Synergistic anti-hepatoma effect of bufalin combined with sorafenib via mediating the tumor vascular microenvironment by targeting mTOR/VEGF signaling

Sorafenib inhibits tumor growth primarily by inhibiting vessel formation, however, its efficacy requires improvement, therefore, the development of strategies which augment its antiangiogenic effect are of primary concern. Bufalin inhibits tumor cell proliferation and metastasis, and induces apoptosis. In our previous study, it was demonstrated that the antiangiogenic effect of sorafenib was improved by bufalin in human umbilical vein endothelial cells (HUVECs). However, whether bufalin synergizes with sorafenib by affecting the tumor vascular microenvironment remains to be elucidated. In the present study, it was found that hepatocellular carcinoma (HCC) cell proliferation was inhibited by either bufalin or sorafenib following incubation for 24 h, and the inhibition was enhanced upon treatment with a combination of the two. Conditioned medium (CM), comprising supernatant from HCC cells was collected from each of the treatment groups. The migration and tubule formation were suppressed the most in the combination-CM treated HUVECs. The secretion of vascular endothelial growth factor (VEGF) was decreased in HCC cells treated with the combination-CM, as determined by an angiogenesis array, enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The inhibition of tube formation in HUVECs treated with the combination-CM was reversed by incubation with VEGF. The in vivo experiments demonstrated that subcutaneous HCC cell tumors from mice treated with the combination treatment expressed the lowest levels of VEGF, as evidenced by immunohistochemistry and ELISA. Additionally, the level of phosphorylated mechanistic target of rapamycin (mTOR) was reduced in HUVECs pretreated with the phosphoinositide 3-kinase inhibitor PI103. Furthermore, the migration of HCC cells and HUVEC tube formation were attenuated by PI103 pretreatment. In conclusion, the results revealed a synergistic anti-hepatoma effect of bufalin combined with sorafenib via affecting the tumor vascular microenvironment by targeting mTOR/VEGF signaling.