Restoration of tissue factor pathway inhibitor-2 in a human glioblastoma cell line triggers caspase-mediated pathway and apoptosis

Clin Cancer Res. 2007 Jun 15;13(12):3507-17. doi: 10.1158/1078-0432.CCR-06-3023.


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-alpha, 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-alpha 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenoviridae / genetics
  • Apoptosis / physiology*
  • Blotting, Western
  • Brain Neoplasms / genetics
  • Brain Neoplasms / metabolism*
  • Caspases / metabolism
  • Cell Line, Tumor
  • Flow Cytometry
  • Fluorescent Antibody Technique
  • Gene Expression
  • Genetic Therapy / methods*
  • Genetic Vectors
  • Glioblastoma / genetics
  • Glioblastoma / metabolism*
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Humans
  • In Situ Nick-End Labeling
  • Reverse Transcriptase Polymerase Chain Reaction


  • Glycoproteins
  • tissue-factor-pathway inhibitor 2
  • Caspases