Methyl selenium regulation of Angiogenic switch Mech

描述（申请人提供）：血管生成是实体癌发生和发展的必要条件，是癌症化学预防的逻辑目标。该资助申请将测试甲基硒醇或相关单甲基硒代谢物通过多个过程抑制血管内皮血管生成反应的假设：a)抑制有丝分裂信号导致G1细胞周期停滞；B)通过caspase驱动执行诱导细胞凋亡；c)抑制降解组织基质所需的基质金属蛋白酶(MMP)-2的表达。我们将使用生化、激酶药理学抑制剂和活性激酶突变体转染方法，研究甲基硒抑制磷脂酰肌醇3-激酶（PI3K）信号级联在血管细胞G1阻滞中的中介作用；2)明确甲基硒诱导血管内皮细胞凋亡的途径，特别是哪些半胱天冬酶被激活，线粒体和细胞色素C在其激活中的作用，以及PI3K和/或有丝分裂激活蛋白激酶（MAPKJERK1/2）抑制是否介导凋亡信号；3)明确甲基硒靶向MMP-2表达的哪个阶段，并阐明其中的生化/分子机制。特别是，我们将测试甲基硒醇是否会破坏MMP-2和模型蛋白/肽分子中的二硫键。人脐静脉内皮细胞及其端粒化不朽克隆将用于细胞培养模型来实现这些目标。通过完成前两个目标，我们期望验证PI3K和/或MAPK1/2通路在甲基硒抑制内皮细胞循环以及导致caspase驱动的凋亡信号传导中的中介作用。这可以为进一步研究甲基硒如何与这些靶蛋白发生反应/相互作用从而产生生化和细胞效应奠定基础。此外，阐明MMP-2在Aim 3中的表达调控机制，特别是二硫化物破坏作为其降解的潜在原因，可能对硒抑制自分泌/旁分泌介导的肿瘤血管生成、生长、存活和侵袭性具有更广泛的机制意义。

DESCRIPTION (provided by applicant): Angiogenesis is obligatory for the genesis and progression of solid cancers and is a logical target for cancer chemoprevention. This grant application will test the hypothesis that methylselenol or related monomethylselenium metabolites inhibit vascular endothelial angiogenic responses by multiple processes: a) inhibiting mitogenic signaling leading to G1 cell cycle arrest; b) inducing apoptosis through caspase-driven execution; c) inhibiting the expression of matrix metalloproteinase (MMP)-2 that is required for degrading tissue matrix. We will 1) investigate the mediator role of a methylselenium-inhibition of the phosphatidylinositol 3- kinase (PI3K) signaling cascade in vascular cell G1 arrest using biochemical, kinase pharmacologic inhibitor and active kinase mutant transfection approaches; 2) define the pathways through which methy-seLenium induces vascular endothelial cell apoptosis, especially what caspases are activated and any role of the mitochondria and cytochrome C in their activation as well as whether PI3K and/or mitogenic activated protein kinase (MAPKJERK1/2) inhibition mediate apoptosis signaling; 3) define what stage(s) of MMP-2 expression is targeted by methylselenium and elucidate the biochemical/molecular mechanisms involved. In particular, we will test whether methylselenol disrupts disulfide bonds in MMP-2 and model protein/peptide molecules. Human umbilical vein endothelial cells and their telomerized immortal clones will be used in cell culture models to accomplish these aims. By accomplishing the first 2 aims, we expect to validate a mediator role of PI3K and/or MAPK1/2 pathways for methylselenium inhibition of endothelial cell cycling as well as for apoptosis signaling leading to caspase-driven execution. This can lay the foundation for future work to investigate how methylselenium reacts/interacts with these target proteins to bring about the biochemical and cellular effects. In addition, elucidating the mechanisms of MMP-2 expression regulation in Aim 3 and especially disulfide disruption as a potential cause of its degradation may have broader mechanistic implications for inhibiting autocrine/paracrine-mediated tumor angiogenesis, growth, survival and invasiveness by selenium.