Study On The Chemical Structure Of Oligosaccharides From Fucosylated Glycosaminoglycan And Their Heparanase Inhibitory Activities

Invasion and metastasis of tumor cells is an important feature of malignant tumors and also the major obstacle to its clinical treatment.Extracellular matrix（ECM）and basement membrane（BM）serve as the main histological barriers for cell metastasis and angiogenesis.Heparan sulfate（HS）contained in them can be blocked by heparanase（HPSE）degradation.Studies have found that inhibiting or blocking HPSE activity can inhibit tumor cell invasion and metastasis.Further studies have confirmed that HPSE is closely related to angiogenesis and tumor invasion and metastasis.Additionally,venous thromboembolism（VTE）is a common malignant tumor complication.In theory,drugs with both anti-tumor and anti-thrombotic activities are beneficial to tumor treatment.Heparin（Hep）,as a classic anticoagulant,is a common drug for clinical treatment of malignant tumor thromboembolism complications.However,the clinical application of Hep is severely limited due to the serious risk of bleeding.Fucosylated glycosaminoglycan（FG）extracted from sea cucumber is a glycosaminoglycan derivative fucose branches.Depolymerized FG and its purified fragments（oligosaccharides）showed potent anticoagulant and antithrombotic activities with low bleeding tendency through selectively inhibiting intrinsic factor Xase（i Xase）.Given that FG has a similar but different backbone structure of glycosaminoglycans from HS,it may have heparin-related activities.Purpose:This dissertation explores the effect of purified oligosaccharides from FG（o FGs）on HPSE activity,and then further analyzed the effects of o FGs on angiogenesis and tumor cell metastasis.The purpose was to discover active ingredients that both have anti-tumor and anti-thrombotic effects with potential clinical applications and without serious bleeding risk.Methods:（1）Usingβ-elimination combined with gel column chromatography method to separate and purify series of oligosaccharides（o FGs）.Then using NMR analysis combined with mass spectrometry data to confirm the structure of the oligosaccharides.（2）Homogeneous time resolved fluorescence resonance energy transfer（HTRF）method was used to analyze the effect of FGs on HPSE activity,and surface plasmon resonance（SPR）method and computer simulation technology were used to analyze its interaction with target protein mechanism.（3）Cell culture in vitro combined with Transwell chamber and Matrigel were used to analyze the effects of o FGs on the migration and invasion of tumor cells and vascular endothelial cells.（4）Matrigel tubule formation and chick embryo chorioallantoic models were studied the effects of o FGs on angiogenesis in vitro and in vivo,respectively.（5）Animal experimental model was used to study the effects of o FGs on tumor cell metastasis and colonization in mice.Results:（1）Preparation and structure analysis of o FGs:The native fucosylated glycosaminoglycan isolated from sea cucumber Holothuria fuscopunctata（n HG）was depolymerized by aβ-eliminative method.The resulting depolymerized product（d HG）was subjected to repeated gel column chromatography to obtain a series of purified oligosaccharides（o FGs）:hs5,hs8,hs11,hs14 and hs17.2D-NMR and ESI-MS spectral analysis showed that the general structure of o FGs is:L-Fuc_3S4S-α1,3-L-ΔU-α1,3-{D-Gal NAc_4S6S-β1,4-[L-Fuc_3S4S-α1,3-]-D-Glc UA-β1,3-}_n-D-Gal NAc_4S6S-β1,4-[L-Fuc_3S4S-α1,3-]-D-Glc UA-ol.（2）The effects of o FGs on HPSE activity:n HG,d HG and o FGs could inhibit HPSE activity in a dose-dependent manner.n HG,d HG and o FGs could bind to HPSE with high affinity.Molecular docking of computer-aided drug mechanism analysis showed that o FGs could bind the substrate binding site of HPSE to achieve its enzyme inhibitory activity.Further analysis showed that the side chain Fuc_3S4S of o FGs had an important contribution to the anti-HPSE activity of o FGs.（3）The effect of o FGs on migration and invasion of vascular endothelial cells:o FGs could inhibit HUVECs invasion and migration in vitro.The inhibitory activity increased with the increase in dp.In addition,at the same concentration,o FGs had a stronger inhibitory effect on HUVECs invasion than HUVECs migration.This result suggested that the inhibitory effect of o FGs on heparanase limited the ability of vascular endothelial cells to break through the tissue barrier.（4）The effects of o FGs on angiogenesis:o FGs could inhibit the formation of Matrigel tubules in a dose-dependent manner in vitro.The results of chick embryo chorioallantoic model showed that o FGs could significantly inhibit angiogenesis.As the degree of polymerization of o FGs increased,the inhibitory activity gradually increased.The above process involved the hydrolysis of HS by HPSE and the migration of HUVECs.These tests verified the inhibitory effect of o FGs on angiogenesis in vitro and in vivo,suggesting that it may affect the process of tumor metastasis.（5）The effects of o FGs on tumor cell migration,invasion and metastasis:o FGs could inhibit the invasion and migration of Luc-4T1 mammary carcinoma cells in vitro.And the activity trend was consistent with the effect of o FGs on HUVECs.The results of animal experiments showed that the bioluminescence detected in the lungs of mice was significantly reduced after hs17（0.2 mg/kg）treatment,indicating that it significantly inhibited the lung metastasis of Luc-4T1 cells,and the activities of hs11and hs8 were relatively weak.This result was consistent with the aforementioned activity trend of o FGs inhibiting HPSE,vascular endothelial cells and tumor cell invasion and angiogenesis,suggesting that o FGs inhibiting tumor metastasis in mice is closely related to these processes.Conclusion:（1）The native FG isolated from sea cucumber H.fuscopunctata possessed a regular chemical structure,with the backbone composition of alternating 4-linked Glc UA and 3-linked Gal NAc residues within disaccharide repeating units,and mono-Fuc_3S4S were branched at O-3 of Glc UA.d HG,prepared from n HG by a selective-bondβ-eliminative depolymerization method,consisted of a series of oligosaccharide homologues with relatively regular chemical structure.From d HG,the series of oligosaccharides（o FGs）:hs5,hs8,hs11,hs14 and hs17 were purified by repeated gel column chromatography.（2）Pharmacological studies have shown that o FGs had concentration dependent HPSE inhibitory activity,and the inhibitory activity strength was related to chain length（degree of polymerization）.Further analysis showed that o FGs exhibit inhibitory effect on vascular endothelial cell invasion that depend on HPSE inhibitory activity,and they could significantly inhibit angiogenesis in vitro and in vivo.Experimental animal model studies have shown that o FGs could significantly inhibit tumor cell metastasis.It is known that d HG and o FGs have anticoagulant and antithrombotic activities with low bleeding tendency.In conclusion,this series of oligosaccharides,as heparanase inhibitors,not only inhibits tumor metastasis but also prevents and inhibits tumor-related thrombosis with low bleeding risk,which may be beneficial for cancer patients.It provides a new treatment strategy for tumor patients with complications of venous thromboembolism.