Background:The Notch signaling pathway has a key role in angiogenesis and Delta-Like Ligand 4 (DLL4) is one of the main ligands of Notch involved in cell proliferation in sprouting vessels.Objective:In this study, we aimed to evaluate the expression of DLL4 in primary breast tumors and to examine the effect of melatonin on DLL4 expression in vitro.Methods:Eighty-five breast tumor and paired adjacent non-tumor tissue samples were collected. Apoptosis assay was performed on breast cancer cells to evaluate melatonin effects. Western blot and quantitative RT-PCR were used to measure DLL4 expression. Then, we investigated the effect of melatonin on the expression of DLL4 in four breast cancer cell lines at RNA and protein levels. We also performed Probabilistic Neural Network analysis to study genes closely associated with DLL4 expression.Results:Our results showed a significantly higher expression of DLL4 in tumor tissues as compared to non-tumor tissues (P = 0.027). Melatonin treatment substantially attenuated DLL4 expression in BT474 and MCF-7 cells, but not in SK-BR3 and MDA-MB-231 cells. Also, melatonin induced apoptosis in all four cell lines. Network analysis revealed a set of 15 genes that had close association and interaction with DLL4. DLL4 was overexpressed in breast cancer tissues as compared to the non-tumor tissues.Conclusion:It can be concluded that melatonin treatment attenuated DLL4 expression only in estrogen-responsive breast cancer cells and is able to induce apoptosis in breast cancer cells.