Ian M. Copple

Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway.

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

BackgroundNrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.ResultsKeap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).ConclusionsExpression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.

The Keap1–Nrf2 Cell Defense Pathway – A Promising Therapeutic Target?

By regulating the basal and inducible expression of an abundance of detoxification enzymes, antioxidant proteins, xenobiotic transporters and other stress response proteins, the Keap1-Nrf2 pathway plays a crucial role in determining the sensitivity of mammalian cells to chemical and oxidative insults that have the capacity to provoke cellular harm. This review highlights historical and recent advances in our understanding of the molecular mechanisms that regulate the activity of the Keap1-Nrf2 pathway. The important role of Nrf2 in protecting against the onset of specific diseases and drug-induced toxicities is also examined, alongside the emerging role of Nrf2 in promoting oncogenesis and chemotherapeutic drug resistance. A particular emphasis is placed on the potential for translation of this mechanistic understanding into clinical strategies that can improve human health, with consideration of the potential applications of targeting Nrf2 therapeutically.

The Role of Heme and the Mitochondrion in the Chemical and Molecular Mechanisms of Mammalian Cell Death Induced by the Artemisinin Antimalarials

The artemisinin compounds are the frontline drugs for the treatment of drug-resistant malaria. They are selectively cytotoxic to mammalian cancer cell lines and have been implicated as neurotoxic and embryotoxic in animal studies. The endoperoxide functional group is both the pharmacophore and toxicophore, but the proposed chemical mechanisms and targets of cytotoxicity remain unclear. In this study we have used cell models and quantitative drug metabolite analysis to define the role of the mitochondrion and cellular heme in the chemical and molecular mechanisms of cell death induced by artemisinin compounds. HeLa ρ0 cells, which are devoid of a functioning electron transport chain, were used to demonstrate that actively respiring mitochondria play an essential role in endoperoxide-induced cytotoxicity (artesunate IC50 values, 48 h: HeLa cells, 6 ± 3 μm; and HeLa ρ0 cells, 34 ± 5 μm) via the generation of reactive oxygen species and the induction of mitochondrial dysfunction and apoptosis but do not have any role in the reductive activation of the endoperoxide to cytotoxic carbon-centered radicals. However, using chemical modulators of heme synthesis (succinylacetone and protoporphyrin IX) and cellular iron content (holotransferrin), we have demonstrated definitively that free or protein-bound heme is responsible for intracellular activation of the endoperoxide group and that this is the chemical basis of cytotoxicity (IC50 value and biomarker of bioactivation levels, respectively: 10β-(p-fluorophenoxy)dihydroartemisinin alone, 0.36 ± 0.20 μm and 11 ± 5%; and with succinylacetone, >100 μm and 2 ± 5%).

The hepatotoxic metabolite of acetaminophen directly activates the Keap1‐Nrf2 cell defense system

The transcription factor Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. We have demonstrated previously that acetaminophen activates Nrf2 in mouse liver following administration of non‐hepatotoxic and hepatotoxic doses in vivo, implying that Nrf2 may have an important role in the protection against drug‐induced liver injury. Nrf2 activation has been proposed to occur through the modification of cysteine residues within Keap1, the cytosolic repressor of Nrf2. We hypothesized that acetaminophen activates Nrf2 via the formation of its reactive metabolite N‐acetyl‐p‐benzoquinoneimine (NAPQI), which may disrupt the repression of Nrf2 through the modification of cysteine residues within Keap1. Here, we show that NAPQI can directly activate the Nrf2 pathway in mouse liver cells, inducing an adaptive defense response that is antagonized by RNA interference targeted against Nrf2. Furthermore, mass spectrometric analysis shows that NAPQI selectively modifies cysteine residues in Keap1, both in recombinant protein in vitro and in cells ectopically expressing Keap1. Using this cell‐based model, we demonstrate that activation of Nrf2 by NAPQI and a panel of probe molecules [dexamethasone 21‐mesylate, 15‐deoxy‐Δ‐(12,14)‐prostaglandin J2, 2,4‐dinitrochlorobenzene, and iodoacetamide] correlates with the selective modification of cysteine residues located within the intervening region of Keap1. However, substantial depletion of glutathione (to less than 15 % of basal levels) by buthionine sulfoximine, which does not directly modify Keap1, is also sufficient to activate Nrf2. Conclusion: Nrf2 can be activated via the direct modification of cysteine residues located within the intervening region of Keap1, but also via the substantial depletion of glutathione without the requirement for direct modification of Keap1. It is possible that both of these mechanisms contribute to the activation of Nrf2 by acetaminophen. (HEPATOLOGY 2008.)

