Restoration of Tissue Factor Pathway Inhibitor-2 in a Human Glioblastoma Cell Line Triggers Caspase-Mediated Pathway and Apoptosis

Abstract

Purpose: The induction of apoptotic pathways in cancer cells offers a novel and potentially useful approach to improve patient responses to conventional chemotherapy. Tissue factor pathway inhibitor-2 (TFPI-2) is a protease inhibitor that is abundant in the extracellular matrix and highly expressed in noninvasive cells but absent or undetectable in highly invasive human glioblastoma cells. Experimental Design: Using a recombinant adeno-associated viral vector carrying human TFPI-2 cDNA, we stably expressed TFPI-2 in U-251 cells, a highly invasive human glioblastoma cell line. Our previous studies showed that restoration of TFPI-2 in glioblastomas effectively prevents cell proliferation, angiogenesis, and tumor invasion. In this study, we determined whether TFPI-2 restoration could induce apoptosis through the caspase-mediated signaling pathway. Results: The results from nuclear chromatin staining, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay, and fluorescence-activated cell sorting analysis showed increased apoptosis in U-251 cells after restoration of TFPI-2. Caspase-9 and caspase-3 activity assays showed increased activity, indicating enhanced apoptosis. Immunofluorescence for cleaved caspase-9 and caspase-3 depicted increased expression and colocalization of both molecules. Western blot analysis showed increased transcriptional activities of Fas ligand, tumor necrosis factor-α, Bax, Fas-associated death domain, and tumor necrosis factor receptor 1–associated death domain as well as elevated levels of cleaved caspases and poly(ADP-ribose) polymerase. Semiquantitative reverse transcription-PCR depicted increased expression of tumor necrosis factor-α and Fas ligand and the related death domains tumor necrosis factor receptor 1–associated death domain and Fas-associated death domain. Conclusions: Taken together, these results show that restoration of TFPI-2 activates both intrinsic and extrinsic caspase-mediated, proapoptotic signaling pathways and induces apoptosis in U-251 cells. Furthermore, our study suggests that recombinant adeno-associated viral vector–mediated gene expression offers a novel tool for cancer gene therapy.