Search

Sofalcone is a synthetic chalcone being used as a gastric mucosa protective agent in Japan. Sofalcone contains a 1,3-diaryl-2-propen-1-one moiety, which is a common chemical scaffold in naturally occurring chalcones. The α,β-unsaturated carbonyl group (Michael reaction acceptor) has electrophilic properties. We investigated the biochemical mechanisms by which sofalcone activated the cytoprotective and anti-inflammatory nuclear factor-erythroid 2 (NF-E2) p45-related factor 2 (Nrf2)-heme oxygenase (HO)-1 pathway. Furthermore, we investigated whether the activation of this pathway was involved in sofalcone -mediated protective effects in an experimental colitis model. Sofalcone induced HO-1 protein expression, which was dependent on increased nuclear accumulation of Nrf2 in human colon carcinoma cells. In addition, Sofalcone reacted with nucleophilic thiol compounds to form Michael adducts. A reduced form of sofalcone (SFCR) in which the Michael reaction acceptor was deactivated, did not exert biological or chemical activity. Biotin-tagged sofalcone bound to Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor of Nrf2. This binding was prevented by pretreatment with sofalcone and a thiol compound but not with SFCR. Furthermore, sofalcone treatment induced dissociation of the Nrf2-KEAP1 complex. Rectal administration of sofalcone alleviated colon damage and inflammation and increased colon nuclear accumulation of Nrf2 and HO-1 levels in a dinitrobenzene sulfonic acid-induced rat colitis model. The protective effects of sofalcone against colon damage and inflammation were significantly inhibited by co-administration of an HO-1 inhibitor. In conclusion, sofalcone activated the Nrf2-HO-1 pathway by covalently binding to KEAP1 via Michael addition, and may confer anti-colitic effects by inducing Nrf2 activation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds have become a significant clinical issue worldwide. Recently, nitric oxide (NO) has emerged as a potent antibacterial agent against MRSA infections and a wound-healing enhancer. Nevertheless, clinical applications of NO have been largely restricted by its gaseous state and short half-life. In this study, our aim was to develop S-nitrosoglutathione (GSNO, an endogenous NO donor)-loaded poly(lactic-co-glycolic acid) [PLGA] microparticles (GSNO-MPs) that release NO over a prolonged period, to accelerate the healing of MRSA-infected wounds with less frequent dosing. GSNO was successfully encapsulated into PLGA microparticles by a solid-in-oil-in-water emulsion solvent evaporation method. Scanning electron microscopy and X-ray diffraction analyses confirmed the successful fabrication of GSNO-MPs. The latter released NO in a prolonged manner over 7 days and exerted a remarkable antibacterial activity against MRSA in a concentration- and time-dependent manner. Moreover, GSNO-MPs had good antibacterial efficacy and were found to accelerate wound healing in a mouse model of MRSA-infected wounds. Therefore, NO-releasing MPs devised in this study may be a promising option for the treatment of cutaneous wounds infected by drug-resistant bacteria such as MRSA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bilirubin (BR) is generated by the reduction of biliverdin (BV), a metabolite that results from the catalytic degradation of heme by the isoforms of heme oxygenase (HO). BV is nontoxic and water-soluble but BR is potentially toxic and lipophilic. Therefore, a further metabolic step is required for BR before excretion is possible. The reductive conversion of BV to BR costs energy and is evolutionarily conserved in human physiology. There must be a compelling reason for this apparently nonsensical evolutionary conservation. In addition to the differences between BR and BV-such as water solubility, antioxidant activity, and participation as a receptor ligand-in the present study, we focused on the chemistry of the two metabolites with regard to an electrophilic functional group called a Michael reaction acceptor (MRA). Our data reveal that the BR reacts with thiol compounds forming adducts, whereas no reaction occurs with BV. Furthermore, the binding of biotin-tagged BR to Kelch-like ECH-associated protein 1 (KEAP1)-a biological electrophile sensor-was prevented by pretreatment with BR or a thiol compound, but was not by pretreatment with BV. In cells, BR could bind to KEAP1 to release and activate nuclear factor-erythroid 2 (NF-E2) p45-related factor 2, a cytoprotective transcription factor, leading to the induction of HO-1. These findings may provide a physiological rationale for the energy-consuming conversion of BV to BR., (Copyright © 2018. Published by Elsevier Inc.)

The biochemical characterization of virulence factors from entomopathogenic bacteria is important to understand entomopathogen-insect molecular interactions. Pseudomonas entomophila is a typical entomopathogenic bacterium that harbors virulence factors against several insects. However, the molecular actions of these factors against host innate immune responses are not clearly elucidated. In this study, we observed that bean bugs (Riptortus pedestris) that were injected with P. entomophila were highly susceptible to this bacterium. To determine how P. entomophila counteracts the host innate immunity to survive within the insect, we purified a highly enriched protein with potential host insect-killing activity from the culture supernatant of P. entomophila. Then, a 45-kDa protein was purified to homogeneity and identified as AprA which is an alkaline zinc metalloprotease of the genus Pseudomonas by liquid chromatography mass spectrometry (LC-MS). Purified AprA showed a pronounced killing effect against host insects and suppressed both host cellular and humoral innate immunity. Furthermore, to show that AprA is an important insecticidal protein of P. entomophila, we used an aprA-deficient P. entomophila mutant strain (ΔaprA). When ΔaprA mutant cells were injected to host insects, this mutant exhibited extremely attenuated virulence. In addition, the cytotoxicity against host hemocytes and the antimicrobial peptide-degrading ability of the ΔaprA mutant were greatly decreased. These findings suggest that AprA functions as an important insecticidal protein of P. entomophila via suppression of host cellular and humoral innate immune responses., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mesalazine (5-aminosalicylic acid, 5-ASA), a currently used drug for anti-inflammatory bowel disease, is easily oxidized by HOCl, a strong oxidant generated in gut inflammation, to produce electrophilic quinones. We investigated whether this chemical feature has an implication in the anti-inflammatory pharmacology of 5-ASA. Human colon carcinoma HCT116 cells were treated with HOCl-reacted 5-ASA. Oxidized 5-ASA activated Nrf2 while 5-ASA itself was not effective. Activation of Nrf2 led to induction of hemeoxygenase (OH)-1, an anti-inflammatory enzyme. Western blot analysis of Keap1, a cytosolic repressor of Nrf2, following precipitation of biotin-labeled proteins in cell lysates treated with biotin-tagged 5-ASA, revealed a much greater amount of Keap1 when biotin-tagged 5-ASA was oxidized with HOCl. Precipitation of Keap1 was attenuated markedly by pretreatment with oxidized 5-ASA or a sulfhydryl donor. In addition, treatment with oxidized 5-ASA in cell lysates reduced the Keap1 amount that coimmunoprecipitated with Nrf2. In parallel, rectal administration of 5-ASA increased the level of HO-1 and nuclear Nrf2 in the inflamed colonic tissues, but not in normal colonic tissues. Moreover, oral gavage of sulfasalazine, a colon-specific prodrug of 5-ASA currently used clinically, activated the Nrf2-HO-1 pathway in the colonic tissues where inflammation was in progress, which was not observed when inflammation subsided. Collectively, our data suggest that Nrf2-HO-1 pathway is involved in the anti-inflammatory pharmacology of 5-ASA, which was likely being exerted exclusively in the inflammatory state., (Copyright © 2017. Published by Elsevier Inc.)

