University of Tabriz Tabriz, Azarbayjan-e Sharqi, Iran
Ali Rajabi (University of Tabriz)| Melika Maydanchi (Zimagene Medical Genetics Laboratory)| Ali Saber (Zimagene Medical Genetics Laboratory)| Mona Bakhshi (Zimagene Medical Genetics Laboratory)| Fereshteh Baniardalan (Zimagene Medical Genetics Laboratory)| Aref Sobhkhizy (Zimagene Medical Genetics Laboratory)| Sepehr Abdolahi (Azerbaijan Shahid Madani University)| Reza Safaralizadeh (University of Tabriz)
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.
Figure 1. ADGRL4 expression and promoter methylation status in breast tumors compared to marginal non-tumor tissue samples a) ADGRL4 expression levels in breast tumors and non-tumor tissue samples. b) ADGRL4 promoter methylation in tumor and non-tumor tissue samples. c) ADGRL4 expression in breast tumors and non-tumor samples using immunoblotting. d) Quantified western blot of ADGRL4.
Figure 2. Effect of melatonin on two breast cancer cell lines (MCF-7 and MDA-MB-231). a) Effect of melatonin on cell viability b) ADGRL4 expression in the absence/presence of melatonin. c) ADGRL4 promoter methylation status. d) ADGRL4 and β-actin expression in melatonin-treated and non-treated cells at protein level. e) Quantified western blot analysis of ADGRL4 and β-actin. f) Apoptosis assay in breast cancer cells following melatonin treatment. g) The mean percentage of total apoptotic cells.