Angiostatin generation by human pancreatic cancer

Background. Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis. In vitro, angiostatin can be generated by pancreatic elastase proteolysis of plasminogen; however, in vivo, the enzymes responsible for angiostatin production are not known. A recent study demonstrates the involvement of a serine protease in angiostatin generation. In this study we sought to determine if the human pancreatic carcinoma cell line ASPC1 produced enzymatic activity capable of converting plasminogen to angiostatin and to determine if urokinase plasminogen activator (uPA) is involved in this system. Methods. ASPC1 cells were grown to near confluence in 20% FBS-RPMI. Media were changed to serum free and cells cultured for an additional 24 h. The serum free conditioned media (SFCM) was obtained. Angiostatin generation was determined by incubating 20 μg of human plasminogen with 100 μl of SFCM for 0, 3, 8, 12, 24, and 48 h. Plasminogen cleavage was assessed in the presence of the following protease inhibitors: pefabloc, aprotinin, phosphoramidon, leupeptin, and EDTA. The effect of uPA on angiostatin generation was determined by incubating plasminogen with antibody to uPA. Angiostatin generation was determined by Western blot. Results. Incubation of plasminogen with SFCM resulted in the generation of immunoreactive bands at 48 kDa corresponding to human angiostatin. Angiostatin generation by ASPC1 SFCM was time dependent; there was a significant decrease in the plasminogen substrate beginning at 3 h with complete conversion to angiostatin by 48 h. Enzymatic activity leading to angiostatin production was found to be due to a serine protease. Antibody to uPA effectively blocked angiostatin production by ASPC1 SFCM in a dose- dependent manner. Conclusion. Human pancreatic cancer cells express enzymatic activity which leads to the generation of angiostatin. Conversion of plasminogen to angiostatin is due to a serine protease. This serine protease is most likely uPA.