Sinomenine inhibits A549 human lung cancer cell invasion by mediating the STAT3 signaling pathway

Abstract

Increasing evidence suggests that the failure of lung cancer treatment may occur as a result of tumor invasion and metastasis. Signal transducer and activator of transcription 3 (STAT3), an epithelial-mesenchymal transition-inducing transcription factor, is a key signaling molecule involved in the proliferation, apoptosis, invasion and metastasis of tumor cells. Sinomenine is an alkaloid compound with an antineoplastic potential against a variety of cancer cells. The aim of the present study was to assess the antitumor mechanisms of sinomenine in the A549 human lung cancer cell line. The results demonstrated that sinomenine manifested dose-dependent cytotoxicity and induced apoptosis in A549 cells. The protein expression of Janus kinase 2, STAT3, phosphorylated-STAT3, Snail, N-cadherin and vimentin decreased in sinomenine-treated cells, while E-cadherin protein expression increased. The regulation of STAT3, N-cadherin and E-cadherin by sinomenine was further confirmed by reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining. It was demonstrated that sinomenine exerts inhibitory effects on A549 human lung cancer cell invasion, possibly through the inhibition of STAT3 signaling. These results provide a novel insight into the role of sinomenine in the treatment of non-small cell lung cancer.

Keywords: epithelial-mesenchymal transition; lung cancer cell invasion; signal transducer and activator of transcription 3; sinomenine.

Figure 1.

Effect of sinomenine on the viability of A549 cells. Cells were treated with 0–2 mM sinomenine or 0.25 uM cucurbitacin I for 24–72 h. Cell viability was analyzed using a colorimetric kit. Data are expressed as the mean ± standard deviation (n=3), *P<0.05 vs. untreated vehicle control.

Figure 2.

Effect of sinomenine on A549 cell apoptosis. A549 cells were treated with sinomenine (0.125, 0.25, 0.5, 1.0 and 2.0 mM) or cucurbitacin I (0.25 µM) for 24 and 48 h, and apoptosis was measured by Annexin V/propidium iodide staining and analyzed by flow cytometry. (A) Representative scatter plots for apoptotic cells. (B) Percentage of apoptotic cells following treatment with sinomenine for 24 h. (C) Percentage of apoptotic cells following treatment with sinomenine for 48 h. Data are expressed as the mean ± standard deviation (n=3). *P<0.05 vs. untreated control. PI, propidium iodide; FITC, fluorescein isothiocyanate.

Figure 3.

Effect of sinomenine on the expression of proteins involved in STAT3 signaling and EMT. Following treatment with sinomenine or cucurbitacin I for 48 h, the expression of proteins involved in the STAT3 signal transduction pathway and EMT were analyzed by western blotting. Band densities were quantified using Image J and adjusted to that of the corresponding loading control. STAT3, signal transducer and activator of transcription 3; EMT, epithelial-mesenchymal transition.

Figure 4.

Effect of sinomenine on the mRNA expression of STAT3, E-cadherin and N-cadherin. A549 cells were treated with 1.0 mM sinomenine or 0.25 µM cucurbitacin I for 48 h. Total RNA was isolated, and the mRNA expression of STAT3, E-cadherin and N-cadherin was determined by reverse transcription-quantitative polymerase chain reaction. (A) Representative agarose electrophoresis bands of N-cadherin, E-cadherin and STAT3 mRNA expression. Quantitative data of (B) STAT3, (C) E-cadherin and (D) N-cadherin gene expression. Data of three independent experiments are expressed as the mean ± standard deviation. *P<0.05 vs. control. STAT3, signal transducer and activator of transcription 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA.

Figure 5.

Immunofluorescence assay of STAT3, E-cadherin, N-cadherin and vimentin expression in A549 cells. Cells were treated with 1.0 mM sinomenine for 24 h. STAT3 and epithelial-mesenchymal transition markers were stained with 4′,6-diamidino-2-phenylindole (blue) and anti-rabbit or anti-mouse immunoglobulin G secondary antibody conjugates (green or red), and visualized by immunofluorescence analysis (magnification, ×400). STAT3, signal transducer and activator of transcription 3.