Studies Of The Influence Of 5-Aza-CdR On The Biological Behaviors And The Mechanism Of Molecular Biology To Human Bladder Carcinoma Cell Line T24

Background:In China, as the most common malignant tumor in the genitourinary system, bladder transitional cell carcinoma (BTCC) has been harmful to people's health heavily and there has been obviously increasing trend of incidence in many cities. Despite of the improvement of operational skills and intravesical chemotherapeutic agents, the recurrent rate of bladder cancer still keeps highly at 50-70% after initial treatment. It is urgent in clinic to develop a new effective therapy on BTCC.Tumorigenesis 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 abnonnal 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 sequenees. 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 modifieation of DNA itself. This theory has been getting more and more attention in the study of tumorigenesis by far. Aberrant DNA methylation pattems go beyond the fields of epigenesis. It's mainly about a reaction of cytosine transformation into 5-meth-ylcytosine 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 proeess, especially in the early stage of tumorgenesis, is in the treatment of tumors. Theoretieally, re-expression of tumor suppressor genes by demethylating agents may contribute to in hibiting 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 bladder cancer line T24 treated with 5-Aza-CdR, a demethylation agent.5-Aza-CdR is one tpye of pyrimidine analogues, which can be obtained by converting the carbon at 5 position of 2'-deoxy-6-axninopyrimidine 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 bladder normal tissue, primary and metastatic tumor, and few reports of the effects of demethylating agents on the chemotherapy and cell growth of bladder cacer have been seen. Part I Expression of XAF1 and XIAP and its correlation in BTCCObjective To investigate the expressions of XAF1 and XIAP in BTCC with regard to the clinical significance.Methods Expressions of XAF1 and XIAP were detected by immunohistochemistry in 56 cases of BTCC and 38 cases of para-carcinoma tissue,18 cases of normal bladder tissues as controls. The relevance between the expressions of XAF1 and XIAP and the clinical pathology of the patients of BTCC were studied.Results Immunohistochemistry demonstrated that the XAF1 score was 3.04±2.61 in BTCC, which significantly down-regulated compared with that in para-carcinoma tissues as 7.32±3.64 and normal bladder tissues as 8.44±3.04 (P<0.01). The XIAP score was 7.13±3.11 in BTCC, which significantly up-regulated compared with that in para-carcinoma tissues as 2.28±1.54 and normal bladder tissues as 1.43±1.48 (P<0.01). The XIAP score had significant differences statistically in comparison Between para-carcinoma tissues and normal bladder tissues P<0.0l), but the XAF1 score had none(p>0.05). The expression of XAF1 was significantly negative correlated with the grade of the tumor (P< 0.05),and XIAP with positive correlation. And no correlation was found between the expression of XAF1 and the sex, age, clinical staging of BTCC (P>0.05), XIAP, too. The expression of XAF1 was significantly negative correlated with the recurrence of BTCC. And expression of XIAP was significantly positive correlated with the recurrence of BTCCConclusion1. The expression of XAF1 was significantly decreased in BTCC tissues, and XIAP was significantly increased in BTCC tissues. 2. The expression of XAF1 was significantly negative correlated with the grade of the tumor, and XIAP with positive correlation. XAF1 was negative correlated with the recurrence of BTCC and XIAP was positive correlated.3. The expression of XAF1 was significantly negative correlated with the expression of XIAP in BTCC. PartⅡEffects of 5-Aza-CdR on biological behavior and chemotherapeutic sensitivity of human bladder cancer cell line T24Objective The present study was undertaken to examine the effects of 5-Aza-CdR on biological behavior of T24 cells, including growth inhibition, cell apoptosis and chemotherapeutic sensitivity.Methods MTT method and flow cytometry were used to detect the growth and apoptosis of human bladder cancer T24 cells after being treated with different dosage of 5-Aza-CdR singly or in combination with other ehemotherapy drugs.Results The inhibition ratio of different dosage(1uM,5uM, 10uM, 15uM,30uM,45uM) of 5-Aza-CdR on human bladder cancer T24 cells were 5.55%,13.27%,15.37%,18.52%,36.50%,44.62%(24 hours),7.47%, 17.86%,21.81%,27.50%,56.67%,67.47%(48 hours); 13.45%,25.82%, 30.81%,44.25%,64.80%,77.26%(72 hours) respectively by MTT assay, we found that 5-Aza-CdR treatment exerted a significant cytotoxic effect upon T24 cells in a dose-dependent manner(p<0.05). The concentration of E-ADM and MCC were respectively 0.25 uM,0.5 uM, 1uM,2uM, the inhibition ratio of T24 cells were respectively 13.64%,26.75%,36.23%, 43.68% and 8.83%,19.86%,26.65%,32.49%, when added 5-Aza-CdR(1.0u M) in same condition,the inhibition ratio were up to 32.50%,46.66%,66.24%,74.53% and 27.83%,42.76%,51.85%, 58.98%.The cell apoptosis index with 5-Aza-CdR treatment at concentrations of 1-30uM after 48 hours ranged from 4.57%-25.58%, increased significantly compared with control group in a dose-dependent manner (P<0.05).Conclusion 1 Our data suggested 5-Aza-CdR treatment resulted in a significant time-dose-dependent Inhibition in the growth of T24 Cells and cell apoptosis induced with a significant dose-dependent.25-Aza-CdR can coordinates with other chemotherapy drugs in the anti-tumor effects and enhance the antitumor effect of these drugs. PartⅢEffects of 5-Aza-CdR on the mechanism of molecular biology of human bladder cancer cell line T24Objective To investigate the effects of 5-Aza-CdR on the mechanism of molecular biology of T24 cells.Methods The effect of 5-Aza-CdR on human bladder cancer T24 cells was evaluated with varying concentration of 5-Aza-CdR (0,1,15 and 30uM) treatment for 48 hours, both RT-PCR and western blot were used to detect the expression of XAF1,XIAP,NF-KBP65. And methylation status of XAF1 from genomic of T24 cells was determined by methylation-specific polymerase chain reaction (MSP).Results MSP showed that after treated with 5-Aza-CdR for 48 hours, the XAF-1 gene hypermethylation may effectively caused demethylation. RT-PCR demonstrated that the expression ratio of XAF1 mRNA and the expression ratio of NF-KBP65 mRNA increased in all groups significantly compared with control group(p<0.01). The expression ratio of XAF1 mRNA was 0.278±0.003 (1Um),0.404±0.020 (15uM), 0.478±0.012 (30uM),0.670±0.050 (0uM) and NF-KBP65 mRNA was 0.364±0.015 (10Um),0.484±0.013 (15uM),0.778±0.018 (30uM), 0.203±0.009 (0uM), respectively. No correlation was found in the expression of XIAP mRNA(P>0.05). We found that 5-Aza-CdR treatment with XAF1 mRNA and NF-KBP65 mRNA upon T24 cells was in a dose-dependent manner. Western blot demonstrated that the expression ratio of XAF1 and NF-KBP65 increased in all test groups significantly compared with control group(P<0.01), both were in a dose-dependent manner. The expression ratio of XAF1 protein was 0.285±0.003 (1Um),0.350±0.003 (15uM),0.982±0.157 (30uM) 0.227±0.002(0uM)and NF-KBP65 protein was 0.262±0.005 (1Um), 0.298±0.004 (15uM), and 0.343±0.003 (30uM),0.195±0.002 (OuM)respectively. No correlation was found in the expression of XIAP protein (P>0.05).Conclusion1.5-Aza-CdR could reverse XAF1 promoter hypermethylation2. The expression of XAF1 mRNA and NF-KBP65 mRNA significantly increased in T24 after 5-Aza-CdR treatment for 48 hours. Both were in a dose-dependent manner. No correlation was found in the expression of XIAP mRNA.3. The expression of XAF1 protein and NF-KBP65 protein significantly increased in T24 after 5-Aza-CdR treatment for 48 hours. Both were in a dose-dependent manner. No correlation was found in the expression of XIAP protein.