Modulation of heparanase expression in myeloma tumor cell lines by defibrotide: A novel mechanism of anti-tumor activity

3100 Introduction: Heparanase is an endo-β-D-glucoronidase that cleaves heparan sulphate, the main polysaccharide component of extracellular matrix and basement membrane. Elevated heparanase expression in humans has been correlated with advanced progression and metastasis of many tumor types, including multiple myeloma (MM), where its presence may be particularly important because MM expresses high levels of heparin sulphate proteoglycan syndecan-1 (CD138). Defibrotide (DF) is a novel polydisperse oligonucleotide with anti-thrombotic, thrombolytic, anti-ischemic, and anti-adhesive properties. Recently, DF has been shown to increase in vivo sensitivity of MM cells to conventional chemotherapy by modulating interactions of MM tumor cells with their local microenvironment. Moreover, early phase clinical studies in MM of DF in combination with melphalan, prednisone and thalidomide have been promising, with increased activity and minimal toxicity reported. We investigated whether DF is effective in regulating the expression and activity of heparanase in two MM cell lines, U266 and RPMI 8226, in vitro. Methods: Immunostaining analysis using Steptavidin-FITC two step method, revealed a high expression of heparanase in U266 and RPMI 8226 cell lines. This result was confirmed by flow cytometric analysis. The potential effect of DF to regulate heparanase gene expression was evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from the two lines of MM cells treated with DF or saline (as a control). Results showed a significant downregulation of heparanase gene expression by DF (at 150µg/ml) after 24h of treatment (p Conclusion: These data suggest DF not only has an effect in downregulation of heparanase gene expression, but also decreases its enzymatic activity in MM cell lines. Thus, DF may suppress tumor-associated angiogenesis and tumor dissemination through suppression of heparanase with a subsequent reduction in the release of stores of growth factors from extra-cellular matrix. This in part may explain DF’s anti-MM activity both in vitro and in vivo. Further studies utilising patient derived MM cells, and murine models to better define this mechanism of action are underway.