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


METHODS
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.
George
Joseph
J
Department of Cancer Biology, University of Illinois, College of Medicine at Peoria, Peoria, Illinois, USA.
Gondi
Christopher S
CS
Dinh
Dzung H
DH
Gujrati
Meena
M
Rao
Jasti S
JS
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D005909:Glioblastoma / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D020133:Reverse Transcriptase Polymerase Chain Reaction
D006023:Glycoproteins; C021240:tissue-factor-pathway inhibitor 2; D020169:Caspases