The KEAP1-NRF2 pathway: Targets for therapy and role in cancer.

The KEAP1-NRF2 pathway is the key regulator of cellular defense against both extrinsic and intrinsic oxidative and electrophilic stimuli. Since its discovery in the 1990s, its seminal role in various disease pathologies has become well appreciated, motivating research to elucidate the intricacies of NRF2 signaling and its downstream effects to identify novel targets for therapy. In this graphical review, we present an updated overview of the KEAP1-NRF2 signaling, focusing on the progress made within the past ten years. Specifically, we highlight the advances made in understanding the mechanism of activation of NRF2, resulting in novel discoveries in its therapeutic targeting. Furthermore, we will summarize new findings in the rapidly expanding field of NRF2 in cancer, with important implications for its diagnostics and treatment.

Gefitinib-Tamoxifen Hybrid Ligands as Potent Agents against Triple-Negative Breast Cancer.

Anticancer drug conjugates may benefit from simultaneous action at two targets potentially overcoming the drawbacks of current cancer treatment, such as insufficient efficacy, high toxicity, and development of resistance. Compared to a combination of two single-target drugs, they may offer an advantage of pharmacokinetic simplicity and fewer drug-drug interactions. Here, we report a series of compounds connecting tamoxifen or endoxifen with the EGFR-inhibitor gefitinib via a covalent linkage. These hybrid ligands retain both ER antagonist activity and EGFR inhibition. The most potent analogues exhibited single-digit nanomolar activities at both targets. The amide-linked endoxifen-gefitinib drug conjugates 17b and 17c demonstrated the most favorable anti-cancer profile in cellular viability assays on MCF7, MDA-MB-231, MDA-MB-468, and BT-549 breast cancer cells. Most importantly, in TNBC cells 17b and 17c displayed nanomolar IC50-values (380 nM - 970 nM) and were superior in their anti-cancer activity compared to their control compounds and combinations thereof.

Profiling the regulatory interplay of BET bromodomains and Sirtuins in cancer cell lines.

Alterations in epigenetic marking, due to changes in expression or activity of epigenetic regulators, may affect cancer development and progression and thus, targeting epigenetic regulators provides potential avenues for cancer treatment. Bromodomain and extra terminal domain (BET) proteins, epigenetic readers recognizing histone acetylation, and Sirtuins (SIRT1-7), histone deacetylases or erasers, affect the chromatin acetylation status, and thus have a vital role in transcriptional regulation of a variety of cancer-related genes. Here, the effects of three BET inhibitors on SIRT expression were screened in a broad set of cancer cell lines to study the potential interplay of these distinct epigenetic factors in gene regulation. We show that BET inhibitors have distinct effects on SIRTs and their target gene expression in cancer cell lines derived from several solid tumour cancers. This functional link may open further avenues for epigenetic combination therapies for different cancers.

Screening of SIRT6 inhibitors and activators: A novel activator has an impact on breast cancer cells.

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates a variety of cellular processes involved in aging, metabolism, and cancer. Dysregulation of SIRT6 is widely observed in different breast cancer subtypes; however, the role and function of SIRT6 in cancer development remain largely unexplored. The aim of this study was to identify novel compounds targeting SIRT6 which may provide a new approach in development of anti-cancer therapy for breast cancer. Virtual screening was utilized to discover potential compounds targeting SIRT6 for in vitro screening. In addition, novel 1,4-dihydropyridine derivatives were synthetized and further subjected for the screening. The impact of the compounds on the deacetylation activity of SIRT6 was determined with HPLC method. The anti-cancer activities were screened for a panel of breast cancer cells. A set of 1,4-dihydropyridine derivatives was identified as SIRT6 inhibitors. A SIRT6 activating compound, (2,4-dihydroxy-phenyl)-2-oxoethyl 2-(3-methyl-4-oxo-2-phenyl-4H-chromen-8-yl)acetate (later called as 4H-chromen), was discovered and it provided 30-40-fold maximal activation. 4H-chromen was proposed to bind similarly to quercetin and place to previously reported SIRT6 activator sites. 4H-chromen was investigated in various breast cancer cells, and it decreased cell proliferation in all cells as well as arrested cell cycle in triple negative cells. Overall, this study describes a highly potent SIRT6 activator and new inhibitors that represent a novel tool to study the mechanism of SIRT6 function.

Metabolism-Disrupting Chemicals and the Constitutive Androstane Receptor CAR.

