Masafumi Tauchi

Pharmacodynamic characterization of chemopreventive triterpenoids as exceptionally potent inducers of Nrf2-regulated genes

Synthetic triterpenoids have been developed, which are potent inducers of cytoprotective enzymes and inhibitors of inflammation, greatly improving on the weak activity of naturally occurring triterpenoids. An imidazolide triterpenoid derivative, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im or TP235), has been previously shown to potently protect against hepatic tumorigenesis, acting in part by inducing cytoprotective genes through Keap1-Nrf2-antioxidant response element (ARE) signaling. In these studies, the pharmacodynamic activity of CDDO-Im is characterized in two distinct lines of ARE reporter mice and by measuring increases in Nqo1 transcript levels as a marker of cytoprotective gene induction. Oral administration of CDDO-Im induces ARE-regulated cytoprotective genes in many tissues in the mouse, including liver, lung, kidney, intestines, brain, heart, thymus, and salivary gland. CDDO-Im induces Nqo1 RNA transcripts in some organs at doses as low as 0.3 μmol/kg body weight (orally). A structure activity evaluation of 15 additional triterpenoids (a) confirmed the importance of Michael acceptor groups on both the A and C rings, (b) showed the requirement for a nitrile group at C-2 of the A ring, and (c) indicated that substituents at C-17 dramatically affected pharmacodynamic action in vivo. In addition to CDDO-Im, other triterpenoids, particularly the methyl ester CDDO-Me (TP155) and the dinitrile TP225, are extremely potent inducers of cytoprotective genes in mouse liver, lung, small intestine mucosa, and cerebral cortex. This pharmacodynamic characterization highlights the chemopreventive promise of several synthetic triterpenoids in multiple target organs. [Mol Cancer Ther 2007;6(1):154–62]

Dietary Sulforaphane-Rich Broccoli Sprouts Reduce Colonization and Attenuate Gastritis in Helicobacter pylori-Infected Mice and Humans

The isothiocyanate sulforaphane [SF; 1-isothiocyanato-4(R)-methylsulfinylbutane] is abundant in broccoli sprouts in the form of its glucosinolate precursor (glucoraphanin). SF is powerfully bactericidal against Helicobacter pylori infections, which are strongly associated with the worldwide pandemic of gastric cancer. Oral treatment with SF-rich broccoli sprouts of C57BL/6 female mice infected with H. pylori Sydney strain 1 and maintained on a high-salt (7.5% NaCl) diet reduced gastric bacterial colonization, attenuated mucosal expression of tumor necrosis factor-α and interleukin-1β, mitigated corpus inflammation, and prevented expression of high salt-induced gastric corpus atrophy. This therapeutic effect was not observed in mice in which the nrf2 gene was deleted, strongly implicating the important role of Nrf2-dependent antioxidant and anti-inflammatory proteins in SF-dependent protection. Forty-eight H. pylori–infected patients were randomly assigned to feeding of broccoli sprouts (70 g/d; containing 420 μmol of SF precursor) for 8 weeks or to consumption of an equal weight of alfalfa sprouts (not containing SF) as placebo. Intervention with broccoli sprouts, but not with placebo, decreased the levels of urease measured by the urea breath test and H. pylori stool antigen (both biomarkers of H. pylori colonization) and serum pepsinogens I and II (biomarkers of gastric inflammation). Values recovered to their original levels 2 months after treatment was discontinued. Daily intake of sulforaphane-rich broccoli sprouts for 2 months reduces H. pylori colonization in mice and improves the sequelae of infection in infected mice and in humans. This treatment seems to enhance chemoprotection of the gastric mucosa against H. pylori–induced oxidative stress.

Constitutive Expression of Aryl Hydrocarbon Receptor in Keratinocytes Causes Inflammatory Skin Lesions

ABSTRACT Occupational and environmental exposure to polycyclic aromatic hydrocarbons (PAHs) has been suggested to provoke inflammatory and/or allergic disorders, including asthma, rhinitis, and dermatitis. The molecular mechanisms of this PAH-mediated inflammation remain to be clarified. Previous studies implied the involvement of PAHs as irritants and allergens, with the reactive oxygen species generated from the oxygenated PAHs believed to be an exacerbating factor. It is also possible that PAHs contribute to the pathogenesis through activation of aryl-hydrocarbon receptor (AhR)-mediated transcription, since PAHs are potent inducers of the AhR. To address this point, we generated transgenic mouse lines expressing the constitutive active form of the AhR in keratinocytes. In these lines of mice, the AhR activity was constitutively enhanced in the absence of ligands, so that any other direct effects of PAHs and their metabolites could be ignored. At birth, these transgenic mice were normal, but severe skin lesions with itching developed postnatally. The skin lesions were accompanied by inflammation and immunological imbalance and resembled typical atopic dermatitis. We demonstrate that constitutive activation of the AhR pathway causes inflammatory skin lesions and suggests a new mechanism for the exacerbation of inflammatory diseases after exposure to occupational and environmental xenobiotics.

Geranylgeranylacetone Protects the Human Gastric Mucosa from Diclofenac-Induced Injury via Induction of Heat Shock Protein 70

Background/Aim: Geranylgeranylacetone (GGA) enhances gastric mucosal protection against nonsteroidal anti-inflammatory drugs by upregulating mucosal heat shock proteins (HSP), but the effects of GGA on the human gastric mucosa have not been well examined. This study was conducted to determine whether a clinical dose of GGA protects the human gastric mucosa from diclofenac (DIC)-induced gastric mucosal injury. Methods: The study group comprised 40 healthy volunteers: 20 subjects were randomly assigned to take either placebo (lactose 1.5 g/day) or GGA (150 mg/day) for 2 weeks (study 1), and 20 subjects were assigned to take DIC (75 mg/day) plus placebo (lactose 1.5 g/day) or DIC (75 mg/day) plus GGA (150 mg/day) for 2 weeks (study 2). In both studies, gastroscopic biopsy specimens were obtained before and after treatment. Mucosal HSP70 expression and DNA damage were analyzed by measuring the levels of HSP70 and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG), respectively. Results: In study 1, GGA increased the mucosal HSP70 expression without increasing the 8-OHdG production. In study 2, DIC treatment increased the 8-OHdG production, whereas the combination of GGA and DIC enhanced the HSP70 expression and attenuated the increase in 8-OHdG induced by DIC. Conclusion: The clinical dose of GGA enhanced the gastric mucosal HSP70 expression and inhibited the DIC-induced gastric mucosal damage in humans.

Role of the nrf-2 gene in protection and repair of gastric mucosa against oxidative stress

Helicobacter pylori infection, as well as NSAIDs induce oxidative stress on gastric mucosa, thereby causing mucosal damage and retarding mucosal repair. Cells can survive against chronic oxidative stress by enhancing activities of anti-oxidant enzymes, thereby protecting cells from DNA damage. Recent studies have clearly shown that the gene encoding Nrf-2 (NF-E2 p45-related factor-2) plays an important role in the induction of antioxidant enzymes against oxidative stress. In this paper, we will describe the cellular mechanisms by which the nrf-2 gene stimulates anti-oxidant enzyme activities during exposure to oxidative stress. Secondly, we will also mention the beneficial effects of sulforaphane, an isothiocyanate family which is abundantly included in broccoli sprouts, on gastric mucosa. Sulforaphane stimulates nrf-2 gene-dependent anti-oxidant enzyme activities, thereby protecting cells from oxidative injury. Finally, we will state our perspective on the efficacy of sulforaphane in protection and repair of gastric mucosa against oxidative stress during H. pylori infection.

Hyperosmotic stress enhances interleukin-1β expression in Helicobacter pylori-infected murine gastric epithelial cells in vitro

Background and Aim:  Gastric cancer is associated not only with Helicobacter pylori (H. pylori) infection, but also with the intake of a high salt diet. Interleukin‐1β (IL‐1β) is highly expressed in H. pylori‐infected gastric mucosa. The aim of the present study was to determine if hyperosmotic stress induces IL‐1β expression in gastric epithelial cells in vitro.

Daily intake of sulforaphane-rich broccoli sprouts prevents progression of high salt diet-induced gastric atrophy in H. pylori-infected C57/BL6 mice in vivo

5170 Background: Sulforaphane, shown to be abundantly present in broccoli sprouts, possesses anticarcinogenic activity, since it strongly induces nrf-2 (NF-E2 p45-related factor-2) gene-dependant antioxidant (or phase 2) enzymes, such as glutathione-S- transferase (GSH) and NADPH:quinone reductase(QR), which exert as free radical scavengers, thereby protecting cells from oxidative injury (PNAS, 94:10367, 1997). It has been reported that nrf-2 is strongly expressed in gastric mucosa (Cancer Res 61:3299,2001). Recent studies have also shown that sulforaphane has strong bactericidal activity against Helicobacter pylori (Hp) in vitro (PNAS 99:7610, 2002). We have previously shown that high salt diet accelerates progression of gastric atrophy in Hp-infected mice in vivo by enhancing oxidative stress and by exacerbating Hp-induced gastritis. (Gastroenterology 122: A1, 2002). Based on these backgrounds, we examined if 1) sulforaphane inhibits Hp colonization in vivo , and if 2) sulforaphane prevents progression of high salt diet-induced gastric atrophy in Hp-infected gastric mucosa via activation of nrf-2 dependent antioxidant ezymes. Methods: C57BL/6 wild type (nrf-2+/+) or nrf-2 gene kockout (nrf-2-/-) female mice were inoculated with Hp Sydney Strain; SS1. Mice were daily fed with high salt diet (7.5% NaCl), supplemented with or without 2.5 mmol sulforaphane or homogenate of broccoli sprouts, which contains 2.5 mmol sulforaphane. Mice were sacrificed at 8 wks after starting the treatment. Degree of gastritis and atrophy was evaluated by updated Sydney system. DNA damage was estimated by measuring mucosal level of 8-OHdG. Expressions of TNF-a, IL-1b, MIP-2, and COX-2 were analyzed by real time PCR. Results: 1. Treatment with either sulforaphane or broccoli sprouts enhanced activities of GSH and quinone reductase in gastric mucosa in nrf2+/+, but not in nrf-2-/- mice in vivo . 2. Treatment with sulforaphane or broccoli sprouts attenuated mucosal expressions of TNF-a, IL-1b, MIP-2, and COX-2, mitigated gastritis and DNA damage, and prevented progression of high salt diet-induced gastric atrophy in nrf2+/+, but not in nrf-2-/- mice. 3. Treatment with either sulforaphane or broccoli sprouts decreased Hp colonization in gastric mucosa, but failed to induce complete eradication of Hp in both nrf2+/+ nrf-2-/- mice in vivo .Conclusions: Daily intake of sulforaphane-rich broccoli-sprouts attenuates gastritis and prevents progression of gastric atrophy in Hp-infected mice fed with high salt diet, effects appear to be mediated in part by enhancing activities of nrf-2 gene dependent anti-oxidant enzymes against oxidative stress induced by Hp infection, and in part by suppressing Hp colonization.