Effects Of DNA Demethylation And Histone Deacetylase Inhibition To Estrogen Receptor Of Prostate Cancer Cell-Lines

IntroductionEpigenetic events are defined as alterations in gene expression without changes in the DNA coding sequence that are heritable through cell division.Study shows that epigenetic changes in tumor happen to play an important role in tomorigenesis,main epigenetic changes in tomorigenes contain DNA methylation and histone modification.Epigenetic is a reversible carcinogenesis event that is different from genetic change.Static genes can reactivate by using DNA methyltransferase inhibitors and histone deacetylase inhibitors,and thus reverse the malignant phenotype of the tumor or slow down the progress of tumor.The reversible characteristics of epigenetic modification hints it can be used as treatment targets for tumor patients.DNA methylation-based regulation of expression and certain tumor suppressor gene hypermethylation and inactivation of tumor-related theory has been widely recognized. DNA methylation in regulating gene expression is closely related to histone acetylation. DNA methylation can induce local histone deacetylation,so that the level of chromatin acetylation reduced.Methylation sequences can collect methy CpG binding protein (MeCP) and histone deacetylase(HADC) complex(MeCP-HADC),exert inhibitory effects on gene expression.If the tumor suppressor gene that inactivated by loss of DNA methylation expression can reexpress by demethylation agents and / or HDAC inhibitors is the purpose of this study.Estrogen receptor belongs to the ligand-activated nuclear transcription factor superfamily.Estrogen receptor is divided into two types,ERa and ERβ;ERa is located in the basement of prostate epithelial cells and stromal space,ERβis located in the prostate gland epithelial gap,the role of estrogen is considered by the prostatic epithelial signaling pathways through ERβ.Pasquali research shows that prostate cancer is found no expression of ERβ.However,Leav reports ERβdecrease in high-level dysplasia,but reemerge in prostate cancer and metastatic tumor.Prostate cancer is considered,like breast cancer,even if there is the expression of Erβ,that are maybe the wild-type ERβvariants.ERβcan regulate cell growth and its mutation may cause anti-estrogen therapy is not sensitive or cause a more invasive cancer.At present, there is only one ER8 antibody in the vast majority of immunohistochemical staining,it is difficult to distinguish subtypes of ERβprotein expression.Related study shows that loss of ERβexpression in the majority of prostate cancer reveals ERβmay be a potential tumor suppressor gene,and ERβis probably the desired objectives treatment of prostate cancer.In prostate cancer cell lines and prostate cancer,there is a wide range of estrogen receptor(ER) gene methylation,the degree of methylation was positively correlated with tumor pathological level,and was negatively correlated with ER expression. Although ER methylation exists in benign prostatic hyperplasia organizations,it was significantly lower than that of prostate tumor.In prostate cancer,at present,a possible reason to make ER gene inactivate is that the ER gene promoter region CpG island methylation,through some mechanism that is not yet known causes inactivation of ER gene transcription.In order to study the association of estrogen receptorβexpression in prostate cancer and DNA methylase inhibitors,HDAC inhibitors,we use DNA methylase inhibitor,5 -aza-2 '- deoxycytidine(5-AZAC) and HDAC inhibitors Trichostatin A(TSA) acts on prostate cancer cell line Du145,PC-3 and 22RV,detect cell apoptosis;detect of estrogen receptorβexpression by fluorescence real-time quantitative PCR,provide a basis for clinical treatmentMethod1.Materials Androgen-independent prostate cancer cells Dul45,PC-3 and androgen independent prostate cancer 22RV purchased from cell bank of China Shanhai Cell Institute;5 - aza-2 '- deoxycytidine(Sigma USA),Trichostatin A(Sigma USA), RT-PCR kit(Takara Company),MTT(Sigma USA),Dimethyl sulfoxide(Sigma USA), Trizol(USA Gibco Company),SBRY-Green(Tiangen),Master-mix(Tiangen).2.Experimental methods (l)Cell CultureDU145,and 22RV cells were maintained in RPMI-1640 medium,PC-3 cell was maintained in HAM/F-12 medium,supplemented with 10%fetal bovine serum(FBS) at 37℃in a humidified,5%CO2 incubator.The cell was passaged every three or four days.The cell on logarithmic growth phase was used to make the study.(2)The effects of 5-AZA and TSAon prostate cell lines by MTTProstate cells were treated with different concentrations of 5-AZA and TSA,and then method of MTT was used to detect the inhibition rate.Survival rate=(A of treated group-A of blank group)/(A of control group- A of blank group)×100%.(3)Detection of ERβexpression by fluorescence real-time quantitative PCRCells were harvested,and total RNA was isolated using Trizol reagent,detection of ERβby RT-PCR kit(Takara Company) and SBRY-Green.(4)Statistic analysisAll the data were analyzed by SPSS11.6 and when the value of p was less than 0.05,it was statistical significance.Result1.The survival rate of prostate cancer cell line after 5-AZAC and TSA treatment decreased by MTT;At a certain range,as the dose increased the survival rate of prostate cancer cells reduce more;prostate cancer treated by association of 5-AZAC and TSA has the lowest survival rate.2.Real-time quantitative PCR showed that after 5-AZAC and TSA treatmenr,mRNA ERβexpression of three types of prostate cancer increased;as drug dose increase, mRNA ERβexpress more;mRNA ERβexpression of prostate cancer treated by association of 5-AZAC and TSA is the most.Conclusion1.5-AZAC and TSA inhibit the growth of prostate cancer cells,in a certain range,as the dose increased,inhibitory effect of prostate cancer cell proliferation was more obvious;TSA had strong inhibitory effect on cells than 5-AZAC;inhibitory effects of association of 5-AZAC and TSA was more obvious than single drug effects.2.5-AZAC and TSA can induce ERβexpression of prostate cancer cells,thereby inhibiting the growth of prostate cancer cells.