The Effect Of Demethylation By Hydralazine On Estrogen Receptor α Negative Human Breast Cancer Cell Line MDA-MB-231

Objective:To explore the demethylation effect of Hydralazine on Estrogen receptor (ER)αnegative human breast cancer cell line MDA-MB-231.Methods:ERαnegative human breast cancer cell line MDA-MB-231 was cultured in vitro. Cells were randomized into four groups and exposed to Hydralazine in different concentrations (0,5,10,20μmol/L) for 96 hours; MDA-MB-231 cells were randomized into four groups according to the various length time (0,72,96,120 h) and separately exposed to the same concentration(10μmol/L) Hydralazine. The growth effect of Hydralazine on MDA-MB-231 cells was observed under the inversed microscope. Total RNA of individual group was extracted. The expression of ERαwas evaluated by reverse transcription polymerase chain reaction (RT-PCR) and image analysis. Genetic DNA of individual group was extracted, purified and modified. DNA methylation status was analysed by methylation specific polymerase chain reaction (MSP).Results:1. RT-PCR results demonstrated that Hydralazine could enhance the expression of ERαmRNA of MDA-MB-231 cells. The ratio of ERαtoβ-actin on optical density is all significantly higher(p<0.05 or p<0.01) in 5,10,20μmol/L Hydralazine groups than that of 0μmol/L group when cells exposed for 96 h; The ratio of ERαtoβ-actin on optical density is also all significantly higher at 72,96,120 hours(p<0.05 or p<0.01) in 10μmol/L Hydralazine than that of 0 h group.2. Hydralazine can reverse DNA methylation status on CpG islands of gene ERα. When cells did not be exposed to Hydralazine, the promoter of gene ERαwas methylated; when cells were exposed to 5μmol/L Hydralazine for 120 hours, the promoter of gene ERαwas partially demethylated; DNA methylation status was completely reversed when cells were exposed to 10μmol/L Hydralazine for 120 hours. Conclusion:1. DNA methylation on CpG islands on gene estrogen receptor (ER)αof ERαnegative human breast cancer cell line MDA-MB-231 possiblely lead to silence of gene ERα.2. Hydralazine can reverse DNA methylation status on CpG islands on gene ERαof MDA-MB-231 cell and induce re-expression of the silenced genes.