Pseudolaric acid B inhibits angiogenesis and reduces hypoxia-inducible factor 1α by promoting proteasome-mediated degradation
Pseudolaric acid B inhibits angiogenesis and reduces hypoxia-inducible factor 1α by promoting proteasome-mediated degradation
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DOI：
10.1158/1078-0432.ccr-04-0951
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发表时间：
2004-12-15
影响因子：
11.5
通讯作者：
Jian, D
中科院分区：
文献类型：
--
作者：
Li, MH;Miao, ZH;Jian, D
目的：从松科植物日本松树根皮中分离得到的天然二萜类化合物太子参B具有较强的抗真菌和抗妊娠作用，可能与血管生成密切相关。本研究旨在检测其对血管生成的抑制作用、对肿瘤细胞分泌血管内皮生长因子(VEGF)的影响以及可能的作用机制。实验设计：通过人脐血管内皮细胞增殖、迁移和管形成试验以及绒毛尿囊膜试验评价其对血管生成的抑制作用。采用ELISA法、逆转录-聚合酶链式反应(RT-PCR)和免疫印迹法(Western Blotting)检测低氧条件下培养的人脐静脉内皮细胞(MDA-MB-468)中血管内皮生长因子(VEGF)的表达，并探讨其可能的作用机制。结果：PAb具有明显的抗血管生成活性，其抑制作用呈浓度依赖关系，可抑制VEGF刺激的人脐静脉内皮细胞的增殖和迁移，抑制胎牛血清刺激的人脐静脉内皮细胞的管状形成。此外，在绒毛尿囊膜实验中，PAB(10nmoL/卵)显著抑制体内血管生成。另一方面，PAB通过减少HIF-1α蛋白表达，抑制缺氧诱导的MDA-MB-468细胞分泌血管内皮生长因子。使用LY294002和U0126的进一步分析表明，低氧诱导因子1(HIF-1)α蛋白水平的增加高度依赖于低氧MDA-MB-468细胞中磷脂酰肌醇3‘-激酶和p42/p44丝裂原活化蛋白激酶的活性。但PAB处理不影响Akt和Erk的活性(磷酸化)形式。值得注意的是，选择性蛋白酶体抑制剂MG-132完全逆转了PAB处理的MDA-MB-468细胞中HIF-1α蛋白的减少。结论：PAb通过促进蛋白酶体介导的降解减少了MDA-MB-468细胞中的HIF-1α蛋白，从而表现出直接抑制内皮细胞生长和消除肿瘤细胞旁分泌刺激血管生成的双重抗血管生成活性，具有潜在的临床意义。
Purpose: Pseudolaric acid B (PAB), the naturally occurring diterpenoid isolated from the root bark of Pseudolarix kaempferi Gordon tree (Pinaceae), possesses potent antifungal and pregnancy-terminating effects that may be tightly associated with angiogenesis. This study was to examine its angiogenic inhibition, impact on vascular endothelial growth factor (VEGF) secretion from tumor cells and the possible mechanism of action.Experimental Design: Angiogenesis inhibition was assessed by the human umbilical vascular endothelial cell proliferation, migration, and tube-formation assays, as well as the chorioallantoic membrane assay. ELISA, reverse transcription-PCR, and Western blotting analyses were performed to examine VEGF protein secretion, mRNA expression, and the possible mechanism in hypoxic MDA-MB-468 cells.Results: PAB displayed potent in vitro antiangiogenic Activity shown by inhibiting VEGF-stimulated proliferation and migration and fetal bovine serum-stimulated tube formation of human umbilical vascular endothelial cells in a concentration-dependent manner. Moreover, PAB (10 nmol per egg) significantly suppressed in vivo angiogenesis in the chorioallantoic membrane assay. On the other hand, PAB abrogated hypoxia-induced VEGF secretion from MDA-MB-468 cells via reducing HIF-1alpha protein. Additional analyses using LY294002 and U0126 indicated that the increase in hypoxia-inducible factor 1 (HIF-1)alpha protein level was highly dependent on phosphatidylinositol 3'-kinase and p42/p44 mitogen-activated protein kinase activities in hypoxic MDA-MB-468 cells. However, PAB treatment did not affect the active (phosphorylated) forms of Akt and Erk. Interestingly, the selective proteasome inhibitor MG-132 completely reversed the reduction of HIF-1alpha protein in the PAB-treated MDA-MB-468 cells.Conclusions: PAB displays the dual antiangiogenic activities of directly inhibiting endothelial cells and abrogating paracrine stimulation of VEGF from tumor cells due to reducing HIF-1alpha protein by promoting its proteasome-mediated degradation in MDA-MB-468 cells, which has potential clinical relevance.