Fig. 1. Inhibition of HeLa tumor growth by ANUP peptides in nude mice. Five million HeLa cells were transplanted per mouse. After 3 days, mice were treated with Pep A (500 or 750 μg), Pep B (500 μg), or buffer only (200 μl volume; PBS+0.05%SDS, Control) on days 3, 6, 10, and 13 (arrows). Tumor growth was measured three times per week and the mice were sacrificed on day 24 post-transplantation. (A) Inhibition of HeLa tumor growth during treatment phase. Results represent the average tumor volume of all mice (n=3). Treatments with Pep A at 500 μg per dose (open square), Pep A at 750 μg per dose (open triangle), and Pep B (open circle) all demonstrate tumor inhibition during the treatment phase while control tumors (solid diamond) remain static. (B) Response of individual tumors to treatment with peptides. Tumors were measured on day 14 (1 day post last treatment). Individual mice tumor volumes demonstrate response of all animals treated with either Pep A or Pep B. (C) Tumor growth profiles post-treatment with Pep A and Pep B. Note slight increase in tumor burden for both Pep A treatment groups and escalation of tumor burden in the control and Pep B-treated groups subsequent to the fourth treatment.

Fig. 2. Dose-dependent suppression of branching vessel formation on day 3.5 chick embryo by Pep A and Pep B. A single topical dose of either peptide induced the suppression of branching vessel formation and development; Pep A showed inhibition beginning at 25 μg vs. Pep B which showed inhibition by 50 μg. The number of embryos treated and the number showing inhibition is indicated in the table on the left.

Fig. 3. Inhibition of bFGF-mediated angiogenesis in early chick embryos by ANUP peptides. Micrographs showing induction of angiogenesis resulting from the topical administration of 100 ng of bFGF on the CAM of day 10 chick embryos. Neo-vascularization along the edges of the filter disks (A) and vascular infiltration (B) are seen after administration of bFGF alone. Inhibition of neo-vascularization and infiltration as well as degradation of pre-existing vessels is demonstrated by co-administration of 200 ng Pep A with 100 ng bFGF (C). Co-administration of 200 ng Pep B with 100 ng bFGF also results in inhibition of bFGF-induced angiogenesis (D).

Fig. 4. Resected CAM tissues topically treated with bFGF in the presence of Pep A and fluorescently labeled to analyze for apoptosis. The condensed apoptotic nuclei will intensely fluoresce (and will appear white) under epifluorescence visualization compared to the staining patter of the viable cells with H-33342 (blue nuclei). (A) 100 ng bFGF plus 200 ng Pep A (100×). Note the intensely staining apoptotic nuclei (white) of vascular cells resulting in the outlining of the vessels against the lighter stained non-vascular cell nuclei which at higher magnification (200×) are clearly apoptotic (B). The Pep B-treated CAM did not demonstrate the peri-vascular apoptosis and the white nuclei were randomly distributed throughout the CAM tissue (C). As expected, bFGF-treated control CAM showed minimal and randomly distributed apoptotic nuclei (not shown). Scale bars, 10 μm.

Fig. 5. Histopathology of tumors isolated from Pep A-treated mice (750 μg). Larger areas of cell death in tumors isolated from Pep A-treated mice are evident by the increased number of dark nuclei. Magnification, 75×; scale bars, 10 μm.

Table 1. Histopathology of tumors isolated from all mice post-sacrifice and analyzed for section size (small, medium, or large), areas of cell death (size and number), tumor infiltration, and neo-vascularization/peripheral vascularization

The numbers represent the group average of the sections analyzed. Note the reduced infiltration and vascularization evident in the treatment groups as compared with the control group.