Transforming growth factor-beta 1 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-beta 1 (TGF-beta 1), 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-beta 1 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 micrograms/mL. Cells were then cultured for an additional 24 hours. The serum-free conditioned medium was obtained. Angiostatin generation was determined by incubating 20 micrograms of plasminogen with 100 microL 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 chemiluminescence.


RESULTS
TGF-beta 1 and PAI-1 inhibited the conversion of plasminogen into angiostatin in a time- and dose-dependent manner. Antibody to PAI-1 completely blocks TGF-beta 1 mediated angiostatin inhibition.


CONCLUSIONS
TGF-beta 1 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.
Department of Surgery, Allegheny University of the Health Sciences, Philadelphia, Pa 19129, USA.
C A
CA
O'Mahony
D
D
Albo
G P
GP
Tuszynski
D H
DH
Berger
D000230:Adenocarcinoma; D043168:Angiostatins; D000906:Antibodies / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000032:analysis / Q000378:metabolism*; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009389:Neovascularization, Pathologic / Q000201:enzymology / Q000503:physiopathology; D010190:Pancreatic Neoplasms; D010446:Peptide Fragments / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D010958:Plasminogen / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology / Q000494:pharmacology; D015842:Serine Proteinase Inhibitors / Q000276:immunology / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000906:Antibodies; D000970:Antineoplastic Agents; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D016212:Transforming Growth Factor beta; D043168:Angiostatins; D010958:Plasminogen