Investigation into the use of histone deacetylase inhibitor MS-275 as a topical agent for the prevention and treatment of cutaneous squamous cell carcinoma in an SKH-1 hairless mouse model.

Cutaneous squamous cell carcinomas are a common form of highly mutated keratinocyte skin cancers that are of particular concern in immunocompromised patients. Here we report on the efficacy of topically applied MS-275, a clinically used histone deacetylase inhibitor, for the treatment and management of this disease. At 2 mg/kg, MS-275 significantly decreased tumor burden in an SKH-1 hairless mouse model of UVB radiation-induced skin carcinogenesis. MS-275 was cell permeable as a topical formulation and induced histone acetylation changes in mouse tumor tissue. MS-275 was also effective at inhibiting the proliferation of patient derived cutaneous squamous cell carcinoma lines and was particularly potent toward cells isolated from a regional metastasis on an immunocompromised individual. Our findings support the use of alternative routes of administration for histone deacetylase inhibitors in the treatment of high-risk squamous cell carcinoma which may ultimately lead to more precise delivery and reduced systemic toxicity.

Sulforaphane Bioavailability from Glucoraphanin-Rich Broccoli: Control by Active Endogenous Myrosinase.

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 ß-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).

Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.

INTRODUCTION: Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (ß-thioglucoside N-hydroxysulphate) precursors. OBJECTIVE: To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. METHODS: A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). RESULTS: Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. CONCLUSION: Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable.

Protection of humans by plant glucosinolates: efficiency of conversion of glucosinolates to isothiocyanates by the gastrointestinal microflora.

Plant-based diets rich in crucifers are effective in preventing cancer and other chronic diseases. Crucifers contain very high concentrations of glucosinolates (GS; ß-thioglucoside-N-hydroxysulfates). Although not themselves protective, GS are converted by coexisting myrosinases to bitter isothiocyanates (ITC) which defend plants against predators. Coincidentally, ITC also induce mammalian genes that regulate defenses against oxidative stress, inflammation, and DNA-damaging electrophiles. Consequently, the efficiency of conversion of GS to ITC may be critical in controlling the health-promoting benefits of crucifers. If myrosinase is heat-inactivated by cooking, the gastrointestinal microflora converts GS to ITC, a process abolished by enteric antibiotics and bowel cleansing. When single oral doses of GS were administered as broccoli sprout extracts (BSE) to two dissimilar populations (rural Han Chinese and racially mixed Baltimoreans) patterns of excretions of urinary dithiocarbamates (DTC) were very similar. Individual conversions in both populations varied enormously, from about 1% to more than 40% of dose. In contrast, administration of ITC (largely sulforaphane)-containing BSE resulted in uniformly high (70%-90%) conversions to urinary DTC. Despite the remarkably large range of conversion efficiencies between individuals, repeated determinations within individuals were much more consistent. The rates of urinary excretion (slow or fast) were unrelated to the ultimate magnitudes (low or high) of these conversions. Although no demographic factors affecting conversion efficiency have been identified, there are clearly diurnal variations: conversion of GS to DTC was greater during the day, but conversion of ITC to DTC was more efficient at night.

Inactivation of tautomerase activity of macrophage migration inhibitory factor by sulforaphane: a potential biomarker for anti-inflammatory intervention.

BACKGROUND: Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine with keto-enol tautomerase activity, rises rapidly in response to inflammation and is elevated in many chronic diseases. Isothiocyanates, such as sulforaphane from broccoli, are very potent inactivators of MIF tautomerase activity. A simple rapid method for determining this activity in tissues and body fluids may therefore be valuable for assessing severity of inflammation and efficacy of intervention. METHODS: Existing spectrophotometric assays of MIF, based on conversion of methyl L-dopachrome to methyl 5,6-dihydroxyindole-2-carboxylate and associated loss of absorption at 475 nm, lack sensitivity. Assay sensitivity and efficiency were markedly improved by reducing the nonenzymatic rate, by lowering pH to 6.2, replacing phosphate (which catalyzes the reaction) with Bis-Tris buffer, and converting to a microtiter plate format. RESULTS: A structure-potency study of MIF tautomerase inactivation by isothiocyanates showed that sulforaphane, benzyl, n-hexyl, and phenethyl isothiocyanates were especially potent. MIF tautomerase could be readily quantified in human urine concentrated by ultrafiltration. This activity comprised: (i) a heat-labile, sulforaphane-inactivated macromolecular fraction (presumably MIF) that was concentrated during ultrafiltration; (ii) a flow-through fraction, with constant activity during filtration, that was heat stable and insensitive to sulforaphane. Administration of the sulforaphane precursor glucoraphanin to human volunteers almost completely abolished urinary tautomerase activity, which recovered over many hours. CONCLUSION: A simple, rapid, quantitative MIF tautomerase assay has been developed as a potential biomarker for assessing inflammatory severity and effectiveness of intervention. IMPACT: An improved assay for measuring MIF tautomerase activity and its applications are described.

