Daphnetin inhibits TNF-α and VEGF-induced angiogenesis through inhibition of the IKKs/IκBα/NF-κB, Src/FAK/ERK1/2 and Akt signalling pathways

Clin Exp Pharmacol Physiol. 2016 Oct;43(10):939-50. doi: 10.1111/1440-1681.12608.

Abstract

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti-angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti-arthritic and anti-inflammatory. The present study was performed to investigate the anti-angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti-angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)-induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)-induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF-induced HUVECs by extracting the mRNA and protein levels using RT-qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor-κB signalling together including TNF-α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF-κB-p65 protein. Furthermore, western blotting revealed that DAP significantly down-regulated the VEGF-induced signalling such as c-Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF-stimulated HUVECs by the caspase-3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi-targeted medication for the anti-angiogenesis and cancer therapy.

Keywords: TNF-α; VEGF; anti-angiogenesis; daphnetin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Angiogenesis Inducing Agents / pharmacology
  • Animals
  • Cell Proliferation / drug effects
  • Cell Proliferation / physiology
  • Cells, Cultured
  • Chick Embryo
  • Chorioallantoic Membrane / drug effects
  • Chorioallantoic Membrane / physiology
  • Focal Adhesion Kinase 1 / antagonists & inhibitors
  • Focal Adhesion Kinase 1 / physiology
  • Human Umbilical Vein Endothelial Cells / drug effects
  • Human Umbilical Vein Endothelial Cells / physiology
  • Humans
  • MAP Kinase Signaling System / drug effects*
  • MAP Kinase Signaling System / physiology
  • Male
  • NF-kappa B / antagonists & inhibitors*
  • NF-kappa B / physiology
  • Organ Culture Techniques
  • Proto-Oncogene Proteins c-akt / antagonists & inhibitors*
  • Proto-Oncogene Proteins c-akt / physiology
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Tumor Necrosis Factor-alpha / antagonists & inhibitors*
  • Tumor Necrosis Factor-alpha / pharmacology
  • Umbelliferones / pharmacology*
  • Vascular Endothelial Growth Factor A / antagonists & inhibitors*
  • Vascular Endothelial Growth Factor A / pharmacology

Substances

  • Angiogenesis Inducing Agents
  • NF-kappa B
  • Tumor Necrosis Factor-alpha
  • Umbelliferones
  • Vascular Endothelial Growth Factor A
  • Focal Adhesion Kinase 1
  • Ptk2 protein, mouse
  • Proto-Oncogene Proteins c-akt
  • daphnetin