Anti-angiogenetic effect of arenobufagin in vitro and in vivo

This study is to investigate the anti-angiogenetic effect of arenobufagin in vitro and in vivo.  The anti-proliferation effect of arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay.  Cell morphological changes of LOVO and HUVECs after arenobufagin treatment were observed by microscopy.  Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner.  Furthermore, it was obviously observed that the subcytotoxic concentration of arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs.  Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of arenobufagin in vivo.  Arenobufagin significantly suppressed the angiogenesis of CAM.  Cell cycle analysis demonstrated that G₂/M phase was arrested and the sub-G₁ peak appeared with the increase of arenobufagin concentration.  PI/Annexin V double staining assay further demonstrated that arenobufagin could induce apoptosis in a dose- and time-dependent manner.  Mitochondrial potential collapse detected by flow cytometric analysis was increased after arenobufagin treatment.  It also observed that PARP was cleaved to p85 active form by Western blotting.  Taken together, arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.