid,gallates and silybinon ethoxyresorufin O -dealkylase(CYP1A1),methoxyresorufin O dealkylase (CYP1A2) and pentoxy- O -dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC = 2.6 mu M. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC = 120 mu M. 3. Protocatechuic acid,chlorogenicandsilybin were moreselectivetowards PRODand MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid protocatechuic acid silybin dodecyl gallate propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-activity or selectivity relationship was observed. 4. Analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature. [ABSTRACT FROM AUTHOR]

The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7-MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P-postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P-postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12-dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P-labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2-dGuo. In addition, a minor dGuo adduct derived from the bay-region syn-diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon. [ABSTRACT FROM PUBLISHER]

Several naturally occurring coumarins to which humans are routinely exposed have been previously found to be potent inhibitors and inactivators of cytochrome P450 (P450) 1A1-mediated monooxygenase in both murine hepatic microsomes and in a reconstituted system using purified human P450 1A1 [Cai et al. (1993) Chem. Res. Toxicol., 6, 872-879 and Cai et al. (1996) Chem. Res. Toxicol., 9, 729-736]. In the present study, several of these coumarins were investigated for their inhibitory effects on the metabolism and metabolic activation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) in cultured mouse keratinocytes. Initial analysis of B[a]P metabolism in cultured keratinocytes showed that imperatorin, isoimperatorin, coriandrin, and bergamottin, at concentrations of 2 nM equal with B[a]P, reduced the formation of water-soluble metabolites of B[a]P by 33% to 57%. Bergamottin and coriandrin were the most potent inhibitors of the compounds examined. HPLC analysis of organic solvent-soluble metabolites of B[a]P indicated that all the coumarins tested significantly reduced the formation of individual B[a]P metabolites (including phenols, diols and tetraols). However, the greatest effect was on the formation of B[a]P tetraols. Additional experiments determined the ability of selected coumarins to block covalent binding of B[a]P and DMBA to DNA in keratinocytes. Bergamottin preferentially inhibited the binding of B[a]P to DNA by 56%, while coriandrin preferentially inhibited the binding of DMBA to DNA by 48%. Notably, analysis of individual DNA adducts formed from B[a]P and DMBA indicated that both bergamottin and coriandrin specifically inhibited the formation of anti diol-epoxide DNA adducts derived from both hydrocarbons. The preferential inhibitory effect of bergamottin and coriandrin on the formation of anti diol-epoxide adducts derived from DMBA was further confirmed by separation of anti- and syn-diol-epoxide-DNA adducts using immobilized boronate chromatography. The current study demonstrates that certain naturally occurring coumarins inhibited metabolic activation of B[a]P and DMBA in cultured mouse keratinocytes and specifically inhibited the formation of DNA adducts derived from the anti diol-epoxide diastereomers from either hydrocarbon. The current data also suggest that certain naturally occurring coumarins may possess anticarcinogenic activity toward polycyclic aromatic hydrocarbons. [ABSTRACT FROM PUBLISHER]

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.

Tannins are a heterogenous class of polyphenolic natural products with promising cancer chemopreventive and therapeutic potential. Studies undertaken over the last 30 years have demonstrated their capacity to target many cellular pathways and molecules important in the development of cancer. Recently, new mechanisms that might be important in anti-carcinogenic activity, such as inhibition of epithelial-to-mesenchymal transition, reduction of cancer stem cell creation, and modulation of cancer cells metabolism have been described. Along with the mechanisms underlying the anti-cancer activity of tannins, this review focuses on their possible application as chemosensitizers in adjuvant therapy and countering multidrug resistance. Furthermore, characteristic physicochemical properties of some tannins, particularly tannic acid, are useful in the formation of nanovehicles for anticancer drugs or the isolation of circulating cancer cells. These new potential applications of tannins deserve further studies. Well-designed clinical trials, which are scarce, are needed to assess the therapeutic effects of tannins themselves or as adjuvants in cancer treatment., (© 2023 British Pharmacological Society.)

