tosylphenylalanyl-chloromethyl-ketone and Adenocarcinoma

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which is abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and cell proliferation via interaction with its receptor, alphavbeta3 integrin. However, the effect of OPN on migration activity in human lung cancer cells is mostly unknown. Here we found that OPN increased the migration via activation of alphavbeta3 integrin in human lung cancer cells (A549 cells). Phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002), Akt inhibitor or ERK inhibitor (PD98059) inhibited the OPN-induced increase in the migration of lung cancer cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), p85 subunit of PI3K, serine 473 of Akt and ERK. In addition, treatment of A549 cells with NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) inhibited OPN-induced migration of lung cancer cells. Stimulation of A549 cells with OPN also induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OPN-mediated increases in IKK alpha/beta, IkappaBalpha and p65 Ser(536) phosphorylation were inhibited by Ly294002, Akt inhibitor and PD98059. Co-transfection with FAK, p85, Akt and ERK mutants also reduced the OPN-induced kappaB-luciferase activity. Taken together, these results suggest that OPN acts through alphavbeta3 integrin, which in turn activates the FAK, PI3K, Akt, ERK and NF-kappaB pathways, contributing to the migration of lung cancer cells.

Topics: Adenocarcinoma; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Cell Movement; Chromones; Flavonoids; Focal Adhesion Kinase 1; Humans; Integrin alphaVbeta3; Lung Neoplasms; Morpholines; NF-kappa B; Oncogene Protein v-akt; Osteopontin; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proline; Signal Transduction; Thiocarbamates; Tosylphenylalanyl Chloromethyl Ketone

Most of cells exhibit low nuclear level of NF-kappaB. However, in some cell lines and tissues aberrantly activated NF-kappaB is playing an important role in cell motility, growth control and survival. Here we describe the result of decrease of constitutive NF-kappaB level in different adenocarcinoma cell lines. Treatment of mouse adenocarcinoma cell line CSML-100 with both synthetic (TPCK or PDTC) or natural (I(kappaB)-alpha) NF-kappaB inhibitors caused apoptotic death. Low doses of TPCK were harmless for CSML100 cells but sensitized them to TNF-induced apoptosis. Death of CSML100 cells in the presence of high concentration TPCK was not accompanied with significant changes in c-myc activity but strongly correlated with rapid decrease in p53 level. Thus, mutual behavior p53 and NF-kappaB represented a unique feature of TPCK-induced apoptosis in CSML-100 adenocarcinoma cells.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Nucleus; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Giant Cells; I-kappa B Proteins; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Proline; Protein Binding; Protein Subunits; Proto-Oncogene Proteins c-myc; Thiocarbamates; Tosylphenylalanyl Chloromethyl Ketone; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential.

Topics: Adenocarcinoma; Cell Adhesion; Dexamethasone; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; NF-kappa B; Pancreatic Neoplasms; Tosylphenylalanyl Chloromethyl Ketone; Transcription Factor RelA; Transcription, Genetic; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

TNF alpha and IL-1 each can activate NF-kappa B and induce gene expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix enzyme which can provide critical protection against hyperoxic lung injury. The regulation of MnSOD gene expression is not well understood. Since redox status can modulate NF-kappa B and potential kappa B site(s) exist in the MnSOD promoter, the effect of thiols (including NAC, DTT and 2-ME) on TNF alpha and IL-1 induced activation of NF-kappa B and MnSOD gene expression was investigated. Activation of NF-kB and increased MnSOD expression were potentiated by thiol reducing agents. In contrast, thiol oxidizing or alkylating agents inhibited both NF-kappa B activation and elevated MnSOD expression in response to TNF alpha or IL-1. Since protease inhibitors TPCK and TLCK can inhibit NF-kappa activation, we also investigated the effect of these compounds on MnSOD expression and NF-kappa B activation. TPCK and TLCK each inhibited MnSOD gene expression and NF-kappa B activation. Since the MnSOD promoter also contains an AP-1 binding site, the effect of thiols and thiol modifying agents on AP-1 activation was investigated. Thiols had no consistent effect on AP-1 activation. Likewise, some of the thiol modifying compounds inhibited AP-1 activation by TNF alpha or IL-1, whereas others did not. Since diverse agents had similar effects on activation of NF-kappa B and MnSOD gene expression, we have demonstrated that activation of NF-kappa B and MnSOD gene expression are closely associated and that reduced sulfhydryl groups are required for cytokine mediation of both processes.

Topics: Adenocarcinoma; Alkylating Agents; Base Sequence; Enzyme Induction; Humans; Interleukin-1; Isoenzymes; Lung Neoplasms; Manganese; Mitochondria; Molecular Sequence Data; NF-kappa B; Oxidants; Oxidation-Reduction; Promoter Regions, Genetic; Protease Inhibitors; Sulfhydryl Compounds; Superoxide Dismutase; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Transcription Factor AP-1; Tumor Necrosis Factor-alpha