Proteinase-activated receptor-2-mediated signaling and inhibition of DNA synthesis in human pancreatic cancer cells.

CONCLUSION: Proteinase-activated receptor-2 (PAR-2)-mediated effects contribute to the intracellular signaling network in pancreatic tumor cells. A role of PAR-2 as negative regulator in human pancreatic tumor growth might be implied. BACKGROUND: Using the human pancreatic tumor cell line MIA PaCa-2, we evaluated cellular effects of trypsin and the PAR-2-activating peptide SLIGRL on [Ca2+]i mobilization, Ins(1,4,5)P3 level, and protein kinase (PKC) activation. Furthermore, PAR-2 involvement in the regulation of cell proliferation has been estimated by measurement of [3H]thymidine incorporation in MIA PaCa-2 cells. RESULTS: Trypsin and the PAR-2 synthetic peptide agonist SLIGRL induced [Ca2+]i mobilization, transient increase in inositol (1,4,5) triphosphate level, and PKC translocation in MIA PaCa-2 cells. In addition, SLIGRL induced a decrease in DNA synthesis in MIA PaCa-2 cells.

Protein kinase C is involved in cholecystokinin octapeptide-induced proliferative action in rat glioma C6 cells.

Chotecystoknin octapeptide (CCK-8) has been shown to stimulate DNA synthesis in rat glioma C6 cells by activation of CCKB type receptors. However, the signalling pathways contributing to this proliferative action in C6 cells have not been investigated thus far. This study demonstrated that stimulation of rat glioma C6 cells with CCK-8S resulted in activation of protein kinase C isozymes betaI, betaII, gamma and zeta. The participation of protein kinase C in the CCK-8S-induced effect on C6 cell growth was demonstrated by measurement of [3H]thymidine incorporation and estimation of cell number. The data indicate that CCK-8S stimulates growth in rat glioma C6 cells by a protein kinase C-dependent mechanism.

Functional thrombin receptor PAR1 in primary cultures of human glioblastoma cells.

In this study we investigated primary cultures obtained from two glioblastomas surgically removed from a 64-year-old man and a 50-year-old woman, respectively. The presence of the tethered ligand thrombin receptor PAR1 (protease-activated receptor 1) in these cells was demonstrated at the level of receptor binding by using immunofluorescence studies with the monoclonal anti-PAR1 antibody Mab 31-2. Stimulation of human glioblastoma cells both with alpha-thrombin and the thrombin receptor activating peptide TRAP-6 resulted in a series of [Ca+]i spikes as shown by confocal laser fluorescence microscopy with fluo-3 as calcium sensitive fluorescence indicator. This effect was completely blocked with the thrombin receptor antagonist peptide T1. Our results demonstrate functional thrombin receptors (PAR1) in primary cultures of human glioblastomas for the first time.

Characterization of functional thrombin receptors in human pancreatic tumor cells (MIA PACA-2).

In this article, the "tethered ligand" thrombin receptor was identified on human pancreatic tumor cells, MIA PaCa-2, using immunofluorescence studies with a monoclonal anti-thrombin receptor antibody. Pharmacological characterization, using 3H-labeled thrombin receptor activating peptide-6 (TRAP-6) as radioligand, demonstrated a single class of high-affinity binding sites (KD = 9.1+/-1.8 x 10(-7) M) and a binding capacity of 13.9+/-0.7 fmol/mg protein. These binding sites represent functional thrombin receptors, as shown by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, protein kinase C translocation from cytosol to the cell membrane, and stimulation of DNA synthesis in MIA PaCa-2 cells. These results provide the first identification of tethered ligand thrombin receptor in human pancreatic cancer cells and suggest thrombin receptor involvement in mechanisms of human pancreatic tumor progression.

Thrombin receptor activation results in calcium signaling and protein kinase C-dependent stimulation of DNA synthesis in HEp-2g laryngeal carcinoma cells.

BACKGROUND: Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined. METHODS: In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of alpha-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated. RESULTS: Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 +/- 2.2 x 10(-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The function of these binding sites was demonstrated by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, alpha-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide. CONCLUSIONS: To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism.

Thrombin has a bimodal effect on glioma cell growth.

Using rat glioma C6 cells as a model, we have found a bimodal effect of alpha-thrombin on cell growth. In C6 cells treated with alpha-thrombin at concentrations from 0.02 nM to 1.0 nM, inhibition of cell proliferation was noted. Because the thrombin receptor agonist peptide TRAP-6 also induced inhibition of cell proliferation and the thrombin receptor antagonist peptide T1 prevented the inhibitory effect of alpha-thrombin on C6 glioma cell growth, thrombin receptor involvement in antiproliferative action of alpha-thrombin in C6 glioma cells is highly likely. However, stimulation of cell proliferation observed when C6 cells were treated with alpha-thrombin at higher doses (> 1.0 nM) seems to be mediated by as yet undefined thrombin receptor-independent biochemical mechanisms.

Cholecystokinin B-type receptor signaling is involved in human pancreatic cancer cell growth.

Cholecystokinin (CCK) is known to stimulate pancreatic cancer cell growth, but no detailed CCK receptor subtype characterization and investigation of CCK receptor-mediated cellular responses in human pancreatic cancer cells have been reported thus far. In this study, CCK binding sites were identified in human pancreatic cancer cells (MIA-PaCa-2) using radioligand binding studies. Pharmacological characterization demonstrated a single class of high-affinity CCK sites on MIA-PaCa-2 cells (326 +/- 18 pM, receptor density 16.9 +/- 2.3 fmol/mg protein). These CCK binding sites displayed a typical CCKB binding profile as shown in competition studies by using different CCK-related compounds and non-peptide CCK antagonists discriminating between CCKA and CCKB sites. CCKB receptor-connected effector systems have been characterized in MIA-PaCA-2 cells, and their involvement in CCK-8S-induced proliferative effects on MIA-PaCa-2 cells has been demonstrated.