HCC is a highly aggressive malignancy with limited treatment options. In this study, novel conjugates of non-steroidal anti-inflammatory drugs (NSAIDs)-Ibuprofen and Ketoprofen-with oleanolic acid oximes derivatives (OAO) were synthesized, and their activity as modulators of signaling pathways involved in HCC pathogenesis was evaluated in normal THLE-2 liver cells, and HCC-derived HepG2 cells. The results demonstrated that conjugation with OAO derivatives reduces the cytotoxicity of parent compounds in both cell lines. In THLE-2 cells, treatment with conjugates resulted in increased activation of the Nrf2-ARE pathway. An opposite effect was observed in HepG2 cells. In the later reduction of NF-κB, it was observed along with modulation of MAPK signaling pathways (AKT, ERK, p38, p70S6K, and JNK). Moreover, STAT3, STAT5, and CREB transcription factors on protein levels were significantly reduced as a result of treatment with IBU- and KET-OAO derivatives conjugates. The most active were conjugates with OAO-morpholide. Overall, the findings of this study demonstrate that IBU-OAO and KET-OAO derivative conjugates modulate the key signaling pathways involved in hepatic cancer development. Their effect on specific signaling pathways varied depending on the structure of the conjugate. Since the conjugation of IBU and KET with OAO derivatives reduced their cytotoxicity, the conjugates may be considered good candidates for the prevention of liver cancer.

Our earlier studies showed that coupling nonsteroidal anti-inflammatory drugs (NSAIDs) with oleanolic acid derivatives increased their anti-inflammatory activity in human hepatoma cells. The aim of this study was to evaluate their effect on the signaling pathways involved in inflammation processes in human pancreatic cancer (PC) cells. Cultured PSN-1 cells were exposed for 24 h (30 µM) to OA oxime (OAO) derivatives substituted with benzyl or morpholide groups and their conjugates with indomethacin (IND) or diclofenac (DCL). The activation of NF-κB and Nrf2 was assessed by the evaluation of the translocation of their active forms into the nucleus and their binding to specific DNA sequences via the ELISA assay. The expression of NF-κB and Nrf2 target genes was evaluated by R-T PCR and Western blot analysis. The conjugation of IND or DCL with OAO derivatives increased cytotoxicity and their effect on the tested signaling pathways. The most effective compound was the DCL hybrid with OAO morpholide (4d). This compound significantly reduced the activation and expression of NF-κB and enhanced the activation and expression of Nrf2. Increased expression of Nrf2 target genes led to reduced ROS production. Moreover, MAPKs and the related pathways were also affected. Therefore, conjugate 4d deserves more comprehensive studies as a potential PC therapeutic agent.

Cancer prevention particularly related to aging can be improved by the use of phytochemicals combinations. In this study, we evaluated the effect of phenethyl isothiocyanate (PEITC), xanthohumol (XAN), indole-3-carbinol (I3C), and resveratrol (RES) and their combinations on the Nrf2 signaling pathway. Human pancreatic cancer cells MIA-Pa-Ca-2 were treated with the phytochemicals alone or their equimolar mixture for 24 h and activation of Nrf2 and expression of its target genes were evaluated. Phytochemicals alone enhanced Nrf2 activation and expression, but their combinations were more efficient. The mixture of XAN and PEITC was found to be the most potent modulator of the Nrf2 pathway. Moreover, increased levels of P-Nrf2 and P-JNK and decreased level of P-GSK-3β suggested possible activation of Nrf2 through modulation of these kinases. The combinations of XAN with PEITC and RES with PEITC increased mostly the expression of SOD , GSTP , CAT , and GPx. XAN and PEITC mixture induced the cell cycle arrest in G0/G1 phase and increased apoptotic and autophagy markers. These results indicate that combinations of phytochemicals resembling that occurring in natural diets may efficiently modulate the signaling pathways, which proper function is important for pancreatic cancer prophylaxis or improving the results of conventional therapy.

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.

