Patrick M. Dolan

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors

Gastric infection with Helicobacter pylori is a cosmopolitan problem, and is especially common in developing regions where there is also a high prevalence of gastric cancer. These infections are known to cause gastritis and peptic ulcers, and dramatically enhance the risk of gastric cancer. Eradication of this organism is an important medical goal that is complicated by the development of resistance to conventional antimicrobial agents and by the persistence of a low level reservoir of H. pylori within gastric epithelial cells. Moreover, economic and practical problems preclude widespread and intensive use of antibiotics in most developing regions. We have found that sulforaphane [(−)-1-isothiocyanato-(4R)-(methylsulfinyl)butane], an isothiocyanate abundant as its glucosinolate precursor in certain varieties of broccoli and broccoli sprouts, is a potent bacteriostatic agent against 3 reference strains and 45 clinical isolates of H. pylori [minimal inhibitory concentration (MIC) for 90% of the strains is ≤4 μg/ml], irrespective of their resistance to conventional antibiotics. Further, brief exposure to sulforaphane was bactericidal, and eliminated intracellular H. pylori from a human epithelial cell line (HEp-2). In complementary experiments, sulforaphane blocked benzo[a]pyrene-evoked forestomach tumors in ICR mice. This protection resulted from induction of phase 2 detoxication and antioxidant enzymes, and was abrogated in mice lacking the nrf2 gene, which regulates phase 2 enzymes. Thus, the dual actions of sulforaphane in inhibiting Helicobacter infections and blocking gastric tumor formation offer hope that these mechanisms might function synergistically to provide diet-based protection against gastric cancer in humans.

Role of Nrf2 in prevention of high-fat diet-induced obesity by synthetic triterpenoid CDDO-Imidazolide

The synthetic oleanolic triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Imidazolide or CDDO-Im) is an extremely potent activator of Nrf2 signaling. In cells undergoing adipogenesis, CDDO-Im prevents lipid accumulation in an Nrf2-dependent manner. However, in vivo evidence for effects of CDDO-Im on obesity is lacking. The goals of these studies were to determine if CDDO-Im can prevent high-fat diet-induced obesogenesis in the mouse, and to elucidate the molecular target of drug action. Wild-type and Nrf2-disrupted C57BL/6J female mice were dosed 3 times per week with 30 micromol/kg CDDO-Im or vehicle by oral gavage, during 95 days of access to a control diet or a high-fat diet. Body weights, organ weights, hepatic fat accumulation and gene expression were measured. Treatment with CDDO-Im effectively prevented high-fat diet-induced increases in body weight, adipose mass, and hepatic lipid accumulation in wild-type mice but not in Nrf2-disrupted mice. Wild-type mice on a high-fat diet and treated with CDDO-Im exhibited higher oxygen consumption and energy expenditure than vehicle-treated mice, while food intake was lower in CDDO-Im-treated than vehicle-treated mice. Levels of gene transcripts for fatty acid synthesis enzymes were downregulated after CDDO-Im treatment in the liver of wild-type mice. This inhibitory effect of CDDO-Im on lipogenic gene expression was significantly reduced in Nrf2-disrupted mice. The results indicate that CDDO-Im is an exceedingly potent agent for preventing obesity, and identify the Nrf2 pathway as a novel target for management of obesogenesis.

Genetic versus chemoprotective activation of Nrf2 signaling: overlapping yet distinct gene expression profiles between Keap1 knockout and triterpenoid-treated mice

