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Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells
Dae Goon Yoo1, Cuk Seong Kim, Sang Ki Lee
1Department of Physiology, Infection Signaling Network Research Center, and Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon 301-131, Korea.
Abstract:
In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-alpha induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-alpha dose-dependent (5~100 ng/ml). TNF-alpha-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-alpha-induced COX-2 expression relative to that seen in the control cells (Adbetagal). Pretreatment with 10 microM of SB203580 suppressed TNF-alpha-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-alpha, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.
Insights
Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) negatively regulates tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression in lung cancer cells. Ref-1 inhibits p38 MAPK phosphorylation, impacting inflammatory gene expression.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- Cyclooxygenase-2 (COX-2) is a key inflammatory mediator implicated in lung cancer.
- Apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) is a multifunctional protein with roles in DNA repair and redox signaling.
- The precise role of Ref-1 in regulating inflammatory gene expression, particularly COX-2, in lung adenocarcinoma remains to be fully elucidated.
Purpose of the Study:
- To investigate the role of Ref-1 in tumor necrosis factor-alpha (TNF-alpha)-induced COX-2 expression in A549 lung adenocarcinoma cells.
- To determine the signaling pathways involved in Ref-1-mediated regulation of COX-2 expression.
- To explore the potential of Ref-1 as a negative regulator of inflammatory gene expression in lung cancer.
Main Methods:
- Utilized A549 lung adenocarcinoma cells and BEAS-2B cells for expression studies.
- Administered TNF-alpha at varying doses to stimulate COX-2 and Ref-1 expression.
- Employed adenoviral transfection with AdRef-1 to overexpress Ref-1.
- Used SB203580, a p38 MAPK inhibitor, to assess pathway involvement.
- Monitored p38 MAPK phosphorylation via Western blotting.
Main Results:
- TNF-alpha induced COX-2 expression in A549 cells in a dose-dependent manner, but not in BEAS-2B cells.
- TNF-alpha stimulation led to increased Ref-1 expression in A549 cells.
- Adenoviral-mediated Ref-1 overexpression inhibited TNF-alpha-induced COX-2 expression.
- p38 MAPK phosphorylation was upregulated by TNF-alpha and markedly suppressed by Ref-1 overexpression.
- SB203580 pretreatment suppressed TNF-alpha-induced COX-2 expression, confirming p38 MAPK involvement.
Conclusions:
- Ref-1 negatively regulates TNF-alpha-induced COX-2 expression in A549 lung adenocarcinoma cells.
- This negative regulation is mediated through the inhibition of p38 MAPK phosphorylation.
- Ref-1 functions as a critical negative regulator of inflammatory gene expression in lung cancer.
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