Naturally occurring pentacyclic triterpenoid oleanolic acid (OA) serves as a good scaffold for additional modifications to achieve synthetic derivatives. Therefore, a large number of triterpenoids have been synthetically modified in order to increase their bioactivity and their protective or therapeutic effects. Moreover, attempts were performed to conjugate synthetic triterpenoids with non-steroidal anti-inflammatory drugs (NSAIDs) or other functional groups. Among hundreds of synthesized triterpenoids, still the most promising is 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), which reached clinical trials level of investigations. The new group of synthetic triterpenoids are OA oximes. The most active among them is 3-hydroxyiminoolean-12-en-28-oic acid morpholide, which additionally improves the anti-cancer activity of standard NSAIDs. While targeting the Nrf2 and NF-κB signaling pathways is the main mechanism of synthetic OA derivatives' anti-inflammatory and anti-cancer activity, most of these compounds exhibit multifunctional activity, and affect cross-talk within the cellular signaling network. This short review updates the earlier data and describes the new OA derivatives and their conjugates in the context of modification of signaling pathways involved in inflammation and cell survival and subsequently in cancer development., Competing Interests: The authors declare there is no conflict of interest.

The study aimed to evaluate the possible modulation of Nrf2, NF-ĸB and STAT3 signaling pathways in the colorectal cancer (CRC) cells line DLD-1 and HCT116 by secondary metabolites of lichens. An attempt was made to indicate the most promising targets in these signaling pathways. Attention was also paid to the effects of the compounds tested on CRC cells using anakoinosis-that is, simultaneous analysis of several signaling pathways. The effects of the tested natural compounds on the activity of selected transcriptional factors related to CRC were analyzed by Western blot and RT-PCR assays. The highest activity against CRC cells was shown by physodic and salazinic acids from the studied secondary metabolites of lichens. As a result, an increase in the activation of transcription factor Nrf2 and the expression of its selected target genes was observed. Physodic and salazinic acids induced the opposite effect in relation to the NF-κB and STAT3 pathways. These results confirmed our earlier observations that lichen-derived compounds have the ability to modulate signaling pathway networks. While caperatic acid affected Wnt/β-catenin to the most extent, salazinic acid was the most potent modulator of Nrf2, NF-κB and STAT3 pathways. Physodic acid seemed to affect all the investigated pathways.

Nrf2 (nuclear factor erythroid 2-related factor 2) and NF-κB (nuclear factor-kappa B) signaling pathways play a central role in suppressing or inducing inflammation and angiogenesis processes. Therefore, they are involved in many steps of carcinogenesis through cooperation with multiple signaling molecules and pathways. Targeting both transcription factors simultaneously may be considered an equally important strategy for cancer chemoprevention and therapy. Several hundreds of phytochemicals, mainly edible plant and vegetable components, were shown to activate Nrf2 and mediate antioxidant response. A similar number of phytochemicals was revealed to affect NF-κB. While activation of Nrf2 and inhibition of NF-κB may protect normal cells against cancer initiation and promotion, enhanced expression and activation in cancer cells may lead to resistance to conventional chemo- or radiotherapy. Most phytochemicals, through different mechanisms, activate Nrf2, but others, such as luteolin, can act as inhibitors of both Nrf2 and NF-κB. Despite many experimental data confirming the above mechanisms currently, limited evidence exists demonstrating such activity in humans. Combinations of phytochemicals resembling that in a natural food matrix but allowing higher concentrations may improve their modulating effect on Nrf2 and NF-κB and ultimately cancer prevention and therapy. This review presents the current knowledge on the effect of selected phytochemicals and their combinations on Nrf2 and NF-κB activities in the above context.

