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Reactive oxygen species regulate urokinase plasminogen activator expression and cell invasion via mitogen-activated protein kinase pathways after treatment with hepatocyte growth factor in stomach cancer cells.

Lee KH, Kim SW, Kim JR - J. Exp. Clin. Cancer Res. (2009)

Bottom Line: We confirmed that Rac-1 regulated ROS production after activation of the AKT pathway with HGF.Exogenously added H2O2 promoted the expression of HGF, but not in a dose-dependent manner and also showed negative expression of HGF after co-treatment with H2O2 and HGF.We clarified the downstream pathways regulated by ROS after treatment with H2O2, which showed negative control between FRK and p38 kinase activities for uPA regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea. lkhee@med.yu.ac.kr

ABSTRACT

Background: Reactive oxygen species (ROS) are closely associated with the intracellular signal cascade, thus strongly implicating involvement in tumor progression. However, the mechanism by which ROS are generated and how ROS target downstream molecules to trigger tumor metastasis is unclear. In this study, we investigated the underlying signal pathways in ROS-induced urokinase plasminogen activator (uPA) expression in the human gastric cancer cells, NUGC-3 and MKN-28.

Methods and results: Intracellular ROS, as determined using the fluorescent probe, 2'-7' dichlorofluorescein diacetate, decreased after treatment with hepatocyte growth factor (HGF). We confirmed that Rac-1 regulated ROS production after activation of the AKT pathway with HGF. Exogenously added H2O2 promoted the expression of HGF, but not in a dose-dependent manner and also showed negative expression of HGF after co-treatment with H2O2 and HGF. Treatment with NAC, an intracellular free radical scavenger, decreased the enhancement of uPA production and tumor invasion in both cells. We clarified the downstream pathways regulated by ROS after treatment with H2O2, which showed negative control between FRK and p38 kinase activities for uPA regulation.

Conclusion: HGF regulates Rac-1-induced ROS production through the Akt pathway and ROS regulates uPA production and invasion via MAP kinase, which provides novel insight into the mechanisms underlying the progression of gastric cancer.

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Effects of HGF and H2O2/LY 294002 on Rac-1 activation. Serum-starved cells was pretreated with or without H2O2 (100 μM) for 30 min and then treated with or without 10 ng/ml HGF (A). Rac-1 dominant positive cells (Q61L) were treated with or without HGF (B). After incubation for 15 min, the cells were collected, washed, and then sonicated. Cell lysates were immunoprecipitated with PAK-1 PBD and Rac-1 activation was measured by Western blotting with a Rac-1 antibody. Representative data from three independent experiments were shown.
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Figure 2: Effects of HGF and H2O2/LY 294002 on Rac-1 activation. Serum-starved cells was pretreated with or without H2O2 (100 μM) for 30 min and then treated with or without 10 ng/ml HGF (A). Rac-1 dominant positive cells (Q61L) were treated with or without HGF (B). After incubation for 15 min, the cells were collected, washed, and then sonicated. Cell lysates were immunoprecipitated with PAK-1 PBD and Rac-1 activation was measured by Western blotting with a Rac-1 antibody. Representative data from three independent experiments were shown.

Mentions: We examined the role of HGF in modulating ROS production, particularly as regulated by Rac-1. Treatment with HGF suppressed the basal activity of Rac-1 and increased Rac-1 activity induced by H2O2 treatment (Figure 2A). In addition, treatment with HGF suppressed also the Rac-1 activity increased in Rac-1 dominant positive cells (Figure 2B). Pretreatment of cells with LY 294002, a PI3-kinase inhibitor, activated Rac-1 (Figure 3). Next, we examined whether Akt is involved in the reduction of the ROS level induced by HGF. Treatment of NUGC-3 and MKN-28 cells with HGF caused Akt activation in a dose-dependent manner (Figure 4A) and pre-incubation of cells with LY 294002 reduced HGF-induced Akt phosphorylation (Figure 4B). Furthermore, inhibition of Akt by LY 294002 treatment increased the ROS levels. More importantly, the effect of LY 294002 was abolished by HGF, as determined using DCF-DA by flow cytometry (Figure 5). These results suggest that PI3-kinase is an essential mediator through which HGF inhibits ROS generation.


