| BackgroundAccording to the American Cancer Society Statistics,2006 in the United States, bladder cancer in men is secondary to prostate cancer, lung cancer and colorectal cancer after 4th of malignant tumors in female ranked No.9-bit. Approximately 6 million people are clinical doctors diagnosed with bladder cancer, every year, more than 1 million people died of bladder cancer, the vast majority of transitional cell carcinoma of the bladder is to infiltrate ways, recurrence rate is 50% to 70%. Although the available local treatment of bladder control, but there is still a 10%-15% of superficial bladder cancer eventually developed into muscle invasive bladder cancer or other parts of the transfer occurs. Despite the superficial bladder cancer survival rates are high, the prognosis is good, but the bladder resection local recurrence rate of 38% to 80%,50% in 1 year, approximately 2/3 in the second year after the operation. Bladder tumor recurrence of the main reasons:(1) tumor cells in surgery residual, falls off one of the reasons for the planting is important (2) the tumor cells along the bladder muscle intramural lymphatics diffusion, actual infringement bladder wall naked eye can see broad, resection of the tumor does not sufficiently. (3) some scholars think that the only bladder tumor recurrence is the TNM prognostic factors. (4) the naked elusive carcinoma in situ and the development of precancerous lesions. (5) bladder continue to be carcinogens in urine. Therefore, the study of bladder cancer pathogenesis, early diagnosis and early treatment has become particularly important. As in recent years the development of molecular biology of cancer, bladder cancer occur the considerable progress. Gene mechanism and epigenetic mechanism of tumor formation mechanism of the two major categories, gene mechanisms include proto-oncogene activation and deactivation of the tumor suppressor gene (mutations, deletions, insertions, rearrange, proliferation) and chromosomal abnormalities, epigenetic mechanism of epigenetic mechanisms include DNA methylation abnormalities, histone modifications, coding RNAs (including microRNA), Chromatin Remodeling, abnormal methylation mainly refers to the methyl group and genome CpG dinucleotide cytosine ring 5 carbon atoms to form covalent bond, forming 5-methylcytosine, causing abnormal gene expression and DNA sequences and gene products. In cancer cells, DNA hypermethylation of local and universal low methylation co-exist, except point mutations, gene deletion, promoter hypermethylation cause transcription inactivating is tumor suppressor gene inactivation of one. Tumor suppressor genes of hypermethylation as gene inactivation of the mechanism first human sporadic retinoblastoma RB confirmed, VonHipple Lindau (VHL) gene 5'CPG islands of new methylation in a sporadic Nephroblastoma disease has also been reported, p16 genes in many kinds of tumor cell lines and primary tumor has experienced the 5'CPG Island hypermethylation and deactivation,5-azido cytosine treated this gene recovery transcription, p15 gene also found in neuroblastoma and Lymphoma remain hypermethylation in addition to the tumor suppressor gene, E-cadherin metastasis suppressor gene in breast cancer and prostate cancer expression is promoter hypermethylation of the results. DNA methylation in tumor formation is multi-faceted, on the one hand,5mC to a high frequency of spontaneous mutations to T, methyltransferase enzyme inhibitory amino and 5mC of DNA mismatch repair is enhanced by C-T of mutation; on the other hand, DNA methylation gene and gene activation, deactivation for tumor suppressor genes. Therefore, DNA methylation changes by gene and gene mechanism, and cell proliferation and differentiation exception for gene expression, causing cells lose control of the normal growth of malignant transformation, which formed the tumor. The incidence of bladder cancer, development is a multi-step process, multiple phase, there are a variety of oncogenes and tumor suppressor genes. In recent years, research on tumor suppressor genes is becoming more and more attention. Where death-associated protein kinase (DAPK) is a means of promoting apoptosis or inhibit growth and functioning of tumor suppressor genes. Located on chromosome DAPK 9q34.1, has been reported