Combining NSAIDs with conventional therapeutics was recently explored as a new strategy in cancer therapy. Our earlier studies showed that novel oleanolic acid oximes (OAO) conjugated with aspirin or indomethacin may enhance their anti-cancer potential through modulation of the Nrf2 and NF-κB signaling pathways. This study focused on the synthesis and biological evaluation of four diclofenac (DCL)-OAO derivative conjugates in the context of these pathways' modification and hepatic cells survival. Treatment with the conjugates 4d , 3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide, and 4c , 3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester significantly reduced cell viability in comparison to the DCL alone. In THLE-2, immortalized normal hepatocytes treated with these conjugates resulted in the activation of Nrf2 and increased expression in SOD-1 and NQO1, while the opposite effect was observed in the HepG2 hepatoma cells. In both cell lines, reduced activation of the NF-κB and COX-2 expression was observed. In HepG2 cells, conjugates increased ROS production resulting from a reduced antioxidant defense, induced apoptosis, and inhibited cell proliferation. In addition, the OAO morpholide derivative and its DCL hybrid reduced the tumor volume in mice bearing xenografts. In conclusion, our study demonstrated that conjugating diclofenac with the OAO morpholide and a benzyl ester might enhance its anti-cancer activity in HCC.

Naturally occurring phytochemicals of different origin and structure, arctigenin, bergenin, usnic acid and xanthohumol, were shown to affect Nrf2 pathway in the context of various diseases, but their effect on this pathway in cancer cells was not extensively investigated. This study aimed to evaluate the effect of these compounds on Nrf2 expression and activation in hypopharyngeal FaDu squamous cell carcinoma cells. FaDu cells were treated with 2 or 10 μM arctigenin, bergenin, (+)-usnic acid or xanthohumol for 24 h. While arctigenin, bergenin, and xanthohumol did not affect either Nrf2 expression or activation, (+)-usnic acid treatment increased its transcript level and increased the nuclear/cytosol Nrf2 protein ratio-the measure of Nrf2 pathway activation. Consequently, (+)-usnic acid enhanced the transcription and translation of Nrf2 target genes: NQO1, SOD, and to a lesser extent, GSTP. The treatment of FaDu cells with (+)-usnic acid decreased both GSK-3β transcript and protein level, indicating its possible involvement in Nrf2 activation. All the tested compounds decreased Bax mRNA but did not change the level of Bax protein. (+)-Usnic acid tended to increase the percentage of early apoptotic cells and LC3 protein, autophagy marker. Significant induction of p53 also was observed after treatment with (+)-usnic acid. In summary, the results of this study indicate that low concentrations of (+)-usnic acid activate Nrf2 transcription factor, most probably as a result of ROS accumulation, but do not lead to FaDu hypopharyngeal carcinoma cells death.

Nrf2 and NF-κB play a key role in inflammation-driven cancers. Conjugation of anti-inflammatory drugs with oleanolic acid oxime (OAO) may enhance their therapeutic potential as a result of downregulation of these pathways. Novel OAO derivatives conjugated with indomethacin (IND) were synthesized, and their effect on the activation and expression of Nrf2 and NF-κB in HepG2 hepatoma cells and THLE-2 immortalized normal hepatocytes was evaluated in relation to cell cycle arrest and apoptosis. Treatment with OAO-IND conjugates reduced the activation of Nrf2 and NF-κB and the expression of their active forms in HepG2 cells, while in normal hepatocytes, the activation of Nrf2 was increased and NF-κB diminished. Compounds 3d , 3-indomethacinoxyiminoolean-12-en-28-oic acid morpholide, and 3c , 3-indomethacinoxyiminoolean-12-en-28-oic acid benzyl ester, were the most efficient. In THLE-2 cells, as opposed to HepG2 cells, the expressions of SOD-1 and NQO1 were significantly enhanced after treatment with these compounds. The COX-2 expression was diminished in both cell lines. OAO-IND derivatives affected the cell cycle arrest at G2/M, leading to increased apoptosis and increased number of resting HepG2 cells. Therefore, the conjugation of IND with OAO derivatives may preserve cancer cells against chemoresistance through the inhibition of the Nrf2-ARE pathway and NF-κB and, at the same time, exert a chemopreventive effect in normal hepatocytes.

