Inhibitors of dermatan sulfate epimerase 1 decreased accumulation of glycosaminoglycans in mucopolysaccharidosis type I fibroblasts.

Genetic deficiency of alpha-L-iduronidase causes mucopolysaccharidosis type I (MPS-I) disease, due to accumulation of glycosaminoglycans (GAGs) including chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) in cells. Currently, patients are treated by infusion of recombinant iduronidase or by hematopoietic stem cell transplantation. An alternative approach is to reduce the L-iduronidase substrate, through limiting the biosynthesis of iduronic acid. Our earlier study demonstrated that ebselen attenuated GAGs accumulation in MPS-I cells, through inhibiting iduronic acid producing enzymes. However, ebselen has multiple pharmacological effects, which prevents its application for MPS-I. Thus, we continued the study by looking for novel inhibitors of dermatan sulfate epimerase 1 (DS-epi1), the main responsible enzyme for production of iduronic acid in CS/DS chains. Based on virtual screening of chemicals towards chondroitinase AC, we constructed a library with 1,064 compounds that were tested for DS-epi1 inhibition. Seventeen compounds were identified to be able to inhibit 27%-86% of DS-epi1 activity at 10 µM. Two compounds were selected for further investigation based on the structure properties. The results show that both inhibitors had a comparable level in inhibition of DS-epi1while they had negligible effect on HS epimerase. The two inhibitors were able to reduce iduronic acid biosynthesis in CS/DS and GAG accumulation in WT and MPS-I fibroblasts. Docking of the inhibitors into DS-epi1 structure shows high affinity binding of both compounds to the active site. The collected data indicate that these hit compounds may be further elaborated to a potential lead drug used for attenuation of GAGs accumulation in MPS-I patients.

Characterization and expression of highly active recombinant human glucuronyl C5-epimerase in mammalian cells.

Heparin, a highly sulfated and epimerized form of heparan sulfate, is a linear polysaccharide with anticoagulant activity widely used in the clinic to prevent and treat thrombotic diseases. However, there are several noteworthy drawbacks associated with animal-sourced heparin during the preparation process. The in vitro enzymatic synthesis of heparin has become a promising substitute for animal-derived heparin. The synthesis of bioengineered heparin involves recombinant expression and preparation of polymerases, sulfotransferases, and an epimerase. D-glucuronyl C5-epimerase (HSepi) catalyzes D-glucuronic acids immediately adjacent to N-sulfo-glucosamine units to L-iduronic acid. Preparation of recombinant HSepi with high activity and production yield for in vitro heparin synthesis has not been resolved as of now. The findings of this study indicate that the catalytic activity of HSepi is regulated using post-translational modifications, including N-linked glycosylation and disulfide bond formation. Further mutation studies suggest that tyrosine residues, such as Tyr168, Tyr222, Tyr500, Tyr560, and Tyr578, are crucial in maintaining HSepi activity. A high-yield expression strategy was established using the lentiviral-based transduction system to produce recombinant HSepi (HSepi589) with a specific activity of up to 1.6 IU/mg. Together, this study contributes to the preparation of highly active HSepi for the enzymatic synthesis of heparins by providing additional insights into the catalytic activity of HSepi.

Hepatic glucuronyl C5-epimerase combats obesity by stabilising GDF15.

