tosylphenylalanyl-chloromethyl-ketone and Lung-Neoplasms

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

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

Three synthetic irreversible enzyme inhibitors (75 microM di-iso-propylphosphorofluoridate (DFP), 310 microM N alpha-p-tosyl-L-lysine (TLCK) and 240 microM L-1-tosylamide-2-phenylethyl (TPCK) chloromethyl ketone), as well as the transition state analogue chymostatin, inhibit the development of Lewis lung adenocarcinoma (3LL) in C57 BI/6 mice, when 3LL cells are treated once and for a limited period (60 min) prior to grafting. These compounds demonstrate divergent protease specificity and, in the case of TLCK and TPCK, convergent reactivity toward the highly conserved protein kinase catalytic subunit. Using 200 microM chymostatin and low doses (25-40 microM) of the irreversible enzyme inhibitors, the antimetastatogenic effect is revealed to be specific, as primary tumor development is not affected. Although no direct experimental evidence can be forwarded, our results fit with the concept that the motile metastatogenic 3LL cells may constitute a phenotype which, in contrast to the resident cells from the primaries, responds to these enzyme inhibitors in a highly sensitive manner.

Topics: Affinity Labels; Animals; Female; Isoflurophate; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; Protease Inhibitors; Protein Kinases; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone