Bong Whan Ahn

Interleukin-1β stimulates IL-8 expression through MAP kinase and ROS signaling in human gastric carcinoma cells

Recent studies have suggested that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. In this study, the effect of IL-1β on IL-8 expression in human gastric cancer TMK-1 cells and the underlying signal transduction pathways were investigated. IL-1β induced the IL-8 expression in a time- and concentration-dependent manner. IL-1β induced the activation of extracellular signal-regulated kinases-1/2 and P38 mitogen-activated protein kinase (MAPK), but not the activation of c-jun amino-terminal kinse and Akt. Specific inhibitors of MEK-1 (PD980590) and P38 MAPK (SB203580) were found to suppress the IL-8 expression and the IL-8 promoter activity. Expression of vectors encoding a mutated-type MEK-1 and P38 MAPK resulted in decrease in the IL-8 promoter activity. IL-1β also induced the production of reactive oxygen species (ROS). N-acetyl cysteine (NAC) prevented the IL-1β-induced ROS production and IL-8 expression. In addition, exogenous H2O2 could induce the IL-8 expression. Deletional and site-directed mutagenesis studies on the IL-8 promoter revealed that activator protein-1 (AP-1) and nuclear factor (NF)-κB sites were required for the IL-1β-induced IL-8 transcription. Electrophoretic mobility shift assay confirmed that IL-1β increased the DNA-binding activity of AP-1 and NF-κB. Inhibitor (PD980590, SB203580) and ROS scavenger (NAC) studies revealed that the upstream signalings for the transcription factors AP-1 and NF-κB were MAPK and ROS, respectively. Conditioned media from the TMK-1 cells pretreated with IL-1β could remarkably stimulate the in vitro growth of HUVEC and this effect was partially abrogated by IL-8-neutralizing antibodies. The above results suggest that MAPK-AP-1 and ROS-NF-κB signaling pathways are involved in the IL-1β-induced IL-8 expression and that these paracrine signaling pathways induce endothelial cell proliferation.

IL-1β induces MMP-9 via reactive oxygen species and NF-κB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2(),7()-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.

Alterations in the activity and expression of endothelial NO synthase in aged human endothelial cells

This study was to investigate factors underlying the age-related decrease in NO production in vascular endothelial cells. The age-related changes in NO production, the activity and expression level of eNOS, and eNOS binding proteins, were studied in HUVECs. NO production in HUVECs significantly decreased in an age-dependent manner. The potentiation of NO production by L-Arg was significantly suppressed by L-NIO (eNOS-specific inhibitor) in young HUVECs and was suppressed by 1400W (iNOS-specific inhibitor) in aged HUVECs. The aged HUVECs had lower eNOS protein levels than young cells. eNOS phosphorylation at Ser-1177 (active) decreased gradually from PDL 23 through 40, and eNOS phosphorylation at Thr-495 (inactive) increased in aged cells. Changes of intracellular eNOS binding proteins, such as caveolin-1, pAkt, and Hsp90, as well as interaction between eNOS and eNOS binding proteins, indicated decreasing enzyme activity in aged HUVECs. Aging might decrease the activity as well as expression level of eNOS in HUVECs. And the decrease in eNOS activity probably implicated to the alterations in the regulatory binding proteins. For further study, it needs to be confirmed that the age-related change in the intracellular distribution of eNOS and the relative contribution of eNOS and iNOS on vascular dysfunction in aged endothelial cells.

Lysophosphatidic acid promotes cell invasion by up-regulating the urokinase-type plasminogen activator receptor in human gastric cancer cells.