The present study aimed to evaluate the cytotoxicity and its mechanism of five synthetic methoxy stilbenes, namely 3,4,4'-trimethoxy, 3,4,2'-trimethoxy, 3,4,2',4'-tetramethoxy, 3,4,2',6'-tetramethoxy, and 3,4,2',4',6'-pentamethoxy-trans-stilbenes (MS), in comparison with resveratrol (RSV). Human promyelocytic (HL-60) and monocytic leukemia (THP-1) cells were treated with the tested compounds for 24 h, and cytotoxicity, cell cycle distribution, and apoptosis were evaluated. Significant differences were found in the susceptibility of these cell lines to all stilbenes, including RSV. The THP-1 cells were more resistant to cytotoxic activity of these compounds than HL-60 cells. Among the tested stilbenes, 3,4,4'-tri-MS and 3,4,2',4'-tetra-MS exhibited higher cytotoxicity toward both cell lines than RSV and the other methoxy stilbenes. This activity might be related to cell cycle arrest at the G2/M phase and induction of apoptosis. In this regard, 3,4,4'-tri-MS and 3,4,2',4'-tetra-MS at highest concentrations increased the p53 protein level particularly in HL-60 cells. Moreover, treatment with these derivatives increased the ratio of the proapoptotic Bax protein to the antiapoptotic Bcl-xl protein, suggesting the induction of apoptosis through the intrinsic mitochondrial pathway in both cell lines. Further studies are required to fully elucidate the mechanism of these activities.

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.)

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.)

Purpose: Activation of the Wnt pathway contributes to the development of head and neck squamous cell carcinomas (HNSCC) and its inhibition has recently emerged as a promising therapeutic strategy. Here, we aimed at identifying suitable molecular targets for down-regulation of canonical Wnt signaling in HNSCC cells., Methods: Candidate target genes (PORCN, WNT3A, FZD2, FZD5, LRP5, DVL1, CIP2A, SET, KDM1A, KDM4C, KDM6A, CBP, CARM1, KMT2A, TCF7, LEF1, PYGO1, XIAP) were silenced using siRNA and selected targets were subsequently blocked using small molecule inhibitors. The effect of this treatment on the expression of β-catenin-dependent genes was assessed by qRT-PCR. The effect of the inhibitors on cell viability was evaluated using a resazurin assay in HNSCC-derived cell lines. A luciferase reporter assay was used for confirmation of the inhibition of Wnt-dependent gene expression. Cell migration was evaluated using a scratch wound healing assay. Cytometric analysis of propidium iodide stained cells was used for cell cycle distribution evaluation, whereas cytometric analysis of caspase 3/7 activity was used for apoptosis induction evaluation., Results: We found that inhibition of Porcupine and CBP/β-catenin interaction by IWP-2 and PRI-724, respectively, most strongly affected β-catenin-dependent gene expression in HNSCC cells. These inhibitors also induced apoptosis and affected HNSCC cell migration., Conclusions: Targeting Porcupine or the CBP/β-catenin interaction seems to be an effective strategy for the inhibition of canonical Wnt signaling in HNSCC cells. Further studies are required to confirm the possible therapeutic effect of IWP-2 and PRI-724 in HNSCC.

The aim of this study was to evaluate the cytotoxicity of a series of seven 4'-methylthio-trans-stilbene derivatives against cancer cells: MCF7 and A431 in comparison with non-tumorigenic MCF12A and HaCaT cells. The mechanism of anti-proliferative activity of the most cytotoxic trans-resveratrol analogs: 3,4,5-trimethoxy-4'-methylthio-trans-stilbene (3,4,5-MTS) and 2,4,5-trimethoxy-4'-methylthio-trans-stilbene (2,4,5-MTS) was analyzed and compared with the effect of trans-resveratrol. All the compounds that were studied exerted a stronger cytotoxic effect than trans-resveratrol did. MCF7 cells were the most sensitive to the cytotoxic effect of trans-resveratrol analogs with IC 50 in the range of 2.1-6.0 µM. Comparing the cytotoxicity of 3,4,5-MTS and 2,4,5-MTS, a significantly higher cytotoxic activity of these compounds against MCF7 versus MCF12A was observed, whereas no significant difference was observed in cytotoxicity against A431 and HaCaT. In the series of 4'-methylthio-trans-stilbenes, 3,4,5-MTS and 2,4,5-MTS were the most promising compounds for further mechanistic studies. The proapoptotic activity of 3,4,5-MTS and 2,4,5-MTS, estimated with the use of annexin-V/propidium iodide assay, was comparable to that of trans-resveratrol. An analysis of cell cycle distribution showed a significant increase in the percentage of apoptotic cells and G2/M phase arrest in MCF7 and A431 as a result of treatment with 3,4,5-MTS, whereas trans-resveratrol tended to increase the percentage of cells in S phase, particularly in epithelial breast cells MCF12A and MCF7. Both trans-stilbene derivatives enhanced potently tubulin polymerization in a dose-dependent manner with sulfur atom participating in the interactions with critical residues of the paclitaxel binding site of β-tubulin.

