Melatonin Inhibits Osteosarcoma Metastasis And Tumor Stem Cells Through SOX9-Mediated Signaling Pathway

Aims:Melatonin(N-acetyl-5-methoxytryptamine)has been reported to suppress epithelial-mesenchymal transition and cancer stem cells in some types of cancer.However,the effects of melatonin on the osteosarcoma stem cells,epithelial-mesenchymal transition and metastasis of osteosarcoma are still not clear.The present study was conducted to dissect the activity of melatonin on the osteosarcoma stem cells and the underlying mechanisms.Main methods:MTT,wound healing,Transwell assay and western blotting were conducted to determine the effect of melatonin on osteosarcoma cell invasion and migration and downregulation of SOX9-mediated signaling.Tumor spheroid assay and FACS analysis were performed to analyze the inhibition of the osteosarcoma stem cells.In vivo model for tumor formation and metastasis from single cell clone was used to evaluate the suppression of osteosarcoma stem cells by melatonin.Key findings:We first determined by MTT assay that melatonin does not affect the concentration of osteosarcoma proliferation in the highest pharmacological concentrations,followed by a scratch test,a Transwell trial to study the effects of melatonin on the migration and invasion of osteosarcoma,and found that,compared with the control group,melatonin reduced the ability to migrate and invade Osteosarcoma.The CD133+labeled flow cytometry and tumor-forming experiments showed that melatonin inhibited the ability of osteosarcoma stem cells.At the same time,the expression of transcription factor SOX9 decreased significantly with melatonin stimulation.After plasmid transfection to express SOX9,the Transwell migration and Invasion experiment,CD 133+ labeled Flow cytometry experiment,tumor stem cell formation experiment were found,the metastasis of osteosarcoma cells after SOX9 expression and the ability of tumor stem cells were enhanced.After siRNA knockdown SOX9,the metastasis of osteosarcoma cells and the ability of stem cell were suppressed.After the expression of SOX9 cells,and then the melatonin stimulation,the metastatic ability of osteosarcoma,tumor stem cell ability is suppressed.Significance:We demonstrated that melatonin potently suppresses the migration and invasion of osteosarcoma cells.Furthermore,melatonin significantly inhibits the sarcosphere formation of osteosarcoma stem cells and regulates EMT markers of osteosarcoma cells.In vivo mice model showed that melatonin significantly inhibits the initiation and metastasis of osteosarcoma.SOX9 is the key transcription factor mediating the effect of melatonin.Melatonin inhibited of cancer stem cell by down-regulation of SOX9-mediated signaling pathway in osteosarcoma.Collectively,these results deepen the understanding of the biological functions of melatonin and provide new insights for the intervention of osteosarcoma stem cells.