Despite the combined use of surgery, radiotherapy, and chemotherapy to treat glioblastoma multiforme, the survival rate for patients with this aggressive, highly resistant cerebral malignancy remains poor. Recent compelling evidence from cellular and molecular studies of the mechanisms underlying the invasiveness of human gliomas has implicated the matrix metalloproteinase (MMP-9) in the invasion process. We propose here to identify the mechanisms that lead to the overexpression of the gene that encodes this enzyme in human glioblastoma and to inhibit the growth of these tumors by using adenoviral constructs carrying antisense message to MMP-9.
The Specific Aims are as follows:
Specific Aim 1 : Determine the effect of the Ad-MMP-9 antisense construct on glioma cell growth, attachment, migration and invasion in in vitro models. (la): Determine the effect of the Ad-MMP-9 antisense construct on the levels of MMP-9 and other proteases in glioma cells. (lb): Determine and compare the effect of the Ad-MMP-9 antisense construct on glioma cell growth, adhesion, and migration with those of mock and Ad-CMV. (lc): Investigate the effects of the Ad-MMP- 9 antisense construct on the invasive behavior of human glioma cells in in vitro models (Boyden chamber/spheroid assays).
Specific Aim 2 : Determine the in vivo efficacy and toxicity of the Ad-MMP-9 antisense construct. (2a): Determine and compare the effects of the Ad-MMP-9 antisense construct with those of mock or the Ad-CMV construct in inhibiting the invasion and growth of human glioma cell lines injected subcutaneously and intracerebrally in nude mice. (2b): Determine MMP-9 activity during glioma invasion and tumor growth in in vitro and in vivo imaging models using a near-infrared probe specific for MMP-9, and correlate these results with traditional immunohistochemical analysis, in situ hybridization and in vitro enzyme assays. (2c): Evaluate the toxicity of the Ad-MMP-9 antisense construct with that of the Ad-CMV construct given as intracerebral injections in Fischer/Wistar rats.
Specific Aim 3 : Determine the molecular mechanisms that regulate cerebral angiogenesis in relation to the inhibition of MMP-9 in co-cultures of endothelial and glioma cells in both in vitro and in vivo models. (3a): Determine the effect of stable antisense MMP-9 transfectants and infection of Ad-MMP-9 antisense constructs during the formation of capillary-like structures in co-cultures of microvascular endothelial cells with antisense MMP-9 stable cells or glioblastoma cells infected with Ad-MMP-9 antisense construct. (3b): Determine the effects of antisense MMP-9 stable transfectants and Ad-MMP-9 infection on tumor angiogenesis in vivo. We anticipate that these results will substantially augment our understanding of how this overexpressed molecule is inhibited; thus, the information gained should help in developing new therapeutic approaches to treat glioblastomas.
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