Expression of Trypsin in Human Cancer Cell Lines and Cancer Tissues and Its Tight Binding to Soluble Form of Alzheimer Amyloid Precursor Protein in Culture

Miyata, Satoshi; Koshikawa, Naohiko; Higashi, Shouichi; Miyagi, Yohei; Nagashima, Yoji; Yanoma, Shunsuke; Kato, Yukio; Yasumitsu, Hidetaro; Miyazaki, Kaoru

doi:10.1093/oxfordjournals.jbchem.a022388

Cited by 72 publications

(82 citation statements)

References 23 publications

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“…In this context, it is worth pointing out that several lines of evidence indicate the importance of locally secreted trypsin at the vicinity of colon tumours: (1) trypsin was shown to be expressed by the large intestine . Normal epithelial cells surrounding colon tumour cells are a likely source of active trypsin; (2) human colon cancer cells themselves were shown to express trypsin and trypsinogen (Bernard-Perrone et al, 1998;Miyata et al, 1999); (3) recent studies on gastric cancer indicate that blood vessels surrounding tumours express trypsin, whereas other blood vessels do not . Trypsin is also present in serum at nanomolar concentrations and could diffuse from blood to tumour cells.…”

Section: Discussionmentioning

confidence: 99%

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Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

et al. 2001

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SummaryThe protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines Cl.19A, SW480,. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 µM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 µM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.

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Section: Discussionmentioning

confidence: 99%

“…Extra-pancreatic production of trypsin was shown in ovarian (Hirahara et al, 1995), lung (Kawano et al, 1997), gastric and colonic tumours (Bernard-Perrone et al, 1998;Miyata et al, 1999); 2. The trypsinogen gene is significantly expressed in vascular endothelial cells around gastric tumours ; 3.…”

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confidence: 99%

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

et al. 2001

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“…Elevated levels of trypsin, the main endogenous PAR2 activator, have been found in a variety of tumors including colorectal carcinomas (8,9,51). Trypsin can also originate from vessels in the vicinity of the tumors (52) or could diffuse from blood to tumor cells.…”

Section: Par2 Transactivates Egf-r In Colon Cancermentioning

confidence: 99%

“…Extra-pancreatic production of trypsin was shown in ovarian (5), lung (6), gastric (7), and colonic tumors (8) and also in colon cancer cell lines (9,10). In addition, overexpression of exogenous trypsinogen cDNA in human gastric cancer cells increases their tumorigenicity in nude mice (7).…”

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confidence: 99%

Protease-activated Receptor 2 in Colon Cancer

Darmoul

Gratio

Devaud

et al. 2004

Journal of Biological Chemistry

190

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Several lines of evidence suggest that tumor-derived trypsin contributes to the growth and invasion of cancer cells. We have recently shown that trypsin is a potent growth factor for colon cancer cells through activation of the G protein-coupled receptor protease-activated receptor 2 (PAR2). Here, we analyzed the signaling pathways downstream of PAR2 activation that lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events upon activation of PAR2 by the serine protease trypsin or the specific PAR2-activating peptide (AP2): (i) a matrix metalloproteinasedependent release of transforming growth factor (TGF)-␣, as demonstrated with TGF-␣-blocking antibodies and measurement of TGF-␣ in culture medium; (ii) TGF-␣-mediated activation of epidermal growth factor receptor (EGF-R) and subsequent EGF-R phosphorylation; and (iii) activation of ERK1/2 and subsequent cell proliferation. The links between these events are demonstrated by the fact that stimulation of cell proliferation and ERK1/2 upon activation of PAR2 is reversed by the metalloproteinase inhibitor batimastat, TGF-␣-neutralizing antibodies, EGF-R ligand binding domain-blocking antibodies, and the EGF-R tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGF-R appears to be a major mechanism whereby activation of PAR2 results in colon cancer cell growth. By using the Src tyrosine kinase inhibitor PP2, we further showed that Src plays a permissive role for PAR2-mediated ERK1/2 activation and cell proliferation, probably acting downstream of the EGF-R. These data explain how trypsin exerts robust trophic action on colon cancer cells and underline the critical role of EGF-R transactivation.Proteases have been increasingly recognized as important factors in pathophysiology of tumor diseases. Besides their contribution to cancer progression by the degradation of extracellular matrix proteins, there is now substantial evidence that certain proteases serve as signal molecules controlling cell functions through specific membrane receptors, the proteaseactivated receptors. PARs 1 are seven transmembrane-spanning domain G protein-coupled receptors comprising four receptors named PAR1, PAR2, PAR3, and PAR4 (1, 2). Thrombin is the physiological activator of PAR1, PAR3, and PAR4, whereas PAR2 is activated by multiple trypsin-like enzymes including trypsin and mast cell tryptase but not thrombin. The mechanism of activation of PARs was initially established for PAR1 (3) and seems to be a paradigm for the other PARs (1, 2, 4). They are irreversibly activated by a proteolytic mechanism in which the protease binds to and cleaves the amino-terminal exodomain of the receptor. This cleavage generates a new amino-terminal sequence that binds intramolecularly to the core receptor and serves as a tethered ligand. Synthetic activating peptides that mimic the tethered ligand domains of PAR1, PAR2, and PAR4 have been developed. The activation of PARs by these synthetic peptides APs is independent of rec...

