Studies On The Mechanism Of Human Endostatin-induced Endothelial Cell Apoptosis

Endostatin is a proteolytic fragment of the C-terminal of collagen XVIII. Since its discovery in 1997, there are more than 1000 papers published on endostatin. It has been found that endostatin can inhibit angiogenesis in vitro and in vivo. Moreover, endostatin has potent therapeutic effect as one of the most powerful endogenous angiogenesis inhibitors in clinical studies.Though the pro-apoptotic activity of endostatin is predictable to its anti-angiogenic function, the exact mechanism remains controversial. In the present study, we elucidate the mechanism of endostatin-induced endothelial cell apoptosis. Our results show that endostatin induces apoptosis in human microvascular endothelial cells (HMECs). In addition, endostatin induces cytochrome c release and caspase-9 activation, which initiate that the apoptosis process is involved in mitochondria. Further ATP production, mitochondrial membrane potential, ROS release and tubule formation assays show that endostatin promotes the mitochondrial permeability transition pore (mPTP) opening via voltage dependent anion channel 1 (VDAC1), a major component of mitochondrial outer membrane. Knocking down VDAC1 attenuates endostatin-induced apoptosis, while over-expression of VDAC1 enhances the sensitivity of endothelial cells to endostatin. These results indicate VDAC1 play an important role in regulating endostatin-induced endothelial cell apoptosis. Moreover, we reveal that endostatin induces the reduction of hexokinase 2, which in turn promotes VDAC1 accumulation and the subsequent apoptosis. We also show that endostatin induces VDAC1 phosphorylation, which may inhibit the degradation of intracellular VDAC1. Further mPTP opening and caspase-3 activation assays indicate that two serine residues of VDAC1, Ser-12 and Ser-103, can modulate VDAC1 protein level and thus the sensitivity to apoptosis stimuli. Our findings are novel and significant, and will have a strong impact to the understanding of the molecular mechanisms of angiogenesis inhibitors such as endostatin. Furthermore, the identification of VDAC1 and HK2 in angiogenesis regulation provides more potential targets for anti-cancer therapeutics.