trypsinogen and Ovarian-Neoplasms

Proteolytic enzymes have shown efficacy in cancer therapy. We present a combination of the two pro-enzymes Trypsinogen and Chymotrypsinogen A with potent in vitro and in vivo anti-tumour efficacy. A synergetic anti-tumour effect for Trypsinogen and Chymotrypsinogen A was determined at a ratio 1:6 (named PRP) using 24 human cancer cell lines. The antiangiogenic effect of PRP was analysed by matrigel-based tube formation and by fibrous capsule formation assays. Furthermore, cell invasion and wound healing assays together with qRT-PCR determination of epithelial-to-mesenchymal transition (EMT) markers were performed on human cancer cells treated with PRP. Additionally, in vivo pharmacokinetic studies were implemented and the PRP's anti-tumour efficacy was explored against orthotopic pancreatic and ovarian cancer tumours. PRP formulation was proven to inhibit in vitro angiogenesis, tumour growth, cancer cell migration and invasiveness; and to be an effective and well tolerated in vivo anti-tumour treatment. Finally, the clinical efficacy of a suppository formulation containing both pancreatic pro-enzymes in the context of a UK Pharmaceuticals Special Scheme was evaluated in advanced cancer patients. Consequently, PRP could have relevant oncological clinical applications for the treatment of advanced or metastatic pancreatic adenocarcinoma and advanced epithelial ovarian cancer.

Topics: Animals; Apoptosis; Cell Proliferation; Chymotrypsinogen; Enzyme Precursors; Female; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Ovarian Neoplasms; Pancreas; Pancreatic Neoplasms; Pilot Projects; Trypsinogen; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The purpose is to study the prognostic significance of tissue expression of trypsinogen-1, trypsinogen-2, and tumor-associated trypsin inhibitor (TATI) and serum concentration of trypsinogen-2, trypsin-2-API (complex of trypsin-2 with alpha-1-proteinase inhibitor), and TATI in epithelial ovarian cancer.. Expression of trypsinogen-1, trypsinogen-2, and TATI was determined by immunohistochemistry with monoclonal antibodies in tissue sections of tumors from 119 patients with untreated primary epithelial ovarian cancer. Preoperative serum concentrations of trypsinogen-2, trypsin-2-API and TATI were analyzed using specific immunoassays.. Fifty-four percent of the tumors expressed trypsinogen-1, 45% expressed trypsinogen-2, and 30% expressed TATI. In patients with stage III and IV disease, TATI tissue expression (P = 0.002) and elevated TATI concentration in serum (P = 0.048) were associated with adverse cancer-specific and progression-free survival in univariate analysis. In multivariate analysis, TATI tissue expression (P = 0.005), tumor grade (P = 0.0001), histological type (P = 0.02), and stage (P = 0.0005) were independent prognostic factors for adverse cancer-specific survival and TATI tissue expression (P = 0.006) and grade (P = 0.0003) for progression-free survival. In multivariate analysis of all patients and those with advanced disease, serum trypsin-2-API concentration was an adverse prognostic factor for cancer-specific and progression-free survival, and it was independent of stage and histological type of the tumor (P

Topics: Adult; Aged; Aged, 80 and over; Female; Humans; Immunoassay; Immunohistochemistry; Middle Aged; Multivariate Analysis; Neoplasm Staging; Ovarian Neoplasms; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Survival Analysis; Trypsin; Trypsin Inhibitors; Trypsinogen

Proteolysis mediated by matrix metalloproteinases (MMPs) and serine proteinases is associated with cancer invasion and metastasis. Activation of latent proMMPs, and especially the proforms of the type IV collagen degrading gelatinases A and B (proMMP-2 and proMMP-9), is thought to be a critical step in this process. We have recently found that human tumour-associated trypsin-2 is a potent activator of proMMP-9 and it also activates proMMP-2 in vitro. Trypsinogen, MMP-2, and MMP-9 are expressed in ovarian cancer. To elucidate the function of trypsin in vivo, we studied whether high concentrations of trypsinogen-1, trypsinogen-2, their alpha(1)-proteinase inhibitor (API) complexes, and tumour-associated trypsin inhibitor (TATI) are associated with proMMP-2 and proMMP-9 activation in ovarian tumour cyst fluids. Zymography and immunofluorometric analysis of 61 cyst fluids showed a significant association between high trypsin concentrations and the activation of MMP-9 (P = 0.003-0.05). In contrast, the trypsin concentrations were inversely associated with the activation of MMP-2 (P = 0.01-0.02). Immunohistochemical analysis of ovarian tumour tissue demonstrated expression of trypsinogen-2 and TATI in the secretory epithelium. MMP-2 was detected both in stromal and epithelial cells whereas MMP-9 was detected in neutrophils and macrophage-like cells in stromal and epithelial areas. These results suggest that trypsin may play a role in the regulation of the MMP-dependent proteolysis associated with invasion and metastasis of ovarian cancer.

