Twenty four hours later, the culture medium was removed and the cells were exposed to fresh medium containing 10% FBS (control) or dilutions of 8-hydroxyquinaldic acid (0.0001; 0.001; 0.05; 0.1; 0.5; 1 mM) and incubated for 96 h in standard conditions. Moreover, 8-hydroxyquinaldic acid inhibited migration of colon cancer HT-29 and LS-180 cells and increased the expression of -catenin and E-cadherin. Importantly, antiproliferative and anti-migratory concentrations of 8-hydroxyquinaldic acid were non-toxic in vitro and in vivo. We reported for the first time antiproliferative and anti-migratory activity of 8-hydroxyquinaldic acid against colon cancer HT-29 and LS-180 cells. < 0.05 (one-way ANOVA with post hoc Tukey test). Open in a separate window Physique 2 The effect of 8-hydroxyquinaldic acid on development (a) and viability (b) of zebrafish larvae. Fertilized zebrafish (AB line) eggs (= 20) were exposed to E3 medium (control, C) or serial dilutions of 8-hydroxyquinaldic acid (0.000001; 0.001; 0.5, 1 mM) in E3 medium for 96 h. The medium was changed every 24 h. The microscopic observations were made in selected time points, i.e., 4, 24, 48, 72, 96 h. To determine the biological effect of 8-hydroxyquinaldic acid on colon cancer cells, several in vitro experiments on human HT-29 and LS-180 colon cancer cells were performed. The effect of 8-hydroxyquinaldic acid on colon cancer cell viability and proliferation was assessed by MTT and BrdU assays, which measure the activity of mitochondrial metabolism and DNA synthesis, respectively. HT-29 and LS-180 cells were exposed either to fresh medium or to 8-hydroxyquinaldic acid (0.0001C1 mM) for 48 h (BrdU assay) or 96 h (MTT assay). 8-Hydroxyquinaldic acid inhibited the viability and mitochondrial metabolism of HT-29 (IC50 = 0.175 mM) and LS-180 (IC50 = 0.349 mM) cells (Figure 3a). Importantly, the inhibitory effect in LS-180 cells was observed in a wide range of concentrations (0.001C1 mM). However, only the highest doses of 8-hydroxyquinaldic acid (0.5C1 mM) decreased DNA synthesis in HT-29 and LS-180 cells up to 90.9% (IC50 = 0.774 mM) and 76.1% Raltitrexed (Tomudex) (IC50 = 0.567 mM), respectively (Figure 3b). Open in a separate window Figure 3 The effect of 8-hydroxyquinaldic acid on proliferation (a) and DNA synthesis (b) of human colon adenocarcinoma cells. HT-29 and LS-180 cells were exposed to Raltitrexed (Tomudex) culture medium (control) or serial dilutions of 8-hydroxyquinaldic acid (0.0001; 0.001; 0.05; 0.1; 0.5; 1 mM) in culture medium. The effect of tested compound LAMB3 on proliferation and metabolic activity of HT-29 and LS-180 cells was assessed by MTT assay after 96 h of incubation (a). The effect of 8-hydroxyquinaldic acid on DNA synthesis of HT-29 and LS-180 cells was assessed by BrdUassay after 48 h of incubation (b). Mean percentage values (% of control) SEM of Raltitrexed (Tomudex) six independent experiments (the mean value of control = 100%) are presented. Values were reported as statistically significant in comparison to the control at * 3. The data were normalized to -actin and calculated fold changes in protein expression marked (fold changes 0.20 were considered as significant [*]). To verify whether 8-hydroxyquinaldic acid has any effect on the migration of colon cancer cells, the ability of HT-29 and LS-180 cells exposed to tested compound to invade the endothelial cell monolayer was analyzed by means of QCM? tumor cell transendothelial migration assay. 8-Hydroxyquinaldic acid in the concentration of 0.5 mM, but not 1 mM, inhibited migration of HT-29 cells (Figure 6). Statistically significant inhibitory effect was also observed in LS-180 cells exposed to 8-hydroxyquinaldic acid (1 mM) (Figure 6). Open in a separate window Figure 6 The effect of 8-hydroxyquinaldic acid on migration of human colon adenocarcinoma cells. The migration of HT-29 Raltitrexed (Tomudex) and LS-180 cells through a monolayer of human umbilical vein endothelial HUVEC cells was assessed byMillipores QCM tumor cell transendothelial cell migration assay. Data represent the OD value (570 nm) SEM of three independent.