Melatonin Suppresses ADGRL 4 Expression and Induces Promoter Methylation in Estrogen‐Responsive Breast Cancer Cells

Abstract

Background Breast cancer (BC) is the second most common malignancy worldwide. Angiogenesis plays a significant role during tumor formation and progression. ADGRL4, as a modulator of angiogenesis, undergoes various epigenetic modifications affecting its biological functions. Here, we aimed to assess ADGRL4 promoter methylation and its expression level in breast tumors and to examine the effect of melatonin on ADGRL4 expression and promoter methylation in vitro. Materials and methods Fifty breast tumor and corresponding adjacent non-tumor tissue samples were collected, followed by DNA isolation, bisulfite conversion, qRT-PCR, qMSP assay and immunoblotting. Then, the effect of melatonin on the ADGRL4 expression and promoter methylation was investigated in two BC cell lines (MCF-7 and MDA-MB-231). Apoptosis assay was performed using annexin v and flow cytometry. Results ADGRL4 was overexpressed in tumors in comparison with normal tissue samples (p<0.0001). We found a significant correlation between ADGRL4 expression level and increased HER2 expression (p=0.021). We observed a significant decrease in ADGRL4 promoter methylation level in tumors as compared to marginal non-tumor samples (p<0.0001). Melatonin treatment significantly attenuated ADGRL4 expression (p<0.0001) and elevated promoter methylation (p<0.001) in MCF-7 cells, but not in MDA-MB-231 cell line. However, melatonin treatment induced apoptosis in both cell lines. Conclusion ADGRL4 was overexpressed and its promotor was hypomethylated in breast tumors. Melatonin treatment attenuated ADGRL4 expression only in estrogen-responsive BC cells (MCF-7) and induced apoptosis in BC cells.