Leptin induces epithelial-to-mesenchymal transition via activation of the ERK signaling pathway in lung cancer cells

Abstract

Previous studies revealed that leptin induces the growth and proliferation and inhibits the apoptosis of lung cancer cells. However, the effect of leptin on epithelial-to-mesenchymal transition (EMT) is not yet clear. In the present study, the effect of leptin on EMT was investigated as well as its underlying mechanisms in A549 cells. The ability of leptin to induce EMT was investigated by microscopic examination and western blotting. The impacts of leptin on cell migration, invasion and tumorigenesis were evaluated by wound healing, Transwell and colony formation assays, respectively. It was demonstrated that leptin induced EMT-associated morphological changes, namely a decrease in cell-cell contact and a more elongated morphological shape. Leptin decreased the expression levels of epithelial phenotype markers E-cadherin and keratin, increased the expression of mesenchymal phenotype marker Vimentin, and raised the expression of EMT-induced transcription factor ZEB-1. In addition, leptin activated the extracellular signal regulated kinase (ERK) signaling pathway and did not affect the activation of the protein kinase B signaling pathway in A549 cells. Leptin also promoted EMT-induced migration, invasion and tumorigenesis in vitro in A549 cells. The present study provides evidence that leptin induced EMT via the activation of the ERK signaling pathway and increased EMT-induced tumor phenotypes in lung cancer cells. These findings suggest that leptin may be a promising target for lung cancer treatment through the regulation of EMT.

Keywords: epithelial-to-mesenchymal transition; extracellular signal regulated kinase; invasion; leptin; metastasis; tumorigensis.

Figure 1.

Leptin induces morphological changes of epithelial-to-mesenchymal transition in A549 cells. (A) Control group. A549 cells display classical epithelial morphology, a pebble-like shape and tight cell-cell adhesion. (B) TGF-β1 and (C) leptin groups. A549 cells were treated with TGF-β1 (5 ng/ml) or leptin (100 ng/ml) for 48 h and morphological changes were evaluated under a microscope. A549 cells had a decrease in cell-cell contacts and adopt a more elongated morphological shape. Scale bar, 20 µm. TGF, tumor growth factor.

Figure 2.

Leptin regulates the expression of epithelial-to-mesenchymal transition markers in A549 cells (n=6). The protein expression of (A) epithelial markers E-cadherin and Keratin and the (C) mesenchymal markers, Fibronectin and Vimentin were measured by western blot analysis after A549 cells were treated with TGF-β1 or leptin for 48 h. (B) Relative content of E-cadherin and Keratin and (D) Fibronectin and Vimentin was calculated against β-actin. Values are expressed as mean ± standard error of the mean. ^#P<0.05, ^##P<0.01 vs. the control group.

Figure 3.

Leptin increases the expression of epithelial-to-mesenchymal transition-induced transcription factors in A549 cells (n=6). (A) Protein levels of ZEB-1 and Twist-1 were measured by western blot analysis after A549 cells were treated with TGF-β1 or leptin for 48 h. The relative content of (B) ZEB-1 and (C) Twist-1 were calculated against β-actin. Values are expressed as mean ± standard error of the mean. ^#P<0.05, ^##P<0.01 vs. the control group.

Figure 4.

Leptin activates epithelial-to-mesenchymal transition-related ERK signaling pathway in A549 cells. (A) Protein levels of p-ERK and total-ERK were measured by western blot analysis after A549 cells were treated with TGF-β1 or leptin for 48 h (n=6). (B) The relative content of p-ERK was calculated against β-actin. (C) Protein levels of p-AKT and total-AKT were measured by western blot analysis after A549 cells were treated with TGF-β1 or leptin for 48 h. (D) The relative content of p-AKT was calculated against β-actin. Values are expressed as mean ± standard error of the mean. ^#P<0.05, ^##P<0.01 vs. the control group. AKT, protein kinase B; TGF, tumor growth factor; ERK, extracellular signal regulated kinase.

Figure 5.

Leptin enhances epithelial-to-mesenchymal transition-induced tumor phenotypes in A549 cells (n=6). (A) Cell migration was measured by a wound-healing assay. The cells were incubated for 24 h after cell wounding and then the images were captured. (B) The invasive activity of cells was assayed by a Matrigel-coated Transwell assay. The cells that invaded through the filter were quantified 24 h following plating. Data were expressed relative to the invasive ability of the control cells. (C) The tumorigenic phenotype was measured by colony formation assay. All the plotted values are relative to A549 cells in the control group. Values are expressed as mean ± standard error of the mean. ^#P<0.05, ^##P<0.01 vs. the control group.