During the last two decades, the constitutive androstane receptor (CAR; NR1I3) has emerged as a master activator of drug- and xenobiotic-metabolizing enzymes and transporters that govern the clearance of both exogenous and endogenous small molecules. Recent studies indicate that CAR participates, together with other nuclear receptors (NRs) and transcription factors, in regulation of hepatic glucose and lipid metabolism, hepatocyte communication, proliferation and toxicity, and liver tumor development in rodents. Endocrine-disrupting chemicals (EDCs) constitute a wide range of persistent organic compounds that have been associated with aberrations of hormone-dependent physiological processes. Their adverse health effects include metabolic alterations such as diabetes, obesity, and fatty liver disease in animal models and humans exposed to EDCs. As numerous xenobiotics can activate CAR, its role in EDC-elicited adverse metabolic effects has gained much interest. Here, we review the key features and mechanisms of CAR as a xenobiotic-sensing receptor, species differences and selectivity of CAR ligands, contribution of CAR to regulation hepatic metabolism, and evidence for CAR-dependent EDC action therein.

Effects of galloflavin and ellagic acid on sirtuin 6 and its anti-tumorigenic activities.

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates distinct cellular functions; genome stability, DNA repair, and inflammation related diseases. Recently, we demonstrated that anthocyanidins in berries induce the catalytic activity of SIRT6. In this study, we explored the effects of Galloflavin and Ellagic acid, the most common polyphenols in berries, on SIRT6. SIRT6 deacetylation was investigated using HPLC and immunoblotting assays. The expression levels of SIRT6, glycolytic proteins and cellular metabolism were studied on human colon adenocarcinoma cells (Caco2). Molecular docking studies were carried out to study possible interactions of the compounds with sirtuins. Ellagic acid increased the deacetylase activity of SIRT6 by up to 50-fold; it showed moderate inhibition of SIRT1-3. Galloflavin and Ellagic acid showed anti-proliferative effects on Caco2. The compounds also upregulated SIRT6 expression whereas key proteins in glycolysis were downregulated. Galloflavin decreased glucose transporter 1 (GLUT1) expression, and Ellagic acid affected the expression of protein dehydrogenase kinase 1 (PDK1). Interestingly, both compounds caused reduction in glucose uptake and lactate production. Both Galloflavin and Ellagic acid were able to form hydrogen bonds with Asp188 and Gly6 in SIRT6. In this study, we showed that Galloflavin and Ellagic acid increased SIRT6 activity and decreased the expression of SIRT6 associated proteins involved in cancer development. Taken together, Galloflavin and Ellagic acid targeting SIRT6 activity may provide a new insight in the development of anti-cancer therapy.

The EDCMET Project: Metabolic Effects of Endocrine Disruptors.

Endocrine disruptors (EDs) are defined as chemicals that mimic, block, or interfere with hormones in the body's endocrine systems and have been associated with a diverse array of health issues. The concept of endocrine disruption has recently been extended to metabolic alterations that may result in diseases, such as obesity, diabetes, and fatty liver disease, and constitute an increasing health concern worldwide. However, while epidemiological and experimental data on the close association of EDs and adverse metabolic effects are mounting, predictive methods and models to evaluate the detailed mechanisms and pathways behind these observed effects are lacking, thus restricting the regulatory risk assessment of EDs. The EDCMET (Metabolic effects of Endocrine Disrupting Chemicals: novel testing METhods and adverse outcome pathways) project brings together systems toxicologists; experimental biologists with a thorough understanding of the molecular mechanisms of metabolic disease and comprehensive in vitro and in vivo methodological skills; and, ultimately, epidemiologists linking environmental exposure to adverse metabolic outcomes. During its 5-year journey, EDCMET aims to identify novel ED mechanisms of action, to generate (pre)validated test methods to assess the metabolic effects of Eds, and to predict emergent adverse biological phenotypes by following the adverse outcome pathway (AOP) paradigm.

The Basis for Strain-Dependent Rat Aldehyde Dehydrogenase 1A7 (ALDH1A7) Gene Expression.

