FIGURE 4.

Anticancer activity of ALA in vitro. (A) ALA inhibits the proliferation of NCI-H460 cells in a dose-dependent manner. Cell viabilities were determined using the CCK-8 assay. (B) The effects of ALA on migration potential of NCI-H460 cells were examined using cell wound healing assay. NCI-H460 cells were seeded and treated with ALA at indicated concentrations (3, 10, and 30 μM) for 24 h. The cell-free gaps were respectively photographed at 0 and 24 h on each plate. Quantitative analysis of the gap distance in the wound healing assay. *p < 0.05, **p < 0.01, ***p < 0.001, vs control. (C) The effects of ALA on invasion potential of NCI-H460 cells were examined using Transwell assay. NCI-H460 cells were pretreated with ALA at indicated concentrations for 24 h and seeded in the Transwell insert (upper chambers were pre-coated with ECM gel). The cells that passed the Transwell insert membrane were stained with crystal violet and counted under a light microscope. Quantitative analysis of the migrated number of cells in the Transwell invasion assay. *p < 0.05, **p < 0.01, ***p < 0.001, vs control. (D) The apoptosis cells were assessed using flow cytometry assays. The number of cells undergoing apoptosis in the ALA treatment group was significantly higher than number of cells in the control group. ***p < 0.001 vs. control. (E) ALA inhibits STAT3 transcriptional activity in human NCI-H460 cell lines. NCI-H460 cells were transfected with pGM-STAT3 and pRL-TK-Luc plasmids, and the impact of different concentrations of ALA on the luciferase activity was determined. Data were presented as mean ± SD, *p < 0.05, **p < 0.01, vs untreated control. (F,G) Western blot analysis displayed that ALA inhibited the expression of AKR1C1 and phosphorylation of STAT3 in the time- and dose-dependent manners. GAPDH was used as loading control.