BACKGROUND & AIMS: Disturbed hepatic metabolism frequently results in excessive lipid accumulation in the adipose tissue. However, the specific role of the liver-adipose axis in maintaining lipid homeostasis, as well as the underlying mechanism, has not yet been fully elucidated. In this study, we investigated the role of hepatic glucuronyl C5-epimerase (Glce) in the progression of obesity. METHODS: We determined the association between the expression of hepatic Glce and body mass index (BMI) in obese patients. Obesity models were established in hepatic Glce-knockout and wild-type mice fed a high-fat diet (HFD) to understand the effect of Glce on obesity development. The role of Glce in the progression of disrupted hepatokine secretion was examined via secretome analysis. RESULTS: Hepatic Glce expression was inversely correlated with BMI in obese patients. Moreover, Glce level was found to be decreased in the liver of a HFD murine model. Hepatic Glce deficiency led to impaired thermogenesis in adipose tissue and exacerbated HFD-induced obesity. Interestingly, decreased level of growth differentiation factor 15 (GDF15) was observed in the culture medium of Glce-knockout mouse hepatocytes. Treatment with recombinant GDF15 obstructed obesity progression derived from the absence of hepatic Glce, similar to the effect of Glce or its inactive mutant overexpressed both in vitro and in vivo. Furthermore, liver Glce deficiency led to diminished production and increased degradation of mature GDF15, resulting in reduced hepatic GDF15 secretion. CONCLUSIONS: Hepatic Glce deficiency facilitated obesity development, and decreased Glce expression further reduced hepatic secretion of GDF15, thereby perturbing lipid homeostasis in vivo. Therefore, the novel Glce-GDF15 axis plays an important role in maintaining energy balance and may act as a potential target for combating obesity. IMPACT AND IMPLICATIONS: Evidence suggests that GDF15 plays a key role in hepatic metabolism; however, the molecular mechanism for regulating its expression and secretion is largely unknown. Our work observes that hepatic Glce, as a key Golgi-localised epimerase, may work on the maturation and post-translational regulation of GDF15. Hepatic Glce deficiency reduces the production of mature GDF15 protein and facilitates its ubiquitination, resulting in the aggravation of obesity development. This study sheds light on the new function and mechanism of the Glce-GDF15 axis in lipid metabolism and provides a potential therapeutic target against obesity.

Re-expression of glucuronyl C5-epimerase in the mutant MEF cells increases heparan sulfate epimerization but has no influence on the Golgi localization and enzymatic activity of 2-O-sulfotransferase.

Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure-function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.

Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts.

Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

A Purified Glucomannan Oligosaccharide from Amorphophallus konjac Improves Colonic Mucosal Barrier Function via Enhancing Butyrate Production and Histone Protein H3 and H4 Acetylation.

A structurally defined konjac glucomannan oligosaccharide (KGMOS) with a relatively high molecular weight and narrow molecular weight distribution (molecular weight ranging from 3000 to 4000 Da, degree of polymerization (dp) 8-11) was prepared from native konjac glucomannan (KGM), and the beneficial effects and molecular mechanisms of KGMOS on colonic functions were investigated in C57BL/6 mice. The results are the first to reveal that KGMOS regulated intestinal microflora composition to facilitate the production of colonic butyrate. Elevated butyrate production further increased the acetylation of histone proteins H3 and H4 and thus enhanced the transcription of the major colonic mucin gene Muc2 and the secretion of mucin elements, which represents a new molecular mechanism of KGM oligosaccharide consumption. The findings indicate that KGM oligosaccharides with specific molecular sizes have highly desirable functional properties and potentially could improve gut health by promoting the barrier function of the colonic mucosa.

Structural and functional study of D-glucuronyl C5-epimerase.

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling.

AIM: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice. METHODS: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured. RESULTS: In RAW264.7 cells and BMMs, WSS25 (2.5, 5, 10 µg/mL) did not affect the cell viability, but dose-dependently inhibited RANKL-induced osteoclastogenesis. Furthermore, WSS25 potently suppressed RANKL-induced expression of TRAP, NFATc1, MMP-9 and cathepsin K in RAW264.7 cells. Treatment of RAW264.7 cells with RANKL increased BMP-2 expression, Smad1/5/8 phosphorylation and Id1 expression, which triggered osteoclast differentiation, whereas co-treatment with WSS25 or the endogenous BMP-2 antagonist noggin suppressed the BMP-2/Smad/Id1 signaling pathway. In RAW264.7 cells, knockdown of Id1 attenuated RANKL-induced osteoclast differentiation, which was partially rescued by Id1 overexpression. In conformity to the in vitro experiments, chronic administration of WSS25 significantly reduced the bone loss in ovariectomized mice. CONCLUSION: WSS25 inhibits RANKL-induced osteoclast formation in RAW264.7 cells and BMMs by blocking the BMP-2/Smad/Id1 signaling pathway. WSS25 administration reduces bone loss in ovariectomized mice, suggesting that it may be a promising therapeutic agent for osteoporosis.

GLCE regulates PC12 cell neuritogenesis induced by nerve growth factor through activating SMAD/ID3 signalling.

