Inhibition of ERK1/2 Signaling Pathway is Involved in Melatonin's Antiproliferative Effect on Human MG-63 Osteosarcoma Cells

Background: In a previous study, we found that melatonin inhibits MG-63 osteosarcoma cell proliferation; however, the underlying mechanisms remain elusive. Mitogen-activated protein kinase (MAPK) and Akt signaling pathways play key roles in the anticancer effects of melatonin. Aims: The present study investigated whether MAPK and Akt signaling pathways are involved in melatonin's antiproliferative actions on the human MG-63 osteosarcoma cells. Methods/Results: Western blot analysis confirmed that melatonin significantly inhibited phosphorylation of ERK1/2 but not p38, JNK, or Akt. The expression of ERK1/2, p38, JNK, and Akt was not altered by melatonin. PD98059 and melatonin alone, and especially in combination, significantly inhibited cell proliferation. The changes included G ₁ and G ₂ /M phase arrest of the cell cycle, and a downregulation of the expression at both the protein and mRNA levels of cyclin D1 and CDK4 (related to the G ₁ phase) and of cyclin B1 and CDK1 (related to the G ₂ /M phase) as measured by flow cytometry after propidium iodide staining, and both western blot and real-time PCR, respectively. Furthermore, the combination of PD98059 and melatonin synergistically and markedly augmented the action of either agent alone. Co-immunoprecipitation further confirmed that there was an interaction between p-ERK1/2 and cyclin D1, CDK4, cyclin B1, or CDK1, which was blunted in the presence of melatonin or PD98059. Conclusion: These findings suggest that melatonin's antiproliferative action is mediated by inhibition of the ERK1/2 signaling pathway rather than the p38, JNK, or Akt pathways.