Wenfeng Liao

A glucan isolated from flowers of Lonicera japonica Thunb. inhibits aggregation and neurotoxicity of Aβ42

Inhibition of Aβ aggregation and attenuation of its cytotoxicity are considered to valuable therapeutics for Alzheimers 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 Aβ42 aggregation was measured by fluorescence spectroscopic analysis with thioflavine T (ThT) and atomic force microscopy (AFM). We showed that polysaccharide LJW0F2 could inhibit Aβ42 aggregation in a dose-dependent-manner. Besides, LJW0F2 could attenuate the cytotoxicity induced by Aβ42 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 Aβ42 aggregation directly and reduce its toxicity in SH-SY5Y cells.

Structural characterization and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme.

A fucoidan FP08S2 was isolated from the boiling-water extract of Sargassum fusiforme, purified by CaCl2 precipitation and chromatography on DEAE-cellulose and Sephacryl S-300. FP08S2 contained fucose, xylose, galactose, mannose, glucuronic acid, and 20.8% sulfate. The sulfate groups were attached to diverse positions of fucose, xylose, mannose, and galactose residues. The backbone of FP08S2 consisted of alternate 1,2-linked α-D-Manp and 1,4-linked β-D-GlcpA. Sugar composition analysis and ESI-MS revealed that the oligosaccharides from branches contained fucose, xylose, galactose, glucuronic acid and sulfate. FP08S2 could significantly inhibit tube formation and migration of human microvascular endothelial cells (HMEC-1) dose-dependently. These results suggested that the fucoidan FP08S2 from brown seaweeds S. fusiforme could be a potent anti-angiogenic agent.

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.

Structure and activities of a novel heteroxylan from Cassia obtusifolia seeds and its sulfated derivative

COB1B1S2 was isolated from an alkaline extract of Cassia obtusifolia seeds, and purified by anion-exchange and gel permeation chromatography. It contains arabinose, xylose, and glucuronic acid, in the molar ratio of 5:81:14, with an apparent molecular weight estimated to be 70.4 kDa. Elucidated by using chemical and spectroscopic methods, COB1B1S2 was shown to have a backbone consisting of 1,4-linked β-D-Xylp, with one single-unit terminal α-D-GlcpA or α-L-Araf substituted at O-2 for nearly every five 1,4-linked Xylp. COB1B1S2 is structurally different from typical glucuronoxylans by its absence of methylation at O-4 of GlcA. The native COB1B1S2 showed no significant inhibition on the tube formation of human microvascular endothelial cells (HMEC) and on the growth of liver and colon cancer cells. On the contrary, COB1B1S2-Sul, prepared as the sulfated derivative of COB1B1S2, exhibited a significant inhibition on tube formation of HMEC in a dose-dependent manner, and on the growth of Bel7402 liver cancer cells. These results indicated that the introduction of sulfate groups significantly enhanced the biological activity of glucuronoxylan.

Structure and biological activities of an alginate from Sargassum fusiforme, and its sulfated derivative.

An alginate fraction, 04S2P, was isolated from the brown seaweed Sargassum fusiforme and was structurally characterized by the ratio (M/G) of β-d-mannuronic acid residues (M) to α-l-guluronic acid residues (G) via (1)H and (13)C NMR spectroscopy. When compared to commercial alginate (Alg) and alginates from other brown algae, 04S2P has a higher M/G ratio of 9.0:1.0 as determined by a modified high-performance liquid chromatography method after pre-column derivatization with PMP. Furthermore, the sulfated polysaccharides 04S2P-S and Alg-S were prepared by the chlorosulfonic acid-pyridine method. Both C-2 and/or C-3 of M and G residues of 04S2P-S were substituted by sulfate groups, with C-3 of M residues preferentially substituted. Their effects on tube formation of HMEC-1 cells were examined, and the results indicated that the sulfated Alg, Alg-S, exhibited a strong anti-angiogenic effect on HMEC-1 cells. The anti-tumor activity of native and sulfated alginates was tested on five different tumor cell lines. Alg-S demonstrated significant anti-tumor effects on the Bel7402, SMMC7721, and HT-29 cell lines, whereas 04S2P-S showed a distinct anti-tumor effect only on the Bel7402 cell line.

