Serpin-derived peptides are antiangiogenic and suppress breast tumor xenograft growth

Abstract

Angiogenesis is the formation of neovasculature from preexisting microvessels. Several endogenous proteins regulate the balance of vessel formation and regression in the body including pigment epithelium-derived factor (PEDF), which has been shown to be antiangiogenic and to suppress tumor growth. Using sequence homology and bioinformatics, we previously identified several peptide sequences homologous to an active region of PEDF existing in multiple proteins in the human proteome. These short 11-mer peptides are found in a DEAH box helicase protein, CKIP-1 and caspase 10, and show similar activity in altering endothelial cell adhesion, migration and inducing apoptosis.We tested the peptide derived from DEAH box helicase protein in a triple-negative MDA-MB-231 breast orthotopic xenograft model in severe combined immunodeficient mice and show significant tumor suppression.

Figure 1

Serpin sequences and their activities in cell adhesion and migration assays. (A) Bioinformatics methodology showing novel sequences in the human proteome termed SP6001, SP6023, and SP6024. We generated a scrambled sequence based on the original SP6001 peptide for test in adhesion assays. (B) SP6001, SP6023, and SP6024 promote endothelial cell adhesion. Adhesion in the presence of SP6001 is significant over the complete medium control at 100 µM. (C) Oris Pro endothelial cell migration assay for HUVECs. Cells were plated for 2 hours in the presence of SP6001 or complete medium around stoppers, which were then removed and cells were then allowed to migrate. After 20 hours, cells were stained with calcein AM and fluorescence intensity measured with a Victor V plate reader. (D) Oris Pro endothelial migration assay testing SP6001SCR. (E) Western blot analysis showing that SP6001 promotes endothelial cell adhesion by sustaining phosphorylation of FAK over time in the presence of VEGF for 24 hours versus untreated cells and VEGF-treated cells. The scrambled peptide did not show this same increase. (F) pFAK quantification for SP6001 (*P < .05) and (G) SP6001SCR.

Figure 2

Serpin-derived peptides induce apoptosis in HUVECs. (A) Caspase 3/7 assay showing an increase in caspase 3/7 for peptide-treated cells in the presence of bFGF and VEGF (30/10 ng/ml) after 48 hours. Values are normalized to 1 (bFGF/VEGF control). **Significant compared to bFGF/VEGF controls for P < .01. (B) Western blot analysis showing JNK-1 stress kinase phosphorylation increases on SP6001 administration over SP6001SCR at 4 hours (1, 10, and 30 µM SP6001 or SP6001SCR treatment). Quantification of pJNK-1 increase for SP6001 (C) and SP6001SCR (D).

Figure 3

MDA-MB-231 breast xenograft assay. SP6001 significantly suppresses breast tumor growth by two methods of administration—intraperitoneal (i.p.) (A) and subcutaneous (s.c.) (B) peptide delivery. Tumors were measured every fourth day. Peptides were injected daily at 1 or 5 mg/kg. (C) CD34 immunohistochemistry for endothelial cells in tumor cross sections. (D) SP6001 at 5 mg/kg subcutaneous administration significantly decreases CD34 below PBS-treated controls (**P < .01).