Because gut symbiotic bacteria affect host biology, host insects are expected to evolve some mechanisms for regulating symbiont population. The bean bug, Riptortus pedestris, harbors the Burkholderia genus as a gut symbiont in the midgut organ, designated as the M4 region. Recently, we demonstrated that the lysate of M4B, the region adjacent to M4, harbors potent antibacterial activity against symbiotic Burkholderia but not to cultured Burkholderia. However, the bona fide substance responsible for observed antibacterial activity was not identified in the previous study. Here, we report that cathepsin-L-like protease purified from the lysate of M4B showed strong antibacterial activity against symbiotic Burkholderia but not the cultured Burkholderia. To further confirm this activity, recombinant cathepsin-L-like protease expressed in Escherichia coli also showed antibacterial activity against symbiotic Burkholderia. These results suggest that cathepsin-L-like protease purified from the M4B region plays a critical role in controlling the population of the Burkholderia gut symbiont., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization-Pluronic F127 and chitosan-for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (∼268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC50 values of NPs-P/C1 were 2.03 and 0.67 μm, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step-fabricated, dual-surface-functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy., (Published by Elsevier Ltd.)

Inhibition of hypoxia inducible factor-prolyl hydroxylase-2 (HPH), leading to activation of hypoxia inducible factor (HIF)-1 is a potential therapeutic strategy for the treatment of colitis. Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid is a naturally occurring polyphenolic compound with two catechols, a or inhibition of HPH. To improve accessibility of highly hydrophilic RA to HPH, an intracellular target, RA was chemically modified to decrease hydrophilicity. Of the less-hydrophilic derivatives, rosmarinic acid methyl ester (RAME) most potently inhibited HPH. Accordingly, RAME prevented hydroxylation of HIF-1α and consequently stabilized HIF-1α protein in cells. RAME inhibition of HPH and induction of HIF-1α were diminished by elevated doses of the required factors of HPH, 2-ketoglutarate and ascorbate. RAME induction of HIF-1α led to activation of an ulcer healing pathway, HIF-1-vascular endothelial growth factor (VEGF), in human colon carcinoma cells. RAME administered rectally ameliorated TNBS-induced rat colitis and substantially decreased the levels of pro-inflammatory mediators in the inflamed colonic tissue. In parallel with the cellular effects of RAME, RAME up-regulated HIF-1α and VEGF in the inflamed colonic tissue. Thus, lipophilic modification of RA improves its ability to inhibit HPH, leading to activation of the HIF-1-VEGF pathway. RAME, a lipophilic RA derivative, may exert anti-colitic effects via activation of the ulcer healing pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The clinical usefulness of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, for treatment of inflammatory bowel disease (IBD) is controversial in terms of efficacy and toxicity. To overcome these problems, colon-specific drug delivery was adopted, which generally confers therapeutic and toxicological advantages of drugs for treatment of colonic diseases. N-succinylaspart-1-yl celecoxib (SA1C), a colon-specific prodrug of celecoxib, was administered orally to rats with experimental colitis, and the anti-colitic effects and a molecular mechanism were investigated and compared to those of conventional celecoxib. SA1C, which delivered a much greater amount of celecoxib to the inflamed colon, alleviated the colonic injury, lowered myeloperoxidase activity in the inflamed colonic tissues and was much more effective than conventional celecoxib. SA1C but not conventional celecoxib significantly attenuated expression of NFκB target gene products in the inflamed tissues. Consistent with this, SA1C effectively prevented nuclear accumulation of p65 in the inflamed tissues. Moreover, while conventional celecoxib lowered the serum level of 6-keto-PGF1α, an inverse indicator of cardiovascular toxicity, SA1C did not change its serum level. Our data suggest that colonic delivery of celecoxib is a feasible strategy for treatment of IBD with improved therapeutic and toxicological properties.

