Effect Of The 5Aza-dc On RASSF1A Gene Demethylation In DU145 Prostate Cancer Line At Transcriptional Level

IntroductionTumorigenesis is a process of multiple factors and multiple stages. The bionomics of it appearance act as infiltrating growth and metastasis. The mechanism of tumorigenesis is caused by the abnormal change of genetic structure and function. The activation of oncogenes or inactivation of tumor suppressor genes is one of the key steps in the molecular process of tumorogenesis. There are two mechanisms concluded in tumorigenesis. One is genetics, which can be understood as the mechanism of mutation caused by the change of DNA sequences. The other is epigenetics, which can be understood as the mechanism that initiates and maintains heritable patterns of gene expression in an inheritable manner by reduction mitosis without changing the sequence of DNA. Although there is no the change of DNA sequence, it could regulate gene expression and control gene function in transcriptional level through chemical modification of DNA itself. This theory has been getting more and more attention in the study of tumorigenesis by far. Aberrant DNA methylation patterns go beyond the fields of epigenesis. It's mainly about a reaction of cytosine transformation into S-methylcytosine mediated by methyltransferases. Transcriptional silencing of many Tumor-supressing Gene for promoter hypermethylation of tumor suppressor genes is a well-recongized mechanism. In contrast to the genetic alterations, DNA methylation is classified as the epigenetic alterations which can be replicated stably and inherited druing cell proliferation. The aberrant pattern of methylation could be reversed more easily, and such reverse process, especially in the early stage of tumorgenesis, is in the treatment of tumors. Theoretically, re-expression of tumor suppressor genes by demethylating agents may contribute to inhibiting the development of tumor. It has been suggested that DNA hypermethylation results in resistance to chemotherapy and consequently reduces the efficacy of chemotherapeutic treatment. Therefore, utilization of demethylating agents synergistically in enhancing the sensitivity of chemotherapy becomes a new and hot spot.It has been reported that the DNA methylation alters the expression of certain genes. Methylation-silenced tumor suppressor genes are detected in some cancers. Our study used prostate cancer line DU-145 treated with 5Aza-dc, a demethylation agent. 5Aza-dc is one type of pyrimidine analogues, which can be obtained by converting the carbon at S position of 2'-deoxy-6-aminopyrimidine into nitrogen. It can inhibit the transmethylation activity through conjugating with DNMT1 and forming a covalent compound when conjugated with replicating DNA molecule. Then the formation of hypomethylated daughter chain makes the demethylation function ture. It is found that we can re-express some silenced tumor suppressor genes containing CpG Island via demethylating pathway and reverse their tumor suppressing function in vitro. These observations provide a novel therapeutic strategy in anticancer treatment. However, there is few systematic study on DNA methylation in prostate normal tissue, primary and metastatic tumor, and few reports of the effects of demethylating agents on the chemotherapy and cell growth of prostate cancer have been seen.RASSF1A gene is a new type of anti-oncogene confirmed in 2000. It has been reported that the transcription factor of RASSF1A can inhibit tumor growth from inhibiting the accumulation of CyclinD₁ and controlling the generation cycle from G₁ stage to S stage. The inactivation of RASSF1A makes the expression deletion and lose the adverse regulation function to generation cycle. These changes appear to have a role in the development and progression of malignant tumor. The inactivation of RASSF1A exists in the tumorigenesis of different kinds of neoplasms; but RASSF1A gene deletion is rarely detected. The hypermethylation of 5' CpG in promoter region serves as a mechanism of the inactivation of RASSF1A gene. This mechanism has been widely confirmed in multiple tumorgenesis including prostate carcinoma cell line DU145.ObjectOur study is to disclose the epigenic mechanism of the carcinogenesis of prostate cancer. Including:1. We use demethylation agents in combination with chemotherapeutic drugs, to disclose whether demethylation agents can enhance the anticancer effect of chemotherapeutic drugs.2. to study whether demethylation drugs can affect the methylation level of RASSF1A gene in.3. To study whether demethylation drugs can affect RASSF1A protein level in prostate cancer cell line Dul45, thus we can further our study in clinical and pathological characteristics to find the molecular mechanism of prostate cancer carcinogenesis and a new way of gene therapy.METHODS1. MTT method and flow cytometry were used to detect the growth and apoptosis of Du145 after being treated with different dosage of 5Aza-dc singly or in combination with other chemotherapy drugs.2. RASSF1A gene DNA, mRNA and protein were determined by MSP, RT-PCR, Western blot.RESULTS1. 5Aza-dc increases DU145 cell apoptosis rate. The concentrations of 5Aza-dc were 1.0, 2.0 and 4.0μmol/L respectively. Suppression of cellular growth appeared when the drug concentration is up to 1.0μmol/L, the drug concentration had positive correlation with suppression function within a certain extent. After treated with 5Aza-dc for 48h, the survival rate of DU145 cells was 83.4% 62.8% 43.2% respectively. In all, 5Aza-dc could significantly increase apoptosis rate of DU145 cell. The effect manifested dose-dependent and when the concentration reached 4.0μmol/L, It achieved the largest effect.2. MSP, RT-PCR, Western blot showed that the RASSF1A gene of Dul45 cell was in the state of methylation before treated with 5Aza-dc. The mRNA expression and protein expression was deletion. After treated with 5Aza-dc for 48h, The RASSF1A gene hypermethylation may effectively caused demethylation. The expression of DU145 cell RASSF1A mRNA treated with 5Aza-dc recovered. Western blot indicated that 5Aza-dc could recover the RASSF1A mRNA gene protein expression.3. 5Aza-dc can coordinates with other chemotherapy drugs in the anti-tumor effects and enhance the antitumor effect of these drugs, we used MTT method to examine, the concentration of 5-FU were respectively 30μg /mL ,60μg /mL, 120μg /mL and 240μg /mL, the concentration of ADM were respectively 2μg/mL ,4μg /mL ,8μg /mL ,16μg /mL, and after treated with them for 48h, the survival rate of DU145 cell were respectively 92.3% 80.2% 73.5% 68.3% and 86.7% 73.5% 64.6% 58.2%. when added 5Aza-dc(1.0μmol/L) in the same condition ,the survival rate decreased to 72.5% 57.1% 49.4% 42.5% and 68.6% 53.2% 36.6% 28.7%.Conclusions1. Methylation of RASSF1A gene promoter leads to deactivation of the gene.2. 5Aza-dc can reverse the abnormal methylation of prostate cancer cell line Du145, activate the silenced gene and induce its expression.3. 5Aza-dc can induce the apoptosis of prostate cancer cell Du145 effectively.4. 5Aza-dc can cooperate with other chemotherapeutic drugs; enhance the sensitivity of prostate cancer cell line Du145 to chemotherapeutic drugs.