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Exemestane (6-methyleneandrosta-1,4-diene-3,17-dione) is a synthetic steroidal inhibitor of the aromatase reaction that catalyzes the terminal and rate-limiting step of the biosynthesis of estrogens. It is active clinically in preventing, delaying progression of, and treating mammary cancers, many of which are estrogen receptor-positive. A striking feature of the structure of exemestane is an extended system of conjugated Michael reaction functions, which is also characteristic of inducers of a broad network of chemoprotective genes regulated by the Keap1 (Kelch-like ECA-associated protein)/Nrf2 (nuclear factor E2-related factor 2)/ARE (antioxidant response element) signaling system. These genes are largely involved in xenobiotic metabolism and antioxidative and anti-inflammatory protection, as well as the synthesis and reduction of glutathione. We show here that exemestane transcriptionally activates NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1), typical chemoprotective gene products, in a wide variety of mouse, rat, and human cells. It protects several cell lines against oxidative toxicity of tert -butyl hydroperoxide and 4-hydroxynonenal, against free radical damage arising from hypoxia–reoxygenation, and against UVA radiation damage. Exemestane also inhibits the inflammatory increases in inducible nitric oxide synthase (iNOS) in mouse macrophages exposed to LPS (lipopolysaccharide), thereby resembling the isothiocyanate sulforaphane derived from broccoli. Remarkably, combinations of exemestane and sulforaphane act highly synergistically, and this property is also displayed by several other phytochemicals. Thus, exemestane has a wide range of previously unrecognized protective activities, probably unrelated to aromatase inhibition. Its potential for reducing the risk, not only of breast cancer, but also of other chronic diseases that arise from inflammation, oxidative stress, and DNA-damaging electrophiles, requires exploration, particularly in view of the synergism with other phytochemicals.

Significance Autism spectrum disorder (ASD), encompassing impaired communication and social interaction, and repetitive stereotypic behavior and language, affects 1–2% of predominantly male individuals and is an enormous medical and economic problem for which there is no documented, mechanism-based treatment. In a placebo-controlled, randomized, double-blind clinical trial, daily oral administration for 18 wk of the phytochemical sulforaphane (derived from broccoli sprouts) to 29 young men with ASD substantially (and reversibly) improved behavior compared with 15 placebo recipients. Behavior was quantified by both parents/caregivers and physicians by three widely accepted measures. Sulforaphane, which showed negligible toxicity, was selected because it upregulates genes that protect aerobic cells against oxidative stress, inflammation, and DNA-damage, all of which are prominent and possibly mechanistic characteristics of ASD. Autism spectrum disorder (ASD), characterized by both impaired communication and social interaction, and by stereotypic behavior, affects about 1 in 68, predominantly males. The medico-economic burdens of ASD are enormous, and no recognized treatment targets the core features of ASD. In a placebo-controlled, double-blind, randomized trial, young men (aged 13–27) with moderate to severe ASD received the phytochemical sulforaphane ( n = 29)—derived from broccoli sprout extracts—or indistinguishable placebo ( n = 15). The effects on behavior of daily oral doses of sulforaphane (50–150 µmol) for 18 wk, followed by 4 wk without treatment, were quantified by three widely accepted behavioral measures completed by parents/caregivers and physicians: the Aberrant Behavior Checklist (ABC), Social Responsiveness Scale (SRS), and Clinical Global Impression Improvement Scale (CGI-I). Initial scores for ABC and SRS were closely matched for participants assigned to placebo and sulforaphane. After 18 wk, participants receiving placebo experienced minimal change (<3.3%), whereas those receiving sulforaphane showed substantial declines (improvement of behavior): 34% for ABC ( P < 0.001, comparing treatments) and 17% for SRS scores ( P = 0.017). On CGI-I, a significantly greater number of participants receiving sulforaphane had improvement in social interaction, abnormal behavior, and verbal communication ( P = 0.015–0.007). Upon discontinuation of sulforaphane, total scores on all scales rose toward pretreatment levels. Dietary sulforaphane, of recognized low toxicity, was selected for its capacity to reverse abnormalities that have been associated with ASD, including oxidative stress and lower antioxidant capacity, depressed glutathione synthesis, reduced mitochondrial function and oxidative phosphorylation, increased lipid peroxidation, and neuroinflammmation.

