Thromboxane synthase inhibitors induce apoptosis in migration-arrested glioma cells.

Abstract

Because of the wide dissemination of malignant glioma cells by the time that malignant glioma is diagnosed, anti-invasive strategies that are designed to limit their further spread may be of little value unless mechanisms of the invasive cascade can be used to render invasive cells susceptible to cytoreductive treatments. We recently determined that elevated thromboxane synthase gene expression and enzymatic activity are associated with a highly migratory phenotype of glioma cells in vitro and that specific inhibitors of this enzyme block cell migration. Interference with this inherent phenotype of malignant gliomas also affects glioma cell proliferation and apoptosis. To study the effect of thromboxane synthase inhibitors on motility, metabolic activity, and cell death, we used five human glioma cell lines, four glioblastoma-derived, low-passage cell cultures, normal human astrocytes, and fibroblasts. Motility was measured in a monolayer migration assay. Caspase activation as an early event in apoptotic cell death was assessed using a caspase 3 cleavage assay. Intracellular deoxyribonucleic acid (DNA) fragmentation was detected by enzyme-linked immunosorbent assay quantification of histone-complexed DNA. Subsequent cell death was scored by trypan blue exclusion. In this study, we demonstrate that the treatment of human glioma cells with the specific thromboxane synthase inhibitor furegrelate leads first to caspase activation (detectable 6 h after treatment), then to DNA fragmentation (24-48 h after treatment) and subsequent cell death. Caspase inhibitors abrogate this effect. Furthermore, the inhibition of thromboxane synthase by furegrelate increases cells' susceptibility to the induction of DNA fragmentation by camptothecin, etoposide, N,N'-bis(2-chloroethyl)-N-nitrosourea, and anti-CD95 antibodies. No induction of apoptosis was observed in normal astrocytes and fibroblasts. These data indicate that thromboxane synthase may represent a vortex of divergent signaling cascades that regulate motility and apoptosis in glioma cells. This paradigm may offer a novel perspective in the treatment of patients with malignant gliomas.