The Effects of Methyltransferase Inhibitor on the Growth of Human Lung Cancer Cell Lines and the Expression of XAF1 Gene
|Course||Department of Cardiothoracic Surgery|
|Keywords||5 - azacytidine lung cancer A549 cell line cell proliferation cell apoptosis methylation XAF1 gene|
Objective: 5-Aza-CdR(5-aza-2’-deoxycytidin)is a methyltransferase inhibitor agent.Some study have recently shown that 5-Aza-CdR has strongly antitumor activity by inhibiting the proliferation and inducing cell apoptosis,it also can induce several tumor inhibitors expression in several human tumors.XAF1 is a new tumor suppressor,was found recently,low expression in tumors because of gene hypermethylation. The aim of this study is to observe the effects of methyltransferase inhibitor agent ,5-Aza-CdR,on the proliferation,apoptosis and XAF1 mRNA expression of human lung adenocarcinoma A549 cells. Preliminary study the effects of methyltransferase inhibitor agent on the proliferation,apoptosis of adenocarcinoma cells and the possible mechanism.Methods: Human lung adenocarcinoma A549 cells were treated with 1.0μmol/L, 5.0μmol/L and 10.0μmol/L 5-azacytidine, respectively, as divided into low concentration, the middle concentration, high concentration of three groups, and the same volume of drug dissolution medium, PBS interventions cells were used as the control group and blank group. the proliferation of A549 cells treated by different concentrations of 5-Aza-CdR was detected by MTT assay at 24,36,48 hours after the intervention,assessment of cell apoptosis were performed by flow cytometry(FCM) with Annexin V / PI double staining method.48h respectively after the intervention by semiquantitative reverse transcription - polymerase chain reaction (RT-PCR) detection of cells the expression of XAF1 mRNA levels and by protein immunoblotting (Western Blot) to detect the changes of XAF1 protein expression levels;Results: The results of MTT showed that three different concentrations of cell proliferation rate were 0.35±0.03, 0.53±0.04, 0.64±0.02 .Compared with the blank group and the control group increased (P <0.01), blank group and control group have no statistical significance (P> 0.05),and the same concentration in a time dependent manner (P <0.05).FCM results showed that apoptosis of three different concentrations rate were 7.85%±1.39%,15.06%±2.07%,22.43%±2.45%. Compared with the blank group (5.06%±1.03%) and the control group(4.99%±1.08%)increased in a dose dependent manner (P <0.05), the proliferation rate between blank group and control group was not significant (P> 0.05).RT-PCR results showed that the XAF1mRNA expression levels of 1.0μmol /L, 5.0μmol/L, 10.0μmol/L group were 3.19±0.71,2.11±0.86,1.41±0.24, compared with blank group (3.96±0.78) and control group (3.99±0.41) increased (P <0.05),the XAF1mRNA expression level of high concentration group increased compared with the low concentration group(P <0.05),the expression level between the media concentration group and the other two groups ,blank group and control group have no statistical significance (P> 0.05). Western-Blot results showed that the XAF1 protein expression levels of three different groups were 0.32±0.04, 0.37±0.01, 0.47±0.06 compared with blank group (0.08±0.01) and control group (0.09±0.03) increased (P <0.05), the XAF1 protein expression level of high concentration group increased compared with the low concentration group(P <0.05),the expression level between the middle concentration group and the other two groups ,blank group and control group have no statistical significance (P> 0.05).Conclusion: 5-Aza-CdR can inhibit the proliferation and induce cell apoptosis of adenocarcinoma A549 cells. 5-Aza-CdR can up-regulate the expression of XAF1 gene on human lung adenocarcinoma A549 cells. The mechanism of biological behavior reversing maybe related to the up-regulation of XAF1 expression .