Glucoraphanin from broccoli and its sprouts and seeds is a water soluble and relatively inert precursor of sulforaphane, the reactive isothiocyanate that potently inhibits neoplastic cellular processes and prevents a number of disease states. Sulforaphane is difficult to deliver in an enriched and stable form for purposes of direct human consumption. We have focused upon evaluating the bioavailability of sulforaphane, either by direct administration of glucoraphanin (a glucosinolate, or β-thioglucoside-N-hydroxysulfate), or by co-administering glucoraphanin and the enzyme myrosinase to catalyze its conversion to sulforaphane at economic, reproducible and sustainable yields. We show that following administration of glucoraphanin in a commercially prepared dietary supplement to a small number of human volunteers, the volunteers had equivalent output of sulforaphane metabolites in their urine to that which they produced when given an equimolar dose of glucoraphanin in a simple boiled and lyophilized extract of broccoli sprouts. Furthermore, when either broccoli sprouts or seeds are administered directly to subjects without prior extraction and consequent inactivation of endogenous myrosinase, regardless of the delivery matrix or dose, the sulforaphane in those preparations is 3- to 4-fold more bioavailable than sulforaphane from glucoraphanin delivered without active plant myrosinase. These data expand upon earlier reports of inter- and intra-individual variability, when glucoraphanin was delivered in either teas, juices, or gelatin capsules, and they confirm that a variety of delivery matrices may be equally suitable for glucoraphanin supplementation (e.g. fruit juices, water, or various types of capsules and tablets).

The retinal pigment epithelial cell (RPE cell) layer protects the photoreceptors of the retina against oxidative stress. The decline of this capacity is believed to be a major factor in the impairment of vision in age-related macular degeneration. Exposure of human adult RPE cells to UV light at predominantly 320-400 nm (UVA light) in the presence of all-trans-retinaldehyde results in photooxidative cytotoxicity. Significant protection of RPE cells was obtained by prior treatment with phase 2 gene inducers, such as the isothiocyanate sulforaphane or a bis-2-hydroxybenzylideneacetone Michael reaction acceptor. The degree of protection was correlated with the potencies of these inducers in elevating cyto-protective glutathione levels and activities of NAD(P)H:quinone oxidoreductase. In embryonic fibroblasts derived from mice in which the genes for the transcription factor Nrf2, the repressor Keap1, or both Nrf2 and Keap1 were disrupted, the magnitude of resistance to photooxidative damage paralleled the basal levels of glutathione and NAD(P)H:quinone oxidoreductase in each cell type. Demonstration of protection of RPE cells against photooxidative damage by induction of phase 2 proteins may shed light on the role of oxidative injury in ocular disease. Moreover, the finding that dietary inducers provide indirect antioxidant protection suggests novel strategies for preventing chronic degenerative diseases, such as age-related macular degeneration.

Background It is widely recognized that deep inspiration (DI), either before methacholine (MCh) challenge (Bronchoprotection, BP) or after MCh challenge (Bronchodilation, BD) protects against this challenge in healthy individuals, but not in asthmatics. Sulforaphane, a dietary antioxidant and antiinflammatory phytochemical derived from broccoli, may affect the pulmonary bronchoconstrictor responses to MCh and the responses to DI in asthmatic patients. Methods Forty-five moderate asthmatics were administered sulforaphane (100 μmol daily for 14 days), BP, BD, lung volumes by body-plethsmography, and airway morphology by computed tomography (CT) were measured pre- and post sulforaphane consumption. Results Sulforaphane ameliorated the bronchoconstrictor effects of MCh on FEV 1 significantly (on average by 21 %; p = 0.01) in 60 % of these asthmatics. Interestingly, in 20 % of the asthmatics, sulforaphane aggravated the bronchoconstrictor effects of MCh and in a similar number was without effect, documenting the great heterogeneity of the responsiveness of these individuals to sulforaphane. Moreover, in individuals in whom the FEV 1 response to MCh challenge decreased after sulforaphane administration, i.e., sulforaphane was protective, the activities of Nrf2-regulated antioxidant and anti-inflammatory genes decreased. In contrast, individuals in whom sulforaphane treatment enhanced the FEV 1 response to MCh, had increased expression of the activities of these genes. High resolution CT scans disclosed that in asthmatics sulforaphane treatment resulted in a significant reduction in specific airway resistance and also increased small airway luminal area and airway trapping modestly but significantly. Conclusion These findings suggest the potential value of blocking the bronchoconstrictor hyperresponsiveness in some types of asthmatics by phytochemicals such as sulforaphane.