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“…14 -19 Trypsin also can stimulate tumor growth and enhance tumor adhesion to extra- cellular matrix components. 20,21 In previous studies, we demonstrated in pancreatic cancer cell lines that hepatic metastasis and invasive ability were correlated with amounts of cationic trypsinogen and were inhibited by a serine protease inhibitor. 24,25 Considering the results of this earlier work, secreted cationic trypsin is likely to degrade extracellular matrix components in several ways, including activating trypsinogen itself as well as latent proenzyme forms of other proteases.…”

Section: Discussionmentioning

confidence: 99%

Enhanced invasiveness of pancreatic adenocarcinoma cells stably transfected with cationic trypsinogen cDNA

Tajima¹,

Ohta²,

Elnemr³

et al. 2001

Int. J. Cancer

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Various studies have described increased expression of cationic trypsinogen in malignant tumor cells. To explore the role of secreted cationic trypsinogen in invasion by cancer cells, we introduced cationic trypsinogen cDNA into Panc-1, a pancreatic adenocarcinoma-derived cell line that lacks expression of endogeneous trypsinogen. Four independent clones (designated Panc-1-Try-7, -9, -11 and -24) stably expressing cationic trypsinogen mRNA were isolated and processed for further study. In a zymographic analysis, gelatinolytic activity for cationic trypsinogen was detectable in serum-free conditioned media obtained from all 4 transfectants but not in media from mock-transfected or parental Proteolytic degradation of extracellular matrix components is a process essential for tumor invasion and metastasis. 1-4 A variety of proteases produced by tumor cells are involved in matrix degradation. 1-4 These enzymes include 2 major categories: matrix metalloproteinases (MMPs) and serine proteases. Although the role of MMPs in tumor malignancy has been studied extensively, 5-9 much less is known about serine proteases, except for urokinase-type plasminogen activator (u-PA). 10 -13 Although trypsin, another member of the serine protease family, also has been implicated in the spread of malignant tumor cells, the underlying molecular mechanism has not been fully elucidated. Several previous reports have described increased amounts of trypsinogen-like enzymes in malignant tumor cells. 14 -21 Koshikawa et al. 22 identified 1-and 2-chain forms of cationic trypsinogen in a gastric carcinoma cell line. Under the acidic condition that prevails in the extracellular space between malignant tumor cells, cationic trypsinogen is spontaneously converted to trypsin, an active form. 23 Trypsin not only degrades extracellular matrix glycoproteins 22 but also activates proenzymes of various MMP and serine proteases. 19 We previously reported that under acidic conditions cationic trypsinogen produced by human pancreatic ductal cancer spontaneously becomes activated and acquires gelatinolytic activity. 24 Furthermore, we found that serine protease inhibitors had a suppressive effect on invasion and metastasis by human pancreatic cancer cell lines. 25 However, the importance of trypsin in initiating invasion of tumor cells is still unclear; 1 major reason is that no available serine protease inhibitor can produce sufficiently selective inhibition of trypsin.To obtain more direct evidence that trypsin potentiates tumor invasion, we transfected cationic trypsinogen cDNA into Panc-1, a pancreatic adenocarcinoma-derived cell line that normally is deficient in expression of trypsinogen. The resulting transfectants acquired enhanced invasiveness. MATERIAL AND METHODS Cell cultureHuman pancreatic adenocarcinoma cell lines, (Capan-1, BxPC-3, AsPC-1, Panc-1 and MIAPaCa-2), were obtained from the American Type Culture Collection (Rockville, MD). These cell lines were maintained at 37°C in a 5% CO 2 incubator and grown in RPMI-1640 medium supplemented w...

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Expression of Trypsin in Human Cancer Cell Lines and Cancer Tissues and Its Tight Binding to Soluble Form of Alzheimer Amyloid Precursor Protein in Culture

Cited by 72 publications

References 23 publications

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

Initiation of human colon cancer cell proliferation by trypsin acting at protease-activated receptor-2

Protease-activated Receptor 2 in Colon Cancer

Enhanced invasiveness of pancreatic adenocarcinoma cells stably transfected with cationic trypsinogen cDNA

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