Suppression of A549 cell proliferation and metastasis by calycosin via inhibition of the PKC‑α/ERK1/2 pathway: An in vitro investigation

Abstract

The migration and invasion of lung cancer cells into the extracellular matrix contributes to the high mortality rates of lung cancer. The protein kinase C (PKC) and downstream signaling pathways are important in the invasion and migration of lung cancer cells. Calycosin (Cal), an effector chemical from Astragalus has been reported to affect the recurrence and metastasis of cancer cells via the regulation of the protein expression of matrix metalloproteinases (MMPs). The inhibition of Cal on the migration and invasion of A549 cells was investigated in the present study. Cell viability and apoptosis assays were performed using MTT and flow cytometric analyses. A wound healing assay and Transwell invasion assay were performed to evaluate the effect of Cal on A549 cell migration and invasion. Invasion‑associated proteins, including MMP‑2, MMP‑9, E‑cadherin (E‑cad), integrin β1, PKC‑α and extracellular signal‑regulated kinase 1/2 (ERK1/2) were detected using western blotting. In addition, PKC‑α inhibitor, AEB071, and ERK1/2 inhibitor, PD98059, were used to determine the association between the suppression of PKC‑α /ERK1/2 and invasion, MMP‑2, MMP‑9, E‑cad and integrin β1. Cal was observed to suppress cell proliferation and induce apoptosis. There were significant differences between the phorbol‑12‑myristate‑13‑acetate (TPA)‑induced A549 cells treated with Cal and the untreated cells in the rates of migration and invasion. The levels of MMP‑2, MMP‑9, E‑cad and integrin β1 in the TPA‑induced A549 cells changed markedly, compared with the untreated cells. In addition, the suppression of Cal was affected by the PKC inhibitor, AEB071, an ERK1/2 inhibitor, PD98059. The results of the present study indicated that Cal inhibited the proliferation, adhesion, migration and invasion of the TPA‑induced A549 cells. The Cal‑induced repression of PKC‑α/ERK1/2, increased the expression of E‑Cad and inhibited the expression levels of MMP‑2, MMP‑9 and integrin β1, which possibly demonstrates the mechanism underlying the biological anticancer effects of Cal.

Figure 1

Effect of Cal on the proliferation and apoptosis of A549 cells. (A) Chemical structure of Cal. (B) A549 cells were treated with Cal at various concentrations (0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 µM) for 24 h, and cell viability was measured using an MTT assay. The results are expressed as a percentage of the control and presented as the mean ± standard deviation (n=6). (C) A549 cells were treated with Cal (20, 30 and 40 µM) and incubated for 24 h. The control group received the same volume of dimethyl sulfoxide. Apoptotic cells were detected using Annexin V and PI staining. (D) Apoptotic rate obtained from three independent experiments and presented the mean ± standard deviation (n=3). ^**P≤0.01, vs. control. Cal, calycosin; Cont, control; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 2

Effect of Cal on the adhesion, migration and invasion of TPA-induced A549 cells. The A549 cells were treated with 0, 20, 30 or 40 µM Cal, in the presence or absence of TPA (80 nM) for 24 h, and were analyzed for (A) adherent ability and (B) wound healing. (C) Migration ability was determined by the closure rate of migrating cells at 24 h, vs. 0 h. (D) A549 cells were inoculated in Transwell chambers treated with Cal for 10 h to assess migration with an IX73 microscope and crystal violet staining (magnification, ×100). (E) Permeated cells, compared with the TPA-induced group. (F) A549 cells were inoculated into Matrigel-coated Transwell chambers and treated with Cal for 24 h to assess cell invasiveness with an IX73 microscope following staining with crystal violet (magnification, ×100). (G) Rate of cell invasion through the membrane, compared with the TPA-induced group. The results were obtained from triplicate experiments and are presented the mean ± standard deviation (n=3). ^*P≤0.05, vs. control; ^**P≤0.01 vs. control. Cal, calycosin; TPA, phorbol-12-myristate-13-acetate.

