Korean Red Ginseng suppresses bisphenol A-induced expression of cyclooxygenase-2 and cellular migration of A549 human lung cancer cell through inhibition of reactive oxygen species

Abstract

Background: Korean Red Ginseng (KRG) is a natural product with antiinflammatory and anticarcinogenic effects. We have previously reported that the endocrine-disrupting compound bisphenol A (BPA)-induced cyclooxygenase-2 (COX-2) via nuclear translocation of nuclear factor-kappa B (NF-κB) and activation of mitogen-activated protein kinase and promoted the migration of A549. Here, in this study, we assessed the protective effect of KRG on the BPA-induced reactive oxygen species (ROS) and expression of COX-2 and matrix metalloproteinase-9 (MMP-9) in A549 cells.

Methods: The effects of KRG on the upregulation of ROS production and COX-2 and MMP-9 expression by BPA were evaluated by fluorescence-activated cell sorting (FACs) analysis, quantitative reverse transcription polymerase chain reaction, and western blotting. Antimigration ability by KRG was evaluated by migration assay in A549 cells.

Results: KRG significantly suppressed the BPA-induced COX-2, the activity of NF-κB, the production of ROS, and the migration of A549 cells. These effects led to the downregulation of the expression of MMP-9.

Conclusions: Overall, our results suggest that KRG exerts an antiinflammatory effect on BPA-treated A549 cells via the suppression of ROS and downregulation of NF-κB activation and COX-2 expression which leads to a decrease in cellular migration and MMP-9 expression. These results provide a new possible therapeutic application of KRG to protect BPA-induced possible inflammatory disorders.

Keywords: Bisphenol A; Cyclooxygenase-2; Korean Red Ginseng; Matrix metalloproteinase-9; Reactive oxygen species.

Fig. 1

Effects of KRG on BPA-induced COX-2 expression. (A) A549 cells were preincubated with KRG for 1h and treated with BPA. After 24 h incubation, cell viability was measured by MTT assay. (B) A549 cells were pretreated with KRG (500 μg/mL) for 1 h and treated with BPA for 24 h. The levels of COX-2 mRNA were determined by qRT-PCR. (C) A549 cells were treated as described in (A). COX-2 and β-actin were evaluated by western blot analysis. ##p < 0.01, CON vs. BPA; *p < 0.05 and **p < 0.01, BPA vs. BPA + KRG. KRG, Korean Red Ginseng; BPA, bisphenol A; MTT, thiazolyl blue tetrazolium bromide; qRT-PCR, quantitative reverse transcription polymerase chain reaction; COX-2, cyclooxygenase-2.

Fig. 2

Effects of KRG on BPA-induced NF-κB activation. (A) A549 cells were transfected with the NF-κB luciferase reporter gene. The next day, A549 cells were pretreated with KRG for 1 h and then treated with BPA for 24 h. Luciferase activities of NF-κB were measured. (B) A549 cells were treated as described in (A). The cytoplasmic and nuclear protein extracts were isolated with different lysis buffers. NF-κB p65, lamin B, and β-actin were evaluated by western blot analysis. #p < 0.05 and ##p < 0.01, CON vs. BPA; *p < 0.05, BPA vs. BPA + KRG. NF-κB, nuclear factor-kappa B; KRG, Korean Red Ginseng; BPA, bisphenol A.

Fig. 3

Effects of KRG on BPA-induced cellular migration. A549 cells were coincubated with BPA or KRG or celecoxib in the upper chamber of transwell for 24 h. The migrated cells were counted using light microscopy. The bar graph shows the cells that have been migrated relatively. Scale bar represents 100 μm. ##p < 0.01, CON vs. BPA; **p < 0.01, BPA vs. BPA + KRG or BPA + celecoxib. KRG, Korean Red Ginseng; BPA, bisphenol A.

Fig. 4

Effects of KRG on BPA-induced ROS production. A549 cells were pretreated with KRG for 1 h and treated with BPA. After 24h incubation, A549 cells were stained with cell-permeable dye 2′,7′-dichlorofluorescin diacetate (DCF-DA) (1 μM). ROS production was measured by flow cytometry. ##p < 0.01, CON vs. BPA; **p < 0.01, BPA vs. BPA + KRG or BPA + NAC. KRG, Korean Red Ginseng; BPA, bisphenol A; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine.

Fig. 5

Effects of KRG on involvement of ROS in COX-2 and MMP-9 expression. (A) A549 cells were preincubated with KRG and NAC for 1 h and treated with BPA for 24 h. The levels of COX-2 mRNA were determined by qRT-PCR. (B) A549 cells were treated as described in (A). COX-2 and β-actin were evaluated by western blot analysis. (C) A549 cells were treated and total RNA was extracted as described in (A). The expression of MMP-9 mRNA was determined by qRT-PCR. (D) A549 cells were treated as described in (A). MMP-9 and β-actin were evaluated by western blot analysis. ##p < 0.01, CON vs. BPA; *p < 0.05 and **p < 0.01, BPA vs. BPA + KRG or BPA + NAC. KRG, Korean Red Ginseng; BPA, bisphenol A; ROS, reactive oxygen species; COX-2, cyclooxygenase-2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; MMP-9, matrix metalloproteinase-9; NAC, N-acetyl-L-cysteine.