Inhibition of Glioma Growth by a Novel Molecule

描述（由申请人提供）：本研究的目的是了解rK 5诱导微血管内皮细胞（MvEC）和恶性胶质瘤细胞凋亡的机制，放射线使这些细胞对rK 5敏感的机制，并确定放射线是否使恶性胶质瘤脑内小鼠模型中的脑MvEC和肿瘤细胞对rK 5诱导的凋亡敏感。rK 5（也称为Abbott-828）是纤溶酶原第五kringle结构域的重组形式。我们的初步数据表明，rK 5，特别是当与先前的辐射结合使用时，有可能杀死增殖的人脑MvEC，并减少小鼠模型中恶性胶质瘤肿瘤的大小。它似乎至少部分地通过低密度脂蛋白受体相关蛋白（LRPI）介导的细胞表面受体GRP 78的内化来起作用。我们已经证实GRP 78在人恶性星形细胞肿瘤活检中的MvEC和神经胶质瘤细胞上表达（5/5）。我们假设，照射将敏感脑MvEC和恶性胶质瘤细胞治疗rK 5导致诱导凋亡的机制，需要GRP 78和LRP 1，这种策略将抑制这些肿瘤在体内的生长。我们将：（1）确定rK 5杀死脑MvEC和神经胶质瘤细胞的最佳条件，并使用抑制剂和siRNA技术鉴定所利用的凋亡途径，使用阻断抗体和siRNA确定rK 5是否通过GRP 78起作用，并通过分析磷酸化状态、小分子抑制剂，以及siRNA下调的效果。(2)使用siRNA、免疫共沉淀和免疫电镜确定LRP 1介导的GRP 78内化在致敏中的作用，以及LRP 1胞质尾的磷酸化的作用。(3)然后，我们将确定rK 5治疗，加/减照射，对恶性胶质瘤生长，侵袭和血管生成在体内同基因的，免疫活性，脑内小鼠模型，并在人异种移植脑内裸鼠模型。相关性：这项工作将直接导致一种新的治疗策略，用于治疗恶性星形细胞肿瘤，这将代表治疗这种毁灭性疾病的重大进展。

DESCRIPTION (provided by applicant): 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.