Aldehyde hydrogenases (ALDHs) belong to a large gene family involved in oxidation of both endogenous and exogenous compounds in mammalian tissues. Among ALDHs, the rat ALDH1A7 gene displays a curious strain dependence in phenobarbital (PB)-induced hepatic expression: the responsive RR strains exhibit induction of both ALDH1A7 and CYP2B mRNAs and activities, whereas the nonresponsive rr strains show induction of CYP2B only. Here, we investigated the responsiveness of ALDH1A1, ALDH1A7, CYP2B1, and CYP3A23 genes to prototypical P450 inducers, expression of nuclear receptors CAR and pregnane X receptor, and structure of the ALDH1A7 promoter in both rat strains. ALDH1A7 mRNA, associated protein and activity were strongly induced by PB and modestly induced by pregnenolone 16α-carbonitrile in the RR strain but negligibly in the rr strain, whereas induction of ALDH1A1 and P450 mRNAs was similar between the strains. Reporter gene and chromatin immunoprecipitation assays indicated that the loss of ALDH1A7 inducibility in the rr strain is profoundly linked with a 16-base pair deletion in the proximal promoter and inability of the upstream DNA sequences to recruit constitutive androstane receptor-retinoid X receptor heterodimers. SIGNIFICANCE STATEMENT: Genetic variation in rat ALDH1A7 promoter sequences underlie the large strain-dependent differences in expression and inducibility by phenobarbital of the aldehyde dehydrogenase activity. This finding has implications for the design and interpretation of pharmacological and toxicological studies on the effects and disposition of aldehydes.

A Reverse Transfection Method for Screening of Nuclear Receptor Activators.

Reporter assays are useful to study nuclear receptor activation and for example to evaluate the propensity of novel drug candidates to cause induction of drug-metabolizing cytochrome P450 enzymes. Here, we describe a protocol for a reverse transfection system to study the activation of human nuclear receptors constitutive androstane receptor and pregnane X receptor. The system provides long-term stability and uniformity of DNA-carrier complexes, thus avoiding the inherent variation in conventional transfection methods. Further, the system is easily adaptable for different studies. It offers reproducible and reliable results for early drug development and mechanistic studies related to nuclear receptor activation and resulting changes in gene expression.

DNA elements for constitutive androstane receptor- and pregnane X receptor-mediated regulation of bovine CYP3A28 gene.

The regulation of cytochrome P450 3A (CYP3A) enzymes is established in humans, but molecular mechanisms of its basal and xenobiotic-mediated regulation in cattle are still unknown. Here, ~10 kbp of the bovine CYP3A28 gene promoter were cloned and sequenced, and putative transcription factor binding sites were predicted. The CYP3A28 proximal promoter (PP; -284/+71 bp) contained DNA elements conserved among species. Co-transfection of bovine nuclear receptors (NRs) pregnane X and constitutive androstane receptor (bPXR and bCAR) with various CYP3A28 promoter constructs into hepatoma cell lines identified two main regions, the PP and the distal fragment F3 (-6899/-4937 bp), that were responsive to bPXR (both) and bCAR (F3 fragment only). Site-directed mutagenesis and deletion of NR motif ER6, hepatocyte nuclear factor 1 (HNF-1) and HNF-4 binding sites in the PP suggested either the involvement of ER6 element in bPXR-mediated activation or the cooperation between bPXR and liver-enriched transcription factors (LETFs) in PP transactivation. A putative DR5 element within the F3 fragment was involved in bCAR-mediated PP+F3 transactivation. Although DNA enrichment by anti-human NR antibodies was quite low, ChIP investigations in control and RU486-treated BFH12 cells, suggested that retinoid X receptor α (RXRα) bound to ER6 and DR5 motifs and its recruitment was enhanced by RU486 treatment. The DR5 element seemed to be recognized mainly by bCAR, while no clear-cut results were obtained for bPXR. Present results point to species-differences in CYP3A regulation and the complexity of bovine CYP3A28 regulatory elements, but further confirmatory studies are needed.

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Regulation of Human Pluripotent Stem Cell-Derived Hepatic Cell Phenotype by Three-Dimensional Hydrogel Models.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes are anticipated as important surrogates for primary human hepatocytes in applications ranging from basic research to drug discovery and regenerative medicine. Although methods for differentiating hepatocyte-like cells (HLCs) from hiPSCs have developed remarkably, the limited yield of fully functional HLCs is still a major obstacle to their utility. A three-dimensional (3D) culture environment could improve the in vitro hepatic maturation of HLCs. Here we compare 3D hydrogel models of hiPSC-derived HLCs in agarose microwells (3D Petri Dish; 3DPD), nanofibrillar cellulose hydrogels (Growdex; 3DNFC), or animal extracellular matrix-based hydrogels (3D Matrigel; 3DMG). In all the tested 3D biomaterial systems, HLCs formed aggregates. In comparison with two-dimensional monolayer culture, 3DPD and 3DMG models showed both phenotypic and functional enhancement in HLCs over 2.5 weeks of 3D culture. Specifically, we found higher hepatocyte-specific gene expression levels and enhanced cytochrome P450 functions. Our work suggests that transferring HLCs into 3D hydrogel systems can expedite the hepatic maturation of HLCs irrespective of the biochemical nature of the 3D hydrogel. Both plant-based nonembedding and animal-based embedding 3D hydrogel models enhanced the maturation.

