Pseudolaric acid B inhibits angiogenesis and reduces hypoxia-inducible factor 1alpha by promoting proteasome-mediated degradation.
OBJECTIVE
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.


METHODS
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.
Li
Mei-Hong
MH
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.
Miao
Ze-Hong
ZH
Tan
Wen-Fu
WF
Yue
Jian-Min
JM
Zhang
Chao
C
Lin
Li-Ping
LP
Zhang
Xiong-Wen
XW
Ding
Jian
J
D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000187:drug effects / Q000378:metabolism; D004224:Diterpenes / Q000627:therapeutic use*; D004365:Drugs, Chinese Herbal / Q000627:therapeutic use*; D004531:Eggs / Q000032:analysis; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000517:prevention & control*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D028201:Pinaceae / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000378:metabolism*; D014471:Umbilical Veins / Q000187:drug effects / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D004224:Diterpenes; D004365:Drugs, Chinese Herbal; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C058391:pseudolaric acid B; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex