Fig 9. Effect of continuous exposure to 5-FU, 4-HC and 5-FU+4-HC on viability and invasiveness of 231/LM2-4 cells.
(A) Effect of prolonged continuous exposures (six days) to different concentrations of 5-FU (0.1, 0.5, 1 and 10μM), 4-HC (0.01, 0.05, 0.1 and 1μM), and to the combination 1μM 5-FU + 0.01μM 4-HC, on in vitro viability of 231/LM2-4 cells. The relative absorbance (A490nm-A650nm) to control is represented; A490nm is directly proportional to the number of living cells and A650nm eliminates any kind of background. (B) Assessment of the invasive capacity of 231/LM2-4 cells, under untreated and treated conditions, in a transwell chemoinvasion assay using collagen type IV as coating. The protracted treatments with low dose and non-toxic doses of 1μM 5-FU, 0.01μM 4-HC and the 1μM 5-FU + 0.01μM 4-HC combination are detailed in Materials and methods. Cells that invaded and passed through membrane pores were stained with 0.1% crystal violet and counted. The numbers are the mean ± standard error of the mean (SEM), of the cell counts of eight random fields (x20 magnification) per sample of a representative experiment. The experiment was repeated three times. A significant reduction in invasive capacity of the cells was observed in all treated groups (***p<0.0001 vs. control). Significance was analyzed by one-way ANOVA with Dunnett’s post-test.