Bufalin enhances anti-angiogenic effect of sorafenib via AKT/VEGF signaling

Sorafenib mainly exerts its anti-hepatoma effect by inhibiting tumor angiogenesis. However, its curative effect is limited. Thus, application of drugs which could augment its anti-angiogenic effect is necessary. Bufalin has been reported to possess anticancer properties. In the present study, we investigated the synergistic anti-angiogenic effect of sorafenib combined with bufalin. The enhanced anti-angiogenic effect of the combination treatment was firstly assessed in nude mice bearing human HCC intradermal tumors. In addition, we found that proliferation was significantly inhibited and the morphology was obviously changed in the combination-treated human umbilical vein endothelial cells (HUVEC) at 48 h of treatment. In addition, the combination treatment was found to suppress vessel formation potently as proved in the tube formation, chick chorioallantoic membrane and rat aortic rings. Mechanistically, HUVEC incubated with the combination treatment showed increased apoptosis, decreased migration, which might account for its capacity against angiogenesis. Vascular endothelial cells have been reported to secrete cytokines to affect angiogenesis. Therefore, suspensions from HUVECs with different treatments were collected as conditioned medium (CM). The combination-treated CM significantly inhibited the migration of HUVEC and blood vessel formation in vitro. Importantly, multiple cytokines associated with angiogenesis were downregulated in the combination-treated CM. Furthermore, we verified that the secretion of VEGF was downregulated and revealed that the reduction might be regulated through the inhibition of the PI3K/AKT pathway. Taken together, our findings demonstrated for the first time that bufalin can enhance anti-angiogenic effect of sorafenib via modulating the AKT/VEGF signaling pathway.