An elaborate network of highly inducible phase 2 proteins protects aerobic cells against the cumulative damaging effects of reactive oxygen intermediates and toxic electrophiles, which are the major causes of malignancy and chronic degenerative diseases. Many chemical and phytochemical agents, all of which react with thiol groups, induce the phase 2 response through their reactivity with critical cysteine thiols of Keap1. We recently found that the anti-inflammatory potencies (suppression of iNOS and COX-2 expression) of a series of triterpenoids with Michael reaction centers were closely correlated with the potencies of these agents to induce the phase 2 response. We now report that representatives of seven recognized chemical classes of inducers, including isothiocyanates, bisbenzylidenes, arsenicals, heavy metals, and vicinal dithiols, showed highly correlated inducer and anti-inflammatory potencies spanning more than six orders of magnitude of concentrations in established cells and in primary mouse peritoneal macrophages. Potency measurements were expressed as the Dm values (median effect concentration) by use of the Median Effect Equation. Whereas the phase 2 induction required the functional integrity of both the repressor Keap1 and the transcription factor Nrf2, the effectiveness of inducers in blocking the up-regulation of iNOS by inflammatory cytokines was related to the nature of the cytokine and the inducer concentration. These studies identify suppression of inflammation as a consistent property of inducers of the phase 2 response and strongly suggest that this property is a central aspect of their chemoprotective actions.

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

Induction of a family of phase 2 genes encoding for proteins that protect against the damage of electrophiles and reactive oxygen intermediates is potentially a major strategy for reducing the risk of cancer and chronic degenerative diseases. Many phase 2 genes are regulated by upstream antioxidant response elements (ARE) that are targets of the leucine zipper transcription factor Nrf2. Under basal conditions, Nrf2 resides mainly in the cytoplasm bound to its cysteine-rich, Kelch domain-containing partner Keap1, which is itself anchored to the actin cytoskeleton and represses Nrf2 activity. Inducers disrupt the Keap1-Nrf2 complex by modifying two (C273 and C288) of the 25 cysteine residues of Keap1. The critical role of C273 and C288 was established by (i) their high reactivity when purified recombinant Keap1 was treated with dexamethasone mesylate and the dexamethasone-modified tryptic peptides were analyzed by mass spectrometry, and (ii) transfection of keap1 and nrf2 gene-deficient mouse embryonic fibroblasts with constructs expressing cysteine to alanine mutants of Keap1, and measurement of the ability of cotransfected Nrf2 to repress an ARE-luciferase reporter. Reaction of Keap1 with inducers results in formation of intermolecular disulfide bridges, probably between C273 of one Keap1 molecule and C288 of a second. Evidence for formation of such dimers was obtained by 2D PAGE of extracts of cells treated with inducers, and by the demonstration that whereas C273A and C288A mutants of Keap1 alone could not repress Nrf2 activation of the ARE-luciferase reporter, an equal mixture of these mutant constructs restored repressor activity.

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated. Here we designed sulforaphane analogs with replacement of the reactive isothiocyanate by the more gentle electrophilic sulfoxythiocarbamate group that also selectively targets cysteine residues in proteins but forms stable thiocarbamate adducts. Twenty-four sulfoxythiocarbamate analogs were synthesized that retain the structural features important for high potency in sulforaphane analogs: the sulfoxide or keto group and its appropriate distance to electrophilic functional group. Evaluation in various cell lines including hepatoma cells, retinal pigment epithelial cells, and keratinocytes as well as in mouse skin shows that these analogs maintain high potency and efficacy for phase 2 enzyme induction as well as the inhibitory effect on lipopolysaccharide-induced nitric oxide formation like sulforaphane. We further show in living cells that a sulfoxythiocarbamate analog can label Keap1 on several key cysteine residues as well as other cellular proteins offering new insights into the mechanism of chemoprotection.

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-γ of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2-/-and keap1-/-mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC50for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r2=0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.