Role of protease activated receptor-2 in lymph node metastasis of uterine cervical cancers

<p>Abstract</p> <p>Background</p> <p>Protease activated receptor-2 (PAR-2) has been implicated in cellular proliferation, invasion and metastasis in various tumors. Lymph node metastasis is an important patient prognostic factor for uterine cervical cancers. This prompted us to study the role of PAR-2 in lymph node metastasis of uterine cervical cancers.</p> <p>Methods</p> <p>Thirty patients underwent surgery for uterine cervical cancers. PAR-2 histoscores and mRNA levels were determined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. Patient prognosis was analyzed with a 48-month survival rate.</p> <p>Results</p> <p>PAR-2 histoscores and mRNA levels significantly (<it>P </it>< 0.05) increased in 12 of 30 metastatic lymph node lesions from the corresponding primary tumor. The 48-month survival rate of the 12 patients with increased PAR-2 levels in metastatic lymph nodes was 42%, while the rate of the other 18 patients with no change in PAR-2 levels was 82%, regardless of histopathological type.</p> <p>Conclusion</p> <p>PAR-2 might work on lymph node metastasis of uterine cervical cancers, and is considered to be a novel prognostic indicator for uterine cervical cancers.</p

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

<p>Abstract</p> <p>Background</p> <p>In addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.</p> <p>Methods</p> <p>We established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH₂). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).</p> <p>Result</p> <p>PAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.</p> <p>Conclusion</p> <p>Our data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.</p

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.</p> <p>Methods</p> <p>Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.</p> <p>Results</p> <p>Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.</p> <p>Conclusions</p> <p>While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.</p

Epithelial NEMO links innate immunity to chronic intestinal inflammation

Deregulation of intestinal immune responses seems to have a principal function in the pathogenesis of inflammatory bowel disease(1-4). The gut epithelium is critically involved in the maintenance of intestinal immune homeostasis-acting as a physical barrier separating luminal bacteria and immune cells, and also expressing antimicrobial peptides(3,5,6). However, the molecular mechanisms that control this function of gut epithelial cells are poorly understood. Here we show that the transcription factor NF kappa B, a master regulator of pro-inflammatory responses(7,8), functions in gut epithelial cells to control epithelial integrity and the interaction between the mucosal immune system and gut microflora. Intestinal epithelial-cell-specific inhibition of NF-kappa B through conditional ablation of NEMO ( also called I kappa B kinase-gamma ( IKK gamma)) or both IKK1 ( IKK alpha) and IKK2 ( IKK beta)-IKK subunits essential for NF-kappa B activation(7-9)-spontaneously caused severe chronic intestinal inflammation in mice. NF-kappa B deficiency led to apoptosis of colonic epithelial cells, impaired expression of antimicrobial peptides and translocation of bacteria into the mucosa. Concurrently, this epithelial defect triggered a chronic inflammatory response in the colon, initially dominated by innate immune cells but later also involving T lymphocytes. Deficiency of the gene encoding the adaptor protein MyD88 prevented the development of intestinal inflammation, demonstrating that Toll-like receptor activation by intestinal bacteria is essential for disease pathogenesis in this mouse model. Furthermore, NEMO deficiency sensitized epithelial cells to tumour-necrosis factor ( TNF)-induced apoptosis, whereas TNF receptor-1 inactivation inhibited intestinal inflammation, demonstrating that TNF receptor-1 signalling is crucial for disease induction. These findings demonstrate that a primary NF-kappa B signalling defect in intestinal epithelial cells disrupts immune homeostasis in the gastrointestinal tract, causing an inflammatory-bowel-disease-like phenotype. Our results identify NF-kappa B signalling in the gut epithelium as a critical regulator of epithelial integrity and intestinal immune homeostasis, and have important implications for understanding the mechanisms controlling the pathogenesis of human inflammatory bowel disease.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62858/1/nature05698.pd

Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection

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

The 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 [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. 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. © 2001 Cancer Research Campaign http://www.bjcancer.co

GPR54 (KISS1R) Transactivates EGFR to Promote Breast Cancer Cell Invasiveness

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness