rottlerin and acetovanillone

The NADPH oxidase (NOX) is a significant determinant for the expression and activity of γ-glutamyltransferase (GGT), which is frequently upregulated after increased levels of reactive oxygen species (ROS) and oxidative stress. Earlier studies on human colon carcinoma HT-29 cells have shown that treatment with phorbol 12-myristate 13-acetate (PMA) activates NOX thus increasing the intracellular level of ROS and upregulating GGT. Another important source of cellular ROS is the mitochondria, and treatment with the mitochondria uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) results in increased ROS levels. The present study shows that when HT-29 cells were simultaneously treated with both agents, a significant and synergic increase in intracellular ROS was detected. NOX activity contributed at least 50 % of this increase as inhibiting NOX activity with apocynin or downregulating the NOX activity using siRNA against p22 phox reduced the synergic ROS production. The combined FCCP and PMA treatment also provoked highly increased GGT mRNA levels after 24 h whereas only minor and delayed increases in GGT protein and enzyme activity levels were detected. The results strongly indicate that ROS production by both mitochondria and NOX is involved in the regulation of GGT expression in colon carcinoma cells.

Topics: Acetophenones; Benzopyrans; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Colonic Neoplasms; Drug Combinations; Drug Synergism; Enzyme Inhibitors; gamma-Glutamyltransferase; HT29 Cells; Humans; Mitochondria; NADPH Oxidases; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tetradecanoylphorbol Acetate; Uncoupling Agents

We investigate the cell signal transduction pathway protein kinase C (PKC) and the role of NADPH subunits in the process of TNF-α-induced endothelial apoptosis. Human umbilical vein endothelial cells (HUVEC) were treated with one of these: 1 mM PKC β(2) inhibitor CGP53353, 10 mM PKC δ inhibitor rottlerin, combination CGP53353 with rottlerin, 3 ×10(-4)M NADPH oxidase inhibitor apocynin, 5 × 10(-6)M NADPH oxidase peptide inhibitor gp91ds-tat. The apoptosis process was assessed by Hoechst 33342 stain, flow cytometry and Western blot analysis, while intracellular reactive oxygen species (ROS) production was detected by 2,7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The NADPH oxidase subunit gene and protein expression were assessed by quantitative real-time PCR and Western blot analysis, respectively. TNF-α significantly induced HUVEC apoptosis and ROS production, accompanying with dramatic upregulation of NADPH oxidase subunits: NOX2/gp91(phox), NOX4, p47(phox) and p67(phox), whereas these enhancements were abolished by the treatment with PKC inhibitors. High TNF-α level exposure induces HUVEC apoptosis, as well as a ROS generation increase via the PKC β(2)-dependent activation of NADPH oxidase. Although the PKC δ pathway may enhance TNF-α-induced HUVEC apoptosis, it does not involve the ROS pathway. Upregulation of expression of NADPH subunits is important in this process, which leads to a new target in antioxidative therapy for vascular disease prevention.

Topics: Acetophenones; Apoptosis; Benzopyrans; Human Umbilical Vein Endothelial Cells; Humans; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidases; Oxidative Stress; Phosphoproteins; Phthalimides; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-delta; Protein Subunits; Signal Transduction; Tumor Necrosis Factor-alpha

Chronic inflammation incited by bacteria in the saccular lung of premature infants contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). LPS-mediated type II alveolar epithelial cell (AEC) injury induces the expression of pro-inflammatory cytokines that trigger pulmonary neutrophil influx, alveolar matrix degradation and lung remodeling. We hypothesized that NADPH oxidase (Nox)-dependent mechanisms mediate LPS-induced cytokine expression in AEC. We examined the role of p47phox in mediating LPS-dependent inflammatory cytokine expression in A549 cells (which exhibit phenotypic features characteristic of type II AEC) and elucidated the proximal signaling events by which Nox is activated by LPS. LPS-induced ICAM-1 and IL-8 expression was associated with increased superoxide formation in AEC. LPS-mediated oxidative stress and cytokine expression was inhibited by apocynin and augmented by PMA demonstrating that Nox-dependent redox signaling regulates LPS-dependent pro-inflammatory signaling in AEC. In LPS-treated cells, p47phox translocated from the cytoplasm to the perinuclear region and co-localized with gp91phox. LPS also induced a temporal increase in p47phox serine304 phosphorylation in AEC. While inhibition of classical PKC and novel PKC with calphostin and rottlerin did not inhibit ICAM-1 or IL-8 expression, the myristolyated PKCζ pseudosubstrate peptide (a specific inhibitor of PKCζ) inhibited LPS-induced cytokine expression in AEC. Inhibition of PKCζ also attenuated LPS-mediated p47phox phosphorylation and perinuclear translocation in AEC. Consistent with these data, LPS activated PKCζ in AEC as evidenced by increased threonine410 phophorylation. We conclude that PKCζ-mediated p47phox activation regulates LPS-dependent cytokine expression in AEC. Selective inhibition of PKCζ or p47phox might attenuate LPS-mediated inflammation and alveolar remodeling in BPD.

Topics: Acetophenones; Benzopyrans; Cell Line, Tumor; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Intercellular Adhesion Molecule-1; Interleukin-8; Lipopolysaccharides; NADPH Oxidases; Naphthalenes; Oxidative Stress; Protein Kinase C; Pulmonary Alveoli; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Superoxides; Tetradecanoylphorbol Acetate