Peptides derived from type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins inhibit neovascularization and suppress tumor growth in MDA-MB-231 breast cancer xenografts

Abstract

Angiogenesis or neovascularization, the process of new blood vessel formation from preexisting microvasculature, involves interactions among several cell types including parenchymal, endothelial cells, and immune cells. The formation of new vessels is tightly regulated by a balance between endogenous proangiogenic and antiangiogenic factors to maintain homeostasis in tissue; tumor progression and metastasis in breast cancer have been shown to be angiogenesis-dependent. We previously introduced a systematic methodology to identify putative endogenous antiangiogenic peptides and validated these predictions in vitro in human umbilical vein endothelial cell proliferation and migration assays. These peptides are derived from several protein families including type IV collagen, CXC chemokines, and thrombospondin-1 domain-containing proteins. On the basis of the results from the in vitro screening, we have evaluated the ability of one peptide selected from each family named pentastatin-1, chemokinostatin-1, and properdistatin, respectively, to suppress angiogenesis in an MDA-MB-231 human breast cancer orthotopic xenograft model in severe combined immunodeficient mice. Peptides were administered intraperitoneally once per day. We have demonstrated significant suppression of tumor growth in vivo and subsequent reductions in microvascular density, indicating the potential of these peptides as therapeutic agents for breast cancer.

Figure 1

In vitro proliferation results for pentastatin-1 on MDA-MB-231 breast tumor cells and 3T3 fibroblasts. Cell viability was determined using a WST-1 colorimetric assay at the end of a 72-hour exposure to peptide. Data are scaled so that 100% represents the signal from the experimental control and thus maximum viability. Cells were treated at concentrations of 1, 10, 20, 30, 40, 50, 80, and 100 µg/ml of peptide. Pentastatin-1, at high concentrations, decreased viability of 3T3 fibroblasts and MDA-MB-231 breast cancer cell lines in vitro in this assay. Statistical significance is determined at P < .01, and the vertical error bars represent the SEM.

Figure 2

(A) MDA-MB-231 breast xenograft growth in SCID mice with application of three peptides derived from three protein families. Cells were inoculated orthotopically into the mammary fat pad with 2 x 10⁶ cells/mouse, and tumors were allowed to grow for 14 to 21 days to an average of 75 to 100 mm³ on peptide administration. The average tumor volume (n = 8) is plotted every fourth day after treatment. Tumor volumes are statistically different from the control at P < .05 by Student's t-test for all days except chemokinostatin-1 on day 13. Scrambled peptide equivalents were also tested in vivo and were not statistically different from the experimental control. (B) Tumor percent change is also shown from the starting tumor values on peptide administration.

Figure 3

H&E staining of tumor cross sections at 1x and 2x for the experimental control, pentastatin-1, chemokinostatin-1, and properdistatin. Pentastatin-1- and properdistatin-treated tumors show increased necrotic regions (arrows), whereas chemokinostatin-1 is most similar to the control.

Figure 4

(A) Immunohistochemistry showing CD31 antibody staining in several conditions as a marker for endothelial cells and vessel density. Images were taken at 20x magnification for the experimental control, pentastatin-1-, chemokinostatin-1-, and properdistatin-treated tumors. (B) Quantification of endothelial cells and vessel density for resulting cross sections scaled to the experimental control. Each condition was quantified by pixel intensity representing the quantity of endothelial cells. The type IV collagen-derived peptide, pentastatin-1, and CXC chemokine-derived peptide, chemokinostatin-1, were statistically different from the control at P < .05. Chemokinostatin-1 was also statistically significant from its scrambled peptide control, showing a reduction in endothelial cells and vasculature.

Figure 5

(A) Fluorescence costain for Ki67 proliferating cells (green) and murine cells by telomeric DNA (red). Composite images are made with DAPI (blue) as a stain for cell nuclei (left panel). Cell counts are approximated using pixel intensity with Image J and Adobe Photoshop CS3 software. Qualitatively, the pentastatin-1-, chemokinostatin-1-, and properdistatin-treated tumors contain fewer proliferating cells than the control. (B) Quantification of proliferating cells and mouse stromal cells. Controls contained an average of 38% proliferating cells and 21% mouse stromal cells. Properdistatin contained a statistically significant reduction in proliferating cells with 14% proliferating. Chemokinostatin-1 contained a statistically significant increase in mouse stromal cells from the controls, with 67% of the tumor microenvironment of this type, suggesting an infiltration of murine-derived cells into the tumor microenvironment.