Anti-Heparanase Aptamers as Potential Diagnostic and Therapeutic Agents for Oral Cancer

Peer reviewe

Development of novel single-stranded nucleic acid aptamers against the pro-Angiogenic and metastatic enzyme heparanase (HPSE1)

Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis. This leads to increased survival and further proliferation of the tumour. Aptamers are single or double stranded oligonucleotides that recognise specific small molecules, peptides, proteins, or even cells or tissues and have shown great potential over the years as diagnostic and therapeutic agents in anticancer treatment. For the first time, single stranded DNA aptamers were successfully generated against the active heterodimer form of heparanase using a modified SELEX protocol, and eluted based on increasing affinity for the target. Sandwich ELISA assays showed recognition of heparanase by the aptamers at a site distinct from that of a polyclonal HPSE1 antibody. The binding affinities of aptamer to immobilised enzyme were high (7×107 to 8×107 M−1) as measured by fluorescence spectroscopy. Immunohistochemistry and immunofluorescence studies demonstrated that the aptamers were able to recognise heparanase with staining comparable or in some cases superior to that of the HPSE1 antibody control. Finally, matrigel assay demonstrated that aptamers were able to inhibit heparanase. This study provides clear proof of principle concept that nucleic acid aptamers can be generated against heparanase. These reagents may serve as useful tools to explore the functional role of the enzyme and in the future development of diagnostic assays or therapeutic reagents

Reactive oxygen species mediate high glucose-induced heparanase-1 production and heparan sulphate proteoglycan degradation in human and rat endothelial cells: a potential role in the pathogenesis of atherosclerosis.

Item does not contain fulltextAIMS/HYPOTHESIS: The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions. METHODS: HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined. RESULTS: High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions. CONCLUSIONS/INTERPRETATION: Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis.1 juni 201