Lung cancer is one of the most common cancer in cancer-related deaths worldwide, which is characterized by its strong metastatic potential. The melatonin hormone secreted by the pineal grand has an antioxidant effect and protects cells against carcinogenic substances. However, the effects of melatonin in lung cancer stemness are largely unknown. We found that melatonin reduces CD133 expression by~50% in lung cancer cell lines, while results of a sphere formation assay showed that melatonin inhibits lung cancer stemness. These effects of melatonin were reversed when the cell lines were incubated with phospholipase C (PLC), ERK/p38, and a β-catenin activator. Transfection with Twist siRNA augmented the inhibitory effects of melatonin, indicating that melatonin suppresses lung cancer stemness by inhibiting the PLC, ERK/p38, β-catenin, and Twist signaling pathways. We also found CD133 expression is positively correlated with Twist expression in lung cancer specimens. Melatonin shows promise in the treatment of lung cancer stemness and deserves further study. K E Y W O R D S lung cancer, melatonin, stemness, Twist 1 | INTRODUCTION Lung cancer is a major occasion of cancer mortality. Non-small cell lung cancer (NSCLC) is the most general type of lung cancer, accounting for more than 85% of all lung cancers. 1,2 Although chemotherapy and radiotherapy have improved lung cancer treatment over the past few decades, survival rates remain poor, with approximately 15% of patients remaining alive at 5 years after diagnosis. This poor prognosis is due to metastasis in the late stage of disease. Cells with a stem-like, dormant phenotype, endowed with unlimited self-renewal and high tumorigenic capability, are deemed responsible for post-therapy relapse and metastatic dissemination in various cancers, including lung cancer. 3-5 Therefore, the drugs that attack the cancer stem cells (CSCs) population have therapeutic benefit.Melatonin (N-acetyl-5-methoxytryptamine), a hormone secreted by the pineal grand and other organs, is produced in a circadian rhythm in reply to photic signaling from the retina. 6,7 Melatonin is important for controlling the immune system 8-10 and has an antioxidant effect. 11,12 This agent protects cells against carcinogenic substances and inhibits the development, metastasis, invasion and angiogenesis of many types

The epithelial–mesenchymal transition (EMT) phenotype, whereby mature epithelial cells undergo phenotype transition and differentiate into motile, invasive cells, has been indicated in tumor metastasis. The melatonin hormone secreted by the pineal gland has an antioxidant effect and protects cells against carcinogenic substances that reduce tumor progression. However, the effects of melatonin in EMT and lung cancer metastasis are largely unknown. We found that melatonin down-regulated EMT by inhibiting Twist/Twist1 (twist family bHLH transcription factor 1) expression. This effect was mediated by MT1 receptor, PLC, p38/ERK and β-catenin signaling cascades. Twist expression was positively correlated with tumor stage and negatively correlated with MT1 expression in lung cancer specimens. Furthermore, melatonin inhibited EMT marker expression and lung cancer metastasis to liver in vivo. Finally, melatonin shows promise in the treatment of lung cancer metastasis and deserves further study.