Nutr Res Pract.  2017 Apr;11(2):83-89. 10.4162/nrp.2017.11.2.83.

Wheatgrass extract inhibits hypoxia-inducible factor-1-mediated epithelial-mesenchymal transition in A549 cells

• Do NY
• Shin HJ
• Lee JE

• KMID: 2412019
• DOI: http://doi.org/10.4162/nrp.2017.11.2.83

Abstract

BACKGROUND/OBJECTIVES
Epithelial-mesenchymal transition (EMT) is involved in not only cancer development and metastasis but also non-cancerous conditions. Hypoxia is one of the proposed critical factors contributing to formation of chronic rhinosinusitis or nasal polyposis. Wheatgrass (Triticum aestivum) has antioxidant, anti-aging, and anti-inflammatory effects. In this study, we analyzed whether wheatgrass has an inhibitory effect on the EMT process in airway epithelial cells.
MATERIALS/METHODS
A549 human lung adenocarcinoma cells were incubated in hypoxic conditions (COâ‚‚ 5%/Oâ‚‚ 1%) for 24 h in the presence of different concentrations of wheatgrass extract (50, 75, 100, and 150 µg/mL) and changes in expression of epithelial or mesenchymal markers were evaluated by immunoblotting and immunofluorescence. Accordingly, associated EMT-related transcriptional factors, Snail and Smad, were also evaluated.
RESULTS
Hypoxia increased expression of N-cadherin and reduced expression of E-cadherin. Mechanistically, E-cadherin levels were recovered during hypoxia by silencing hypoxia inducible factor (HIF)-1α or administering wheatgrass extract. Wheatgrass inhibited the hypoxia-mediated EMT by reducing the expression of phosphorylated Smad3 (pSmad3) and Snail. It suppressed the hypoxia-mediated EMT processes of airway epithelial cells via HIF-1α and the pSmad3 signaling pathway.
CONCLUSION
These results suggest that wheatgrass has potential as a therapeutic or supplementary agent for HIF-1-related diseases.

Keyword

Triticum; epithelial-mesenchymal transition; sinusitis; hypoxia inducible factor 1; cadherins

MeSH Terms

Adenocarcinoma
Anoxia
Cadherins
Epithelial Cells
Epithelial-Mesenchymal Transition*
Fluorescent Antibody Technique
Humans
Hypoxia-Inducible Factor 1
Immunoblotting
Lung
Neoplasm Metastasis
Sinusitis
Snails
Triticum
Cadherins
Hypoxia-Inducible Factor 1

Figure

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  Fig. 1 Wheatgrass inhibits the hypoxia-mediated EMT process in airway epithelial cells.(A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h and total cell lysates were analyzed by immunoblotting with anti-E-cadherin or N-cadherin antibodies. (B and C) E-cadherin levels were visualized by immunofluorescence analysis. Green fluorescence indicates E-cadherin expression, blue denotes DAPI staining of the nucleus, and the right panel is a merged image of the two panels. Scale bar represents 20 µm. (D and E) N-cadherin expression levels were visualized by immunofluorescence analysis. Red fluorescence indicates E-cadherin expression (*P < 0.01). WG, wheatgrass; EMT, epithelial mesenchymal transition; HPF, high power field.

• 

  Fig. 2 Wheatgrass inhibits the hypoxia-mediated EMT process by inhibiting the activation of HIF-1α in airway epithelial cells.(A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h, and nuclear protein fractions were analyzed by immunoblotting with an anti-HIF-1α antibody. (B and C) Cells were transfected with either si-HIF-1α, si-negative control (NC), wheatgrass (150 µg/mL), or hypoxia only as indicated. E-cadherin expression levels (green) were analyzed by immunofluorescence analysis. (D and E) HIF-1α expression levels (red) were visualized by immunofluorescence analysis (*P < 0.01). WG, wheatgrass; EMT, epithelial mesenchymal transition; HIF, hypoxia inducible factor; HPF, high power field.

• 

  Fig. 3 Wheatgrass inhibits the hypoxia-mediated EMT process by reducing Smad activation.(A and B) Cells were treated and cultured with wheatgrass at the indicated concentrations and times. (C and D) Smad3 expression levels (green) were analyzed by immunofluorescence (*P < 0.01). WG, wheatgrass; EMT, epithelial mesenchymal transition; HPF, high power field.

• 

  Fig. 4 Wheatgrass negatively regulates Snail expression in airway epithelial cells.(A) A549 cells were treated with wheatgrass at the indicated concentrations for 24 h, and total cell lysates were analyzed by immunoblotting with anti-E-cadherin, N-cadherin, and Snail antibodies. (B and C) Snail expression levels (green) were analyzed by immunofluorescence (*P < 0.01). WG; wheatgrass; HPF, high power field.

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