Role of Nrf2 in protection against acute kidney injury.

Multifaceted cell defense pathways perform a critical role in the maintenance of homeostasis at the cellular, tissue, and organism levels. The Keap1-Nrf2 pathway is one of the most important of these cytoprotective pathways, with Nrf2 serving as a master transcriptional regulator of the basal and inducible expression of a multitude of genes encoding detoxification enzymes, antioxidant proteins, xenobiotic transporters, and other stress-response mediators. An increasing body of evidence supports a vital physiological role for Nrf2 in protection of the kidney against a number of diseases, and the pharmacological induction of Nrf2 by bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate, CDDO-Me) has shown promise for the management of such pathologies. Acute kidney injury, induced by drugs and other stimuli, is a significant clinical problem, and accounts for the cessation of development of many promising drug candidates. A better understanding of the molecular mechanisms that underlie acute kidney injury, and the biological facets that determine the balance between renal adaptation and dysfunction, is therefore vital to reducing clinical burden and patient suffering. The focus of this review is to highlight recent work that has demonstrated an ability of Nrf2 to determine the sensitivity of the kidney to acute injury invoked by environmental insults such as heavy metals and ischemia, as well as xenobiotics such as cyclosporin A and cisplatin.

Brusatol provokes a rapid and transient inhibition of Nrf2 signaling and sensitizes mammalian cells to chemical toxicity-implications for therapeutic targeting of Nrf2.

The transcription factor Nrf2 regulates the basal and inducible expression of a battery of cytoprotective genes. Whereas numerous Nrf2-inducing small molecules have been reported, very few chemical inhibitors of Nrf2 have been identified to date. The quassinoid brusatol has recently been shown to inhibit Nrf2 and ameliorate chemoresistance in vitro and in vivo. Here, we show that brusatol provokes a rapid and transient depletion of Nrf2 protein, through a posttranscriptional mechanism, in mouse Hepa-1c1c7 hepatoma cells. Importantly, brusatol also inhibits Nrf2 in freshly isolated primary human hepatocytes. In keeping with its ability to inhibit Nrf2 signaling, brusatol sensitizes Hepa-1c1c7 cells to chemical stress provoked by 2,4-dinitrochlorobenzene, iodoacetamide, and N-acetyl-p-benzoquinone imine, the hepatotoxic metabolite of acetaminophen. The inhibitory effect of brusatol toward Nrf2 is shown to be independent of its repressor Keap1, the proteasomal and autophagic protein degradation systems, and protein kinase signaling pathways that are known to modulate Nrf2 activity, implying the involvement of a novel means of Nrf2 regulation. These findings substantiate brusatol as a useful experimental tool for the inhibition of Nrf2 signaling and highlight the potential for therapeutic inhibition of Nrf2 to alter the risk of adverse events by reducing the capacity of nontarget cells to buffer against chemical and oxidative insults. These data will inform a rational assessment of the risk:benefit ratio of inhibiting Nrf2 in relevant therapeutic contexts, which is essential if compounds such as brusatol are to be developed into efficacious and safe drugs.

Identification and quantification of the basal and inducible Nrf2-dependent proteomes in mouse liver: biochemical, pharmacological and toxicological implications.

The transcription factor Nrf2 is a master regulator of cellular defence: Nrf2 null mice (Nrf2(−/−)) are highly susceptible to chemically induced toxicities. We report a comparative iTRAQ-based study in Nrf2(−/−) mice treated with a potent inducer, methyl-2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate (CDDO-me; bardoxolone -methyl), to define both the Nrf2-dependent basal and inducible hepatoproteomes. One thousand five hundred twenty-one proteins were fully quantified (FDR < 1%). One hundred sixty-one were significantly different (P < 0.05) between WT and Nrf2(−/−) mice, confirming extensive constitutive regulation by Nrf2. Treatment with CDDO-me (3 mg/kg; i.p.) resulted in significantly altered expression of 43 proteins at 24 h in WT animals. Six proteins were regulated at both basal and inducible levels exhibiting the largest dynamic range of Nrf2 regulation: cytochrome P4502A5 (CYP2A5; 17.2-fold), glutathione-S-transferase-Mu 3 (GSTM3; 6.4-fold), glutathione-S-transferase Mu 1 (GSTM1; 5.9-fold), ectonucleoside-triphosphate diphosphohydrolase (ENTPD5; 4.6-fold), UDP-glucose-6-dehydrogenase (UDPGDH; 4.1-fold) and epoxide hydrolase (EPHX1; 3.0-fold). These proteins, or their products, thus provide a potential source of biomarkers for Nrf2 activity. ENTPD5 is of interest due to its emerging role in AKT signalling and, to our knowledge, this protein has not been previously shown to be Nrf2-dependent. Only two proteins altered by CDDO-me in WT animals were similarly affected in Nrf2(−/−) mice, demonstrating the high degree of selectivity of CDDO-me for the Nrf2:Keap1 signalling pathway. Biological significance The Nrf2:Keap1 signalling pathway is attracting considerable interest as a therapeutic target for different disease conditions. For example, CDDO-me (bardoxolone methyl) was investigated in clinical trials for the treatment of acute kidney disease, and dimethyl fumarate, recently approved for reducing relapse rate in multiple sclerosis, is a potent Nrf2 inducer. Such compounds have been suggested to act through multiple mechanisms; therefore, it is important to define the selectivity of Nrf2 inducers to assess the potential for off-target effects that may lead to adverse drug reactions, and to provide biomarkers with which to assess therapeutic efficacy. Whilst there is considerable information on the global action of such inducers at the mRNA level, this is the first study to catalogue the hepatic protein expression profile following acute exposure to CDDO-me in mice. At a dose shown to evoke maximal Nrf2 induction in the liver, CDDO-me appeared highly selective for known Nrf2-regulated proteins. Using the transgenic Nrf2(−/−) mouse model, it could be shown that 97% of proteins induced in wild type mice were associated with a functioning Nrf2 signalling pathway. This analysis allowed us to identify a panel of proteins that were regulated both basally and following Nrf2 induction. Identification of these proteins, which display a large magnitude of variation in their expression, provides a rich source of potential biomarkers for Nrf2 activity for use in experimental animals, and which may be translatable to man to define individual susceptibility to chemical stress, including that associated with drugs, and also to monitor the pharmacological response to Nrf2 inducers.

A tetraoxane-based antimalarial drug candidate that overcomes PfK13-C580Y dependent artemisinin resistance.

K13 gene mutations are a primary marker of artemisinin resistance in Plasmodium falciparum malaria that threatens the long-term clinical utility of artemisinin-based combination therapies, the cornerstone of modern day malaria treatment. Here we describe a multinational drug discovery programme that has delivered a synthetic tetraoxane-based molecule, E209, which meets key requirements of the Medicines for Malaria Venture drug candidate profiles. E209 has potent nanomolar inhibitory activity against multiple strains of P. falciparum and P. vivax in vitro, is efficacious against P. falciparum in in vivo rodent models, produces parasite reduction ratios equivalent to dihydroartemisinin and has pharmacokinetic and pharmacodynamic characteristics compatible with a single-dose cure. In vitro studies with transgenic parasites expressing variant forms of K13 show no cross-resistance with the C580Y mutation, the primary variant observed in Southeast Asia. E209 is a superior next generation endoperoxide with combined pharmacokinetic and pharmacodynamic features that overcome the liabilities of artemisinin derivatives.

Integrated transcriptomic and proteomic analyses uncover regulatory roles of Nrf2 in the kidney

The transcription factor Nrf2 exerts protective effects in numerous experimental models of acute kidney injury, and is a promising therapeutic target in chronic kidney disease. To provide a detailed insight into the regulatory roles of Nrf2 in the kidney, we performed integrated transcriptomic and proteomic analyses of kidney tissue from wild-type and Nrf2 knockout mice treated with the Nrf2 inducer methyl-2-cyano-3,12-dioxooleano-1,9-dien-28-oate (CDDO-Me, also known as bardoxolone methyl). After 24 hours, analyses identified 2561 transcripts and 240 proteins that were differentially expressed in the kidneys of Nrf2 knockout mice, compared to wild-type counterparts, and 3122 transcripts and 68 proteins that were differentially expressed in wild-type mice treated with CDDO-Me, compared to vehicle control. In light of their sensitivity to genetic and pharmacological modulation of renal Nrf2 activity, genes/proteins that regulate xenobiotic disposition, redox balance, the intra/extracellular transport of small molecules, and the supply of NADPH and other cellular fuels were found to be positively regulated by Nrf2 in the kidney. This was verified by qPCR, immunoblotting, pathway analysis and immunohistochemistry. In addition, the levels of NADPH and glutathione were found to be significantly decreased in the kidneys of Nrf2 knockout mice. Thus, Nrf2 regulates genes that coordinate homeostatic processes in the kidney, highlighting its potential as a novel therapeutic target.