Melatonin inhibits the migration of human lung adenocarcinoma A549 cell lines involving JNK/MAPK pathway

Abstract

Objective: Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. But, the mechanisms for the anti-cancer effects are unknown. The present study explored the effects of melatonin on the migration of human lung adenocarcinoma A549 cells and its mechanism.

Methods: MTT assay was employed to measure the viability of A549 cells treated with different concentrations of melatonin. The effect of melatonin on the migration of A549 cells was analyzed by wound healing assay. Occludin location was observed by immunofluorescence. The expression of occludin, osteopontin (OPN), myosin light chain kinase (MLCK) and phosphorylation of myosin light chain (MLC), JNK were detected by western blots.

Results: After A549 cells were treated with melatonin, the viability and migration of the cells were inhibited significantly. The relative migration rate of A549 cells treated with melatonin was only about 20% at 24 h. The expression level of OPN, MLCK and phosphorylation of MLC of A549 cells were reduced, while the expression of occludin was conversely elevated, and occludin located on the cell surface was obviously increased. The phosphorylation status of JNK in A549 cells was also reduced when cells were treated by melatonin.

Conclusions: Melatonin significantly inhibits the migration of A549 cells, and this may be associated with the down-regulation of the expression of OPN, MLCK, phosphorylation of MLC, and up-regulation of the expression of occludin involving JNK/MAPK pathway.

Figure 1. Melatonin inhibits the migration of A549 cells.

(A) The migration of A549 cells at 0.1, 0.75, 2.5, 5.0 mmol/L melatonin groups respectively when A549 cells were treated for 0 h, 12 h and 24 h. (B) Analysis of migration rate (%), compared with control group (DMSO): *P<0.05, ^#P<0.05.

Figure 2. The effect of melatonin, SP600125 and PMA on migration of A549 cells.

(A) The effect of melatonin, SP600125 and PMA on migration of A549 cells after 0 h, 12 h and 24 h. (B) Analysis of migration rate, compared with control group: *P<0.05, ^#P<0.05; compared with PMA group: ^▴P<0.05, ^△P<0.05.

Figure 3. Immunofluorescence detection of tight junction correlated protein occludin in A549 cells.

(A) DMSO control. (B) melatonin (0.1 mmol/L). (C) melatonin (2.0 mmol/L).

Figure 4. Effect of melatonin on the expression of occludin, OPN, MLCK and phosphorylation of MLC, JNK.

(A) occludin, (B) OPN, (C) MLC and MLCK, (D) JNK. Results are presented as mean ± SD of three independent experiments. *P<0.05, in comparison to control group.

Figure 5. The Effect of melatonin, SP600125 and PMA on the expression of occludin, OPN, MLCK and phosphorylation of MLC, JNK.

(A) occludin, (B) OPN, (C) MLC and MLCK, (D) JNK. Results are presented as mean ± SD of three independent experiments. *P<0.05, ^#P<0.05, in comparison to control; ^▴P<0.05, ^△P<0.05, in comparison to PMA group.