The esophageal cancer-related gene 2 (ECRG2) is a novel gene that shows sequence similarity to KAZAL-type serine protease inhibitor. In this study, the migration and invasion of PG cancer cells were inhibited by ectopic expression of ECRG2 in vitro, and metastases decreased after injecting PG/pcDNA3.1-ECRG2 cells into the tail veins of nude mice. Control mice were injected with PG/pcDNA3.1 cells. To test the hypothesis that ECRG2 interacts with proteases and inactivates extracellular matrix degradation, binding affinity and co-immunoprecipitation experiments were performed using serum-free conditioned medium. The results showed that ECRG2 bound to two species of urokinase-type plasminogen activator (uPA) with molecular weights of 55 and 33 kDa. Furthermore, analysis of the uPA/plasmin activity showed that expression of ECRG2 reduced proteolysis of the plasmin substrate D-Val-Phe-Lys-p-nitroanilide, which was seen by a decrease of absorbance at 405 nm. Taken together, these results suggested that ECRG2 inhibits aggressiveness of cancer cell, possibly through the down-regulation of uPA/plasmin activity.
Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), an enzyme involved in prostaglandin (including PGE(2)) bio-inactivation, is down-expressed in several epithelial malignancies including CRC. Although its role in the suppression of colon tumorigenesis has been well learned, little is known about the role of 15-PGDH in the process of tumor metastasis. Here, we tested the hypothesis that 15-PGDH over-expression in CRC cells results in decreased cell motility and invasion. In this study, 15-PGDH was re-expressed in SW480 cells by the use of gene transient transfection with eukaryotic expression vector pcDNA3.1-PGDH. We confirmed the over-expression of 15-PGDH protein by Western blot and enzymatic activity assay. The cell motility was tested by counting the number of cells crossing an 8-micron pore size PET membrane and by measuring cells migration distance through wound healing assay. Furthermore, cell invasive activity was evaluated by counting the number of cells invading through a Matrigel-coated membrane simulating basement membrane. The effects of 15-PGDH on the adhesion were investigated by MTT assay. Ectopic expression of 15-PGDH in SW480 cancer cells significantly inhibited the cell migratory and invasive capacity in vitro by approximately 1.9- and 8.4-fold, respectively. To test the hypothesis that 15-PGDH affects proteases and inactivates extracellular matrix (ECM), Western blot and gelatin zymography were performed by using serum-free conditioned medium. The results showed that re-expression of 15-PGDH suppresed matrix metalloproteinase-2 (MMP2) synthesis and secretion. In addition, the analysis of the MMP2 activity indicated that re-expression of 15-PGDH could inhibit activation of MMP2. Furthermore, we found that 15-PGDH inhibited cell adhesion to ECM and reduced CD44 expression in SW480 cell. Taken together, these results suggest that induced 15-PGDH expression may contribute to the inhibition of the invasive and metastatic capacity of colon cancer cells in vitro.
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