Development of Novel Single-Stranded Nucleic Acid Aptamers against the Pro-Angiogenic and Metastatic Enzyme Heparanase (HPSE1)

Abstract

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 (7×10 7 to 8×10 7 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.

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

“…ELISAs and fluorescence titrations separated the two longer aptamers as showing higher affinity and recognition of heparanase in placental cells, whereas placental tissue staining favoured ‘1.5 M long’. This was confirmed in a Matrigel invasion assay using ovarian carcinoma cells previously shown to require heparanase for invasion [46] . Two additional aptamers, termed ‘pink’ and ‘yellow’ were selected against the linker peptide sequence of pro-heparanase, as these could have a function in inhibiting the formation of the active heterodimer enzyme, by blocking peptide protease excision.…”

“…The cytotoxicity of the selected aptamers on HSC-3 cells was studied, to verify that the inhibition of invasion observed in the organotypic model was a result of the inhibition of the heparanase, as previously verified [46] and not cell cytotoxicity. The MTS assay was performed over 72 hrs, with a single addition of the aptamer in the beginning of the assay, and measurements over the period intervals of 24, 48 and 72 hrs.…”

Effects of ionizing radiation and HPSE1 inhibition on the invasion of oral tongue carcinoma cells on human extracellular matrices in vitro

“…For comparison, an unirradiated control group (0 Gy) was included in each experiment. Cells were treated with 0.7 mM/10 µg/ml of an antibody against heparanase (Anti-HPSE1) [26], 1 µM 1.5 M short protected heparanase aptamer (inverted 3′-base dT5) [28] and 10 µg/ml of Erbitux (Merck Serono).…”

Targeting Heparanase in Cancer: Inhibition by Synthetic, Chemically Modified, and Natural Compounds

“…The aptamer binds to heparanase with high affinity and inhibits its enzymatic activity. The enzyme recognition by the aptamer is in some cases superior over polyclonal anti-heparanase antibody (Simmons et al., 2012). Furthermore, a short anti-heparanase aptamer (30 bases) inhibits tissue invasion, does not exhibit cytotoxicity on oral carcinoma cells, and is stable in human serum without significant binding to serum proteins suggesting, the potential application of aptamers as therapeutic agents (Simmons et al., 2014, Vayrynen et al., 2018).…”