Despite many therapeutic strategies for glioblastoma multiforme, the survival rate in patients with this aggressive cerebral malignancy remains poor. These gliomas are highly resistant even to combinations of different therapies such as surgery, radiotherapy, and chemotherapy. A more effective therapy for these gliomas is to restore specific tumor suppressor genes and to downregulate genes that are overexpressed because such treatment is directed against the basic neoplastic mechanisms rather than the tumor proper. Because multiple genes are altered in these tumors, an efficient combination of genes should effectively inhibit tumor growth and invasion and activate the various pathways involved in programmed cell death. Specific hypotheses to be tested in this project are: 1) whether the administration of an antisense message that downregulates the urokinase-type plasminogen activator-receptor (uPAR)-mediated proteolytic cascade involved in invasion, in combination with a tumor suppressor gone using adenovirus vectors, has additive or synergistic anticancer effects on gliomas invasion and tumor growth; and 2) whether inhibition of integrin levels in gliomas by a bicistronic construct regulates cell spreading, invasion, tumor growth, and other signaling pathways.
The specific aims are: 1) Evaluate the effects of a bicistronic construct (Ad-uPAR-p16), with the antisense Upar gene and the sense tumor suppressor gene p16, on glioma cell growth, attachment, migration, and invasion in vitro, la) Determine the effect of the bicistronic Ad-uPAR-p16 construct on uPAR and p16 levels in glioma cells; lb) Compare the effect of the bicistronic Ad-uPAR-p16 construct on glioma cell growth, adhesion, and migration with that of mock, Ad-CMV, Ad-uPAR (antisense), and Ad-p16 (sense) constructs; and 1c) Investigate the effect of the bicistronic Ad-uPAR-p16 construct on the invasive behavior of human glioma cells in vitro models (Matrigel/spheroids). 2) Determine the in vivo efficacy (Inhibition of invasive characteristics and tumorigenicity) and toxicity of the bicistronic Ad-uPAR-p16 gene construct. 2a) Compare the ability of the bicistronic Ad-uPAR-p16 construct to that of the single-gene (Ad-uPAR or Ad-p16) construct to inhibit the invasion and growth of human glioma cell lines injected subcutaneously and intracerebrally in nude mice; and 2b) evaluate and compare the toxicity of the single-gene adenovirus constructs (Ad-uPAR and Ad-p16) with that of the bicistronic gene construct (Ad-uPAR-p16) given as intracerebral injections in Fischer or Wistar rats. 3) Assess the effect of the bicistronic Ad-uPAR-p16 construct on the expression of integrins (particularly avb3) and molecules involved in signaling pathways mediated by integrins. 3a) Assess the effect of the bicistronic Ad-uPAR-p16 construct on avb33 and other integrins that mediate the spreading of glioma cells; 3b) determine the effect of the bicistronic Ad-uPAR-p16 construct on the signaling pathway molecules mediated by integrins (FAK, MAPK); and 3c) identify those molecules that are involved in the absence of integrin-mediated adhesion (JNK, BAX, and Bcl2). In summary, the present study will examine anti-cancer strategies consisting of the administration of gene combinations that generate cytostatic and cytotoxic actions by acting on distinct mechanisms that inhibit tumor growth and invasion and cause cell death. ? ?
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