Caffeic acid phenethyl ester (CAPE) is a polyphenolic natural product that possesses numerous biological activities including anti-inflammatory effects. CAPE-mediated nuclear factor-erythroid 2 p45 (NF-E2)-related factor 2 (Nrf2) activation is likely responsible for some of its biological effects. CAPE was chemically modified to yield CAPE analogues that were subjected to experiments examining cellular Nrf2 activity. CAPE and the CAPE analogue with a catechol moiety, but not the other analogues, activated the Nrf2 pathway. In addition, only biotin-labeled CAPE analogues with the catechol moiety precipitated Kelch-like ECH associated protein 1 (Keap1) when incubated with cell lysates and streptavidin agarose beads. Sodium hypochlorite (NaOCl) oxidation of the catechol moiety in CAPE produced an oxidized, electrophilic form of CAPE (Oxi-CAPE) and greatly enhanced the ability of CAPE to activate Nrf2 and to bind to Keap1. Rectal administration of CAPE ameliorated 2,4,6-trinitrobenzene sulfonic acid-induced rat colitis and activated the Nrf2 pathway in the inflamed colon, and incubation of CAPE in the lumen of the inflamed distal colon generated Oxi-CAPE. However, these biological effects and chemical change of CAPE were not observed in the normal colon. Our data suggest that CAPE requires the catechol moiety for the oxidation-enhanced activation of the Nrf2 pathway and has potential as a pathologically targeted Nrf2-activating agent that is exclusively activated in pathological states with oxidative stress such as colonic inflammation., (© 2013 Elsevier Inc. All rights reserved.)

To investigate the mechanisms underlying the biological activity of piceatannol (PCT), a hydroxystilbene natural product that has anti-colitic properties, we examined whether PCT could modulate hypoxia-inducible factor (HIF)-1 activity in human colon carcinoma cells. PCT induced HIF-1α protein, leading to induction of its target gene products, vascular endothelial growth factor and heme oxygenase-1, which are involved in amelioration of colitis. PCT induction of HIF-1α resulted from HIF-1α protein stabilization, which occurred through inhibition of HIF-prolyl hydroxylase-2 (HPH-2). PCT inhibition of HPH-2 was reversed by addition of ascorbate, a cofactor of HPH-2, but not the cosubstrate, 2-ketoglutarate, to the reaction mixture of an in vitro von Hippel-Lindau (VHL) capture assay, and pretreatment with ascorbate abrogated PCT induction of cellular HIF-1α. Moreover, PCT prevented hydroxylation of cellular HIF-1α and attenuated coimmunoprecipitation of Flag-VHL protein and HA-HIF-1α over-expressed in human embryonic kidney 293 cells. Structural analysis using derivatives of PCT revealed that the catechol moiety in PCT was required for the stabilization of HIF-1α protein. Taken together, PCT activation of HIF-1 resulting from inhibition of HPH-2 may be a molecular mechanism for an anti-colitic effect of the natural product., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Prolyl 4-hydroxylases (P4Hs), such as collagen prolyl-4-hydroxylases (CPHs) and hypoxia inducible factor prolyl-4-hydroxylases (HPHs), have recently been recognized as promising drug targets for the treatment of fibrotic and ischemic diseases. CPHs and HPHs catalyze identical metabolic reactions, yet lead to quite different physiological consequences, collagen synthesis and the regulation of oxygen homeostasis. Selective modulation of the two enzymes should provide a therapeutic benefit upon pharmacotherapy. In an in vitro VHL capture assay, hydroxylation of the 19mer HIF peptide (corresponding to HIF-1α residues 556-574) by HPH-2 was effectively prevented by nitric oxide (NO) donors, (±)-S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione. The NO donors also caused inhibition of HPHs and accumulation of nonhydroxylated HIF-1α protein in A549 human lung adenocarcinoma cells. Hyperoxia (100% O(2)) attenuated both NO donor-induced accumulation of HIF-1α and inhibition of HPH-mediated hydroxylation. In the presence of a proteasome inhibitor, MG132, the hyperoxia-mediated degradation of HIF-1α was deterred and hydroxylated HIF-1α was detected. SNAP, while being an effective inhibitor of proline 4-hydroxylation of HIF-1α by HPH-2, did not diminish proline hydroxylation of collagen by CPHs. Our data suggest that NO inhibits HPH-2 via competing with dioxygen and that the