Topics: Adolescent; Adult; Aged; Collagenases; Cystadenocarcinoma, Mucinous; Enzyme Activation; Enzyme Precursors; Female; Fluorescent Antibody Technique; Gelatinases; Humans; Immunohistochemistry; Isoenzymes; Matrix Metalloproteinase 9; Metalloendopeptidases; Middle Aged; Ovarian Cysts; Ovarian Neoplasms; Trypsin; Trypsinogen

We have determined the concentrations of two tumor-associated trypsinogen (TAT) isoenzymes, called TAT-1 and TAT-2, in human ovarian tumor cyst fluid using monoclonal antibody-based immunofluorometric assays specific for each isoenzyme. TAT-1 and TAT-2 are immunologically indistinguishable from the two pancreatic trypsinogen isoenzymes, cationic trypsinogen (-1) and anionic trypsinogen (-2). Our results show that of the two isoenzymes TAT-2 is the predominant form in cyst fluid and its concentrations are significantly higher than the levels of the trypsinogen isoenzymes in serum and in preovulatory follicular fluid from hyperstimulated ovaries. The median concentration of TAT-2 was higher in mucinous than in serous cyst fluid as has been found previously for the specific trypsin inhibitor, tumor-associated trypsin inhibitor. Most notably, in mucinous cyst fluids the median level of TAT-2 was higher in borderline and malignant (2640 micrograms/liter) than in benign cases (84 micrograms/liter). Also in serous cyst fluids the TAT-2 level was higher in borderline and malignant (median 345 micrograms/liter) than in benign cases (median 18 micrograms/liter). In fluids from other types of malignant ovarian carcinomas slightly elevated levels of TAT-2 were also observed (median 62 micrograms/liter). The identity of the trypsinogens was verified by isolating them by immunoaffinity chromatography using monoclonal antibodies. The increased levels in association with malignancy suggest that TAT is involved in ovarian tumor dissemination and breakage of tissue barriers.

Topics: Adult; Aged; Antibodies, Monoclonal; Biomarkers, Tumor; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Exudates and Transudates; Female; Fluorescent Antibody Technique; Humans; Isoenzymes; Middle Aged; Molecular Weight; Ovarian Cysts; Ovarian Neoplasms; Radioimmunoassay; Trypsinogen

We have developed sensitive time-resolved immunofluorometric assays for the two trypsinogen isoenzymes, trypsinogen-1 and trypsinogen-2, which also are called cationic and anionic trypsinogen, respectively. The assays use monoclonal antibodies produced by immunization with tumor-associated trypsinogen that is isolated from mucinous ovarian cyst fluid. In each assay, one antibody is immobilized onto the walls of polystyrene microtiter strip wells and the other is labeled with an europium(III) chelate. The cross-reaction of each trypsinogen isoenzyme in the assay for the other isoenzyme is less than 1%. The detection limits are 0.1 micrograms/L for trypsinogen-1 and 0.3 micrograms/L for trypsinogen-2. In sera of healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher (median, 21 micrograms/L) than that of trypsinogen-2 (median, 17 micrograms/L), but in acute pancreatitis the ratio is reversed. In acute pancreatitis the concentration of trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentrations is only 15-fold. The corresponding difference in immunoreactive trypsin measured by a commercially available radioimmunoassay was also only 10-fold.

Topics: Acute Disease; Adult; Aged; Antibodies, Monoclonal; Biomarkers; Cross Reactions; Female; Fluoroimmunoassay; Humans; Isoenzymes; Male; Middle Aged; Ovarian Neoplasms; Pancreatitis; Radioimmunoassay; Trypsin; Trypsinogen