trypsinogen and Colonic-Neoplasms

In this work, we showed that human colon cancer cell lines produce trypsin which can activate a receptor for trypsin, the protease-activated receptor-2 (PAR-2), in these cells. RT-PCR experiments showed that trypsinogen transcripts were present in four colon cancer cell lines: T84, Caco-2, HT-29 and C1.19A. By Western blot analysis we found a 25 kDa immunoreactive band identified as trypsinogen I in cell lysates and in the corresponding culture media. Concentrations of trypsin in cell media were found in nanomolar range, thus compatible with activation of protease-activated receptor 2 (PAR-2). This was further demonstrated in a colon cancer cell line (H-29) Ca2+i assay since increases in Ca2+i were observed in response to media from T84, Caco-2 or C1.19A cells that were similar to that observed with 2-5 nM trypsin and were abolished by trypsin inhibitor. Altogether, these data show that colon cancer cell lines produce and secrete trypsin at concentrations compatible with activation of PAR-2. They support possible autocrine/paracrine regulation of PAR-2 activity by trypsin in colon cancer cells.

Topics: alpha-Amylases; Blotting, Western; Calcium; Colonic Neoplasms; Culture Media, Conditioned; DNA Primers; Dose-Response Relationship, Drug; Humans; Nanotechnology; Plant Proteins; Receptor, PAR-2; Receptors, Thrombin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Trypsin; Trypsin Inhibitors; Trypsinogen; Tumor Cells, Cultured

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.

Topics: Amyloid beta-Protein Precursor; Animals; Breast Neoplasms; Colonic Neoplasms; Culture Media, Conditioned; Female; Humans; Immunohistochemistry; Mice; Neoplasms; Protein Binding; RNA, Messenger; RNA, Neoplasm; Solubility; Stomach Neoplasms; Trypsin; Trypsin Inhibitors; Trypsinogen; Tumor Cells, Cultured

Pancreatic trypsin has been found to induce tight junction or dome formation in some colon cancer cell lines (HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type II, has been isolated from another colon cancer cell line (COLO 205). We have tried to determine if trypsinogen is present and how its expression varies during cell culture in HT-29 Glc+/- and Caco-2 cells, which exhibit enterocytic differentiation, and in HT-29 Glc+ cells, which never differentiate. Trypsinogen mRNA presence and expression were demonstrated in these cells by mRNA hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot analysis, and gel filtration. Trypsinogen was found to be trypsinogen type I and was mainly in zymogen form in culture media. Differentiating cells exhibited variations in trypsinogen I expression, but cells that remained undifferentiated did not. In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the first steps of differentiation.

Topics: Adenocarcinoma; Blotting, Western; Cell Differentiation; Cell Division; Chromatography, Gel; Colonic Neoplasms; Fluorescent Antibody Technique; Gene Expression; Humans; Nucleic Acid Hybridization; Pancreas; Polymerase Chain Reaction; RNA-Directed DNA Polymerase; RNA, Messenger; Trypsinogen; Tumor Cells, Cultured

The concentrations of trypsinogen-1 and -2 in serum samples from patients who have undergone pancreatectomy were measured by highly sensitive and specific time-resolved immunofluorometric assays. The isoenzyme pattern was determined by ion-exchange chromatography and determination of immunoreactivity in the fractions. All samples contained trypsinogen-2, the mean level being one fifth of that in healthy controls. Trypsinogen-1 was detected in one of nine samples. In addition to the main trypsinogen isoenzymes, we observed in normal serum two trypsinogen isoenzymes previously found in mucinous ovarian cyst fluid. Our results suggest that trypsinogen is not exclusively expressed by the pancreas and certain tumors but that it also may be produced by normal extrapancreatic tissues. This should be considered when an assay of trypsinogen in serum is used for clinical purposes.

Topics: Adenocarcinoma; Adult; Aged; Chromatography, Ion Exchange; Colonic Neoplasms; Culture Media, Conditioned; Female; Humans; Immunoradiometric Assay; Isoenzymes; Male; Middle Aged; Osmolar Concentration; Ovarian Cysts; Pancreatectomy; Pancreatitis; Postoperative Period; Reference Values; Trypsinogen; Tumor Cells, Cultured

To test the diagnostic utility of pancreatic digestive enzyme immunohistochemistry in liver cancers, the expression of three pancreatic digestive enzymes (trypsinogen, chymotrypsinogen and pancreatic lipase) was investigated in cholangiocarcinoma (CC) (n = 42), hepatocellular carcinoma (HCC) (n = 35), combined HCC-CC (n = 11) and metastatic adenocarcinoma (MA) of the liver (n = 34; 4 gastric cancer, 5 pancreatic cancer and 25 colon cancer). In CC, 15 (36%) expressed one or more of these enzymes, while the remaining 27 (64%) did not express any enzymes. In MA, 13 (38%) expressed one or more of these enzymes, while the remaining 21 (62%) did not express any enzymes. Expression of trypsinogen, chymotrypsinogen and lipase was noted in 15 CC (36%), 11 CC (25%) and 15 CC (36%), respectively, and in 9 MA (26%), 6 MA (18%) and 13 MA (38%), respectively. There was no significant difference in the positive ratio of each enzyme between CC and MA. In positive cases, the enzymes were expressed with a cytoplasmic granular pattern. In MA, there was no significant difference in the positive ratio of the enzymes among the primary sites. In contrast to CC and MA, these enzymes were not expressed in any cases of HCC and combined HCC-CC. These data suggest that pancreatic digestive enzyme immunohistochemistry may be useful for differential diagnosis between HCC and CC or MA as well as between combined HCC-CC and CC or MA, but it is not useful for differential diagnosis between CC and MA. A positive reaction for these enzymes is indicative of CC or MA and is against the diagnosis of HCC or combined HCC-CC, and a negative reaction is noncontributory to the differential diagnosis.

Topics: Adenocarcinoma; Aged; Carcinoma, Hepatocellular; Cholangiocarcinoma; Chymotrypsinogen; Colonic Neoplasms; Diagnosis, Differential; Female; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Pancrelipase; Stomach Neoplasms; Trypsinogen

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.

Topics: Carcinoma; Colonic Neoplasms; Dexamethasone; Extracellular Matrix; Fibrosarcoma; Humans; Isoenzymes; Leukemia; Trypsinogen; Tumor Cells, Cultured