Figure 2.

Characterization of NGR sequence‐modified endostatin (NGR‐endostatin). (A) Aminopeptidase N (APN)‐inhibitory activity. APN was extracted from human umbilical vein endothelial cell (HUVEC) cultures. Endostatin preparations, bestatin (positive control), and leupeptin (negative control) were used at a concentration of 5 μM. Endostatin‐RGD: endostatin containing an RGD sequence. (B) Cell attachment assay. HUVEC or WM35 cells that were prelabeled with 5‐(and‐6)‐carboxy fluorescein diacetate, succinimidyl ester, were added into triplicate wells coated with either endostatin or NGR‐endostatin at a concentration of 1 nmole per well. Wells coated with 0.2% gelatin were used as maximum attachment (100%). The attached cells were quantified by a fluorescence plate reader. Values represent the mean of two independent experiments. (C) The effect of endostatin and NGR‐endostatin on endothelial cell proliferation: Endostatin and NGR‐endostatin were used at a concentration of 2.5 μg/mL. Basic fibroblast growth factor (5 ng/mL) was used to induce proliferation of endothelial cells. The proliferation was determined by 5′‐bromo‐2′‐deoxyuridine uptake. (D) The effect of endostatin (open bar) and NGR‐endostatin (solid bar) on HUVEC migration. NGR‐containing peptide (SR‐1) plus endostatin (hatched bar) did not enhance the basal level of inhibition seen with endostatin alone (open bar). (E) Tumor localization: Human colon carcinoma cells (LS174T) were injected subcutaneously into female athymic nude mice. When the tumors reached a size of ≈ 500 mm³ (10 days after inoculation), endostatin (open bars) or NGR‐endostatin (solid bars) was injected at a dose of 20 mg/kg subcutaneously. Tumor, liver, and lung tissues were resected and homogenized. Endostatin levels in the tissues and the sera were determined by enzyme‐linked immunoadsorbent assay in the soluble fraction. Endostatin levels are expressed as a relative concentration to serum levels of endostatin. Error bars indicate the standard error. Statistical significance was determined with a Student t test. An asterisk indicates P < 0.05.