Figure 3

Effect of Cal on the expression levels of PKC-α, p-ERK1/2, E-Cad, integrin β1, MMP-2 and MMP-9. (A) A549 cells were treated with various concentrations (0, 20, 30 and 40 µM) of Cal in the presence or absence of TPA (80 nM) for 24 h, and then subjected to western blotting to analyze the protein levels of E-cad and integrin β1. (B) Quantification of the protein level of E-cad and integrin β1. (C) A549 cells were treated with various concentrations (0, 20, 30 and 40 µM) of Cal in the presence or absence of TPA (80 nM) for 24 h, and then subjected to western blotting to analyze the protein levels of MMP-2 and MMP-9. (D) Quantification of the protein level of MMP-2 and MMP-9. (E) A549 cells were treated with various concentrations (0, 20, 30 and 40 µM) of Cal in the presence or absence of TPA (80 nM) for 24 h, and then subjected to western blotting to analyze the protein levels of PKC-α. (F) Quantification of the protein level of PKC-α. (G) A549 cells were treated with various concentrations (0, 20, 30 and 40 µM) of Cal in the presence or absence of TPA (80 nM) for 24 h, and then subjected to western blotting to analyze the protein levels of p-ERK1/2 and ERK1/2 (H) Quantification of the proteins level of p-ERK1/2 and ERK1/2. Values are presented as the mean ± standard deviation of three independent experiments, performed in triplicate. ^*P≤0.05 and ^**P≤0.01, vs. TPA-induced group. Cal, calycosin; TPA, phorbol-12-myristate-13-acetate; PCK, protein kinase C; p-ERK, phosphorylated extracellular signal-regulated kinase; E-Cad, E-cadherin; MMP, matrix metalloproteinase.

Figure 4

Cal inhibits invasion by suppressing PKC-α. (A and B) Cells were pretreated with AEB071 (0.1 µM) for 30 min and then incubated in the presence or absence of Cal (30 µM) for 24 h. The A549 cells were then subjected to western blotting to analyze the protein levels of PKC-α and (C and D) E-Cad, integrin β1, MMP-2 and MMP-9. (E) Cells were pretreated with AEB071 (0.1 µM) for 30 min and then incubated in the presence or absence of Cal (30 µM) for 24 h. Cellular invasiveness was measured using a Transwell invasion assay and an IX73 microscope following staining with crystal violet (magnification, ×100). (F) Invasion rate is expressed as a percentage of the TPA-induced group. Values are presented as the mean ± standard deviation of three independent experiments, performed in triplicate. ^*P≤0.05 and ^**P≤0.01, vs. TPA-induced group. ^#P≤0.05 and ^##P≤0.01, vs. Cal⁺ group. Cal, calycosin; TPA, phorbol-12-myristate-13-acetate; PCK, protein kinase C; E-Cad, E-cadherin; MMP, matrix metalloproteinase.

Figure 5

Cal inhibits invasion by suppressing ERK1/2 phosphorylation. (A and B) Cells were pretreated with PD98059 (20 µM) for 30 min and then incubated in the presence or absence of Cal (30 µM) for 24 h. A549 cells were then subjected to western blotting to analyze the protein levels of p-ERK1/2 and ERK1/2 and (C and D) E-Cad, integrin β1, MMP-2 and MMP-9. (E) Cells were pretreated with PD98059 (20 µM) for 30 min and then incubated in the presence or absence of Cal (30 µM) for 24 h. Cell invasiveness was measured using a Transwell invasion assay and an IX73 microscope following staining with crystal violet (magnification, ×100). (F) Invasion rate is expressed as a percentage of the TPA-induced group. Values are presented as the mean ± standard deviation of three independent experiments, performed in triplicate. ^*P≤0.05 and ^**P≤0.01, vs. TPA-induced group; ^#P≤0.05 and ^##P≤0.01, vs. Cal⁺ group. Cal, calycosin; TPA, phorbol-12-myristate-13-acetate; PCK, protein kinase C; p-ERK, phosphorylated extracellular signal-regulated kinase; E-Cad, E-cadherin; MMP, matrix metalloproteinase.