A role for protease-activated receptor-2 in pancreatic cancer cell proliferation

The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin and tryptase. The purpose of this study was to clarify the role of PAR-2 in proliferation of human pancreatic cancer cells. PAR-2 mRNA and protein expression were detected by RT-PCR and Western blotting in three cell lines, SW1990, Capan-2, and Panc-1. The PAR-2 agonist peptide, SLIGKV (25, 50 micro g/ml) and trypsin (10, 100 ng/ml) significantly increased cell proliferation. Enhancement of MAP kinase also was observed in cancer cells treated with SLIGKV and trypsin. In vivo, subcutaneous xenografted tumors showed significantly enhanced growth after treatment with SLIGKV. Tumor-associated trypsinogen (TAT) mRNA and protein expression was detected in SW1990 and Capan-2, suggesting autocrine trypsin production. PAR-2 activated by trypsin plays an important role in promoting proliferation of pancreatic cancer.     

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S?phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.     

Expression of protease activated receptor-2 (PAR-2) in gastric cancer

BACKGROUND AND OBJECTIVES: Recently, the four-kind cloning of the protease-activated receptor (PAR) had been carried out. PAR-2 is activated by trypsin and it is supposed that PAR-2 participated in proliferation of the endothelial cell or in neovascularization. We considered whether the expression of PAR-2 has relevance to progression in gastric cancer. METHODS: Immunohistochemical study by the envision method was carried out on 183 samples of gastric cancer in the first department of surgery, University of Fukui, using anti-PAR-2 mouse monoclonal antibody and on 95 samples of them that were pointed out advanced gastric cancers by pathological diagnosis using anti-trypsin rabbit polyclonal antibody. Tissues, which were stained more than 20% of the tumor cells, were classified as PAR-2 protein-positive. Correlation with immunostainings and clinicopathological factors was analyzed statistically. RESULTS: There were 77 (42.1%) carcinomas positive for PAR-2 expression. The PAR-2 expression was intensely strong on the cell membrane of primary cancer tissues. The expression of PAR-2 correlated with the depth of wall invasion, lymphatic invasion, venous invasion, and liver metastasis. The patients with PAR-2 expression-positive tumors had a significant poorer prognosis than those with expression-negative tumors. Univariate analyses identified PAR-2 expression as negative predictors. Multivariate analyses indicated that PAR-2 expression was not an independent factor. A positive reaction for trypsin was obtained in 45 (47.4%) patients. We found a significant correlation between PAR-2 immunostaining and trypsin immunostaining. CONCLUSION: The results of this study lead us to believe that expression of PAR-2 is concerned with progression of gastric cancer.     

Protease-activated receptor-2 regulates cell proliferation and enhances cyclooxygenase-2 mRNA expression in human pancreatic cancer cells

BACKGROUND AND OBJECTIVES: Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by trypsin. Recent studies have suggested that PAR-2 activity correlates with inflammatory processes and cell proliferation and that PAR-2 activation in non-neoplastic cells induces expression of cyclooxygenase-2 (COX-2). In the present study, we examined whether PAR-2 activation regulates cell proliferation and COX-2 expression by pancreatic cancer cells. METHODS: We analyzed PAR-2 expression immunohistochemically in 40 intraductal papillary-mucinous neoplasms (IPMNs) and 73 invasive ductal carcinomas (IDCs) of the pancreas. We used four pancreatic cancer cell lines (Panc1, T3M4, BxPC3, and MIApaca2) to measure cell proliferation and COX-2 mRNA expression after PAR-2 activation. RESULTS: PAR-2 protein was detected immunohistochemically in 85.0% of IPMNs and 65.8% of IDCs. Trypsin and a PAR-2 agonist peptide, SLIGKV, stimulated proliferation of each cell line in a dose-dependent manner. Exposure of cells to anti-PAR-2 neutralizing antibody prior to PAR-2 activation suppressed cell proliferation. In COX-2-positive cell lines (T3M4 and BxPC3), PAR-2 activation significantly increased COX-2 mRNA expression. CONCLUSIONS: Our results suggest that PAR-2 activation is associated with cell proliferation and COX-2 expression in pancreatic cancer cells. Blockade of the PAR-2 signaling pathway may be a novel strategy for suppressing pancreatic tumor growth.     

Expression of proteinase-activated receptor-2 in human pancreatic cancer: a possible relation to cancer invasion and induction of fibrosis

The proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. To clarify the presence of PAR-2 in human pancreatic cancer, the expression of PAR-2 was analyzed by RT-PCR, immunoblotting and immunocytochemistry using 5 human pancreatic cancer cell lines. And to evaluate the biological significance, immunohistochemical expression of PAR-2 in malignant and non-malignant human pancreatic tissues was examined using paraffin-embedded sections. The presence of PAR-2 was confirmed in all 5 pancreatic cancer cell lines and all 21 paraffin-embedded specimens from human pancreatic cancer examined. The expression of PAR-2 was found to be higher in the tissues with infiltrative growth pattern than those with expansive growth pattern. Moreover, significantly higher expression of PAR-2 was observed in the tissues which were accompanied by severe fibrosis. Even in the same specimen, the intensity of immunoreactivity tended to be stronger in the part with severe fibrosis than that with mild fibrosis. Similarly, the higher expression of PAR-2 was observed in chronic pancreatitis with severe fibrosis than with mild fibrosis. Taken together, these results suggest that the activation of PAR-2 is involved in cancer invasion and the induction of fibrosis in human pancreatic cancer.     

Signal of proteinase-activated receptor-2 contributes to highly malignant potential of human pancreatic cancer by up-regulation of interleukin-8 release

Proteinase-activated receptor-2 (PAR-2) is expressed in various tissues, including cancer lesions. However, the functional consequences of PAR-2 expression in cancer cells, especially in pancreatic cancer cells, are poorly understood. To clarify the biological significance of PAR-2 signaling in pancreatic cancer, we examined the production of growth factors and cytokines, such as IL-6, IL-8, bFGF, TGF-beta1, and VEGF, by specific ELISAs. Two human pancreatic cancer cell lines, SUIT2 and MiaPaCa2, which have been shown to express PAR-2, were stimulated by trypsin and PAR-2 activating peptide (PAR-2AP: SLIGKV-NH2). After 24 h, the culture supernatants were collected and specific ELISAs were performed. Although no significant changes were observed in the release of IL-6, bFGF, TGF-beta1, or VEGF, that of IL-8 was significantly up-regulated by PAR-2 agonists in a dose-dependent manner. In addition, IL-8 receptor expression was found in pancreatic cancer cells and fibroblasts. These results suggest that the PAR-2 signal up-regulates IL-8 release from pancreatic cancer cells. This up-regulated IL-8 has an effect on the pancreatic cancer cells in an autocrine manner and on the fibroblasts in a paracrine manner. Thus, this signal might contribute to tumor progression and characteristic fibrosis in pancreatic cancer.     

A small interfering RNA targeting proteinase-activated receptor-2 is effective in suppression of tumor growth in a Panc1 xenograft model

Proteinase-activated receptor-2 (PAR-2), which is a G protein-coupled receptor, is activated in inflammatory processes and cell proliferation. We previously demonstrated that an anti-PAR-2 antibody suppresses proliferation of human pancreatic cells in vitro. However, there have been no studies of PAR-2 signaling pathways in vivo. The aim of this study was to determine whether blockade of PAR-2 by RNA interference influences pancreatic tumor growth. We originally constructed small interfering RNAs (siRNAs) targeting human PAR-2, and performed cell proliferation assays of Panc1 human pancreatic cancer cell line with these siRNAs. Intratumoral treatment with these PAR-2 siRNAs and atelocollagen was also performed in a xenograft model with nude mice and Panc1 cells. siRNAs against human PAR-2 inhibited proliferation of Panc1 cells, whereas control scramble siRNAs had no effect on proliferation. The PAR-2 siRNAs dramatically suppressed tumor growth in the xenograft model. PAR-2-specific siRNA inhibited growth of human pancreatic cancer cells both in vitro and in vivo. Blockade of PAR-2 signaling by siRNA may be a novel strategy to treat pancreatic cancer.     

Meizothrombin,an intermediate of prothrombin cleavage potently activates renal carcinoma cells by interaction with PAR-type thrombin receptors

Recently, meizothrombin (MT), an intermediate enzyme in the prothrombin cleavage cascade has been shown to activate cells of a brain tumor cell line by interaction with PAR-1-type thrombin receptors with a potency comparable to that of thrombin. In this study, we investigated the effect of recombinant human MT (rMT) on calcium mobilization in primary cultures established from surgically resected human renal cell carcinomas. Meizothrombin induced very rapidly transient calcium mobilization in RCC cells comparable to that observed with thrombin. RCC cells stimulated with thrombin after rMT challenge were unable to elicit a new calcium response and vice versa. Therefore, rMT and thrombin seem to activate calcium signaling in primary RCC cultures by similar mechanisms including PAR-1- and PAR-3-type thrombin receptors as shown by using PAR-type specific antibodies. Our results demonstrate rMT as a potent activator of human RCC cells suggesting a function of not only thrombin but also of this catalytically active thrombin precursor enzyme in human renal cell carcinoma.     