discriminative effect of NO on CPHs and HPH-2 is attributable to the difference in the affinity of the two enzymes toward dioxygen., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated potential molecular mechanisms underlying CAPE-mediated nuclear factor kappa beta (NFkappaB) inhibition and analyzed structure of CAPE for its biological effect. CAPE attenuated expression of NFkappaB dependent luciferase stimulated with TNF-alpha or LPS and suppressed LPS-mediated induction of iNOS, a target gene product of NFkappaB. In HCT116 cells, CAPE interfered with TNF-alpha dependent IkappaBalpha degradation and subsequent nuclear accumulation of p65, which occurred by direct inhibition of inhibitory protein kappaB kinase (IKK). CAPE increased the expression of Nrf2-dependent luciferase and heme oxygenase-1, a target gene of Nrf2, and elevated the nuclear level of Nrf2 protein, indicating that CAPE activated the Nrf2 pathway. In HCT116 cells with stable expression of Nrf2 shRNA, CAPE elicited a reduced inhibitory effect on TNF-alpha-activated NFsmall ka, CyrillicB compared to scramble RNA expressing control cells. On the other hand, the NFkappaB inhibitory effect of CAPE was diminished by removal or modification of the Michael reaction acceptor, catechol or phenethyl moiety in CAPE. These data suggest that CAPE inhibits TNF-alpha-dependent NFkappaB activation via direct inhibition of IKK as well as activation of Nrf2 pathway, in which the functional groups in CAPE may be involved., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated a potential molecular mechanism underlying a CAPE-mediated protective effect against ischemia/reperfusion (I/R) injury and analyzed the structure contributing to the CAPE effect. CAPE induced hypoxia-inducible factor-1 (HIF-1) alpha protein, concomitantly transactivating the HIF-1 target genes vascular endothelial growth factor and heme oxygenase-1, which play a protective role in I/R injury. CAPE delayed the degradation of HIF-1alpha protein in cells, which occurred by inhibition of HIF prolyl hydroxylase (HPH), the key enzyme for von Hippel-Lindau-dependent HIF-1alpha degradation. CAPE inhibition of HPH and induction of HIF-1alpha protein were neutralized by an elevated dose of iron. The catechol moiety, a chelating group, is essential for HPH inhibition, while hydrogenation of the double bond (-C=C-) in the Michael reaction acceptor markedly reduced potency. Removal of the phenethyl moiety of CAPE (substitution with the methyl moiety) severely deteriorated its inhibitory activity for HPH. Our data suggest that a beneficial effect of CAPE on I/R injury may be ascribed to the activation of HIF-1 pathway via inhibition of HPH and reveal that the chelating moiety of CAPE acted as a pharmacophore while the double bond and phenethyl moiety assisted in inhibiting HPH., (Copyright 2010 Elsevier Inc. All rights reserved.)

Previously, we reported that oral administration of taurine conjugated 5-aminosalicylic acid, a colon-specific prodrug of 5-aminosalicylic acid (5-ASA), is effective in ameliorating experimental colitis and taurine elicits an additive anti-inflammatory effect upon cotreatment with 5-ASA. To explore a molecular mechanism for the anti-inflammatory property of the prodrug, we investigated the effect of the conjugate on IL-1beta-mediated NFkappaB activation. In human colon carcinoma Caco-2 and HCT116 cells, NFkappaB activity was accessed by a luciferase reporter assay and IL-6 secretion. Protein levels were determined by Western blotting. IL-6 levels were monitored by an Elisa kit. Treatment with either 5-ASA or taurine chloramine (TauCl) inhibited IL-1beta-mediated NFkappaB dependent luciferase expression and IL-6 secretion. In HCT116 cells, the inhibitory effect by TauCl or 5-ASA was through preventing IL-1beta-induced IkappaB kinase activation and subsequently interfering with IkappaBalpha degradation and p65 nuclear accumulation. Furthermore, combined TauCl/5-ASA treatment interfered additively with the activation process, leading to additive inhibitory effect on IL-1beta-mediated NFkappaB activation. Our results suggest that the anti-inflammatory effect of the prodrug on experimental colitis is attributed to the inhibition of the IL-1beta-mediated NFkappaB activation and the taurine effect is through TauCl potentiating the ability of 5-ASA to inhibit IL-1beta dependent NFkappaB activation.