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.

Background: Cancer cells are dependent on aerobic glycolysis for energy production and increased glutamine consumption. HIF-1α and c-MYC transcription factors regulate the expression of glycolytic and glutaminolytic genes. Their activity may be repressed by SIRT6. Head and neck carcinomas show frequent activation of c-MYC function and SIRT6 down-regulation, which contributes to a strong dependence on glucose and glutamine availability., Objectives: The aim of this study was to compare the influence of HIF-1α and c-MYC inhibitors (KG-548 and 10058-F4, respectively) and potential SIRT6 inducers - resveratrol and its synthetic derivative DMU-212 with the effect of glycolysis and glutaminolysis inhibitors (2-deoxyglucose and aminooxyacetic acid, respectively) on the metabolism and expression of metabolic enzymes in FaDu hypopharyngeal carcinoma cells., Material and Methods: Cell viability was assessed by means of an MTT assay. Quantitative PCR was performed to evaluate the expression of SIRT6, HIF-1α, c-MYC, GLUT1, SLC1A5, HK2, PFKM, PKM2, LDHA, GLS, and GDH. The release of glycolysis and glutaminolysis end-products into the culture medium - lactate and ammonia, respectively - was assessed using standard colorimetric assays., Results: Lactate production was significantly inhibited by 10058-F4, KG-548, and 2-deoxyglucose. Moreover, 10058-F4 strongly reduced the amount of ammonia release. The effects of 10058-F4 activity can be attributed to a reduction in the expression of PKM2 and LDHA. On the other hand, the induction of SIRT6 expression by resveratrol and DMU-212 was not associated with significant modulation of the expression of metabolic enzymes., Conclusions: Overall, the results of this study indicate that the inhibition of c-MYC may be considered to be a promising strategy of the modulation of cancer-related metabolic changes in head and neck carcinomas.

Cytochromes P450 family 1 (CYP1) are responsible for the metabolism of procarcinogens, for example polycyclic aromatic hydrocarbons and aromatic and heterocyclic amines. The inhibition of CYP1 activity is examined in terms of chemoprevention and cancer chemotherapy. We designed and synthesized a series of trans-stilbene derivatives possessing a combination of methoxy and methylthio functional groups attached in different positions to the trans-stilbene skeleton. We determined the effects of synthesized compounds on the activities of human recombinant CYP1A1, CYP1A2 and CYP1B1 and, to explain the variation of inhibitory potency of methoxystilbene derivatives and their methylthio analogues, we employed computational analysis. The compounds were docked to CYP1A1, CYP1A2 and CYP1B1 binding sites with the use of Accelrys Discovery Studio 4.0 by the CDOCKER procedure. For CYP1A2 and CYP1B1, values of scoring functions correlated well with inhibitory potency of stilbene derivatives. All compounds were relatively poor inhibitors of CYP1A2 that possess the most narrow and flat enzyme cavity among CYP1s. For the most active CYP1A1 inhibitor, 2-methoxy-4'-methylthio-trans-stilbene, a high number of molecular interactions was observed, although the interaction energies were not distinctive., (© 2017 John Wiley & Sons A/S.)

