Interfering with UDP-GlcNAc Metabolism and Heparan Sulfate Expression Using a Sugar Analogue Reduces Angiogenesis

Abstract

Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analog of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analog was activated intracellularly into UDP-4-deoxy-GlcNAc and HS expression was inhibited up to ~96% (IC50 = 16 μM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analog, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Micro-injection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis which hampers pro-angiogenic signaling and neo-vessel formation.

The glycocalyx and its significance in human medicine

“…Van Wijk et al [126] tested 11 sugar analogues for their capacity to interfere with GAG chain elongation or GAG sulphation and discovered that peracetylated 6-fluoro-GalNAc was particularly effective in inhibiting the biosynthesis of sulphated GAGs. This is in line with data showing that GAG biosynthesis is inhibited by two other peracetylated sugar analogues: 4-deoxyGlcNAc [127] and 4F-GlcNAc [128].…”

‘Immunosequencing’ of heparan sulfate from human cell lines and rat kidney: the (GlcNS6S-IdoA2S)3 motif, recognized by antibody NS4F5, is located towards the non-reducing end

“…Heparinase I and III from Flavobacterium heparinum were purchased from IBEX Pharmaceuticals. The synthesis of non-peracetylated and peracetylated 4-deoxy-GlcNAc was as described previously in [9] and [10] respectively.…”

“…SKOV3 ovarian carcinoma cells were cultured for 3 days in the presence of 3-200 μM peracetylated 4-deoxy-GlcNAc. Staining for HS and analysis by flow cytometry was performed as described previously [10]. MFI (mean fluorescence intensity) was plotted as a percentage of inhibition, and IC 50 values including 95 % confidence intervals were calculated in GraphPad Prism using the non-linear regression model log(inhibitor) against the normalized response.…”

“…Staining for HS and analysis by flow cytometry was performed as described previously [10]. MFI was plotted as a percentage of undigested controls.…”

“…Two approaches were taken: one based on inhibition of HS synthesis followed by resynthesis, and the other based on limited enzymatic digestion of HS. In the first approach, cells were incubated with 4-deoxy-GlcNAc, a sugar analogue that inhibits HS synthesis and reduces HS chain length [8][9][10]. Next, cells were allowed to resynthesize HS and the sequence of the reappearance of antibody-defined HS structures was established.…”