Neurodevelopment is orchestrated by a series of growth factor-HS (heparan sulfate) interactions which are involved in neuritogenesis. GLCE (glucuronic acid epimerase) is a critical enzyme involved in HS synthesis, which converts GlcA (D-glucuronic acid) into IdoA (L-iduronic acid). However, the function of GLCE in neuritogenesis is largely unknown. In the present study we showed that GLCE depletion caused arrested PC12 cell growth and promoted the cell neuritogenesis and differentiation induced by NGF (nerve growth factor). PC12 cell growth was boosted by overexpression of GLCE, and neuritogenesis was impaired when GLCE depletion was rescued. Interestingly, overexpression of wild-type GLCE with Y168A and Y222A mutations led to enhanced PC12 cell growth and attenuated the neuritogenesis triggered by GLCE silencing. We showed further that GLCE depletion blocked SMAD1/5/8 phosphorylation; however, this signalling could be restored by GLCE or the mutation of its active enzymatic site. In addition, the downstream effector of SMAD1/5/8, ID3 (inhibitor of DNA binding/differentiation 3) was induced by GLCE. ID3 silencing inhibited PC12 cell growth and induced cell neuritogenesis and differentiation. In addition, ectopic expression of ID3 partially rescued the phenotype caused by GLCE silencing. The results of the present study suggest that GLCE plays a key role in PC12 cell growth and neuritogenesis through SMAD/ID3 signalling.

Multi-scale study of load-bearing mechanism of uplift piles based on model tests and numerical simulations.

The uplift pile is an anti-uplift measure in engineering widely used in practice. In order to study the mechanical parameters of the pile and the surrounding soil under the uplift load, a pile uplift model test and relevant numerical test were conducted. Image analysis technique was applied to the model test to investigate the soil displacements caused by pulling the pile. The load-displacement and pile axial force-lateral friction resistance relationships were investigated at three burial depths. Comparing the model test and numerical test results, it reveals that the pile primarily underwent four stages under the influence of uplift load: initial stage of loading, strain-hardening stage, peak of loading stage, and the strain-softening stage; the soil displacements around the pile exhibited inverted conical shape as the uplift load increases; and obvious soil arching effects could be observed near the ground surface. In addition, the development of force chains and major principal stresses indicated that the pile lateral frictional resistance first increased to its maximum value and then decreased sharply along the depth direction.

Heparin impairs angiogenesis through inhibition of microRNA-10b.

Heparin, which has been used as an anticoagulant drug for decades, inhibits angiogenesis, whereas thrombin promotes tumor-associated angiogenesis. However, the mechanisms underlying the regulation of angiogenesis by heparin and thrombin are not well understood. Here, we show that microRNA-10b (miR-10b) is down-regulated by heparin and up-regulated by thrombin in human microvascular endothelial cells (HMEC-1). Overexpression of miR-10b induces HMEC-1 cell migration, tube formation, and angiogenesis, and down-regulates homeobox D10 (HoxD10) expression via direct binding of miR-10b to the putative 3' UTR of HoxD10. In addition, HMEC-1 cell migration and tube formation are induced by HoxD10 knockdown, whereas angiogenesis is arrested when HoxD10 expression is increased after anti-miR-10b or heparin treatments. Furthermore, expression of miR-10b and its transcription factor Twist are up-regulated by thrombin, whereas HoxD10 expression is impaired by thrombin. Using quartz crystal microbalance analysis, we show that heparin binds to thrombin, thereby inhibiting thrombin-induced expression of Twist and miR-10b. However, the expression of miR-10b is not attenuated by heparin any more after thrombin expression is silenced by its siRNA. Interestingly, we find that heparin attenuates miR-10b expression and induces HoxD10 expression in vivo to inhibit angiogenesis and impair the growth of MDA-MB-231 tumor xenografts. These results provide insight into the molecular mechanism by which heparin and thrombin regulate angiogenesis.

Structure-activity relationship study of WSS25 derivatives with anti-angiogenesis effects.

WGEW, an α(1-4) linked glucan with an α(1-4) linked branch attached to C-6, was isolated from the rhizoma of Gastrodia elata Bl. WSS25, a sulfated derivative of WGEW, was reported to inhibit angiogenesis by disrupting BMP2/Smad/Id1 signaling pathway. However, the structure-activity relationship (SAR) for WSS25 is not known. To study the SAR, seven sulfated saccharides derived from WGEW degradation products, six sulfated polysaccharides with varying degrees of substitution, and four aminopropylated, carboxymethylated, phosphorylated, and acetylated derivatives of WGEW were prepared. A sulfated, unbranched product of polysaccharide was also obtained. The structural features of these derivatives were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. An HMEC-1 cell tube formation assay was employed to measure the antiangiogenic effect of the derivatives. The results indicated that only sulfated polysaccharides with molecular weights of more than 41,000 Da could inhibit HMEC-1 cell tube formation. The inhibition effect was dependent on the presence of a sulfate group, since the tube formation was not blocked by aminopropylated, carboxymethylated, phosphorylated, or acetylated WGEW. A higher degree of sulfate substitution on the polysaccharide led to a stronger inhibitory effect, and the degree of sulfate substitution between 0.173 and 0.194 was found to be optimal. Interestingly, the WGEW side chain was not required for anti-tube formation activity. All these preliminary results may provide a clue for further modification of the core structure of WSS25 to discover polysaccharide derivatives as novel anti-angiogenic inhibitors.

