1. Abstract. We synthesized a novel set of non-toxic compounds that kill proliferating and nonproliferating glioma cells residing in perinecrotic tumor microenvironments. These hypoxic glioma populations have increased resistance to conventional radiation therapy coupled with alkylating agents such that cancer recurs in greater than 90% of individuals with high grade gliomas. The design and syntheses of these novel compounds were derived from our recent demonstration that urokinase plasminogen activator (uPA) becomes enzymatically activated intracellularly in hypoxic and acidotic high grade human gliomas. Amiloride is an FDA-approved drug that selectively inhibits uPA, but not other proteases. We synthesized and evaluated a series of amiloride-based compounds that inhibit either total or extracellular uPA activity in human glioma cells. Interestingly, forms of the drugs excluded from the cell interior and act only on surface uPA are cytostatic. Significantly, congeners that permeate the plasma membrane and also inhibit intracellular uPA trigger glioma cell death, yet do not affect normal brain cell types. Our pharmacological findings are consistent with RNA interference experiments demonstrating that inhibition of uPA mRNA initiates apoptosis of glioma cells by unknown cellular mechanisms. These observations point to the novel notion that infiltrative, hypoxic tumor cells can become reliant on intracellular uPA for their survival. Since intracellular uPA activation is not observed in normal tissue cell types or in normal adult brain, these observations suggest that intracellular uPA may represent an important drug target of malignant glioma cells that survive and recur in hypoxic-ischemic microenvironments. The selective anti-glioma cytotoxicities and absence of CNS toxicities of our lead compounds in rat orthotopic glioma xenografts are encouraging given the extremely poor outcome of most individuals having high grade gliomas.
Aim 1 will investigate the in vivo efficacies of the lead compounds in a NOD/SCID-gamma murine intracerebral glioma xenograft model. We will investigate the efficacies of our lead compounds on {1} primary glial tumor growth kinetics and on {2} recurrent glioma following radiation therapy and temozolamide (TMZ) treatment. Because uPA inhibition impedes glioma neovascularization, we will also investigate {3} the potential synergism of our lead compounds to prevent or retard the previously described disseminated intracerebral growth of VEGF-depleted glioma cells in mice.
Aim 2 will utilize these novel small molecule uPA inhibitors to identify the intracellular mechanisms contributing to their selective anti-glioma cytotoxicity. We will also target total uPA using RNA interference and compare resultant glioma cell death pathways to those identified using our small molecule inhibitors.
The incidence of high-grade gliomas is 5-7/100,000, with 10-15,000 new cases in the US per year. These are the most common form of adult-onset primary brain tumor, with a mean life expectancy of less than 24 months at the time of diagnosis and with a cancer recurrence rate of more than 90% in the high- grade forms (Anticancer Res. 2007 Jul-Aug;27(4C):2993-6). Currently, radiation therapy, followied by alkylating agents, BCNU or temozolamide (TMZ) is associated with a small advantage in grade 3 (7%) but not grade 4 gliomas (Health Technol Assess 45(11) 1-222 (2007). It is recognized that new therapeutic agents must be developed to treat these brain cancers. High-grade malignant gliomas are highly infiltrative with poorly defined surgical borders preventing surgical resection for cure. These glioma cells survive in avascular tumor regions and are resistant to conventional radiation therapy and treatment with either BCNU or TMZ. Recently, we identified a relative non-toxic set of compounds that kill proliferating and non-proliferating hypoxic-ischemic glioma cells by a novel intracellular mechanism. We propose to synthesize and evaluate the efficacies of additional compounds using both human glioma cell lines and animal models of intracranial human glial tumors. Additionally, this research will utilize this chemical and biological information to better understand the novel cellular mechanisms by which these potential drug agents kill infiltrative, hypoxic glioma cells. The goal is to create a new set of therapeutic agents, which to be used in conjunction with conventional therapies to prevent or retard glioma recurrence.
