Novel Anti-angiogenic Peptide Derived from Canstatin Induces Apoptosis In Vitro and In Vivo

Canstatin, the NC1 domain of the alpha 2 chain of collagen IV, prevents tumor growth through angiogenesis inhibition and apoptosis induction. N-terminal 1-89 amino acid fragment of canstatin induces apoptosis much higher than the C-terminal fragment. Recently, we demonstrated that the amino acids 78-86 of canstatin, so-called Cans, have more anti-migration, anti-tube formation, and anti-tumor activities than other collagen IV derived peptides. Here, we evaluated HUVEC, MCF10A, and L929 cell viability, the percentage of apoptotic cells by Annexin V-FITC/PI staining, caspase-3 activity, Bcl-2, and caspase-8 gene expression using RT-qPCR in endothelial cells, and Bax and Bcl-2 expression in tumors by immunohistochemistry to investigate the apoptotic effect of Cans. Results showed that the peptide reduced the percentage of viable HUVE cells with EC50 of 21 mu M and was not toxic for normal cell lines. 30 and 50 mu M of Cans induced 34.6% and 50.7% early and late apoptosis in HUVECs compared to 16.5% in control. In addition, caspase-3 activity was amplified up to threefold compared to the untreated cells. Cans down-regulated Bcl-2 and caspase-8 gene expression. This result may show that this peptide acts through the intrinsic pathway and cannot affect the extrinsic pathway of apoptosis. However, this hypothesis requires more investigation. Besides, Bcl-2 reduction and Bax elevation in tumor sections indicated that this peptide could stimulate apoptosis in vivo. In conclusion, we showed that the short canstatin peptide induces apoptosis in endothelial and tumor cells as one of its anti-angiogenic and anti-tumor mechanisms.