Abstract
Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.
Highlights
Human colon cancer is the most frequent cancer worldwide as well as in china, and which is a malignant tumor arising from the inner wall of the large intestine has become the third global leading cause of death from different cancers and its metastasis at distant sites is the major cause of death (André et al, 2004)
Our studies showed that the anti-proliferation and antimigration activity of melatonin in RKO cells were accompanied by inhibition of p38 Mitogen-activated protein kinase (MAPK) pathways, and the inhibition of myosin light chain kinase (MLCK) meditated by melatonin resulted in the depression of migration of colon cancer RKO cells through cross-talk between MLCK and down-regulated p38, in which both phosphorylated myosin light chains (MLC) serve as essential downstream effector
Our findings indicated that melatonin induced the down-regulation of p38 MAPK signaling pathway in a dose- and time-dependent manner, suggesting that p38 MAPK may be suppressed the migration of RKO cells by decreasing the expression and activity of MLCK
Summary
Human colon cancer is the most frequent cancer worldwide as well as in china, and which is a malignant tumor arising from the inner wall of the large intestine has become the third global leading cause of death from different cancers and its metastasis at distant sites is the major cause of death (André et al, 2004). We investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. Materials and Methods: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. Results: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Conclusions: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK
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