An exceptionally potent inducer of cytoprotective enzymes: elucidation of the structural features that determine inducer potency and reactivity with Keap1.

The Keap1/Nrf2/ARE pathway controls a network of cytoprotective genes that defend against the damaging effects of oxidative and electrophilic stress, and inflammation. Induction of this pathway is a highly effective strategy in combating the risk of cancer and chronic degenerative diseases, including atherosclerosis and neurodegeneration. An acetylenic tricyclic bis(cyano enone) bearing two highly electrophilic Michael acceptors is an extremely potent inducer in cells and in vivo. We demonstrate spectroscopically that both cyano enone functions of the tricyclic molecule react with cysteine residues of Keap1 and activate transcription of cytoprotective genes. Novel monocyclic cyano enones, representing fragments of rings A and C of the tricyclic compound, reveal that the contribution to inducer potency of the ring C Michael acceptor is much greater than that of ring A, and that potency is further enhanced by spatial proximity of an acetylenic function. Critically, the simultaneous presence of two cyano enone functions in rings A and C within a rigid three-ring system results in exceptionally high inducer potency. Detailed understanding of the structural elements that contribute to the reactivity with the protein sensor Keap1 and to high potency of induction is essential for the development of specific and selective lead compounds as clinically relevant chemoprotective agents.

Safety, tolerance, and metabolism of broccoli sprout glucosinolates and isothiocyanates: a clinical phase I study.

Broccoli sprouts are widely consumed in many parts of the world. There have been no reported concerns with respect to their tolerance and safety in humans. A formal phase I study of safety, tolerance, and pharmacokinetics appeared justified because these sprouts are being used as vehicles for the delivery of the glucosinolate glucoraphanin and its cognate isothiocyanate sulforaphane [1-isothiocyanato-(4R)-(methylsulfinyl)butane] in clinical trials. Such trials have been designed to evaluate protective efficacy against development of neoplastic and other diseases. A placebo-controlled, double-blind, randomized clinical study of sprout extracts containing either glucosinolates (principally glucoraphanin, the precursor of sulforaphane) or isothiocyanates (principally sulforaphane) was conducted on healthy volunteers who were in-patients on our clinical research unit. The subjects were studied in three cohorts, each comprising three treated individuals and one placebo recipient. Following a 5-day acclimatization period on a crucifer-free diet, the broccoli sprout extracts were administered orally at 8-h intervals for 7 days (21 doses), and the subjects were monitored during this period and for 3 days after the last treatment. Doses were 25 micromol of glucosinolate (cohort A), 100 micromol of glucosinolate (cohort B), or 25 micromol of isothiocyanate (cohort C). The mean cumulative excretion of dithiocarbamates as a fraction of dose was very similar in cohorts A and B (17.8 +/- 8.6% and 19.6 +/- 11.7% of dose, respectively) and very much higher and more consistent in cohort C (70.6 +/- 2.0% of dose). Thirty-two types of hematology or chemistry tests were done before, during, and after the treatment period. Indicators of liver (transaminases) and thyroid [thyroid-stimulating hormone, total triiodothyronine (T3), and free thyroxine (T4)] function were examined in detail. No significant or consistent subjective or objective abnormal events (toxicities) associated with any of the sprout extract ingestions were observed.

Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein.

Induction of the phase 2 response, a major cellular reaction to oxidative/electrophile stress depends on a protein triad: actin-tethered Keap1 that binds to Nrf2. Inducers react with Keap1 releasing Nrf2 for nuclear translocation and activation of the antioxidant response element (ARE), which regulates phase 2 genes. The primary sensors for inducers are certain uniquely reactive cysteine thiols of Keap1. Recombinant murine Keap1 contains 0.9 zinc atoms per monomer as determined by inductively coupled plasma-optical emission spectrometry: its zinc content depends on the metal composition of the overexpression medium. Simultaneous direct measurement of bound zinc using a pyridazoresorcinol chelator and protein thiol groups using 4,4'-dipyridyl disulfide has established that (i) zinc is bound to reactive cysteine thiols of Keap1 and is displaced stoichiometrically by inducers, (ii) with these cysteines mutated to alanine, the affinity for zinc is reduced by nearly 2 orders of magnitude, and (iii) the association constant of Keap1 for zinc is 1.02 (+/-0.19) x 10(11) M(-)(1), consistent with a Zn(2+) metalloprotein. Co(2+) substitution for Zn(2+) yields an optical spectrum consistent with tetrahedral metal coordination. Coincident binding of inducers and release of zinc alters the conformation of Keap1, as shown by a profound decline of its tryptophan fluorescence and depression of fluorescence of a hydrophobicity probe. Thus, regulation of the phase 2 response involves chemical modification of critical cysteine residues of Keap1, whose reactivity is modulated by zinc binding. Keap1 is a zinc-thiol protein endowed with a delicate switch controlled by both metal-binding and thiol reactivity.

Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress.

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma 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 IC(50) for 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 (r(2) = 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.

Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.

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.

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.

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 [(3)H]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 (C(257), C(273), C(288), and C(297)) 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.