Genetically Modified Caco-2 Cells With Improved Cytochrome P450 Metabolic Capacity.

The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells.

Characterization of ligand-dependent activation of bovine and pig constitutive androstane (CAR) and pregnane X receptors (PXR) with interspecies comparisons.

1. Nuclear receptors CAR (NR1I3) and PXR (NR1I2) are major ligand-activated transcriptional regulators of xenobiotic metabolism and disposition and modulators of endobiotic metabolism. Differences in xenobiotic selectivity between the human and rodent receptors are well recognized but there is lack of such information on properties of CAR and PXR in important domestic animals. 2. The pig and bovine receptors were cloned and their ligand profiles were systematically compared to corresponding human and mouse forms utilizing a panel of xenobiotics and structural analysis. 3. Pig CAR and PXR resemble their human counterparts which can be rationalized by only modest amino acid changes between critical residues of the human ligand-binding pockets (H203Q for CAR, L210V and M243I for PXR). 4. In contrast, bovine CAR shows a blunted response to CAR agonists and inverse agonists. These changes are likely due to disruptive mutations at or near critical hydrogen bond-forming residues (N165I, Y326F). The unresponsiveness of bovine PXR to human- and mouse-selective agonists may be related to substitutions at important ligand-contacting residues R410Q and F305V, respectively. 5. Our findings have implications for regulation of drug-metabolizing enzymes and transporters and pharmacokinetics in cattle and pigs.

Improved assays for xenosensor activation based on reverse transfection.

Discovery of receptor-dependent mechanisms for regulation of drug metabolism has provided a new way to evaluate the propensity of drug candidates to cause induction of cytochrome P450 enzymes. Therefore, receptor-based reporter assays have become common in early stages of drug development projects and in mechanistic studies. Here, we report a reverse transfection system to conduct activation assays for human xenosensors AhR, CAR and PXR. The assay format is based on long-term stability and uniformity of DNA/carrier complexes on culture plates, avoiding multiple stages and variation inherent in conventional transfection methods. Consequently, these improved assays are streamlined, reproducible and formally validated with Z' factors exceeding 0.5. This novel reverse transfection system is expected to find use in diverse areas of early drug development such prediction of CYP induction, evaluation of species differences and in mechanistic studies.

Direct and rapid transcript analysis assay for CYP mRNA expression and inducibility in human primary hepatocytes.

Induction of cytochrome P450 (CYP) enzymes is commonly analyzed in cultured human primary hepatocytes (HPHs) by measuring CYP1A2, CYP2B6 and CYP3A4/3A5 activities after exposure to test and reference compounds. Because chemicals can both inhibit and induce CYP enzymes, this traditional approach fails to distinguish such simultaneous effects. Regulatory authorities have therefore suggested that measurement of CYP expression levels should complement activity measurements. We aimed to compare a hybridization and bead-based assay termed transcript analysis with the aid of affinity capture (TRAC) with the routinely used quantitative real-time PCR (qRT-PCR) assay and to study its suitability for CYP induction studies on mRNA level. HPHs from three donors were treated with vehicle, reference substances omeprazole, phenobarbital and rifampicin and six test compounds on 48-well plates. The mRNA expression of ten CYP isoforms important for drug metabolism was determined by TRAC and qRT-PCR methods in order to validate the novel TRAC method. The fold-increases of CYP mRNA levels showed a good correlation between the assays. With TRAC, the marker CYP mRNAs for induction could be easily detected from about 10 000 hepatocytes per sample, with a coefficient of variation below 10% between triplicates. Time spent for TRAC analysis was significantly shorter. Thus, TRAC is a sensitive and reproducible high-throughput assay, which enables accurate and direct detection of multiple mRNA targets simultaneously from large number of samples without enzymatic reactions inherent to qRT-PCR. It is a valuable method to study CYP induction and expandable to other genes relevant for drug metabolism and toxicity.

Preclinical pharmacology of FL442, a novel nonsteroidal androgen receptor modulator.

The preclinical profiles of two most potent compounds of our recently published cycloalkane[d]isoxazole pharmacophore-based androgen receptor (AR) modulators, FL442 (4-(3a,4,5,6,7,7a-hexahydro-benzo[d]isoxazol-3-yl)-2-(trifluoromethyl)benzonitrile) and its nitro analog FL425 (3-(4-nitro-3-(trifluoromethyl)phenyl)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazole), were explored to evaluate their druggability for the treatment of AR dependent prostate cancer. The studies revealed that both compounds are selective to AR over other closely related steroid hormone receptors and that FL442 exhibits equal inhibition efficiency towards the androgen-responsive LNCaP prostate cancer cell line as the most widely used antiandrogen bicalutamide and the more recently discovered enzalutamide. Notably, FL442 maintains antiandrogenic activity with enzalutamide-activated AR mutant F876L. In contrast to bicalutamide, FL442 does not stimulate the VCaP prostate cancer cells which express elevated levels of the AR. Distribution analyses showed that [(14)CN]FL442 accumulates strongly in the mouse prostate. In spite of its low plasma concentration obtained by intraperitoneal administration, FL442 significantly inhibited LNCaP xenograft tumor growth. These findings provide a preclinical proof for FL442 as a promising AR targeted candidate for a further optimization.

Interactions of sesquiterpenes zederone and germacrone with the human cytochrome P450 system.

Misclassification of Curcuma species (family Zingiberaceae) may lead to unwanted human exposure to Curcuma elata sesquiterpenes zederone and germacrone which have caused hepatotoxicity and changes in CYP expression in laboratory animals. We investigated how these compounds interact with the human cytochrome P450 (CYP) system, in order to evaluate their potential for human liver toxicity and herb-drug interactions. We found that both sesquiterpenes (1-30 µM) greatly induced expression of CYP2B6 and CYP3A4 but not CYP1A2 mRNAs in human primary hepatocytes (HPHs). This induction profile correlated with activation of constitutive androstane and pregnane X receptors. Cytotoxicity was also observed in exposed HPHs. CYP inhibition studies with pooled human liver microsomes (HLMs) indicated that zederone and germacrone moderately inhibited CYP2B6 and CYP3A4 activities in vitro, with IC50 values below 10 µM. When zederone was incubated with HLMs and NADPH, one di-epoxide metabolite was formed and by using glutathione trapping, five epoxide-derived conjugates were detected. Germacrone produced two oxidized metabolites and four glutathione conjugates. The results suggest that enzymes in HLMs convert sesquiterpenes into reactive, electrophilic compounds which may be causative for the reported liver injuries. These findings provide insight on the safety and drug-herb interactions of the Curcuma species.

An update on the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR; NR1I3) has emerged as one of the main drug- and xenobiotic-sensitive transcriptional regulators. It has a major effect on the expression of several oxidative and conjugative enzymes and transporters, and hence, CAR can contribute to drug/drug interactions. Novel functions for CAR are also emerging: it is able to modulate the metabolic fate of glucose, lipids, and bile acids, and it is also involved in cell-cell communication, regulation of the cell cycle, and chemical carcinogenesis. Here, we will review the recent information available on CAR and its target gene expression, its interactions with partner proteins and mechanisms of action, interindividual and species variation, and current advances in CAR ligand selectivity and methods used in interrogation of its ligands.

Towards personalized medicine with a three-dimensional micro-scale perfusion-based two-chamber tissue model system.

A three-dimensional micro-scale perfusion-based two-chamber (3D-µPTC) tissue model system was developed to test the cytotoxicity of anticancer drugs in conjunction with liver metabolism. Liver cells with different cytochrome P450 (CYP) subtypes and glioblastoma multiforme (GBM) brain cancer cells were cultured in two separate chambers connected in tandem. Both chambers contained a 3D tissue engineering scaffold fabricated with biodegradable poly(lactic acid) (PLA) using a solvent-free approach. We used this model system to test the cytotoxicity of anticancer drugs, including temozolomide (TMZ) and ifosfamide (IFO). With the liver cells, TMZ showed a much lower toxicity to GBM cells under both 2D and 3D cell culture conditions. Comparing 2D, GBM cells cultured in 3D had much high viability under TMZ treatment. IFO was used to test the CYP-related metabolic effects. Cells with different expression levels of CYP3A4 differed dramatically in their ability to activate IFO, which led to strong metabolism-dependent cytotoxicity to GBM cells. These results demonstrate that our 3D-µPTC system could provide a more physiologically realistic in vitro environment than the current 2D monolayers for testing metabolism-dependent toxicity of anticancer drugs. It could therefore be used as an important platform for better prediction of drug dosing and schedule towards personalized medicine.