Loss of NF-E2-related factor 2 (Nrf2) signaling increases susceptibility to acute toxicity, inflammation and carcinogenesis in mice due to the inability to mount adaptive responses. In contrast, disruption of Keap1 (a cytoplasmic modifier of Nrf2 turnover) protects against these stresses in mice, although inactivating mutations in Keap1 have been identified recently in some human cancers. Global characterization of Nrf2 activation is important to exploit this pathway for chemoprevention in healthy, yet at-risk individuals and also to elucidate the consequences of hijacking the pathway in Keap1-mutant human cancers. Liver-targeted conditional Keap1-null, Albumin-Cre:Keap1((flox/-)) (CKO) mice provide a model of genetic activation of Nrf2 signaling. By coupling global gene expression analysis of CKO mice with analysis of pharmacologic activation using the synthetic oleanane triterpenoid 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), we are able to gain insight into pathways affected by Nrf2 activation. CDDO-Im is an extremely potent activator of Nrf2 signaling. CKO mice were used to identify genes modulated by genetic activation of Nrf2 signaling. The CKO response was compared with hepatic global gene expression changes in wild-type mice treated with CDDO-Im at a maximal Nrf2 activating dose. The results show that genetic and pharmacologic activation of Nrf2 signaling modulates pathways beyond detoxication and cytoprotection, with the largest cluster of genes associated with lipid metabolism. Genetic activation of Nrf2 results in much larger numbers of detoxication and lipid metabolism gene changes. Additionally, analysis of pharmacologic activation suggests that Nrf2 is the primary mediator of CDDO-Im activity, though other cell-signaling targets are also modulated following an oral dose of 30 micromol/kg.

Biochemical effects of dithiolthiones

Administration of dithiolthiones to mice in single intragastric doses (2-4 mmol/kg body weight) or in the diet (0.5% for 14 days), and to rats in the diet (0.1% for 14 days) was found to increase glutathione levels and the activities of a number of enzymes in various tissues including the liver and lung. The enzymes affected were glutathione transferases (with chlorodinitrobenzene or dichloronitrobenzene), quinone and glutathione reductase, and glucose-6-phosphate and 6-phosphogluconate dehydrogenase, all of which are involved directly or indirectly in the detoxication of xenobiotics, including carcinogens. The dithiolthiones tested in mice were oltipraz, ADT, 116L and 129L, and in rats, oltipraz. Intragastric administration of dithiolthiones (oltipraz, ADT or 116L; two doses each of 1 g/kg body weight) did not increase glutathione levels or enzyme activities in murine mammary adenocarcinoma transplants. Increases in glutathione levels and enzyme activities similar to those found with dithiolthiones were observed when a semi-synthetic diet containing 10-40% lyophilized cabbage was fed to mice for 30 days. Dithiolthiones that are present in cabbage may play a role in the protective actions of diets high in vegetables against the toxic actions of xenobiotics. The biochemical effects of dithiolthiones reported here may account for the protective actions of these compounds.

Increased colonic inflammatory injury and formation of aberrant crypt foci in Nrf2‐deficient mice upon dextran sulfate treatment

Chronic inflammation has been associated with increased risk of developing cancer. The transcription factor NF‐E2‐related factor 2 (Nrf2) controls the expression of numerous antioxidative enzymes that have been shown to attenuate acute inflammation. The present study investigated the role of Nrf2 genotype in modulating inflammation‐promoted colorectal tumorigenesis. Nrf2 wild‐type (WT) and Nrf2‐deficient (N0) mice were administered a single dose of azoxymethane followed by a 1‐week dose of drinking water with or without 1% dextran sulfate sodium (DSS). Aberrant crypt foci were counted 3 weeks after the cessation of DSS treatment. DSS treatment significantly increased numbers of aberrant crypt foci in N0 mice, but not WT mice. The extent of inflammation over the course of DSS treatment was analyzed in both genotypes. Histological analysis of colon sections revealed that N0 mice had markedly increased inflammation and mucosal damage when compared to WT mice beginning on Day 6 of DSS treatment. Although similar levels of inflammatory and oxidative damage biomarkers were evident in colons from WT and N0 mice at the start of DSS treatment, increased colonic proinflammatory cytokine mRNA transcript levels, myeloperoxidase activity and 3‐nitrotyrosine immunoreactivity were observed on Day 6 of DSS treatment in N0 mice, but not WT mice. Additionally, DSS treatment resulted in increased lipid peroxidation and loss of aconitase activity in N0 mice, but not WT mice, reflecting increased oxidative damage in colons from N0 mice. Taken together, these results clearly illustrate the role of Nrf2 in regulating an adaptive response that protects against early‐phase inflammation‐mediated tumorigenesis.

Potent, selective and low-calcemic inhibitors of CYP24 hydroxylase: 24-sulfoximine analogues of the hormone 1α, 25-dihydroxyvitamin D3

A dozen 24-sulfoximine analogues of the hormone 1alpha,25-dihydroxyvitamin D(3) were prepared, differing not only at the stereogenic sulfoximine stereocenter but also at the A-ring. Although these sulfoximines were not active transcriptionally and were only very weakly antiproliferative, some of them are powerful hydroxylase enzyme inhibitors. Specifically, 24-(S)-NH phenyl sulfoximine 3a is an extremely potent CYP24 inhibitor (IC(50) = 7.4 nM) having low calcemic activity. In addition, this compound shows high selectivity toward the CYP24 enzyme in comparison to CYP27A1 (IC(50) > 1000 nM) and CYP27B (IC(50) = 554 nM).

Conceptually new deltanoids (vitamin D analogs) inhibit multistage skin tumorigenesis

Development of vitamin D analogs (deltanoids) as chemopreventive agents requires separation of desirable antiproliferative and pro-differentiating activities from the undesirable calcemic activity also found in the hormone calcitriol (1 alpha, 25-dihydroxyvitamin D(3)). Therefore, several conceptually new deltanoids were synthesized with modifications to the 1alpha- and/or 25-hydroxyl groups, positions traditionally considered essential for stimulating biological responses. In this study, 1 beta-hydroxymethyl-3-epi-25-hydroxyvitamin D(3), a non-calcemic CH(2) homolog of the natural hormone with antiproliferative activity in vitro, was ineffective as an inhibitor of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced induction of ornithine decarboxylase activity in mouse epidermis. However, a hybrid analog incorporating not only the calcemia-ablating 1 beta-hydroxymethyl alteration, but potentiating C,D ring 16-unsaturation and side chain 24,24-fluorination and 26, 27-homologation was found to be as effective as calcitriol. Several non-calcemic 24- or 25-t-butyl sulfones, some containing side chain fluorination but all lacking the 25-hydroxyl group, were also shown to be active in this assay. Three sulfones and the 1 beta-hydroxymethyl hybrid were evaluated as inhibitors of multistage carcinogenesis in mouse skin. Female CD-1 mice were initiated with a single dose of 7,12-dimethylbenz[a]anthracene and then promoted twice weekly for 20 weeks with TPA. Deltanoids were applied topically 30 min before TPA. Unlike calcitriol, none of the atypical deltanoids affected body weight gain in these animals. Minimal effects on urinary calcium excretion were observed following chronic treatment with these analogs. All deltanoids inhibited the incidence and multiplicity of papilloma formation, with the hybrid analog showing the greatest efficacy. With this deltanoid, tumor incidence was significantly reduced by 28% and tumor multiplicity by 63%. These results, coupled with the rich chemical diversity available in side chain sulfur-containing deltanoids, particularly when combined with A ring modifications such as 1 beta-hydroxylalkyl groups, provide important new advances in the fundamental understanding of chemical structure-biological activity relationships as well as more potent and safe vitamin D analogs for cancer chemoprevention and other medicinal uses.

Trioxane dimers have potent antimalarial, antiproliferative and antitumor activities in vitro

A series of tetracyclic and tricyclic trioxane dimers has been prepared with ether and ester tethers of varying length and flexibility. Several of these trioxane dimers have been found to have potent and potentially therapeutically valuable antimalarial, antiproliferative, and antitumor activities in vitro.

Potent, low-calcemic, selective inhibitors of CYP24 hydroxylase: 24-sulfone analogs of the hormone 1α,25-dihydroxyvitamin D3

The new 24-phenylsulfone 4a, a low-calcemic analog of the natural hormone 1alpha,25-dihydroxyvitamin D(3), is a potent (IC(50) = 28nM) and highly selective inhibitor of the human 24-hydroxylase enzyme CYP24.