Structure of a ß-glucan from Grifola frondosa and its antitumor effect by activating Dectin-1/Syk/NF-κB signaling.

A soluble homogeneous ß-glucan, GFPBW1, with a molecular mass of 300 kDa was purified from the fraction of the fruit bodies of Grifola frondosa extracted with 5% NaOH. Using various methods, such as infrared spectroscopy, NMR, methylation and monosaccharide composition analysis, its structure was determined to be a ß-D-(1-3)-linked glucan backbone with a single ß-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. It induced TNF-α and IL-6 production and the activation of Syk and NF-κB signaling in resident peritoneal macrophages from ICR mice, which could be significantly inhibited by the blocking reagent laminarin. A competitive phagocytosis assay with FITC-zymosan indicated that GFPBW1 could bind to DC-associated C-type lectin 1 (Dectin-1). The TNF-α secretion and activation of Syk/NF-κB signaling triggered by GFPBW1 were enhanced in RAW264.7 cells overexpressing wild but not mutant (Δ38 and Y15S) Dectin-1. Furthermore, GFPBW1 potentiated the Concanavalin A-induced proliferative response of splenocytes and inhibited Sarcoma-180 growth allografted in ICR mice but not in immunodeficient BALB/c nu/nu mice. These results suggested that the antitumor activity of GFPBW1 was partially associated with the activation of macrophages via the Dectin-1/Syk/NF-κB signaling pathway. This molecule could be a promising biological response modifier with clear application for antitumor therapies.

Identification and synthesis of substituted pyrrolo[2,3-d]pyrimidines as novel firefly luciferase inhibitors.

A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based NF-κB luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus pyralis with an IC(50) value of 0.36 ± 0.05 µM. Kinetic analysis of 3a inhibition showed that it is predominantly competitive with respect to D-luciferin and uncompetitive with respect to ATP. Therefore, several pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relationship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC(50) value of 0.06 ± 0.01 µM. In addition, molecular docking studies suggested that both 3a and 4c could be accommodated in the D-luciferin binding pocket, which is expected for a predominantly competitive inhibitor with respect to D-luciferin.

Lentivirus-mediated RNA silencing of c-Met markedly suppresses peritoneal dissemination of gastric cancer in vitro and in vivo.

AIM: To investigate the expression of c-Met in peritoneal free cancer cells isolated from human gastric cancer ascites, and its relationship to peritoneal dissemination of gastric cancer. METHODS: Peritoneal free cancer cells (PFCCs) were isolated from ascites specimens of gastric cancer patients. c-Met expression in PFCCs was detected with immunocytochemistry. In human gastric cancer cell line SGC7901, c-Met expression was detected using RT-PCR and Western blot, and was suppressed with lentivirus-mediated RNAi. The proliferation of SGC7901 cells was measured using MTT assay, and the invasion ability was detected with invasion assay. The adhesion of SGC7901 cells to peritoneum was observed in human peritoneal mesothelial cells (HPMCs) monolayer in vitro and in mice in vivo. RESULTS: PFCCs were isolated from ascites of 6 out of 10 gastric cancer patients. c-Met expression in PFCCs was detected in 5 of the 6 gastric cancer patients. In SGC7901 cells, Lentivirus-mediated RNAi significantly reduced both c-Met mRNA and protein expression, which resulted in suppressing the cell proliferation, invasion and adhesion to peritoneum. The expression of α3ß1 integrin and E-cadherin was significantly inhibited in SGC7901 cells transfected with Lenti-miRNAc-Met. In the peritoneal dissemination model of gastric cancer, intraperitoneal injection of Lenti-miRNAc-Met markedly suppressed the tumor Progression of SGC7901 cells. CONCLUSION: c-Met is expressed in PFCCs from the ascites of gastric cancer patients. Down-regulation of c-Met expression markedly suppresses the multistep process of peritoneal dissemination, thus may be a potential target for the treatment of gastric cancer.

