Peptides inhibiting heparanase procoagulant activity significantly reduce tumour growth and vascularisation in a mouse model

Summary

Heparanase is implicated in angiogenesis and tumour progression. We previously demonstrated that heparanase might also affect the haemostatic system in a non-enzymatic manner. It forms a complex and enhances the activity of the blood coagulation initiator tissue factor (TF). Peptides that we generated from TF pathway inhibitor (TFPI)-2, which inhibit heparanase procoagulant activity, were recently demonstrated to attenuate inflammation in a sepsis mouse model. The present study was designated to explore peptides effects on tumour growth and vascularisation. Cell lines of mouse melanoma (B16), mouse breast cancer (EMT-6), and human breast cancer (MDA-231) were injected subcutaneously to mice. Inhibitory peptides 5, 6 and 7 were injected subcutaneously in the area opposite to the tumour side. In the three tumour cell lines, peptides 5, 6 and 7 inhibited tumour growth and vascularisation in a dose-dependent manner, reaching a 2/3 reduction compared to control tumours (p<0.001). Additionally, a survival advantage (p<0.05) and reduced plasma thrombin-antithrombin complex (p<0.05) were observed in the treatment groups. Peptides delayed tumour relapse by six days and inhibited relapsed tumour size (p<0.001). In vitro, peptides did not inhibit tumour cell proliferation, migration or heparanase degradation of heparan sulfate chains, but significantly decreased tube formation. In conclusion, peptides inhibiting heparanase procoagulant activity significantly reduced tumour growth, vascularisation, and relapse. The procoagulant domain in heparanase protein may play a role in tumour growth, suggesting a new mechanism of coagulation system involvement in cancer.

Keywords

Heparanase - tumour - inhibitory peptides - tissue factor

• References

• 1 Freeman C, Parish CR.. Human platelet heparanase: purification, characterisation and catalytic activity. Biochem J 1998; 330: 1341-1350.
• 2 Pikas DS, Li JP, Vlodavsky I. et al. Substrate specificity of heparanases from human hepatoma and platelets. J Biol Chem 1998; 273: 18770-18777.
• 3 Parish CR, Freeman C, Hulett MD.. Heparanase: a key enzyme involved in cell invasion. Biochim Biophys Acta 2001; 1471: M99-108.
• 4 Vlodavsky I, Friedmann Y.. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 2001; 108: 341-347.
• 5 Gingis-Velitski S, Zetser A, Flugelman MY. et al Heparanase induces endothelial cell migration via protein kinase B/Akt activation. J Biol Chem 2004; 279: 23536-23541.
• 6 Doviner V, Maly B, Kaplan V. et al. Spatial and temporal heparanase expression in colon mucosa throughout the adenoma-carcinoma sequence. Mod Pathol 2006; 19: 878-888.
• 7 Zetser A, Bashenko Y, Miao HQ. et al. Heparanase affects adhesive and tumour-igenic potential of human glioma cells. Cancer Res 2003; 63: 7733-7741.
• 8 Cohen-Kaplan V, Naroditsky I, Zetser A. et al Heparanase induces VEGF C and facilitates tumour lymphangiogenesis. Int J Cancer 2008; 123: 2566-2573.
• 9 Nadir Y, Brenner B, Zetser A. et al. Heparanase induces tissue factor expression in vascular endothelial and cancer cells. J Thromb Haemost 2006; 04: 2443-2451.
• 10 Nadir Y, Brenner B, Gingis-Velitski S. et al. Heparanase induces tissue factor pathway inhibitor expression and extracellular accumulation in endothelial and tumour cells. Thromb Haemost 2008; 99: 133-141.
• 11 Nadir Y, Brenner B, Fux L. et al. Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII. Haematologica 2010; 95: 1927-1934.
• 12 Axelman E, Henig I, Crispel Y. et al. Novel peptides that inhibit heparanase activation of the coagulation system. Thromb Haemost 2014; 112: 466-477.
• 13 Zetser A, Levy-Adam F, Kaplan V. et al. Processing and activation of latent he-paranase occurs in lysosomes. J Cell Sci 2004; 117: 2249-2258.
• 14 Nadir Y, Kenig Y, Drugan A. et al. An assay to evaluate heparanase procoagulant activity. Thromb Res 2011; 128: e3-8.
• 15 Hammond E, Li CP, Ferro V.. Development of a colorimetric assay for hepara-nase activity suitable for kinetic analysis and inhibitor screening. Anal Biochem 2010; 396: 112-116.
• 16 McKenzie E, Young K, Hircock M. et al. Biochemical characterisation of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells. Biochem J 2003; 373: 423-435.
• 17 Smorenburg SM, Van Noorden CJ.. The complex effects of heparins on cancer progression and metastasis in experimental studies. Pharmacol Rev 2001; 53: 93-105.
• 18 Klerk CP, Smorenburg SM, Otten HM. et al. The effect of low molecular weight heparin on survival in patients with advanced malignancy. J Clin Oncol 2005; 23: 2130-2135.
• 19 Lee AY, Rickles FR, Julian JA. et al. Randomized comparison of low molecular weight heparin and coumarin derivatives on the survival of patients with cancer and venous thromboembolism. J Clin Oncol 2005; 23: 2123-2129.
• 20 Nadir Y, Sarig G, Axelman E. et al. Heparanase procoagulant activity is elevated and predicts survival in non-small cell lung cancer patients. Thromb Res 2014; 134: 639-642.