Cholesterol sequestration by nystatin enhances the uptake and activity of endostatin in endothelium via regulating distinct endocytic pathways.

Specific internalization of endostatin into endothelial cells has been proved to be important for its biologic functions. However, the mechanism of endostatin internalization still remains elusive. In this study, we report for the first time that both caveolae/lipid rafts and clathrin-coated pits are involved in endostatin internalization. Inhibition of either the caveolae pathway or the clathrin pathway with the use of chemical inhibitors, small interfering RNAs, or dominant-negative mutants alters endostatin internalization in vitro. Intriguingly, cholesterol sequestration by nystatin, a polyene antifungal drug, significantly enhances endostatin uptake by endothelial cells through switching endostatin internalization predominantly to the clathrin-mediated pathway. Nystatin-enhanced internalization of endostatin also increases its inhibitory effects on endothelial cell tube formation and migration. More importantly, combined treatment with nystatin and endostatin selectively enhances endostatin uptake and biodistribution in tumor blood vessels and tumor tissues but not in normal tissues of tumor-bearing mice, ultimately resulting in elevated antiangiogenic and antitumor efficacies of endostatin in vivo. Taken together, our data show a novel mechanism of endostatin internalization and support the potential application of enhancing the uptake and therapeutic efficacy of endostatin via regulating distinct endocytic pathways with cholesterol-sequestering agents.