Induction Of Apoptosis On Human Lung Cancer A549 Cell Line Via Reversing The Supermethylation Of P53 Gene By 8- Bromo-7-methoxy-chrysin

ObjectiveTo investigate whether 8-bromo-7-methoxychrysin(BrMC), a derivative of chrysin induces growth inhibition, and apoptosis in human adenocarcinoma of lung A549 cells in vitro, and evaluate BrMC whether reverses the supermethylation of p53 gene in A549 cells, in order to provide a laboratory and theoretic evidence for searching for a novel potent and promising candidate for lung carcinoma chemotherapy.MethodsHuman lung carcinoma A549 cells were cultured in vitro. Plate-colony formation assay was used to test colony formation inhibitory effect of A549 by BrMC. Flow cytometry(FCM) with PI stain was used to analyse the apoptosis after treatment with BrMC.DNA agarose electrophoresis was used to detect the fragmentation of DNA of A549 cell. Methylation specific polymerase chain reaction (MSP) detected the effect of BrMC on the reversing supermethylation of p53 gene in A549 cells.ResultsPlate-colony formation assay showed that the colony inhibitory rate was 7.9%,19.7%, 28.1%, 39.8%, 51.9% and 65.4% after treatment with BrMC at of various concentration 0.1, 0.3, 1.0, 3.0, 10.0, 30.0μmol/L for 48h respectively. IC50 was 7.6μmol/L; DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with BrMC at 10.0μmol/L for 48 h. FCM with PI stain indicated that the rate of apoptosis was 2.8%±0.60%, 5.1%±0.11%, 19.8%±1.74% respectively after treatment with BrMC in the concentrations of 0.3, 3.0, 30.0μmol/L for 48h. And the exposure of BrMC at the concentration of 10.0μmol/L for 0, 24, 48, 72h to A549 cells, the rate of apoptosis was 0.83%±0.13%, 5.36%±0.11%, 8.78%±0.05%, 22.67%±1.16% respectively. MSP indicated that A549 cells has the characteristics with hypermethylation of p53 gene and BrMC could reverse the hypermethylation of p53 gene.Conclusion1. BrMC inhibits the growth of A549 cells, in a dose-dependent manner in vitro.2. BrMC significantly induces apoptosis of A549 cells, in a time- and dose- dependent manner.3. Induction of the growth inhibition and apoptosis by BrMC might be associated with reversing the hypermethylation of p53 gene in A549 cells.