Daphnetin inhibits TNF-α and VEGF-induced angiogenesis through inhibition of the IKKs/IκBα/NF-κB, Src/FAK/ERK1/2 and Akt signalling pathways.
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.
Division of Advanced Pharmacology, Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology (BIT), Mesra, Ranchi, India.
Abhishek
A
Kumar
Government Pharmacy Institute, Department of Health, Family Welfare and Medical Education, Government of Jharkhand, Bariatu, Ranchi, India.
Priyashree
P
Sunita
Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology University, Mesra, Ranchi, India.
Shivesh
S
Jha
Division of Advanced Pharmacology, Department of Pharmaceutical Sciences & Technology, Birla Institute of Technology (BIT), Mesra, Ranchi, India.
Shakti Prasad
SP
Pattanayak
D043925:Angiogenesis Inducing Agents / Q000494:pharmacology; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000187:drug effects / Q000502:physiology; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D008297:Male; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000502:physiology; D009924:Organ Culture Techniques; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D014468:Umbelliferones / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D043925:Angiogenesis Inducing Agents; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D014468:Umbelliferones; D042461:Vascular Endothelial Growth Factor A; D051417:Focal Adhesion Kinase 1; C496051:Ptk2 protein, mouse; D051057:Proto-Oncogene Proteins c-akt; C039952:daphnetin
TNF-α; VEGF; anti-angiogenesis; daphnetin