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-1β, 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 IKKβ and TGFβ-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.

Sulfated fucoidan FP08S2 inhibits lung cancer cell growth in vivo by disrupting angiogenesis via targeting VEGFR2/VEGF and blocking VEGFR2/Erk/VEGF signaling.

Fucoidan may inhibit angiogenesis. However, its functional target molecule and the underlying mechanism are still vague. In the present study, we showed that sulfated fucoidan FP08S2 from Sargassum fusiforme inhibited tube formation as well as migration and invasion of human microvascular endothelial cells (HMEC-1). In addition, FP08S2 was confirmed to disrupt VEGF-induced angiogenesis both in vitro and in vivo. Further study indicated that FP08S2 could bind to both VEGF and VEGFR2 to interfere with VEGF-VEGFR2 interaction. Moreover, VEGFR2/Erk/VEGF signaling pathway was blocked by FP08S2 in HMEC-1 cells. Importantly, FP08S2 impeded the growth and microvessel formation of A549 cancer cell xenograft in nude mice. These results suggested that FP08S2 presented remarkable anti-angiogenic activity via blocking VEGF signaling and could be a potential novel leading compound to inhibit lung cancer cell growth.

microRNA-149 targets caspase-2 in glioma progression

Malignant gliomas are the most common form of intrinsic primary brain tumors worldwide. Alterations in microRNAs play a role in highly invasive malignant glioma, but detail mechanism still unknown. In this study, the role and mechanism of microRNA-149 (miR-149) in glioma are investigated. We show that miR-149 is expressed at substantially higher levels in glioma than in normal tissues. Stable overexpression of miR-149 augments potent prosurvival activity, as evidenced by promotion of cell viability, inhibition of apoptosis, and induced xenografted tumor growth in vivo. We further show that Caspase-2 is identified as a functional target of miR-149 and expression of caspase-2 is inversely associated with miR-149 in vitro. In addition, miR-149 promotes tumor survival in the U87-MG and A172 cell lines and it targets caspase-2 via inactivation of the p53 and p21 pathways. There results support a special role for miR-149 by targeting Caspase-2 to impact on p53 signaling pathway. We speculate that miR-149 has distinct biological functions in p53 wild type cells and p53 mutation cells, and the mechanisms involved remain to be explored in future. Our study suggests that targeting miR-149 may be a novel therapy strategy for treating p53 wild type glioma tumors in humans.

An arabinogalactan from fruits of Lycium barbarum L. inhibits production and aggregation of A β 42

The β amyloid (Aβ) induced neurodegeneration is believed to be one of pathological mechanisms of Alzheimers disease (AD). The inhibition of Aβ production or aggregation is one of the promising therapeutic strategies for anti-AD drug discovery. Here, a homogeneous neutral polysaccharide designated LBP1A1-1 with an average molecular weight of 45.0 kDa was purified from fruits of Lycium barbarum L. Its structure was characterized to possess a backbone of 1, 3-linked β-Galp, 1, 6-linked β-Galp, 1, 4-linked α-Glcp with branches substituted at C-3 position of 1, 6-linked β-Galp or C-6 position of 1, 3-linked β-Galp. The branches contained terminal (T)-linked β-Galp, T-linked α-Araf, T-linked β-Araf, 1, 5-linked α-Araf and T-linked β-Rhap. The in vitro experiments revealed that LBP1A1-1 could inhibit Aβ42 production and impede Aβ42 aggregation in a dose-dependent-manner without cytotoxicity. These results suggested that LBP1A1-1 might have the multiple potential for the treatment of AD.

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.