Lobolide, a diterpene, blockades the NF-κB pathway and p38 and ERK MAPK activity in macrophages in vitro.

AIM: Recent studies have shown that constitutive activation of the nuclear factor κB (NF-κB) plays a key role in chronic inflammation and cancers. The aim of this study was to characterize lobolide, a cembrane diterpene, as a drug candidate targeting the NF-κB signaling pathway. METHODS: A HEK 293/NF-κB-Luc stable cell line was constructed to evaluate the effect of lobolide on NF-κB activation. THP-1 human monocytes and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were tested. Lipopolysaccharide (LPS)-induced TNFα and IL-1ß production and activation of the TAK1-IKK-NF-κB pathway were studied using ELISA and Western blot analysis. RESULTS: In HEK 293/NF-κB-Luc stable cells, lobolide (0.19-50 µmol/L) inhibited NF-κB activation in a concentration-dependent manner with an IC(50) value of 4.2 ± 0.3 µmol/L. Treatment with lobolide (2.5-10 µmol/L) significantly suppressed LPS-induced production of TNFα and IL-1ß in both THP-1 cells and PBMCs. In THP-1 cells, the suppression was partially caused by blockade of the translocation of NF-κB from the cytoplasm to the nucleus via affecting the TAK1-IKK-NF-κB pathway and p38 and ERK MAPK activity. CONCLUSION: Lobolide is a potential inhibitor of the NF-κB pathway, which blocks the translocation of NF-κB from the cytoplasm to the nucleus. Lobolide inhibits LPS-stimulated TNFα and IL-1ß release, suggesting that the compound might be an anti-inflammatory compound.

A novel small molecule, HK-156, inhibits lipopolysaccharide-induced activation of NF-κB signaling and improves survival in mouse models of sepsis.

AIM: To characterize a small molecule compound HK-156 as a novel inhibitor of the nuclear factor κB (NF-κB) signaling pathway. METHODS: THP-1 monocytes and HEK293/hTLR4A-MD2-CD14 cells were tested. HK-156 and compound 809, an HK-156 analogue, were synthesized. A luciferase assay was used to evaluate the transcriptional activity of NF-κB. The levels of cytokines were measured with cytokine arrays, ELISA and quantitative PCR. An electrophoretic mobility shift assay (EMSA), immunofluorescence, Western blot and mass spectrometry were used to investigate the molecular mechanisms underlying the actions of the agent. BALB/c mice challenged with lipopolysaccharide (LPS, 15 mg/kg, ip) were used as a mouse experimental endotoxemia model. RESULTS: In HEK293hTLR4/NF-κB-luc cells treated with LPS (1000 ng/mL), HK-156 inhibited the transcriptional activity of NF-κB in a concentration-dependent manner (IC50=6.54 ± 0.37 µmol/L). Pretreatment of THP-1 monocytes with HK-156 (5, 10 and 20 µmol/L) significantly inhibited LPS-induced release and production of TNF-α and IL-1ß, attenuated LPS-induced translocation of NF-κB into the nucleus and its binding to DNA, and suppressed LPS-induced phosphorylation and degradation of IκBα, and phosphorylation of IKKß and TGFß-activated kinase (TAK1). Meanwhile, HK-156 (5, 10 and 20 µmol/L) slightly suppressed LPS-induced activation of p38. The effect of HK-156 on LPS-induced activation of NF-κB signaling was dependent on thiol groups of cysteines in upstream proteins. In mouse models of sepsis, pre-injection of HK-156 (50 mg/kg, iv) significantly inhibited TNFα production and reduced the mortality caused by the lethal dose of LPS. CONCLUSION: HK-156 inhibits LPS-induced activation of NF-κB signaling by suppressing the phosphorylation of TAK1 in vitro, and exerts beneficial effects in a mouse sepsis model. HK-156 may therefore be a useful therapeutic agent for treating sepsis.

Heparanase enhances nerve-growth-factor-induced PC12 cell neuritogenesis via the p38 MAPK pathway.

