Inhibition of Glioma Growth by a Novel Molecule

描述(申请人提供)：本研究的目的是了解rK5诱导微血管内皮细胞(MVEC)和恶性胶质瘤细胞凋亡的机制，以及辐射使这些细胞对rK5增敏的机制，并确定辐射是否使脑内恶性胶质瘤小鼠模型的脑MVEC和肿瘤细胞对rK5诱导的凋亡敏感。RK5(也称为Abbott-828)是纤溶酶原第五个kringle结构域的重组形式。我们的初步数据表明，rK5，特别是当与先前的照射联合使用时，具有杀死增殖的人脑MVEC的潜力，并在小鼠模型中缩小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)介导的其细胞表面受体GRP78的内化起作用。我们已经证实GRP78在人恶性星形细胞瘤活检组织中的MVEC和胶质瘤细胞上表达(5/5)。我们假设辐射将使脑MVEC和恶性胶质瘤细胞对rK5治疗敏感，从而通过一种需要GRP78和LRP1的机制诱导细胞凋亡，并且这种策略将在体内抑制这些肿瘤的生长。我们将：(1)确定rK5杀伤脑MVEC和胶质瘤细胞的最佳条件并利用抑制剂和siRNA技术鉴定所利用的凋亡途径，利用阻断抗体和siRNA确定rK5是否通过GRP78发挥作用，并通过分析磷酸化状态、小分子抑制剂以及siRNA下调的作用来确定p38 MAP激酶信号在促凋亡信号中的作用。(2)通过siRNA、免疫共沉淀法和免疫电子显微镜检测LRP1介导的GRP78内化在致敏中的作用，以及LRP1胞浆尾部的磷酸化作用。(3)然后，我们将在同基因、免疫活性的脑内小鼠模型和人异种脑内移植瘤裸鼠模型中，确定rK5治疗加/减照射对体内恶性胶质瘤生长、侵袭和血管生成的影响。相关性：这项工作将直接导致治疗恶性星形细胞肿瘤的新治疗策略，这将代表着这种毁灭性疾病的治疗方面的重大进步。

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.