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.

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.

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.

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.

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.

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.

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.

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.

A ß-glucan from Grifola frondosa effectively delivers therapeutic oligonucleotide into cells via dectin-1 receptor and attenuates TNFα gene expression.

Grifola frondosa is an edible and medicinal mushroom with great nutritional values and bioactivities. In the present study, a soluble homogeneous ß-glucan, GFPS, with high molecular mass of 5.42 × 106 Da was purified from the fruit bodies of Grifola frondosa using 5% cold NaOH. The structure of GFPS was determined with FT-IR, NMR, and monosaccharide composition analysis, and was identified 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. Our results indicated that GFPS had a triple helical structure and could form complex with polydeoxyadenylic acid (poly[A]). Further studies demonstrated that GFPS could interact with poly[A] moiety of a designed antisense oligonucleotide (ASO) targeting the primary transcript of proinflammatory cytokine TNFα (TNFα-A60). This GFPS-based complex could incorporate TNFα-A60 into the macrophage cells via dectin-1 receptor and attenuate lipopolysaccharide-induced secretion of TNFα. Our results suggested that GFPS could be applied to deliver therapeutic oligonucleotides for the treatment of diseases such as inflammation and cancers.

Upregulated BMP-Smad signaling activity in the glucuronyl C5-epimerase knock out MEF cells.

Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce, in mice resulted in neonatal lethality with varied defects in organ development. To understand the underlying molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activitywhen cultured in osteogenic medium. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.

Characterization of a pectin from Lonicera japonica Thunb. and its inhibition effect on Aß42 aggregation and promotion of neuritogenesis.

Pectin is a class of complex polysaccharides and recognized for its potential bioactivities. In this study, we showed that a pectic polysaccharide, LFA03-a, was extracted from Lonicera japonica Thunb. flowers and purified with DEAE-cellulose and Sephacryl S-100HR. LFA03-a was composed of rhamnose, arabinose, galactose and galacturonic acid in the molar ratio of 18.1:25.3:36.8:19.5. Its structure was determined to possess a rhamnogalacturonan I (RG-I) backbone consisting of α-l-1,2-Rhap and α-d-1,4-GalAp disaccharide repeating unit, substituted at O-4 of l-rhamnose. The side chain was involved with ß-d-1,4-Galp, ß-d-1,3-Galp, ß-d-1,3,6-Galp and branched α-l-1,5-Araf. Fluorescence spectroscopic analysis with thioflavine T (ThT) and atomic force microscopy (AFM) results showed that LFA03-a inhibited Aß42 aggregation in a dose dependent manner and impeded Aß42 oligomerization and fibril formation. In addition, LFA03-a mildly induced the differentiation of PC12 cells and promoted neuritogenesis.The results suggested that pectin LFA03-a might be a potential targeted therapeutic drug for Alzheimer's disease.

Overexpression of heparanase attenuated TGF-ß-stimulated signaling in tumor cells.

Heparan sulfate (HS) mediates the activity of various growth factors including TGF-ß. Heparanase is an endo-glucuronidase that specifically cleaves and modifies HS structure. In this study, we examined the effect of heparanase expression on TGF-ß1-dependent signaling activities. We found that overexpression of heparanase in human tumor cells (i.e., Fadu pharyngeal carcinoma, MCF7 breast carcinoma) attenuated TGF-ß1-stimulated Smad phosphorylation and led to a slower cell proliferation. TGF-ß1-stimulated Akt and Erk phosphorylation was also affected in the heparanase overexpression cells. This effect involved the enzymatic activity of heparanase, as overexpression of mutant inactive heparanase did not affect TGF-ß1 signaling activity. Analysis of HS isolated from Fadu cells revealed an increase in sulfation of the HS that had a rapid turnover in cells overexpressing heparanase. It appears that the structural alterations of HS affect the ability of TGF-ß1 to signal via its receptors and elicit a growth response. Given that heparanase expression promotes tumor growth in most cancers, this finding highlights a crosstalk between heparanase, HS, and TGF-ß1 function in tumorigenesis.

Enzyme overexpression - an exercise toward understanding regulation of heparan sulfate biosynthesis.

Biosynthesis of heparan sulfate (HS) involves conversion of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) units catalyzed by glucuronyl C5-epimerase (Hsepi). IdoA units are the favored substrate for 2-O-sulfotransferase (2OST). We used HEK293 cells as a model to investigate the effects of overexpression of these enzymes on HS structure. Overexpression of Hsepi alone resulted in an unexpected increase in HS chain length. A Hsepi point-mutant (Y168A), devoid of catalytic activity, failed to affect chain length. Moreover, the effect of Hsepi overexpression on HS chain length was abolished by simultaneous overexpression of 2OST. These findings raise novel aspects on regulation of HS biosynthesis. We propose a hypothetical enzyme-binding protein (EBP) with distinct, specific and partly overlapping binding sites, the interactions of which will determine levels of enzymes available to the biosynthetic process.

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.

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.

A glucan isolated from flowers of Lonicera japonica Thunb. inhibits aggregation and neurotoxicity of Aß42.

Inhibition of Aß aggregation and attenuation of its cytotoxicity are considered to valuable therapeutics for Alzheimer's disease (AD). Here, a glucan named as LJW0F2 was purified from flowers of Lonicera japonica Thunb. Using monosaccharides composition analysis, methylation analysis, IR and NMR spectroscopy, this polysaccharide was elucidated to be an α-D-(1→4)-glucan with an α-(1→4) linked branch attached to the C-6 position. Its inhibitory effect on Aß42 aggregation was measured by fluorescence spectroscopic analysis with thioflavine T (ThT) and atomic force microscopy (AFM). We showed that polysaccharide LJW0F2 could inhibit Aß42 aggregation in a dose-dependent-manner. Besides, LJW0F2 could attenuate the cytotoxicity induced by Aß42 aggregation in SH-SY5Y neuroblastoma cells. To the best of our knowledge, this was the first report that the exogenous plant-derived polysaccharide might block Aß42 aggregation directly and reduce its toxicity in SH-SY5Y cells.

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.

A heteropolysaccharide, L-fuco-D-manno-1,6-α-D-galactan extracted from Grifola frondosa and antiangiogenic activity of its sulfated derivative.

The tumor growth and metastasis are angiogenesis dependent, thus blockade of angiogenesis is a promising approach for treatment of cancer. Herein we reported the structural and biological features of a novel water-soluble polysaccharide named GFPW from the fruit body of Grifola frondosa. Chemical and spectral analysis revealed that GFPW with an average molecular weight of 15.7 kDa, possessed a backbone consisting of α-1,6-linked galactopyranosyl residues, with branches attached to O-2 of α-1,3-linked fucose residues and α-terminal mannose. By the chlorosulfonic acid-pyridine method, we prepared a sulfated derivative of GFPW, Sul-GFPW, with a substitution degree of 0.33. According to the (13)C NMR spectrum, the substitution position was deduced at C-2 and C-3. The angiogenesis assays in vitro showed that Sul-GFPW significantly inhibited endothelial cell proliferation in a dose- and time-dependent manner, and reduced endothelial cell migration and tube formation as well.

Inducement of cytokine release by GFPBW2, a novel polysaccharide from fruit bodies of Grifola frondosa , through dectin-1 in macrophages.

Polysaccharides, especially ß-glucans isolated from various species of mushrooms, are considered as biological response modifiers (BRMs) to be widely used in the treatment of cancer, especially due to their immunostimulatory activity. We herein characterized the structure of a novel water-soluble homogeneous polysaccharide (GFPBW2) from the fruit bodies of mushroom Grifola frondosa and investigated its immunomodulatory activity in vitro. GFPBW2 was purified from the alkali-extracted fractions by stepwise elution with a molecular weight of 26.2 kDa. On the basis of infrared and NMR spectroscopy, methylation and monosaccharide composition analysis, partial acid hydrolysis, and Smith degradation, its structure was elucidated to possess a backbone consisting of ß-d-1,3- and ß-d-1,4-linked glucopyranosyl residues, with branches attached to O-6 of ß-d-1,3-linked glucopyranosyl residues. Functionally, it is an effective inducer of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion in murine resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW2 could bind dendritic cell-associated C-type lectin-1 (Dectin-1) with an affinity constant (Kd) value of 1.08 × 10 (-7) M, while it could activate Syk and enhance TNF-α production in RAW264.7 cells overexpressing wild type but not mutant Dectin-1. Furthermore, Syk, NF-κB signaling, and cytokine release in resident peritoneal macrophages induced by GFPBW2 could be significantly inhibited by a specific Dectin-1 blocking reagent, Laminarin. These data suggested that GFPBW2 might be a potential ligand of Dectin-1, and the potential of GFPBW2 to activate macrophage through triggering cytokine secretion might be attributed, at least in part, to the involvement of Dectin-1.

Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth.

BACKGROUND: Vascular endothelial growth factor (VEGF)-A and VEGF-C are two important molecules involving in tumor development and metastasis via angiogenesis and lymphangiogenesis. However, the combined effect of VEGF-A and VEGF-C on the growth of gastric cancer (GC) is not clear. METHODS: The correlations of VEGF-A and VEGF-C expressions with clinicopathologic parameters and prognosis were evaluated in patients with GC. Furthermore, lentivirus-mediated RNA interfering (RNAi) targeting VEGF-A and/or VEGF-C was employed to silence their expressions in SGC7901 GC cell line. Cell proliferation and apoptosis were measured in vitro. Suppressive effect lentivirus-mediated VEGF-A and/or VEGF-C silencing on GC growth was evaluated in GC bearing mice. RESULTS: The patients with high expression of both VEGF-A and VEGF-C (A+C+) had larger tumor size, higher peritumoral lymphatic vessel density(P-LVD), microvessel density(MVD), lymphatic vessel invasion (LVI), lymph node(LN) metastasis, and worse prognosis than those with low expression of both VEGF-A and VEGF-C (P<0.05). Lentivirus-mediated RNAi significantly reduced the mRNA and protein expression of VEGF-A and VEGF-C in the SGC7901 cells. The Lenti-miRNA-VEGF-A+VEGF-C significantly inhibited the cell proliferation and tumor growth, compared with Lenti-miRNA-VEGF-A or Lenti-miRNA-VEGF-C (P<0.05). In addition, Lenti-miRNA- VEGF-A+VEGF-C markedly lowered the tumor size in vivo in comparison with Lenti-miRNA-VEGF-A or Lenti-miRNA-VEGF-C (P<0.05). CONCLUSION: Expressions of both VEGF-A and VEGF-C predict worse prognosis of GC patients. Combined silencing of VEGF-A and VEGF-C markedly suppresses cancer growth than silencing of VEGF-A or VEGF-C. Thus, to inhibit the expressions of VEGF-A and VEGF-C may become a novel strategy for the treatment of GC.