Hypertensive Disorders of Pregnancy (HDsP) remain leading causes of maternal and perinatal morbidity and mortality. Growing evidence suggests the involvement of epigenetic factors, such as gene-specific and global DNA methylation changes, both in the etiology and as an effect of HDsP. In this study, we investigated the potential association between placental DNA methylation status in selected CpGs of HSD11B2 cortisol level controlling gene, RUNX3 tumor suppressor gene, and long interspersed nucleotide element-1 (LINE-1) repetitive elements and HDsP-preeclampsia (PE), gestational hypertension (GH), and chronic hypertension (CH). Methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ) were used to analyze placental DNA methylation. Plasma and urine cortisol and cortisone levels were measured using high performance liquid chromatography with fluorescence detection (HPLC-FLD), whereas serum progesterone level was determined by electrochemiluminescence immunoassay. The mean percentage of HSD11B2, RUNX3, and LINE-1 methylation was not altered in the placentas of patients with HDsP, as compared to the controls. However, among patients from PE, GH, and CH groups, several significant correlations were observed between the methylation status of HSD11B2, RUNX3, or LINE-1 and children's birth weight, gestational age at delivery, mother's age, and body mass index as well as hormones levels. These results indicate lack of association between methylation status of HSD11B2, RUNX3, or LINE-1 repetitive elements and HDsP. However, association of these parameters with some clinical variables may suggest the role of placental DNA methylation in fetal development and should be further explored.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a potent chemopreventive and potentially cancer therapeutic agent. Since rapid metabolism limits resveratrol bioavailability, derivatives less prone to metabolic transformation are being sought and tested. We evaluated the effect of resveratrol, and its analogs (pterostilbene and 3,5,4'-trimethoxystilbene) along with tannic acid, on cell cycle and apoptosis in rat C6 and human T98G glioma cells. At concentration ranges both lower and higher than IC 50 calculated based on MTT assay, all these polyphenols affected the cell cycle distribution. However, resveratrol and pterostilbene increased the percentage of the cells in S phase, while trimethoxystilbene (TMS) caused a massive accumulation of cells at the G2/M phase of the cell cycle. Tannic acid had no effect on cell cycle distribution in C6 cells, but increased the number of dead cells in both glioma cell lines. The ability to induce apoptosis by tannic acid and stilbenes was confirmed by phosphatidylserine externalization, the loss of mitochondrial membrane potential and the level of cleaved caspase-3. The apoptosis rate was most significantly increased by TMS and this was related to p53 induction. These results indicate that methoxylated stilbenes are efficient inhibitors of glioma cell proliferation and apoptosis inducers and might be considered adjuvants in glioma therapy., (Copyright © 2017. Published by Elsevier Ltd.)

Nrf2 acts as a sensor of oxidative or electrophilic stress and prevents genome instability. The activation of Nrf2 signaling induces ARE-dependent expression of detoxifying and antioxidant defense proteins. Nrf2-ARE signaling has become an attractive target for cancer chemoprevention. On the other hand, constitutive over-activation of Nrf2 in cancer cells has been implicated in cancer progression as well as in resistance to cancer chemotherapeutics. Two basic Nrf2 activation pathways were described. The canonical pathway is the primary mechanism of Nrf2 activation and is based on dissociation of Nrf2 from its inactive complex with the repressor protein Keap1 and the subsequent translocation of Nrf2 into the nucleus. Numerous proteins which compete with Nrf2 for Keap1 binding stabilize Nrf2 and are involved in non-canonical pathways of Nrf2 activation. However, growing evidence indicates that the regulation of Keap1-Nrf2-ARE is more complex than was previously thought and that other molecular mechanisms are also involved. Among them is epigenetic regulation of Nrf2 and Keap1, which seems to be a particularly interesting subject for future studies. Nrf2 has become an important chemopreventive and therapeutic target, and many natural and synthetic chemicals have been described as its modulators. However, most small molecules which are either inducers or inhibitors of Nrf2 may provoke "off-target" toxic effects because of their electrophilic character. This review highlights Nrf2-ARE activation pathways and their role in cancer prevention and therapy. A critical evaluation of currently available Nrf2 inducers and inhibitors is also presented., (Copyright © 2016. Published by Elsevier Urban & Partner Sp. z o.o.)

