Inhibition of the arachidonic acid metabolism blocks endothelial cell migration and induces apoptosis.

Previous studies have demonstrated that inhibitors of the arachidonic acid metabolism block migration and sensitise human glioma cells to treatment inducing apoptosis. This paradigm may provide a new concept for anti-invasive treatment strategies targeting invasive glioma cells. However, the effect of such treatment on other cellular elements in glial tumours such as endothelial cells is unknown. In this study we have analysed the expression of metabolites of the arachidonic acid pathway in endothelial cells in vitro and in vivo and we have assessed the influence of inhibitors of this pathway on motility, capillary like tube formation, and apoptosis in human endothelial cells. Human endothelial cells (HUVEC) in culture showed expression for thromboxane synthase and both isoforms of cyclo-oxygenase, COX-1 and COX-2. Immunostaining demonstrated low levels of COX-1 expression in capillaries and larger vessels of normal brain and moderately elevated levels of this enzyme in small vessels of brain tumours of various grades. Both thromboxane synthase and COX-2 expression was limited to endothelial cells found in anaplastic gliomas and glioblastomas. Thromboxane synthase inhibitors strongly decreased endothelial cell migration in HUVEC in vitro and capillary like tube formation was strongly inhibited by the compound at a similar dose range. The non-selective cyclo-oxygenase inhibitor ASA and the selective COX-2 inhibitor sulindac only had a minor effect on endothelial cell migration, however, the COX-2 inhibitor sulindac showed a synergistic effect with the thromboxane synthase inhibitor. Thromboxane synthase inhibitors induced apoptosis in endothelial cells as demonstrated by intracellular histone-complexed DNA fragmentation. These data suggest that inhibitors of thromboxane synthase influence migration and apoptosis in both human glioma cells and human endothelial cells. An anti-invasive treatment strategy using this class of compounds may therefore not only sensitise glioma cells to conventional treatments inducing apoptosis but may also be supported by an anti-angiogenic effect.

Cyclo-oxygenase inhibitors and thromboxane synthase inhibitors differentially regulate migration arrest, growth inhibition and apoptosis in human glioma cells.

We have previously identified thromboxane synthase as an important regulator of glioma cell migration. Inhibitors of this enzyme abrogate cell motility and induce apoptosis. However, the formation rate of thromboxanes is indirectly dependent on the activity of cyclo-oxygenase, which represents the rate-limiting step in the synthesis of prostaglandins and thromboxanes. In this study we have analyzed the expression of COX-1 and COX-2 in glioma cell lines and biopsies of glial tumors. In normal glia no expression of both COX isoforms was present, however, reactive astrocytes and glial tumors of all grades demonstrated expression of both COX-1 and COX-2. In contrast to inhibitors of thromboxane synthase, selective and non-selective cyclo-oxygenase inhibitors did not block cell motility. Specific COX-2 inhibitors resulted in growth inhibition and induction of intracellular DNA fragmentation indicative of apoptotic cell death. Treatment of glioma cells with thromboxane synthase inhibitors had a synergistic effect on induction of apoptosis by camptothecin, whereas COX inhibitors had not. Furthermore, combined treatment using COX-2 inhibitors and specific thromboxane synthase inhibitors did not show a synergistic increase of apoptosis. These data indicate that COX inhibitors and thromboxane synthase inhibitors influence apoptosis in glioma cells through different pathways. We hypothesize that, in contrast to the COX-2 inhibitors, thromboxane synthase inhibitors block the invasive phenotype of glioma cells and therefore increase the pro-apoptotic disposition of the cells and increase the susceptibility to induced apoptosis. This effect may be independent of prostaglandin synthesis controlled by cyclo-oxygenases.