The goals of this study are to understand the mechanism by which rK5 induces apoptosis of microvascular endothelial cells (MvEC) and of malignant glioma cells, the mechanism by which radiation sensitizes these cells to rK5, and to determine whether radiation sensitizes brain MvEC and tumor cells in intracerebral mouse models of malignant glioma to rK5-induced apoptosis. rK5 (also known as Abbott-828) is a recombinant form of the fifth kringle domain of plasminogen. Our preliminary data indicate that rK5, especially when used in conjunction with prior irradiation, has the potential to kill proliferating human brain MvEC, and to reduce the size of malignant glioma tumors in a mouse model. It appears to act, at least in part, through the low density lipoprotein receptor-related protein (LRPI)-mediated internalization of its cell surface receptor GRP78. We have confirmed that GRP78 is expressed on MvEC and glioma cells in human malignant astrocytic tumor biopsies (5 of 5). We hypothesize that irradiation will sensitize brain MvEC and malignant glioma cells to treatment with rK5 resulting in induction of apoptosis through a mechanism that requires GRP78 and LRP1, and that this strategy will inhibit the growth of these tumors in vivo. We will: (1) Determine the optimal conditions for rK5 killing of brain MvEC and glioma cells and identify the apoptotic pathways utilized using inhibitors and siRNA technology, determine whether the rK5 acts through GRP78 using blocking antibodies and siRNA, and determine the role of p38 MAP kinase signaling in the pro- apoptotic signaling through analysis of the phosphorylation status, small molecule inhibitors, and the effect of downregulation with siRNA. (2) Determine the role of LRP1-mediated internalization of GRP78 in the sensitization using siRNA, co-immunoprecipitation and immuno-electron microscopy, and the role of phosphorylation of the cytoplasmic tail of LRP1. (3) We will then determine the effect of rK5 treatment, plus/minus irradiation, on malignant glioma growth, invasion and angiogenesis in vivo in a syngeneic, immune-competent, intracerebral mouse model, and in a human xenograft intracerebral nude mouse model. Relevance: This work should lead directly to a novel therapeutic strategy for the treatment of malignant astrocytic tumors, which would represent a major advance in the treatment of this devastating disease.
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