Protease-activating-receptor-2 is frequently expressed in papillary adenocarcinoma of the gallbladder

Gallbladder carcinoma is one of the most devastating malignant tumors in Japan. An important risk factor for gallbladder carcinoma is pancreaticobiliary maljunction (PBM), which allows reciprocal reflux of bile and pancreatic juice. Protease-activated-receptor-2 (PAR-2), which is activated by trypsin, may be a key molecule in the process of carcinogenesis in the gallbladder epithelium. We investigated the relation between the expression of PAR-2 and clinicopathological findings in gallbladder carcinoma. The study group comprised 58 patients with gallbladder carcinoma. PAR-2 expression was identified by immunohistochemical staining of all tumor specimens. PAR-2 was expressed in cancerous gallbladder epithelium in 37 of 58 patients (64%). PAR-2 expression occurred more frequently in papillary adenocarcinoma (15 of 16 patients, 94%) than in non-papillary types (20 of 42 patients, 48%, p=0.005). Neither lymphatic invasion (p=0.03) nor venous invasion (p=0.009) occurred more frequently in gallbladder carcinoma with PAR-2 than in that without PAR-2. PAR-2 expression was not directly related to PBM (p=0.46). Papillary adenocarcinoma was associated with polypoid growth (p=0.01), PBM (p=0.01), decreased invasion to lymphatic (p=0.007) and venous vessels (p=0.005), lower T-factor (p<0.001), and lower clinical stage (p=0.02). PAR-2 is frequently expressed in papillary adenocarcinoma of the gallbladder. Trypsin may play an important role for carcinogenesis of the gallbladder through PAR-2 signaling.     

Actinic cheilitis: epithelial expression of COX-2 and its association with mast cell tryptase and PAR-2

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies, including oral cancers. Recent studies have shown that mast cell-derived protease tryptase can induce COX-2 expression by the cleavage of proteinase-activated receptor-2 (PAR-2). Actinic cheilitis (AC) is a premalignant form of lip cancer characterized by an increased density of tryptase-positive mast cells. To investigate the possible contribution of tryptase to COX-2 overexpression during early lip carcinogenesis, normal lip (n=24) and AC (n=45) biopsies were processed for COX-2, PAR-2 and tryptase detection, using RT-PCR and immunohistochemistry. Expression scores were obtained for each marker and tested for statistical significance using Mann-Whitney and Spearmann's correlation tests as well as multivariate logistic regression analysis. Increased epithelial co-expression of COX-2 and PAR-2, as well as, elevated subepithelial density of tryptase-positive mast cells were found in AC as compared to normal lip (P<0.001). COX-2 overexpression was found to be a significant predictor of AC (P<0.034, forward stepwise, Wald), and to be correlated with both tryptase-positive mast cells and PAR-2 expression (P<0.01). The results suggest that epithelial COX-2 overexpression is a key event in AC, which is associated with increased tryptase-positive mast cells and PAR-2. Therefore, tryptase may contribute to COX-2 up-regulation by epithelial PAR-2 activation during early lip carcinogenesis.     

环氧化酶-2 及COX-2mRNA 在脑膜瘤中的表达及意义

  目的 观察环氧化酶-2(COX-2)蛋白及COX-2mRNA在人脑膜瘤组织中的表达，探讨COX-2基因表达与脑膜瘤病理分型的相关性。方法 收集1995年3月～2003年4月在我院住院的56例脑膜瘤病人经手术治疗切除的肿瘤标本，另选颅脑损伤内减压手术切除的脑组织标本8例作为正常对照标本。石蜡包埋切片后，用免疫组化的方法检测COX-2蛋白在脑膜瘤中的表达，用原位杂交的方法检测COX-2mRNA在脑膜瘤中的表达。结果 脑膜瘤的COX-2蛋白和COX-2mRNA的阳性表达率分别为53．57％和58．93％；正常对照组不表达。恶性脑膜瘤的COX-2蛋白和COX-2mRNA表达率明显高于良性脑膜瘤(P<0．01)。结论 COX-2和COX-2mRNA在脑膜瘤中高度表达，且与脑膜瘤的病理分型有关；从蛋白水平和基因分子水平阐明COX-2可能在脑膜瘤的发生、发展过程中起重要作用。     

PAR-1- and PAR-3-type thrombin receptor expression in primary cultures of human renal cell carcinoma cells.

In this study, we report coexpression of proteinase-activated receptor (PAR)-1- and PAR-3-type thrombin receptors in primary cultures obtained from surgically resected specimens of renal cell carcinomas (RCCs). Receptor expression on RNA level was evaluated by using the RT-PCR technique. Results demonstrated the presence of mRNA encoding PAR-1 and PAR-3, but mRNA encoding PAR-4 could not be found in human RCC cells. The expression of PAR-1 and PAR-3 on protein level was investigated with confocal laser fluorescence and freeze-fracture electron microscopy. Both thrombin receptor types were localized on the cell membrane but were also found on intracellular compartments of RCC cells. On the outer cell membrane, clustering of PAR-1 and PAR-3 molecules was partly observed. This is the first study demonstrating presence of both PAR-1 and PAR-3 in human carcinoma cells.     

Effects of Tissue Factor,PAR-2 and MMP-9 Expression on Human Breast Cancer Cell Line MCF-7 Invasion

Objective: This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2(PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence oninvasiveness. Methods: Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established.TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasivenesswas evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. Results: TFprotein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expressionwas significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR-2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TFShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TFand MMP-9 expression was positively correlated with the invasiveness of tumor cells. Conclusion: TF, PAR-2,and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.