Moreno María J, Ball Marguerite, Andrade Moises F, McDermid Angela, Stanimirovic Danica B
Cerebrovascular Research Group, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
Glia. 2006 Jun;53(8):845-57. doi: 10.1002/glia.20345.
cAMP has been shown to reverse the transformed phenotype of various cancer cells. Human glioblastoma U87MG cells exposed to 500 microM dB-cAMP for 6 days showed reduced proliferation, attenuated invasiveness, and inability to induce angiogenic responses in human brain endothelial cells (HBECs) grown in Matrigeltrade mark. VEGF was the principal mediator of angiogenic actions of U87MG conditioned media (CM), since VEGF neutralizing antibody completely inhibited U87MG-induced angiogenic responses and no detectable levels of IGF, bFGF, and PlGF were found in U87MG CM. VEGF release was induced ( approximately 20%) in dB-cAMP-treated U87MG cells, suggesting a simultaneous induction of anti-angiogenic mediators. Down-stream effectors of dB-cAMP actions in U87MG were investigated by microarray gene expression analysis. Detected increases in differentiation genes, staniocalcin-1 and Wnt-5a, and angiogenesis-related genes, PAI-1, SPARC, IGFBP-4, IGFBP-7, PAPP-A, and PRSS-11 in dB-cAMP-treated U87MG cells were validated by real-time PCR, Western blot, and/or ELISA. A subsequent series of experiments identified IGFBP-4 as the principal anti-angiogenic mediator secreted by glioblastoma cells in response to dB-cAMP. Human recombinant IGFBP-4 inhibited the angiogenic response of HBEC induced by U87MG CM, whereas anti-human IGFBP-4 antibody restored the pro-angiogenic activity of dB-cAMP-treated U87MG CM. Since neither U87MG nor HBEC cells secreted detectable levels of IGF-I, and there are no known cellular IGFBP-4 receptors, the anti-angiogenic effect of IGFBP-4 was likely IGF-I-independent and indirect. IGFBP-4 also antagonized angiogenic effects of VEGF(165), PlGF, and bFGF, and reduced U87MG colony formation in soft-agar. IGFBP-4 is a novel dB-cAMP-induced anti-angiogenic and anti-tumorigenic mediator that may be a promising candidate for glioblastoma therapy.
环磷酸腺苷（cAMP）已被证明可逆转多种癌细胞的转化表型。暴露于500微摩尔二丁酰环磷腺苷（dB - cAMP）6天的人胶质母细胞瘤U87MG细胞，其增殖能力降低，侵袭性减弱，且无法在基质胶中生长的人脑内皮细胞（HBECs）中诱导血管生成反应。血管内皮生长因子（VEGF）是U87MG条件培养基（CM）血管生成作用的主要介质，因为VEGF中和抗体完全抑制了U87MG诱导的血管生成反应，并且在U87MG CM中未检测到胰岛素样生长因子（IGF）、碱性成纤维细胞生长因子（bFGF）和胎盘生长因子（PlGF）的水平。在经dB - cAMP处理的U87MG细胞中，VEGF释放被诱导（约20%），这表明同时诱导了抗血管生成介质。通过微阵列基因表达分析研究了U87MG中dB - cAMP作用的下游效应器。经实时定量聚合酶链反应（PCR）、蛋白质免疫印迹法（Western blot）和/或酶联免疫吸附测定（ELISA）验证，在经dB - cAMP处理的U87MG细胞中，检测到分化相关基因（钙结合蛋白-1和Wnt - 5a）以及血管生成相关基因（纤溶酶原激活物抑制剂-1、富含半胱氨酸的酸性分泌蛋白、胰岛素样生长因子结合蛋白-4、胰岛素样生长因子结合蛋白-7、妊娠相关血浆蛋白-A和丝氨酸蛋白酶-11）增加。随后的一系列实验确定胰岛素样生长因子结合蛋白-4是胶质母细胞瘤细胞响应dB - cAMP分泌的主要抗血管生成介质。重组人胰岛素样生长因子结合蛋白-4抑制了U87MG CM诱导的HBEC血管生成反应，而抗人胰岛素样生长因子结合蛋白-4抗体恢复了经dB - cAMP处理的U87MG CM的促血管生成活性。由于U87MG细胞和HBEC细胞均未分泌可检测水平的胰岛素样生长因子-I，且不存在已知的细胞胰岛素样生长因子结合蛋白-4受体，胰岛素样生长因子结合蛋白-4的抗血管生成作用可能不依赖于胰岛素样生长因子-I且是间接的。胰岛素样生长因子结合蛋白-4还拮抗了VEGF(165)、PlGF和bFGF的血管生成作用，并减少了U87MG在软琼脂中的集落形成。胰岛素样生长因子结合蛋白-4是一种新型的由dB - cAMP诱导的抗血管生成和抗肿瘤介质，可能是胶质母细胞瘤治疗的一个有前景的候选药物。
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