Human heparanase nuclear localization and enzymatic activity.

In previous studies, we have demonstrated that human heparanase (endo-beta-D-glucuronidase) is localized primarily in a perinuclear pattern within lysosomes and late endosomes, and occasionally may be surface associated and secreted. The presence of two potential nuclear localization sequences in human heparanase, led us to investigate heparanase translocation into the nucleus and subsequent degradation of nuclear heparan sulfate. Applying cell fractionation, Western blot analysis, determination of heparanase activity and confocal microscopy, we identified heparanase within the nuclei of human glioma and breast carcinoma cells and estimated its amount to be about 7% of the cytosolic enzyme. Our results indicate that nuclear heparanase colocalizes with nuclear heparan sulfate and is enzymaticaly active. Moreover, following uptake of latent 65 kDa heparanase by cells that do not express the enzyme, an active 50 kDa heparanase was detected in the cell nucleus, capable of degrading both nuclear and extracellular matrix-derived heparan sulfate. Immunohistochemical examination of human squamous cell carcinoma specimens revealed a prominent granular staining of heparanase within the nuclei of the epithelial tumor cells vs no nuclear staining in the adjacent stromal cells. Taken together, it appears that heparanase is translocated into the cell nucleus where it may degrade the nuclear heparan sulfate and thereby affect nuclear functions that are thought to be regulated by heparan sulfate. Nuclear localization of heparanase suggests that the enzyme may fulfill nontraditional functions (ie, regulation of gene expression and signal transduction) apart of its well-documented involvement in cancer metastasis, angiogenesis and inflammation.

A novel mechanism for the inhibition of NF-kappaB activation in vascular endothelial cells by natural antioxidants.

The activation of Nuclear Factor kappa B (NF-kappaB) in vascular endothelial cells, in response to biochemical or biomechanical stimuli, is associated with vascular pathologies such as atherosclerosis. The present manuscript studies the ability of the natural antioxidant-pomegranate wine (PW), to inhibit tumor necrosis factor alpha (TNF-alpha) or shear stress-mediated-NF-kappaB activation in vascular endothelial cells and compares it to that of red wine (RW) and N-acetyl cysteine (NAC). PW and RW act as potent antioxidants in vascular endothelial cells, inhibiting the oxidation of 2',7'-dichloroflurescin diacetate in TNF-alpha treated cells. PW (as well as RW and NAC) acted as potent inhibitors of NF-kappaB activation (migration into the nucleus and DNA binding activity) in vascular endothelial cells. Nevertheless, PW and NAC failed to inhibit TNF-a induced serine 32/36 phosphorylation and IkappaBalpha degradation. Surprisingly, these antioxidants alone induced enhanced IkappaB serine phosphorylation, which was not accompanied by IkappaBalpha degradation, or NF-kappaB nuclear translocation. This phosphorylation did not involve serine 32/36. Furthermore, we show for the first time that NAC inhibited TNF-alpha mediated phosphorylation of p65 (ser536), whereas PW had no effect on this phosphorylation. Thus, natural antioxidants may serve as potent NF-kappaB inhibitors in vascular endothelial cells, yet act through unique and divergent pathways.