DNA Methyltransferase Expression and Proliferation Status of Metastatic Breast Cancer Cell Line After Prolonged and Repeated Rapamycin and Melatonin Application

Abstract

Objective: The aim of this study was to investigate the effects of Rapamycin and Melatonin and their combination on deoxyribonucleic acid (DNA) methylation and cell proliferation in a estrogen receptor (ER)-negative breast cancer cell line (4T1 cell line). Materials and Methods: Four groups were designed with 4T1 cell line depending on drug combination (control, Rapamycin, Melatonin, Rapamycin + Melatonin) and their administration on different time periods (24, 48 and 72 hours). The drugs were administrated for 1, 2 and 3 times, respectively for these time periods. All samples were counted; immunostained (Ki67, DNA methyltransferase-1 (DNMT-1), DNA methyltransferase-3a (DNMT-3a) and p53) and real-time polymerase chain reaction (PCR) (DNMT-1 and DNMT-3a) was performed. Results: The live/dead cell ratios were decreased in the Rapamycin and Rapamycin + Melatonin applied groups. Ki67 immunostaining showed that there was a decreased proliferation in the drug applied groups at 48th hours compared to the 24th hours. Also DNMT-1 expressions were decreased at 72th hour compared to that at 24th hour in all groups, especially in the Rapamycin administrated group. Adversely, DNMT-3a expression was increased at 72th hour compared to that at 24th hour in the groups, especially in the Rapamycin administrated group. Furthermore, an increased expression of p53 was seen in the drug given groups (highest in the Rapamycin applied group) when the time prolonged. Real-time RT-PCR analysis of DNMT-1 gene expression showed a decreased expression level in the Melatonin given group compared to the control group and an increased expression level was seen in the Rapamycin and Rapamycin + Melatonin administrated groups compared to the control group. Conclusion: As a result, it was found that Rapamycin is more effective in metastatic breast cancer cells than Melatonin, both in the manner of cell viability and expressional changes of Ki67, DNMT-1, DNMT-3a and p53.