7-(3-(3-hydroxy-4-(4--iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic-acid and Adenocarcinoma

Matrix metalloproteinases (MMPs) and monocyte chemoattractant protein-1 (MCP-1, CCL2) are known to be upregulated in many tumors. Their roles in tumor invasion and metastasis are being uncovered. How they are related to each other and involved in tumor progression remains to be determined. Earlier it was reported that I-BOP-initiated activation of thromboxane A2 receptor (TP) induced the release of MMP-1, MMP-3, and MMP-9 from lung cancer A549 cells overexpressing TPα (A549-TPα). Herein it was found that MMP-1, but not MMP-3 or MMP-9, induced the expression of MCP-1 in A549 cells. Conditioned medium (CM) from I-BOP activated, MMP-1 siRNA pretreated A549-TPα cells induced greatly attenuated expression of MCP-1 in A549 cells indicating that MMP-1 in the CM contributed significantly to the expression of MCP-1. MMP-1 was shown to activate protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1 to increase the expression of MCP-1 in A549 cells. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, and also PAR2 agonist peptide, was inhibited by a PAR2 antagonist; (2) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was attenuated significantly by pretreatment of cells with PAR2-siRNA. These results suggest that PAR2 is a novel MMP-1 target mediating MMP-1-induced signals in A549 lung cancer cells.

Topics: Adenocarcinoma; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Chemokine CCL2; Culture Media, Conditioned; Fatty Acids, Unsaturated; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Real-Time Polymerase Chain Reaction; Receptor, PAR-2; Receptors, Thromboxane A2, Prostaglandin H2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured

Thromboxane A(2) receptor (TP) has been shown to play an important role in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Here we report that TP mediates cancer cell invasion by inducing expression of matrix metalloproteinases (MMPs). TP agonist, I-BOP, significantly elevated MMP-1, MMP-3, MMP-9 and MMP-10 mRNA levels in A549 human lung adenocarcinoma cells overexpressing TPα or TPβ. The secretion of MMP-1 and MMP-9 in conditioned media was determined using Western blot analysis and zymographic assay. Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549 cells expressing TPα or TPβ was determined by invasion assay. GM6001, a general inhibitor of MMPs, decreased basal and I-BOP-induced cell invasion. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated cell invasion suggesting that other MMPs induced by I-BOP were also involved. Our studies establish the relationship between TP and MMPs in cancer cell invasion and suggest that the thromboxane A(2) (TXA(2))-TP signaling is a potential therapeutic target for cancer invasion and metastasis.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Bridged Bicyclo Compounds, Heterocyclic; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cell Movement; Dipeptides; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Matrix Metalloproteinase 1; Matrix Metalloproteinase 10; Matrix Metalloproteinase 3; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Neoplasm Invasiveness; Protease Inhibitors; Protein Kinase C; Receptors, Thromboxane A2, Prostaglandin H2; RNA Interference; RNA, Messenger; Signal Transduction; Thromboxane A2; Time Factors; Transcription Factor AP-1; Transfection; Up-Regulation

The role of thromboxane receptor alpha (TPalpha) in tumor growth and angiogenesis was investigated in a nude mice model and in cell culture. Stable human lung cancer A549 cells over-expressing TPalpha (A549-TPalpha) was generated and used to inoculate athymic nude mice. A549-TPalpha cells induced greater tumor growth and increased vascularization in tumors than in the control A549 cells. Increased angiogenesis was further verified by studying the induction of vascular endothelial growth factor (VEGF) in A549-TPalpha cells. I-BOP, an agonist of TP, stimulated the expression of VEGF in this cell line as well as in another human lung cancer H157 cells in a time and dose dependent manner. The expression of VEGF was determined at both the mRNA and protein levels. The signaling pathways that are involved in I-BOP-induced VEGF expression were further examined by the use of inhibitors. Inhibition of the extracellular signal-regulated kinase (ERK) activation blocked the induction almost completely indicating that ERK activation was an essential step in the induction. Each of the three upstream kinases, protein kinase A, EGFR kinase and Src kinase, contributed partially to the overall induction. However, PI 3-kinase and protein kinase C had minimal contribution. These results indicate that activation of the TPalpha induces the expression of VEGF through multiple signaling pathways.

Topics: Adenocarcinoma; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cloning, Molecular; Fatty Acids, Unsaturated; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Receptors, Thromboxane; Signal Transduction; Transgenes; Tumor Burden; Vascular Endothelial Growth Factor A

Human lung adenocarcinoma A549 cells stably transfected with TPalpha (A549-TPalpha) were used to study agonist I-BOP-induced expression of cyclooxygenase-2 (COX-2) and the related mechanisms of induced expression. I-BOP, a TP agonist, induced a time- and dose-dependent expression of COX-2 in A549-TPalpha cells. The signaling pathways of I-BOP-induced COX-2 expression were elucidated by using various inhibitors of the signaling molecules. The effects of these inhibitors were assessed at protein level, enzyme activity and promoter activity of COX-2. Within MAPK family, both ERK and p38 MAPK but not JNK/SAPK pathways were involved in the induction. Other pathways such as JAK/Stat3 pathway and beta-catenin/TCF/LEF pathway also participated in the induction. The activation of key signaling molecules, ERK, p38 MAPK, CREB and NF-kappaB, involved in the COX-2 transcription was further studied at the phosphorylation step. Activation of ERK and p38 MAPK appeared to be mediated primarily by transactivation of EGFR, whereas activation of CREB and NF-kappaB was mediated by PKA, PKC and ERK. The role of CREB and NF-kappaB in I-BOP-induced COX-2 expression was further explored at the promoter level. Studies on promoter fragments and mutation of responsive motifs indicated that CRE and NF-kappaB sites are critical for the COX-2 induction. Distal NF-kappaB site is essential for the basal induction of the COX-2 transcription, whereas CRE and proximal NF-kappaB sites are important for the induced transcription. These results indicate that I-BOP-induced COX-2 expression through multiple signaling pathways.

Topics: Adenocarcinoma; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cyclooxygenase 2; Enzyme Induction; Fatty Acids, Unsaturated; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Protein Isoforms; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Signal Transduction; Time Factors