Kosir M A, Wang W, Zukowski K L, Tromp G, Barber J
Department of Surgery, Wayne State University School of Medicine, Detroit, Michigan, 48201, USA.
J Surg Res. 1999 Jan;81(1):42-7. doi: 10.1006/jsre.1998.5519.
The degradation of basement membrane (BM) by cancer is an important event that characterizes invasive biological behavior. A component of BM is heparan sulfate proteoglycan (HSPG). The glycanase(s) that degrade HSPG in BM are not yet isolated. We recently identified HSPG-degrading activity (PC-3M heparanase) in the conditioned media (CM) of malignant prostate carcinoma cells (PC-3M and LNCaP C4-2). Antibodies (Abs) to a recently isolated heparanase from human platelets (CTAP-III), cross-react with PC-3M heparanase although they differ in size; under reduced conditions PC-3M heparanase is 60 kDa whereas CTAP-III is 10 kDa by polyacrylamide gel electrophoresis. PC-3M heparanase therefore shares homology with CTAP-III. The purpose of this study was to test the inhibition of PC-3M heparanase by Abs specific to the N- and C-terminals of CTAP-III.
CM from PC-3M and LNCaP C4-2 cells were tested for heparanase activity. Each reaction contained substrate as [3H]glucosamine-labeled HSPG (>50 kDa) from the BM of the EHS tumor, CM from PC-3M or LNCaP C4-2 cells, and inhibitor or buffer (negative control). Protease inhibitors were present throughout. After incubation for 3-20 h at 37 degreesC and pH 5.8, the reaction was stopped with 0.2% SDS. Each reaction mixture was centrifuged in an Ultrafree-MC 30,000 NMWL filter unit (Millipore) and radioactivity in the filtrate counted by scintillation counting. Results. For both cell lines, there was a linear relationship between the amount (microgram) of CM and degradation of HSPG. Degradation was inhibited by 54.1% (mean) using carrageenan lambda (10 microgram/ml), a nonspecific glycanase inhibitor (P < 0.05 by ANOVA). Ab to the N-terminus of CTAP-III (anti-Hep A) reduced degradation by 10-50% (mean 31.1%) and to the C-terminus (anti-Hep C) by 38.8-64.3% (mean 51.1%) (P < 0.003 by ANOVA).
The degradation of HSPG by malignant prostate cancer cell lines is inhibited by both a nonspecific glycanase inhibitor, and specific Abs to a homologous platelet heparanase. Based upon molecular weight, PC-3M heparanase is different from platelet heparanase and degrades BM.
