Atsuko Inoue

Expression of Wnt-5a Is Correlated with Aggressiveness of Gastric Cancer by Stimulating Cell Migration and Invasion

Wnt-5a is a representative ligand that activates a beta-catenin-independent pathway in the Wnt signaling. Although abnormal activation of beta-catenin-dependent pathway is often observed in human cancer, the relationship between beta-catenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.

HMGB1 inhibitor glycyrrhizin attenuates intracerebral hemorrhage-induced injury in rats.

Thrombin activates immunocompetent microglia and increases release of inflammatory cytokines under intracerebral hemorrhage (ICH) insults. Also, thrombin injection into the striatum evokes acute necrosis and delayed apoptosis of neurons. A nucleoprotein high-mobility group box 1 (HMGB1) that is released from necrotic cells has been suggested to behave like a cytokine and cause over-facilitation of immune functions. Here we examined the effect of glycyrrhizin, known as an inhibitor of HMGB1, on thrombin-induced injury in rat cortico-striatal slice cultures and in vivo rat ICH model. In slice cultures, thrombin-induced a drastic increase in propidium iodide fluorescence indicating necrotic cell death in the cortical region, and robust shrinkage of the striatal tissue. Glycyrrhizin (10-100 μM) attenuated thrombin-induced cortical injury in a concentration-dependent manner. The protective effect of glycyrrhizin was not mediated by glucocorticoid receptors or modulation of nitric oxide production, but was reversed by exogenous HMGB1 application. The injury induced by a high concentration of HMGB1 was suppressed by glycyrrhizin. In vivo, unilateral injection of type IV collagenase into rat striatum induced ICH associated with brain edema formation, contralateral paralysis and neuron death. Once daily intraperitoneal administration of glycyrrhizin attenuated ICH-induced edema in both the cortex and the basal ganglia, and improved behavioral performance of rats in forelimb placing. Moreover, glycyrrhizin partially but significantly ameliorated ICH-induced neuron loss inside hematoma. These findings suggest that an HMGB1 inhibitor glycyrrhizin is a potential candidate for a remedy for ICH.

Sesamin suppresses activation of microglia and p44/42 MAPK pathway, which confers neuroprotection in rat intracerebral hemorrhage.

Thrombin plays important roles in the pathology of intracerebral hemorrhage (ICH). The recruitment of activated microglia, accompanied by thrombin-induced phosphorylation of the mitogen-activated protein kinase (MAPK) family, contributes to ICH-associated neuron loss. Here we investigated the possibility that sesamin, a lignan of sesame seed oil, is a natural candidate as an inhibitor of microglial activation and MAPK pathways under ICH insults. Sesamin (30-100 μM) suppressed thrombin-induced nitric oxide (NO) production by primary-cultured rat microglia via inhibition of inducible NO synthase (iNOS) protein expression, independently of the antioxidative effect. Sesamin selectively inhibited p44/42 MAPK phosphorylation in the MAPK family (p38 and p44/42) involved in iNOS protein expression in primary-cultured rat microglia. An in vivo rat ICH model was prepared by intrastriatal injection of 0.20U collagenase type IV unilaterally. ICH evoked the phosphorylation of p44/42 MAPK, microglial proliferation with morphological change into the activated ameboid form, and neuron loss. The phosphorylation of p44/42 MAPK was inhibited by intracerebroventricular administration of 30-nmol sesamin. Sesamin prevented ICH-induced increase of microglial cells in the perihematomal area. Notably, ramified microglia, the resting morphology, were observed in brain sections of the animals administrated sesamin. Sesamin furthermore achieved neuroprotection in the perihematomal area but not in the hematomal center. These results suggest that sesamin is a promising natural product as a novel therapeutic strategy based on the regulation of microglial activities accompanied by the activated p44/42 MAPK pathway in ICH.

