The synergistic effect of propofol and ulinastatin suppressed the viability of the human lung adenocarcinoma epithelial A549 cell line

Abstract

Ulinastatin and propofol (PPF) are recognized for their anticancer properties. The aim of the present study was to evaluate the synergistic antitumor effect of PPF followed by ulinastatin against A549 cells. In MTT assays, PPF (10, 20 and 30 µM) followed by 200 U/ml ulinastatin was more effective at inhibiting A549 cell viability compared with PPF (10, 20 and 30 µM) or 200 U/ml ulinastatin. PPF (10, 20 and 30 µM) followed by 200 U/ml ulinastatin treatments synergistically increased the number of S cells and synergistically reduced the number of G2/M cells associated with PPF stimulation in a dose-dependent manner. Western blot analysis demonstrated that the antitumor effect of PPF followed by 200 U/ml ulinastatin treatments were associated with the downregulated expression of extracellular signal-regulated kinase 1 and 2 phosphorylation (p-ERK1/2) and matrix metalloproteinases 2 (MMP-2). In conclusion, these data demonstrated that PPF (20 and 30 µM) followed by 200 U/ml ulinastatin treatments synergistically stimulated a significant proportion of A549 cells in S phase. Furthermore, the combination synergistically reduced a significant proportion of A549 cells in G2/M phase and synergistically suppressed the viability of A549 cells, which was possibly related regulation of the expression of p-ERK1/2 and MMP-2 in A549 cells.

Keywords: A549 cells; antitumor; extracellular signal-regulated kinase 1 and 2 phosphorylation; matrix metalloproteinases 2; propofol; ulinastatin.

Figure 1.

Different treatments schedules with PPF and ulinastatin for A549 cells. All compounds were dissolved in the serum-free medium. (A) To evaluate the antitumor effect of different treatment schedules relating to PPF and ulinastatin through high dose treatment, A549 cells were exposed to seven different treatments. 800 U/ml ulinastatin + 100 µM PPF were dissolved in the same serum-free medium. (B) To verify the synergistic antitumor effect of PPF followed by ulinastatin treatments at low concentrations, A549 cells were exposed to nine different treatments. PBS phosphate buffer saline; PPF, propofol.

Figure 2.

Viability assay of ulinastatin and PPF in A549 cells. A549 cells were inhibited using different groups and data were obtained from three independent experiments. Data were presented as mean ± SD of three independent experiments, and analyzed by the Least-Significant-Difference. (A) Antitumor effects of different sequential administration with 800 U/ml ulinastatin (UTI₈₀₀) and 100 µM PPF (PPF₁₀₀); (B) The synergistic effect of PPF→ulinastatin at clinical concentrations. ΩP<0.05 vs. Control; #P<0.05 vs. UTI₈₀₀; ΔP<0.05 vs. 200 U/ml ulinastatin (UTI₂₀₀); ^&P<0.05 vs. PPF₁₀₀; ΨP<0.05 vs. 800 U/ml ulinastatin→100 µM PPF (UTI₈₀₀→PPF₁₀₀); αP<0.05 vs. 30 µM PPF (PPF₃₀); βP<0.05 vs. 10 µM PPF→200 U/ml ulinastatin (PPF₁₀→UTI₂₀₀); εP<0.05 vs. 20 µM PPF (PPF₂₀). PPF, propofol.

Figure 3.

A549 cell proliferation cycle of PPF→ulinastatin at clinical concentrations. A549 cells were inhibited using different groups and data were obtained from three independent experiments. Data were presented as mean ± SD of three independent experiments, and analyzed by the Least-Significant-Difference. (A) Percentage of A549 cells in G0/G1 phase; (B) Percentage of A549 cells in S phase; (C) Percentage of A549 cells in G2/M phase. ΩP<0.05 vs. Control; #P<0.05 vs. 10 µM PPF (PPF₁₀); ΔP<0.05 vs. 200 U/ml ulinastatin (UTI₂₀₀); ΨP<0.05 vs. 20 µM PPF (PPF₂₀); αP<0.05 vs. 30 µM PPF (PPF₃₀); βP<0.05 vs. 10 µM PPF→200 U/ml ulinastatin (PPF₁₀→UTI₂₀₀); δP<0.05 vs. 20 µM PPF→200 U/ml ulinastatin (PPF₂₀→UTI₂₀₀). PPF, propofol.

Figure 4.

Effect of ulinastatin and PPF on invasion and migration of A549 cells. Cells that had migrated (A) and invaded (B) were photographed (magnification, ×200), and photographed in 9 predetermined fields for each treatment. Data were calculated from three independent experiments. Data were presented as mean ± SD of three independent experiments and analyzed by Dunnett' T3. ΩP<0.05 vs. Control; ^&P<0.05 vs. 100 µM PPF (PPF₁₀₀); αP<0.05 vs. 800 U/ml ulinastatin +100 µM PPF (UTI₈₀₀+PPF₁₀₀); βP<0.05 vs. 100 µM PPF→800 U/ml ulinastatin (PPF₁₀₀→UTI₈₀₀). PPF, propofol.

Figure 5.

Effect of ulinastatin and PPF on apoptosis of A549 cells. Annexin V-FITC/PI staining assay. After different treatments, cells were labeled and sorted using flow cytometry. (A) Late apoptotic or necrotic cells in Q2 (B) are depicted in the upper right-hand quadrant of the dot plot; viable cells in Q3 (C) are depicted in the lower left-hand quadrant of the dot plot. Data were calculated from three independent experiments. Data were presented as mean ± SD of three independent experiments and analyzed by the Least-Significant-Difference. For late apoptotic or necrotic cells, ΩP<0.05 vs. Control; #P<0.05 vs. 800 U/ml ulinastatin (UTI₈₀₀); ^&P<0.05 vs. 100 µM PPF (PPF₁₀₀); ΨP<0.05 vs. 800 U/ml ulinastatin→100 µM PPF (UTI₈₀₀→PPF₁₀₀). For early apoptotic and viable cells vs. Control, all P-values of treated groups >0.05. PI, propidium iodide; PPF, propofol.

Figure 6.

Effects of treatment with ulinastatin, PPF, and PPF → ulinastatin on the protein expression of p-ERK1/2 (A) and MMP-2 (B) in A549 cells. Data were calculated from three independent experiments. Data were presented as mean ± SD of three independent experiments, and analyzed by the Least-Significant-Difference. ΩP<0.05 vs. Control; #P<0.05 vs. 200 U/ml ulinastatin (UTI₂₀₀); βP<0.05 vs. 10 µM PPF (PPF₁₀); εP<0.05 vs. 20 µM PPF (PPF₂₀); µP<0.05 vs. 30 µM PPF (PPF₃₀). p-ERK, extracellular signal-regulated kinase phosphorylation; MMP, matrix metalloproteinases; PPF, propofol.