Publishing ID: 1962
A cleaved conformation of the serpin antithrombin, generated by a human pancreatic cancer cell
line, has antiangiogenic activity.
Oliver Kisker, Steven Pirie-Shepherd, Shina Onizuka, Eike Achilles, Christian Becker, Michael O'Reilly, Judah
Folkman, Children's Hosp, Boston, MA; Harvard Med Sch, Boston, MA.
Tumor growth is dependent upon the balance of positive and negative regulators of angiogenesis. Our laboratory recently
reported that the cleaved conformation of antithrombin inhibits endothelial proliferation and angiogenesis, but does not bind
thrombin (O'Reilly et al, Science 285: 1926, 1999). We here show that a human pancreatic cell line (Bx Pc-3) can cleave
antithrombin into its antiangiogenic form. Methods and Results: BxPc-3 conditioned media (containing 5% fetal calf serum) was
applied to a heparin Sepharose column and eluted with a NaCl gradient. Fractions that inhibited capillary endothelial cell
proliferation were further purified using anion exchange and gel filtration chromatography. The inhibitory activity was associated
with a protein of apparent Mr of 53-55 kD. Sequence analysis revealed identity to bovine antithrombin. We next tested the
ability of serum free conditioned media from BxPC3 cells to process human antithrombin by incubating human antithrombin
(1mg/100ml) in conditioned media. Cleaved antithrombin in the media was identified by Western Blot and it specifically
inhibited endothelial cells in vitro. In contrast intact (58 kD) antithrombin had no significant effect on endothelial cell
proliferation. Conclusions: Human pancreatic cancer cells express enzymatic activity that can cleave exogenous antithrombin to
a potent antiangiogenic conformation. These data strongly suggest that the growth of pancreatic cancer and its metastases is
regulated by both stimulators and inhibitors of angiogenesis. Our findings may help to explain the clinical patterns and
coagulation abnormalities observed in patients with pancreatic cancer. |