Retinoic acid induces discrete Wnt-signaling-dependent differentiation in F9 cells

Retinoic acid (RA) induces F9 cells, the mouse teratocarcinoma cells, to differentiate into primitive endoderm and further into visceral and parietal endoderm depending on the culture conditions. To elucidate the instructive mechanisms involved in the differentiation steps we investigated the effects of Wnt-signaling members, Wnt3a and beta-catenin, on the differentiation of F9 cells and beta-catenin-deficient F9 cells (betaT cells). RA up-regulated the expression of differentiation markers for primitive, visceral and parietal endoderm in F9 cells but not for visceral endoderm in betaT cells. Wnt3a or leukemia inhibitory factor (LIF) inhibited the RA-induced differentiation in F9 cells. LIF but not Wnt3a could inhibit differentiation in betaT cells. RA evoked ZO-1alpha+ signals at cell-to-cell contacts in F9 cells in a Wnt3a sensitive manner. The results suggest that Wnt3a inhibits differentiation into endoderm through a pathway involving beta-catenin, and beta-catenin might be necessary in the process leading from primitive to visceral endoderm in F9 cells.

Sesamin increases heme oxygenase-1 protein in RAW 264.7 macrophages through inhibiting its ubiquitination process.

Sesamin is a major component in lignans of sesame seed oil, known to possess potent anti-oxidative capacity. In this study, the variation of heme oxygenase (HO)-1, a kind of anti-oxidative enzyme, by sesamin in murine macrophage cell line RAW 264.7 cells was investigated. Lipopolysaccharide (LPS; 10μg/ml) exposure tended to increase HO-1 protein expression. Co-treatment with 100μM sesamin for 12h up-regulated the HO-1 protein level increased by LPS; however, HO-1 mRNA was unaffected. Sesamin delayed the reversal, by the protein synthesis inhibitor cycloheximide (1μM), of the LPS-induced increase of HO-1 protein level. Meanwhile, sesamin suppressed LPS-induced expression of inducible nitric oxide (NO) synthase (iNOS) protein and associated NO release. LPS-induced increase of iNOS protein expression was also reversed by cycloheximide, which was not affected by sesamin, unlike HO-1. To clarify the mechanisms that underlie the up-regulation of HO-1 protein level by sesamin, the human embryonic kidney (HEK) 293T cell line transfected with Flag-tagged HO-1 was used. A proteasome inhibitor, MG-132 (10μM), stabilized HO-1 protein in HEK 293T cells. Co-treatment with sesamin decreased ubiquitinated HO-1 protein accumulation by MG-132. However, sesamin did not affect the proteasome activity. These findings suggest that sesamin disturbs the degradation of HO-1 protein through inhibiting its ubiquitination, resulting in HO-1 protein up-regulation.

Curcuma sp.‐derived dehydrocurdione induces heme oxygenase‐1 through a Michael reaction between its α, β‐unsaturated carbonyl and Keap1

To elucidate the anti‐inflammatory mechanism of Curcuma sp., we investigated whether dehydrocurdione, a sesquiterpene contained in Curcuma sp., induces heme oxygenase (HO)‐1, an antioxidative enzyme, in RAW 264.7 macrophages. Dehydrocurdione was extracted from the rhizome of Curcuma sp., and its purity was verified by high performance liquid chromatography. Treatment with 10–100 μM dehydrocurdione transiently and concentration‐dependently increased HO‐1 mRNA and protein levels. Docking simulation suggested the presence of the Michael reaction between dehydrocurdione and Kelch‐like ECH‐associated protein (Keap)1 keeping nuclear factor‐erythroid2‐related‐factor (Nrf)2, a transcription factor, in the cytoplasm. Nrf2 that was definitely free from Keap1 was detected in the nuclei after dehydrocurdione treatment. Subsequently, the HO‐1 E2 enhancer, a target of Nrf2, was activated, resulting in HO‐1 expression. Also, an investigation using 6‐shogaol and 6‐gingerol supported the concept that the α, β‐unsaturated carbonyl structure plays an important role in the interaction with Keap1. Dehydrocurdione suppressed lipopolysaccharide‐induced NO release, a marker of inflammation. Clarification of the HO‐1 synthesis increase mechanism revealed in this study will help contribute to the development of novel phytotherapeutic strategies against inflammation‐associated diseases.

Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage

The claudin family comprises four‐pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin‐25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult. For immunostaining, an anti‐CLDND1 antibody that recognizes the specific epitope in the extracellular first loop was prepared. Dual immunohistochemical staining with CD31 using coronal cryosections of intact murine cerebellum tissue revealed that CLDND1 is expressed on endothelial cells. We therefore performed an in vitro permeability test using a human brain endothelial cell (HBEC) line to reveal whether CLDND1 contributes to cell adhesion like other claudins. CLDND1 was expressed on HBECs as well as in murine cerebellum tissue, and a strong signal was observed at TJs. RNA interference against CLDND1 decreased both the mRNA and protein levels without cytotoxicity. The permeability to small molecules, but not to large ones, across confluent HBECs increased on CLDND1 knockdown compared with mock‐treated cells. These results suggest that the transient decrease of CLDND1 after cerebellar hemorrhage is responsible for low‐molecular‐weight selective vascular hyperpermeability.

Fabrication and characterization of water-dispersed chitosan nanofiber/poly(ethylene glycol) diacrylate/calcium phosphate-based porous composites

We evaluated the capacity of chitosan nanofiber (CNF)- and poly(ethylene glycol) (PEG)-based hydrogel/calcium phosphate hybrid (CNF-PEG/CaP) composites to act as scaffolding materials. CNF-PEG/CaP composites were fabricated by mineralization of CNF-PEG hydrogels using an alternate soaking method. The amount of CaP mineralized on CNF-PEG hydrogels increased as the ratio of CNF in the CNF-PEG hydrogel increased. Youngs modulus of CNF-PEG/CaP hydrogels was enhanced by increase in CNF ratio. It was further confirmed that osteoblasts embedded on the CNF-PEG/CaP composites were viable after incubation for 5days and firmly attached to the CaP porous layer, forming an extensive cell-scaffold leading to cell-cell interactions. These results indicated that the micro-porous structure of CNF-PEG hydrogels is suitable for CaP to be utilized as a scaffold for bone regeneration.

Shogaol but not gingerol has a neuroprotective effect on hemorrhagic brain injury: contribution of the α, β-unsaturated carbonyl to heme oxygenase-1 expression

Abstract We investigated the effects of shogaol, which has an &agr;, &bgr;‐unsaturated carbonyl group, and gingerol, which does not, on primary‐cultured microglia to understand how the &agr;, &bgr;‐unsaturated carbonyl interacts with Kelch‐like ECH‐associated protein (Keap)1. Shogaol (1 &mgr;M) but not the same concentration of gingerol significantly increased heme oxygenase (HO)‐1 protein levels in cultured microglia without cytotoxicity. In addition, shogaol suppressed the release of the inflammation marker nitric oxide induced by 30 U/ml thrombin treatment. A docking simulation suggested that the &agr;, &bgr;‐unsaturated carbonyl of shogaol but not gingerol interacts with Keap1. Nuclear import of nuclear factor E2‐related factor 2 and increased binding of the HO‐1 E2 enhancer support the docking‐simulation prediction. The transcription inhibitor actinomycin D (0.1 &mgr;g/ml) markedly blocked the increase of HO‐1 mRNA levels by shogaol. To evaluate whether the &agr;, &bgr;‐unsaturated carbonyl can be used for intracerebral hemorrhage (ICH) therapy, we investigated the effect of shogaol on an in vivo mouse ICH model. Intracerebroventricular injection of 0.2 nmol shogaol increased striatal HO‐1 protein levels and rescued ICH‐induced neuron loss. Thus, the &agr;, &bgr;‐unsaturated carbonyl is necessary for the interaction of compounds, such as shogaol, with Keap1, and these findings may be useful for screening novel ICH therapeutic agents that increase HO‐1 expression.