Effects And Its Probable Mechanism Of N-(3-trifluoromethyl-pheny)-Retinamide On The Migration Ability Of Human Lung Adenocarcinoma A549 Cells

Objective To investigate the effects of All Trans Retinoic Acid (ATRA) derivatives [N-(3-trifluoromethylphenyl)-retinamide, NTFMP] on the morphology and migration ability of human lung adenocarcinoma A549 cells and its possible molecular mechanism.Methods The A549 cells were respectively treated with ATRA at the dose of (1,10) mg/L, as a positive control group and with NTFMP at the dose of (1,7.5) mg/L, as a treated group. At the same time set the cell control group and DMSO control group. The effect of NTFMP on cell morphology was observed by optical microscope. The change of migration of A549 cells was measured by scarification test. The expression of myosin light chain kinase (MLCK), phosphorylation of myosin light chain (p-MLC) and proteins related to mitogen-activated protein kinase (MAPK) signal pathes were analyzed by western-blotting. The possible signal transduct pathway was analyzed by addition of ERK inhibitor (PD98059). Statistical analysis was performed by using SPSS 10.0. Experimental data were represented with Mean±SD. Comparison among group's data was analyzed by One-Way ANOVA analysis. Analysis of correlated data was used by Person method. P<0.05 was considered as significant difference.Results When A549 cells were treated with ATRA (1 mg/L,10mg/L) and NTFMP (1mg/L), the shapes of A549 cells didn't change significantly under the light microscope. While treated with NTFMP (7.5mg/L), A549 cells became lesser, smaller and more circular, and lost the character of adherence growth. The scarification test showed that NTFMP (1mg/L,7.5mg/L) all could inhibit A549 cell migration, the distance of migration was decreased with the increasing of drug concentration. NTFMP(7.5mg/L) could significantly inhibit the expression of MLCK and p-MLC protein but no inhibited effect on MLC protein expression in A549 cells. The results of A549 cells treated with the inhibitor of ERK (40umol/L PD98059) were as the same as that of the cells treated with NTFMP. When NTFMP and PD98059 were combined to treat the cells, the change of cell shapes and the inhibitition of cell migration distance became more significantly, which appeared synergistic effect. Western blot indicated that the expression of p-ERK, MLCK, p-MLC decreased and emerged highly consistent among them. By analyzing the influence of the above correlated proteins on the cell migration, we found NTFMP and the inhibitor of ERK can inhibit the cell migration. Bivariate correlation analysis indicated that the distance of cell migration is positively(P<0.05) correlated with the content of intracellular MLCK(r=0.832), and the degree of phosphorylation MLC(r=0.966) and ERK(r=0.811) (P<0.05), without the content of intracellular MLC(r=0.149, P>0.05). This result indicated that ERK signal transduction pathway was involved in cell migration. NTFMP may down regulate the expression of MLCK and p-MLC to A549 cell migration by inhibiting ERK signal transduction pathway. MLCK and p-MLC are ERK signal transduction pathway downstream proteins. When NTFMP and PD98059 treated together A549 cells, which showed synergistic effect of inhibiting cells migration and the down regulation of the expression of MLCK, p-MLC and p-ERK proteins. It indicated that NTFMP inhibited A549 cells migration by inhibiting the expression of MLCK/p-MLC through ERK signal transduction way.Conclusion NTFMP could inhibit the migration of A549 cells, whose possible mechanism may be related to down-regulation of the expression of MLCK and p- MLC through the ERK signal transduction pathway of MAPK.