There is a strong correlation between the overexpression of urokinase‐type plasminogen activator receptor (uPAR) and gastric cancer invasion. This study examined the effect of phospholipid lysophosphatidic acid (LPA) on uPAR expression in human gastric cancer AGS cells and the underlying signal transduction pathways. Treating human gastric AGS cells with LPA induced the expression of uPAR mRNA and promoter activity in both a time‐ and dose‐dependent manner. Small interfering RNA targeting for LPA receptors, dominant negative Rho‐family GTPase (RhoA, Rac1, and Cdc42) and an expression vector encoding a mutated c‐jun (TAM67) partially blocked the LPA‐induced uPAR expression. Site‐directed mutagenesis and electrophoretic mobility shift studies showed that the transcription factors activation protein‐1 (AP‐1) and nuclear factor (NF)‐κB are essential for the LPA‐induced uPAR transcription. In addition, AGS cells treated with LPA showed enhanced invasion, which was partially abrogated by the uPAR‐neutralizing antibodies and inhibitors of Rho kinase, JNK, and NF‐κB. This suggests that LPA induces uPAR expression through the LPA receptors, Rho‐family GTPase, JNK, AP‐1 and NF‐κB signaling pathways, which in turn stimulates the cell invasiveness of human gastric cancer AGS cells. J. Cell. Biochem. 104: 1102–1112, 2008.

Total peroxyl radical‐trapping ability and anti‐oxidant vitamins of the umbilical venous plasma and the placenta in pre‐eclampsia

Aim:u2002 Our purpose was to investigate lipid peroxide levels, total peroxyl radical‐trapping anti‐oxidative parameter (TRAP) values, and anti‐oxidant vitamin levels in umbilical venous plasma and placental tissues, and to evaluate their roles in the pathophysiology of pre‐eclampsia.

Redox regulation of the tumor suppressor PTEN by glutathione

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Δ and gsh2Δ mutants. Expression of γ‐glutamylcysteine synthetase Gsh1 in the gsh1Δ mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration‐ and time‐dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Hemin inhibits lipid peroxidation induced by ascorbate/FeSO4 and 2,2'-azobis-2-amidino-propane hydrochloride (ABAP)

Hemin blocked lipid peroxidations induced by either ascorbate/FeSO4, a metal-catalyzed oxidation system, or 2,2-azobis-2-amidino-propane hydrochloride (ABAP) which produces peroxy radicals at constant rates. Hemin at very low micromolar concentrations strongly inhibited the ascorbate/FeSO4-induced peroxidation of rat liver phopholipids, soybean phosphatidylcholine and arachidonic acid, and this inhibition was also evident with the use of ABAP, although much higher concentrations of hemin were required than those for the inhibition of ascorbate/FeSO4-induced lipid peroxidation. However, hemoproteins such as hemoglobin, myoglobin and cytochrome C did not show any significant effect on this lipid peroxidation. Hemopexin and albumin abolished the inhibitory action of hemin. During incubation with ascorbate/FeSO4 or ABAP, hemin underwent a change in its absorption spectrum, resulting in a progressive decrease in the peak height of the characteristic absorption band at 385 nm. The above results suggest that hemin may act as an important antioxidant in vivo, protecting lipids from the peroxidative damage.

Gastroprotective Effects of Glutinous Rice Extract against Ethanol-, Indomethacin-, and Stress-induced Ulcers in Rats.

This study was designed to evaluate the efficacy of an orally administered aqueous extract of glutinous rice (GRE) to protect against acute gastric mucosal lesions induced by ethanol, indomethacin, and water immersion restraint stress in rats and to characterize the active substances responsible for the protection. GRE was shown to dose-dependently prevent the gastric lesions induced by the above ulcerogenic treatments at doses of 30 to 300 mg/kg. GRE treatment increased the gastric mucin content and partially blocked the ethanol-induced depletion of the gastric mucus layer. Also, it increased the nonprotein sulfhydryl concentration in the gastric mucosa. The gastroprotective action of GRE was markedly enhanced by co-treatment with 4-8 mg/kg tea extracts. The activity of GRE was completely lost by heat treatment at 80℃ for 3 min or treatment with 0.01% pepsin at 37℃ for 1 h. Protein extraction studies indicated that prolamins are involved in the gastroprotective activity of GRE. Our results suggest that glutinous rice proteins are useful for the prevention and treatment of gastritis and peptic ulcer.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.