Introduction: Epigenetics is a rapidly growing field describing heritable alterations in gene expression that do not involve DNA sequence variations. Advances in epigenetics and epigenomics have influenced pharmacology, leading to the development of a new specialty, pharmacoepigenetics, the study of the epigenetic basis for the individual variation in drug response. Areas covered: We present an overview of the major epigenetic mechanisms and their effects on the expression of drug metabolizing enzymes and drug transporters, as well as the epigenetic status of drug protein targets affecting therapy response. Recent advances in the development of pharmacoepigenetic biomarkers and epidrugs are also discussed. Expert opinion: There is growing evidence that pharmacoepigenetics has the potential to become an important element of personalized medicine. Epigenetic modifications influence drug response, but they can also be modulated by drugs. Moreover, they can be monitored not only in the affected tissue, but also in body fluids. Nevertheless, there are very few examples of epigenetic biomarkers implemented in the clinical setting. Explanation of the interplay between genomic and epigenomic changes will contribute to the personalized medicine approach. Ultimately, both genetic biomarkers and epigenetic mechanisms should be taken into consideration in predicting drug response in the course of successful personalized therapy.

The role of CYP1A1 and CYP1B1 enzymes in the biotransformation and biological activity of the methylated resveratrol analogue, 3,4,5,4'-tetramethoxystilbene (DMU-212) is still elusive. Our recently published data have shown that one of the metabolites of DMU-212, 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214) exerts more potent cytotoxic effects in A-2780 ovarian cancer cell line, as compared to the parent compound. Hence, this study aims to elucidate whether the biological activity of DMU-212 is related to its biotransformation to DMU-214. Furthermore, we aimed to assess which enzymes of CYP1 family are involved in the biotransformation of DMU-212. The human ovarian cancer cell lines A-2780, A-2780CYP1A1(-) and non-cancerous human ovarian surface epithelial (HOSE) cells were employed in the present study. In contrary to other authors' suggestions we have found that CYP1A1 is the major enzyme of CYP1 family involved in the metabolic activation of DMU-212. Since the distinctly weaker anti-proliferative effects of DMU-212 against HOSE and A-2780CYP1A1(-) cells have been associated with the lack of the expression of CYP1A1, we suggest that the biological activity of the parent compound may be related to its metabolic activation to DMU-214 and the level of this enzyme., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

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 deregulation of Wnt signaling is observed in various cancers, including gliomas, and might be related to the methylation of the genes encoding antagonists of this signaling pathway. The aim of the study was to assess the methylation status of the promoter regions of six Wnt negative regulators and to determine their prognostic value in clinical samples of gliomas of different grades. The methylation of SFRP1, SFRP2, PPP2R2B, DKK1, SOX17, and DACH1 was analyzed in 64 glioma samples using methylation-specific polymerase chain reaction (MSP). The results were analyzed in correlation with clinicopathological data. Promoter methylation in at least one of the analyzed genes was found in 81.3 % of the tumors. All benign tumors [grade I according to the World Health Organization (WHO) classification] lacked the methylation of the studied genes, whereas grade II, III, and IV tumors were, in most cases, methylation-positive. The methylation index correlated with the patient's age. The most frequently methylated genes were SFRP1 and SFRP2 (73.4 % and 46.9 %, respectively), followed by SOX17 (20.3 %) and PPP2R2B (10.9 %); DKK1 and DACH1 were basically unmethylated (1.6 %). SFRP1 methylation negatively correlated with patients' survival time, and was significantly more frequent in older patients and those with higher grade tumors. Overall, the results of this study indicate that aberrant promoter methylation of Wnt pathway antagonists is common in gliomas, which may be the possible cause of up-regulation of this signaling pathway often observed in these tumors. Moreover, SFRP1 promoter methylation can be regarded as a potential indicator of glioma patients' survival.