Antitumor And Immunomodulatory Activities Of Heparan Sulfate Mimetics From Escherichia Coli K5 Capsular Polysaccharide

Heparan sulfate（HS） is a member of the glycosaminoglycan family of polysaccharides that are a ubiquitous class of sulfated polysaccharides. HS is present in almost all tissues on the cell surfaces and extracellular matrix in the form of HS proteoglycans（HSPGs）, in which its polysaccharide chains are covalently attached to the core proteins. It is involved in diverse biological processes, such as development, tissue homeostasis, angiogenesis, inflammation, and immunity. Recent findings help to clarify the roles of HS in tumor cell proliferation, metastasis, tumor angiogenesis, and terminal differentiation, identifying novel therapeutic targets and HS-based therapeutic strategies. HS-based drugs can act in several ways by activating（agonists） or inactivating（antagonists） protein-based receptors, competing with endogenous HS and/or inhibiting HS biosynthesis. Escherichia coli K5 capsular polysaccharide（K5PS） has been used as the starting material to synthesize HS analogs with various biological properties because of its similar carbohydrate backbone as the HS biosynthetic precursor. Chemically and/or enzymatically sulfated K5 PSs possess antiproliferative, antiangiogenic, antimetastatic, and antiviral activities. However, neither the antitumor and immunomodulatory activities of the sulfated K5 PSs nor their functional mechanism is clearly elucidated. To clarify the antitumor and immunoregulatory properties of sulfated K5 PSs, this study examined the effect of different concentrations and sulfation patterns of sulfated K5 PSs on Murine B16 melanoma cells, macrophages, and spleen lymphocytes in vitro. It aimed to elucidate the structure-activity relationships among the sulfated K5 PSs, and further illustrate the possible underlying mechanism. The main results obtained in this dissertation were described as follows:（1） K5 capsular polysaccharide and its chemically modified K5 PSs, including O-sulfated K5 PS with the degree of substitution（DS） of 0.10 or 0.33（K5-OS₁, K5-OS₂）, N-sulfated K5 PS with DS of 0.66（K5-NS）, N,O-sulfated K5 PS with DS of 1.04（K5-NS,OS） were synthesized. The modification by N-deacetylation/N-sulfation elevated the levels of △UA-GlcNS and △UA-GlcNS6 S, confirming the structure of K5-NS,OS. K5-OS₂ was more heterogeneous in O-positions, with 20% of its sequence being represented by △UA-GlcNAc6 S disaccharide units, and 10% of the disaccharide △UA2S-GlcNAc6 S.（2） K5-NS,OS significantly inhibited the proliferation, adhesion, and migration of B16 cells. K5 PS and K5-NS presented no clear influence on the B16 cells compared with the control group. These results suggested that K5-NS,OS had remarkable effects on the proliferation, adhesion and migration of B16 melanoma cells, which indicated that the existence of O-sulfate group was required for the effects and the existence of O- and N-sulfate groups was more effective in exhibiting anti-cancer activity.（3） K5-NS,OS could modulate the SDF-1/CXCL12-induced biological effects by binding CXCL12 and thereby blocking both CXCL12-induced CXCR4 receptor activation, internalization and downstream effects such as Akt and MAPK signalling pathways in vitro.（4） Among the four sulfated K5 PSs, K5-OS₂ strongly modulated the immune response by stimulating TNF-α and IL-1β in the mouse macrophage RAW 264.7 cell line. K5-OS₂ increased splenocyte proliferation and enhanced both murine T- and B-cell proliferation and activation. High sulfation in O-position of K5 polysaccharide may be required for their immunomodulatory activities. We conclude that K5-OS₂ may be useful as an immune stimulant and a promising agent with immune-stimulating effects, which may also be indirectly involved in antitumor activity through improving immunologic function.（5） The NO production induced by K5-OS₂ was reduced by anti-TLR4 mAb, NF-κB inhibitors or MAPK inhibitors, including PD98059（ERK inhibitor）, SP600125（JNK inhibitor）, and SB203580（p38 inhibitor）. Western blotting analysis showed that K5-OS₂ suppressed NF-κB activation, and the phosphorylation of ERK, JNK and p38. These results suggested that K5-OS₂ exerted an immunoregulatory property by up-regulating the expression of TLR4 in RAW264.7 cells, and thereby promoting TLR4 mediated the activation of NF-κB and MAPK signaling.