Reactive oxygen species regulate urokinase plasminogen activator expression and cell invasion via mitogen-activated protein kinase pathways after treatment with hepatocyte growth factor in stomach cancer cells.

Lee KH, Kim SW, Kim JR - J. Exp. Clin. Cancer Res. (2009)

Effects of HGF and H2O2/LY 294002 on Rac-1 activation. Serum-starved cells was pretreated with or without H2O2 (100 μM) for 30 min and then treated with or without 10 ng/ml HGF (A). Rac-1 dominant positive cells (Q61L) were treated with or without HGF (B). After incubation for 15 min, the cells were collected, washed, and then sonicated. Cell lysates were immunoprecipitated with PAK-1 PBD and Rac-1 activation was measured by Western blotting with a Rac-1 antibody. Representative data from three independent experiments were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698863&req=5

Figure 2: Effects of HGF and H2O2/LY 294002 on Rac-1 activation. Serum-starved cells was pretreated with or without H2O2 (100 μM) for 30 min and then treated with or without 10 ng/ml HGF (A). Rac-1 dominant positive cells (Q61L) were treated with or without HGF (B). After incubation for 15 min, the cells were collected, washed, and then sonicated. Cell lysates were immunoprecipitated with PAK-1 PBD and Rac-1 activation was measured by Western blotting with a Rac-1 antibody. Representative data from three independent experiments were shown.
Mentions: We examined the role of HGF in modulating ROS production, particularly as regulated by Rac-1. Treatment with HGF suppressed the basal activity of Rac-1 and increased Rac-1 activity induced by H2O2 treatment (Figure 2A). In addition, treatment with HGF suppressed also the Rac-1 activity increased in Rac-1 dominant positive cells (Figure 2B). Pretreatment of cells with LY 294002, a PI3-kinase inhibitor, activated Rac-1 (Figure 3). Next, we examined whether Akt is involved in the reduction of the ROS level induced by HGF. Treatment of NUGC-3 and MKN-28 cells with HGF caused Akt activation in a dose-dependent manner (Figure 4A) and pre-incubation of cells with LY 294002 reduced HGF-induced Akt phosphorylation (Figure 4B). Furthermore, inhibition of Akt by LY 294002 treatment increased the ROS levels. More importantly, the effect of LY 294002 was abolished by HGF, as determined using DCF-DA by flow cytometry (Figure 5). These results suggest that PI3-kinase is an essential mediator through which HGF inhibits ROS generation.

Bottom Line: We confirmed that Rac-1 regulated ROS production after activation of the AKT pathway with HGF.Exogenously added H2O2 promoted the expression of HGF, but not in a dose-dependent manner and also showed negative expression of HGF after co-treatment with H2O2 and HGF.We clarified the downstream pathways regulated by ROS after treatment with H2O2, which showed negative control between FRK and p38 kinase activities for uPA regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemistry and Molecular Biology, College of Medicine, Yeungnam University, Daegu, Korea. lkhee@med.yu.ac.kr

ABSTRACT

Background: Reactive oxygen species (ROS) are closely associated with the intracellular signal cascade, thus strongly implicating involvement in tumor progression. However, the mechanism by which ROS are generated and how ROS target downstream molecules to trigger tumor metastasis is unclear. In this study, we investigated the underlying signal pathways in ROS-induced urokinase plasminogen activator (uPA) expression in the human gastric cancer cells, NUGC-3 and MKN-28.

Methods and results: Intracellular ROS, as determined using the fluorescent probe, 2'-7' dichlorofluorescein diacetate, decreased after treatment with hepatocyte growth factor (HGF). We confirmed that Rac-1 regulated ROS production after activation of the AKT pathway with HGF. Exogenously added H2O2 promoted the expression of HGF, but not in a dose-dependent manner and also showed negative expression of HGF after co-treatment with H2O2 and HGF. Treatment with NAC, an intracellular free radical scavenger, decreased the enhancement of uPA production and tumor invasion in both cells. We clarified the downstream pathways regulated by ROS after treatment with H2O2, which showed negative control between FRK and p38 kinase activities for uPA regulation.

Conclusion: HGF regulates Rac-1-induced ROS production through the Akt pathway and ROS regulates uPA production and invasion via MAP kinase, which provides novel insight into the mechanisms underlying the progression of gastric cancer.

Show MeSH
Related in: MedlinePlus