Therapeutic Effect of Proteinase-Activated Receptor-1 Antagonist on Colitis-Associated Carcinogenesis.

BACKGROUND & AIMS: Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR1) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR1 antagonism on colitis-associated carcinogenesis. METHODS: A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR1 antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn's disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR1 agonist proteinases. RESULTS: AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The ß-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota ß-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR1 mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca2+ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR1 at the thrombin cleavage site. CONCLUSIONS: PAR1 antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

Higher susceptibility of NOD/LtSz-scid Il2rg (-/-) NSG mice to xenotransplanted lung cancer cell lines.

PURPOSE: No lung cancer xenograft model using non-obese diabetic (NOD)-scid Il2rg (-/-) mice has been reported. The purpose of this study is to select a suitable mouse strain as a xenogenic host for testing tumorigenicity of lung cancer. MATERIALS AND METHODS: We directly compared the susceptibility of four immunodeficient mouse strains, c-nu, C.B-17 scid, NOD-scid, and NOD/LtSz-scid Il2rg (-/-) (NSG) mice, for tumor formation from xenotransplanted lung cancer cell lines. Various numbers (10(1)-10(5) cells/head) of two lung cancer cell lines, A549 and EBC1, were subcutaneously inoculated and tumor sizes were measured every week up to 12 weeks. RESULTS: When 10(4) EBC1 cells were inoculated, no tumor formation was observed in BALB/c-nu or C.B-17 scid mice. Tumors developed in two of the five NOD-scid mice (40%) and in all the five NSG mice (100%). When 10(3) EBC1 cells were injected, no tumors developed in any strain other than NSG mice, while tumorigenesis was achieved in all the five NSG mice (100%, P=0.0079) within 9 weeks. NSG mice similarly showed higher susceptibility to xenotransplantation of A549 cells. Tumor formation was observed only in NSG mice after inoculation of 10(3) or fewer A549 cells (40% vs 0% in 15 NSG mice compared with others, respectively, P=0.0169). We confirmed that the engrafted tumors originated from inoculated human lung cancer cells by immunohistochemical staining with human cytokeratin and vimentin. CONCLUSION: NSG mice may be the most suitable strain for testing tumorigenicity of lung cancer, especially if only a few cells are available.

Utility of immediate cytologic diagnosis of lung masses using ultrafast Papanicolaou stain.

PURPOSE: At Kagawa University Hospital we conduct immediate cytology using ultrafast Papanicolaou (U-Pap) stain when collecting cytology specimens by bronchoscopy or CT-guided needle aspiration, and report here an investigation of its utility. METHODS: The subjects were 503 patients for whom a final histopathological diagnosis could be made, taken from among 568 immediate cytology patients between July 2000 and June 2008. In immediate cytology at our hospital, a cytotechnologist goes to the bronchoscopy room just before tissue is collected. U-Pap stain is done immediately and examined microscopically. A tentative report is made orally on the spot as to the specimen adequacy, whether it is benign or malignant, and the presumed histopathological type. RESULTS: Of the 503 patients, the sample was inadequate in only two cases and there were no false positives. With respect to diagnostic accuracy, sensitivity was 82.9%, specificity was 100%, and the correct diagnosis rate was 85.6%. For lesions ≤3 cm, sensitivity was 77.2%, specificity was 100%, and the correct diagnosis rate was 81.3%, and for lesions ≤2 cm sensitivity was 74.7%, specificity was 100%, and the correct diagnosis rate was 81.3%. Diagnostic accuracy was thus maintained even for small lesions. No false positive case was detected. CONCLUSION: Because immediate cytology enables more precise sample collection, it is thought to be a useful technique that leads to improved diagnostic accuracy, while also improving the technical skill of the doctor collecting samples through immediate feedback.