Heparanase is involved in the cleavage of the HS (heparan sulfate) chain of HSPGs (HS proteoglycans) and hence participates in remodelling of the ECM (extracellular matrix) and BM (basement membrane). In the present study we have shown that NGF (nerve growth factor) promoted nuclear enrichment of EGR1 (early growth response 1), a transcription factor for heparanase, and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of the NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 MAPK (mitogen-activated protein kinase) by phosphorylation and enhanced the neurite outgrowth induced by NGF, whereas knock down of heparanase impaired this process. However, overexpression of latent pro-heparanase with a Y156A mutation still led to enhanced NGF-induced neurite outgrowth and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK by SB203580 suppressed the promotion of NGF-induced neuritogenesis by the wild-type and mutant heparanase. The impaired differentiation by knock down of heparanase could be restored by transfection of wild-type or mutant heparanase in PC12 cells. The results of the present study suggest that heparanase, at least in the non-enzymatic form, may promote NGF-induced neuritogenesis via the p38 MAPK pathway.

Liver-Targeted Delivery of Oligonucleotides with N-Acetylgalactosamine Conjugation.

The potential therapeutic application of oligonucleotides (ONs) that selectively suppress target genes through antisense and RNA interference mechanisms has attracted great attention. The clinical applications of ONs have overcome multiple obstacles and become one of the most active areas for the development of novel therapeutics. To achieve efficient and specific cellular internalization, conjugation of a variety of functional groups to ONs has been the subject of intensive investigations over the past decade. Among them, a promising liver-targeted N-acetylgalactosamine (GalNAc) ligand has been evaluated in multiple preclinical and clinical trials for improving the cellular uptake and tissue specific delivery of ONs. GalNAc-based delivery relies on the fact that liver hepatocytes abundantly and specifically express the asialoglycoprotein receptor that binds and uptakes circulating glycoproteins via receptor-mediated endocytosis. In recent years, encouraging progress has been made in the field of GalNAc conjugates. This review aims to provide an overview of GalNAc-mediated liver-targeted delivery of small interfering RNA and antisense oligonucleotides, and the immense effort as well as recent advances in the development of GalNAc-conjugated agents are described.

Different significance between intratumoral and peritumoral lymphatic vessel density in gastric cancer: a retrospective study of 123 cases.

BACKGROUND: Patients with gastric cancer in China have worse outcome and poorer prognosis. Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. The intratumoral and peritumoral lymphatics were supposed to have different biological effects. Three major growth factors, vascular endothelial growth factor- (VEGF)-A, VEGF-C and VEGF-D, are involved in the activation process via their receptors (VEGFRs). The purpose of current study is to investigate the significant difference between intratumoral and peritumoral lymphatic vessel density (LVD) in gastric cancer and their correlations with lymphangiogenetic growth factors. METHODS: Intratumoral LVD (I-LVD) and peritumoral LVD (P-LVD) of 123 patients with primary gastric cancer were assessed after staining with D2-40, and confirmed by double staining with D2-40/CD34. Proliferative activity of lymphatics endothelium was evaluated by double staining with D2-40/Ki-67. The associations were analyzed between I-LVD/P-LVD and the expression level of VEGF-A, VEGF-C, VEGF-D and the receptor VEGFR-3, which was measured by immunohistochemistry (IHC). The correlations of I-LVD and P-LVD with patient prognosis were also valued. RESULTS: (1) The peritumoral lymphatics (PTLs) were relatively enlarged with dilated lumen compared with the intratumoral lymphatics (ITLs). Increased P-LVD was significantly higher than I-LVD (P < 0.05). (2) P-LVD was found significantly associated with lymph node metastasis (LNM) (P < 0.001), lymphatic vessel invasion (LVI) (P < 0.001), VEGF-C (P = 0.003), VEGF-D expression level (P = 0.005) and VEGFR-3 expression level (P < 0.001) in peritumoral tissues, despite no significant association was found between above variants with I-LVD. However, increased I-LVD was demonstrated to be associated with decreased tumor volume (P < 0.001). Neither I-LVD nor P-LVD was correlated with VEGF-A expression (P > 0.05). (3) Proliferative activity of lymphatics endothelium was observed in PTLs, in spite of ITLs. (4) Increased P-LVD, but not I-LVD, was indicated to be an independent risk factor for lymph node metastasis by multivariate logistic regression analysis, and was related to worse disease-free survival and overall survival. CONCLUSIONS: PTLs play roles in gastric cancer progression. Increased P-LVD, but not I-LVD, was significantly associated with VEGF-C/-D/VEGFR-3 system, and could be an independent risk factor for lymph node metastasis and a prognostic factor in gastric cancer.