The Demethylation Effect Of Arsenic Trioxide On RASSFIA Gene In DU145 Prostate Cancer Cells

Background:Prostate Cancer(PCa) is one of the most common malignant tumor in male genitourinary system, the incidence rate of which ranked the first in all male maligant tumor and meanwhile mortality rate ranked second. The incidence rate of PCa in our country has been increasing rapidly in recently years, once diagnosed the middle and late stage occupied the most, so the treatment of anvanced PCa is the enormous challenge that we clinicians will face.RASSF1A(Rasassciation domain family 1A) belongs to the Ras domain family, newly discovered tumor supressor gene which locates on human chromosome 3p21.3 and encodes a group of RAS effector proteins having potential functions in apoptosis signal transduction、microtubulestability and cell cycle. Large number of studies show it can be expressed in normal tissues, while there could be higher frequency of loss of expression in a variety of solid tumors having mechanisms including:methylation of RASSF1A promoter、the absence of the homozygous and heterozygous, and the former is more important.It could be re-expressed if making the gene demethylation.So far, the commonly used methylation inhibitor are cytidine analogues such as 5-aza-deoxycytidine (5-Aza-CdR) and so on, but the cell toxicity and strong mutagenic effect limit its clinical application. In recent years, a large number of studies have shown that arsenic trioxide (As2O3) could be used to cure relapsed and refractory acute myeloid leukemia, and remission can reach up to 72%. At the same time, the efficacy of As2O3 used to treat solid tumors is aslo in clinical researc, which are mainly on inducing apoptosis and cell differentiation and are relatively less on demethylation.This study is to observe whether it make the promoter CpG island of RASSF1A gene in DU145 prostate cancer cells demethylation and re-expression through treated by different concentrations of As2O3. Maybe in this way, we could provide new therapeutic drug for androgen-independent prostate cancer.Objective:To study the effects of the arsenic trioxide on the growth inhibition demethylation effect and gene expression on RASSF1A gene in hormonal independent prostate cancer Du145 cells, and intially explore the possible mechanisms of As2O2 on RASSF1Agene.Materials and Methods:1、To culture hormonal independent prostate cancer Du145 cells, when grow to reach logarithmic phase, dilute the concentration to 4×104 cells/mL, then continuous culture the cells 24、48、and 72 h respectively with 1640 culture medium containing 0、0.5、1.0、2.0、4.0、6.0、12.0、20.0μmol/LAs2O3.2、MTT assay was used to test the growth of DU145 cells after the treatment of As2O3 at 0.5,1.0、2.0、4.0、6.0、12.0 and 20.0μmol/L for 24,48,72 h, and to draw the curve of growth inhibition.3、Methylation-specific PCR (MSP) was used to detect the methylation status on RASSF1A gene in Du145 cells after 72 h treated by different concentrations (0、0.5、1.0、2.0、4.0μmol/L) As2O3.4、Western blot analysis was used to detect the re-expression of RASSF1A gene in Du145 cells after 72 h treated by different concentrations (0、0.5、1.0、2.0、4.0μmol/L) As2O3.5, Experimental data was processed by SPSS 17.0 statistical software, statistical differences were compared using non-parametric test and variance analysis, inspection levelα=0.05.Results:1、DU145 cells proliferation was significantly suppressed after exposure to different concentrations of As2O3, in a certain range,as the heighten of drug concentration,gradually increasing in inhibited effect(F=838.089,P<0.05),and in the same drug concentration,as the prolongation of effect time,the heighten of inhibition ratio(F=8.849/P<0.05), the most significant inhibition was at 20.0μmol/L. 2、RASSF1A gene on DU145 cell was in methylated state before treated by As2O3, while MSP showed reversal of the methylation of the RASSF1A gene after 72 h treated by As2O3.3、Western blot showed the expression of RASSF1A gene on DU145 cell was missing,after 72 h treated by As2O3, which was re-expressed. The difference of RASSF1A gene expression was significant between the treated groups and negative control group (Z=10.385,P<0.001), and there was also significant difference (P<0.05) between any two of the treatment groups.Conclusion:1、As2O3 could suppress the growth and proliferation of prostate cancer DU145 cells, and the inhabition effect was strengthened as increasing the dose or time, which was dose-dependend and time-dependend.2、As2O3 could reverse abnormal methylation status of promoter CpG island of RASSF1A gene in DU145 prostate cancer cells, induce protein re-expression. The protein expression was enhanced with increasing the concentration, which was also dose-dependend.