Sungchae Hong

Caffeic acid phenethyl ester-mediated Nrf2 activation and IκB kinase inhibition are involved in NFκB inhibitory effect: Structural analysis for NFκB inhibition

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.

Colon-targeted cell-permeable NFκB inhibitory peptide is orally active against experimental colitis.

For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.

Caffeic acid phenethyl ester is a potent inhibitor of HIF prolyl hydroxylase: structural analysis and pharmacological implication

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.

Caffeic acid phenethyl ester activation of Nrf2 pathway is enhanced under oxidative state: Structural analysis and potential as a pathologically targeted therapeutic agent in treatment of colonic inflammation

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.

An anti-inflammatory mechanism of taurine conjugated 5-aminosalicylic acid against experimental colitis: Taurine chloramine potentiates inhibitory effect of 5-aminosalicylic acid on IL-1β-mediated NFκB activation

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.

Sulfate-conjugated methylprednisolone as a colon-targeted methylprednisolone prodrug with improved therapeutic properties against rat colitis.

Methylprednisolone (MP) is one of the most widely used corticosteroids for the treatment of inflammatory bowel disease (IBD). However, systemic adverse effects of MP limit its availability for the disease. In present study, sulfate-conjugated methylprednisolone (MPS) was evaluated in vivo as a colon-targeted prodrug of MP and its therapeutic properties against 2,4,6-trinitrobenzenesulfonic acid–induced rat colitis were investigated. Upon oral administration, a large fraction of MPS reached the large intestine, where MPS was converted to MP implying that MPS would deliver MP effectively to the large intestine. The fecal recovery of MP (after MPS administration) was much greater than that after MP administration and the urinary recovery of MP (after MPS administration) was much less than that after MP administration, suggesting that MPS should exhibit enhanced therapeutic activity and reduced systemic adverse effects. Consistent with this notion, MPS was more effective than MP in ameliorating rat colitis. Moreover, the adverse effects of MPS on adrenal function and thymus were much lower than those of MP. Taken together, MPS may be therapeutically superior to MP in IBD treatment.

Piceatannol, a hydroxystilbene natural product, stabilizes HIF-1α protein by inhibiting HIF prolyl hydroxylase

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.

Manganese (II) induces chemical hypoxia by inhibiting HIF-prolyl hydroxylase: implication in manganese-induced pulmonary inflammation.

Manganese (II), a transition metal, causes pulmonary inflammation upon environmental or occupational inhalation in excess. We investigated a potential molecular mechanism underlying manganese-induced pulmonary inflammation. Manganese (II) delayed HIF-1alpha protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1alpha hydroxylation and subsequent von Hippel-Lindau(VHL)-dependent HIF-1alpha degradation. HPH inhibition by manganese (II) was neutralized significantly by elevated dose of iron. Consistent with this, the induction of cellular HIF-1alpha protein by manganese (II) was abolished by pretreatment with iron. Manganese (II) induced the HIF-1 target gene involved in pulmonary inflammation, vascular endothelial growth factor (VEGF), in lung carcinoma cell lines.The induction of VEGF was dependent on HIF-1. Manganese-induced VEGF promoted tube formation of HUVEC. Taken together, these data suggest that HIF-1 may be a potential mediator of manganese-induced pulmonary inflammation.

Synthesis and Evaluation of N-Nicotinoyl-2-{2-(2-Methyl-5-Nitroimidazol-1-yl)Ethyloxy}-D,L-Glycine as a Colon-Specific Prodrug of Metronidazole

Metronidazole (MTZ) is a drug of choice for protozoal infections such as luminal amoebiasis. We designed and synthesized N-nicotinoyl-2-{2-(2-methyl-5-nitroimidazol-1-yl)ethyloxy}-D,L-glycine (NMG) as a colon-specific prodrug of MTZ. The synthetic yield of NMG was about 34%. The apparent partition coefficient of MTZ was greatly reduced by the chemical modification. While (bio)chemically stable in the contents of the upper intestine, NMG was rapidly cleaved to liberate MTZ on incubation with the cecal contents of rats. MTZ metabolized quickly in the cecal contents at least partly by a microbial nitroreductase, suggesting that the metabolism of MTZ is relevant to its bioactivation leading to amoebicidal action. The systemic absorption, analyzed by the blood concentration and urinary recovery of NMG, was very low after oral administration of NMG. In parallel with this, whereas MTZ disappeared mostly during the transit of the proximal small intestine, a substantial amount of NMG remained in the small intestine moving down to the large intestine where it metabolized rapidly. Moreover, comparing systemic absorption of MTZ after oral administration of NMG or MTZ, NMG markedly reduced the systemic absorption. These results suggest that NMG is a potential colon-specific prodrug of MTZ which improves therapeutic and toxicological properties.

Lipophilic modification enhances anti-colitic properties of rosmarinic acid by potentiating its HIF-prolyl hydroxylases inhibitory activity

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.