Transforming growth factor-beta1 inhibits generation of angiostatin by human pancreatic cancer cells

Background. Angiostatin, a proteolytic fragment of plasminogen, is a potent inhibitor of angiogenesis. We have previously shown that the human pancreatic cancer cell line ASPC-1 produces enzymatic activity capable of generating angiostatin. In this study we sought to determine whether angiostatin production by ASPC-1 cells was regulated by the growth factor transforming growth factor-β1 (TGF-β1), a key mediator of tumor angiogenesis. Methods. ASPC-1 cells were grown to 70% to 80% confluence in 20% fetal calf serum-RPMI. Medium was changed to serum free. TGF-β1 was added at concentrations of 0, 1, 5, and 10 ng/mL with or without plasminogen activator inhibitor type-1 (PAI-1) at concentrations of 0, 5, 10, 50, and 100 μg/mL. Cells were then cultured for an additional 24 hours. The serum-free conditioned medium was obtained. Angiostatin generation was determined by incubating 20 μg of plasminogen with 100 IlL of serum-free conditioned medium for 0, 1, 2, 3, 6, 12, and 24 hours. Samples were run on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred. The membrane was probed with a monoclonal antibody to the kringle 1-3 fragment of plasminogen and developed using enhanced chemiluminesence. Results. TGF-β1 and PAI-1 inhibited the conversion of plasminogen into angiostatin in a time- and dose-dependent manner. Antibody to PAId completely blocks TGF-β1 mediated angiostatin inhibition. Conclusions. TGF-β1 inhibits the generation of the antiangiogenic molecule angiostatin by human pancreatic cancer cells in a time- and dose-dependent manner. This effect is mediated through modulation of the plasminogen/plasmin system.