Reverse‐zymographic analysis of protease nexin‐II/amyloid β protein precursor of human carcinoma cell lines, with special reference to the grade of differentiation and metastatic phenotype

Abstract

Trypsin inhibitors in serum-free conditioned media (SFCM) of various human carcinoma cell lines were analyzed by reverse zymography. Most of the cells secreted high-molecular-weight trypsin inhibitors (HMTI) larger than 100 kDa. The cell lines of colorectal carcinoma origin had a tendency to secrete HMTI whose molecular weight was a little higher than that of the other cell lines. Analysis of SFCM of subclones with different histological differentiation and metastatic/invasive potentials derived from a single pancreatic carcinoma cell line SUIT-2 showed that the HMTI activity in SFCM was correlated to the degree of histological differentiation in vivo and tended to be inversely correlated to their metastatic/invasive capabilities. Immunoblotting analysis revealed that these HMTI were protease nexin-II/amyloid beta protein precursors (PN-II/APP). Semi-quantificative reverse-transcriptase/polymerase-chain reaction study for PN-II/APP mRNAs suggested that the differences in PN-II/APP activities in SFCM between the subclones might be post-transcriptional or post-secretional events. In addition, SFCM of a highly metastatic subclone contained 43-kDa protein which reacted to anti-APP monoclonal antibody (MAb) suggesting that the subclone may have APP-degrading activity.

Amyloid β protein precursor is involved in the growth of human colon carcinoma cell in vitro and in vivo

“…DISCUSSION An important goal of our study was to determine whether APP production could influence the growth of human colon carcinoma cells in vitro and in vivo, since we have observed that most carcinoma cells express APP and that a major serine proteinase inhibitor secreted by a number of colon carcinoma cell lines is sAPP with Kunitz-type serine proteinase inhibitor domain. [3][4][5] Here, we demonstrated that down-regulation of APP in SW837 human colon carcinoma cells resulted in markedly reduced cellular growth in vitro, and more importantly, also in vivo. The reduced growth in vitro was restored by the addition of a cultured conditioned medium of parent SW837 cells, whereas the conditioned medium pretreated with anti-APP antibody followed by immunoprecipitation failed to restore the antisense effect.…”

“…Although the precise mechanism underlying this finding is undefined, it may be in accordance with our previous observation using cloned sublines derived from a single pancreatic adenocarcinoma cell line. 4 In that study, the levels of sAPP in SFCM of the subclones correlated to the degree of histological differentiation in vivo. Given the possible roles of APP in cell-ECM and cell-cell interactions as mentioned above and the observations that cellular microenvironment such as cell-ECM interaction could play an important role in glandular differentiation of adenocarcinoma cells, 30 suppression of APP may somehow impair the cell-ECM and/or cell-cell interactions that are involved in the formation of tubular structures.…”

“…4 The resolving gel, 8% polyacrylamide with 0.1% SDS, was polymerized in the presence of 1 mg/ml type-I gelatin (Koken, Tokyo, Japan). Stacking gel did not contain the gelatin.…”

“…Previously, we reported that human tumor cell lines consistently secreted highmolecular weight trypsin inhibitors, which were identical to the secreted form of APP (sAPP) with Kunitz domain. 3,4 Indeed, sAPP represented the major trypsin inhibitor produced by a panel of colon carcinoma cell lines. 5 This evidence suggests that APP and/or sAPP may have important, though undefined, biological functions in tumor cells, including colon carcinoma cells.…”

“…Since all tumor cell lines so far examined express APP mRNA 4 and most colon carcinoma cell lines secrete sAPP with Kunitz-type serine proteinase inhibitor domain as their main cellular serine proteinase inhibitor, 5 it can be hypothesized that APP and/or sAPP may also have a fundamental important function(s) in tumor cells, perhaps being involved in cellular growth, differentiation and tumorigenesis. In an attempt to test this hypothesis, we used the antisense RNA strategy to impair the expression of APP in human colon carcinoma cell line SW837.…”