Chandrasekar Nirmala, Mohanam Sanjeeva, Lakka S Sajani, Dinh Dzung H, Olivero William C, Gujrati Meena, Rao Jasti S
Program of Cancer Biology, University of Illinois College of Medicine at Peoria, Peoria, Illinois 61656, USA.
Clin Cancer Res. 2003 Jun;9(6):2342-9.
Tumor vasculature provides the infrastructure by which malignant tissue can be nourished; therefore, targeting angiogenesis may be an effective means of treating cancer. We showed previously that SNB19 glioblastoma cells modulate bovine retinal endothelial cells in cocultures to form capillary-like network structures, that matrix metalloproteinase-9 (MMP-9) expression is critical for endothelial morphogenesis, and that MMP-9 expression in glioblastoma cells is regulated by extracellular signal-regulated kinase-1 (ERK-1). In the present study, we investigated whether interfering with the activation of this mitogen-activated protein (MAP) kinase would repress MMP-9 synthesis and inhibit capillary formation.
Cocultures of bovine retinal endothelial and SNB19 cells were analyzed for MMP-9 secretion, and phospho- and total ERK levels. These cocultures were treated with PD98059, a specific inhibitor of MAP/ERK kinase 1, or transfected with dominant-negative ERK-1 mutant containing expression vector. Alterations in capillary-like structure formation, and actin cytoskeleton and secretion of vascular endothelial growth factor (VEGF), MMP-9, and tissue inhibitor of metalloproteinase-1 were determined by immunofluorescence, gelatin zymography, and Western blotting.
We found that inhibition of the ERK-1/2 pathway with PD98059 abrogated glial cell-mediated capillary formation by the endothelial cells and reduced the levels of MMP-9 in the coculture. Strikingly, the abrogation of MAP kinase signaling by a dominant-negative ERK-1 mutant inhibited glial-induced capillary network formation by reducing VEGF levels and MMP-9 activity and increasing the levels of tissue inhibitor of metalloproteinase-1. Inhibition of ERK activity also disrupted the formation of the actin cytoskeleton, a prerequisite for endothelial cell migration.
The mechanism underlying activation of ERK is involved in reorganization of the actin cytoskeleton, and induction of VEGF and MMP-9, thereby stimulating endothelial cell morphogenesis. These studies clearly provide experimental evidence that ERK inhibition diminishes glial-induced endothelial-cell morphogenesis; therefore, interfering with ERK signaling may be a viable approach to target angiogenesis.
肿瘤血管为恶性组织提供营养支持，因此，靶向血管生成可能是治疗癌症的有效手段。我们之前的研究表明，在共培养体系中，SNB19胶质母细胞瘤细胞可调节牛视网膜内皮细胞形成毛细血管样网络结构，基质金属蛋白酶-9（MMP-9）的表达对内皮细胞形态发生至关重要，且胶质母细胞瘤细胞中MMP-9的表达受细胞外信号调节激酶-1（ERK-1）调控。在本研究中，我们探究了干扰这种丝裂原活化蛋白（MAP）激酶的激活是否会抑制MMP-9的合成并抑制毛细血管形成。
分析牛视网膜内皮细胞与SNB19细胞共培养体系中MMP-9的分泌情况以及磷酸化和总ERK水平。这些共培养体系用MAP/ERK激酶1的特异性抑制剂PD98059处理，或用含有显性负性ERK-1突变体的表达载体转染。通过免疫荧光、明胶酶谱法和蛋白质印迹法测定毛细血管样结构形成、肌动蛋白细胞骨架的变化以及血管内皮生长因子（VEGF）、MMP-9和金属蛋白酶组织抑制剂-1的分泌情况。
我们发现，用PD98059抑制ERK-1/2信号通路可消除胶质细胞介导的内皮细胞毛细血管形成，并降低共培养体系中MMP-9的水平。引人注目的是，显性负性ERK-1突变体消除MAP激酶信号传导，通过降低VEGF水平和MMP-9活性以及增加金属蛋白酶组织抑制剂-1的水平，抑制胶质细胞诱导的毛细血管网络形成。抑制ERK活性还会破坏肌动蛋白细胞骨架的形成，而肌动蛋白细胞骨架的形成是内皮细胞迁移的前提条件。
ERK激活的潜在机制参与肌动蛋白细胞骨架的重组以及VEGF和MMP-9的诱导，从而刺激内皮细胞形态发生。这些研究明确提供了实验证据，表明抑制ERK可减少胶质细胞诱导的内皮细胞形态发生；因此，干扰ERK信号传导可能是靶向血管生成的可行方法。