that a point in the head and neck tumors can occur point mutations, in digestive cancer occur in the absence of. It is reported that many kinds of cancer, development and expression for DAPK, and DAPK gene CPG Island (CPG second nucleotide accumulation, distribution to the 5'-end of gene promoter hypermethylation regional) is DAPK expression one of the important links. DAPK cancer pathogenesis may be:when DAPK gene genetic altered (or table cell genetic), does not die, constantly forming tumors mitosis. Promoter CPG Islands in a normal state general non-methylation of methylation occurs when its often result in gene transcription silence makes some important genes such as tumor suppressor genes in DNA repair genes, such as loss of functionality, resulting in a normal cell regulation and control arrhythmias as well as DNA damage cannot be repaired in a timely manner, resulting in the formation with a variety of tumors. We can put the CpG methylation as tumor markers, tumor occurs often with varying degrees of DNA methylation patterns change, by promoter CpG Island hypermethylation of the test can detect DNA methylation silent tumor suppressor gene, which has become a search for cancer-related genes. The present experimental techniques can be done to detect tumor suppressor gene promoter hypermethylation, and DNA methylation inhibition range of quantitative, so as to assess the risk of cancer, making prevention policy, access to early diagnosis and to trace the prognosis provide guidance. Caused by the methylation of gene silencing the tumor, available through DNA methyltransferase non-competitive or competitive inhibitors methylation occurs, activating the silence of the tumor suppressor gene, so as to achieve the aim of the treatment of tumors. Demethylation drugs:5-nitrogen cytosine and 5-AZA-2'-Deoxy cytosine can inhibit methyltransferase activity, in some of the intractable cancer, especially in leukemia treatment have better outcomes.5-AZA-2'-deoxycytidine (5-aza-cdr) is a DNA methyltransferase 1 (DNMT1) inhibitors can suppress the DNMT1 functionality to reverse the promoter hypermethylation, gene expression. Many of the in vitro research confirmed that the 5-aza-cdr pass to a variety of CPG Island hypermethylation of the tumor suppressor gene expression, thus restoring tumor suppressor function, such as lymphoma, gastric cancer, breast cancer. But 5-aza-cdr on bladder T24 cell lines DAPK role not seen at home and abroad. This experiment will use different concentrations of the 5-aza-dc at different times with T24 cell lines, testing its proliferation, apoptosis of biological effects and related mechanism for bladder cancer chemotherapy with new ideas.Objective(1) To analyze the proliferation and the apoptosis of T24 cells before and after treatment with 5-Aza-CdR by using different concentrations.(2) To detect the biological effect of the DNA methyltransferase of human bladder cell cancer T24 lines.To investigate the effect and the possible mechanism of 5-Aza-2'-deoxycytidine (5-aza-dc) on the growth of bladder cancer T24 cells, and to find a new target for bladder cancer genetic therapy.MethodsCell culture medium for tumor cells RPMI1640 + 10% fetal bovine serum. At 37℃,5%CO2 conditions in the incubator, each of the 12-24h change 1 time depending on cell growth, in cell close to the shops flasks or training Board (4-5 days) for generations. Digestion cells with 0.25% of pancreatic lipase PBS fluid (usingμm o 1/L). T24 cells in the pancreas to handle 30 seconds after termination of digestion. Apply different concentrations (0.1,0.5,2.5,12.5μm o 1/L) of specific DNA methyltransferase inhibitor 5-aza-dc, to the different roles of time (6,12,24,48h) deal with bladder cancer cell lines T24. Using four methyl blue (MTT) method for the determination of different concentrations of the 5-aza-dc at different times of T24 cell line rate, the MTT joined via different concentrations of 5 a Aza-CdR treatment before and after the bladder T24 cells, DMSO fully dissolved, reset the automatic 570nm wavelength of the enzyme to read absorbance (A) the value, cell proliferation ability to average absorbance (A) analysis of the value to A value of ordinate, time (d) as jian, draw the growth curve. Using flow cytometry different concentrations (0.1, 0.5,2.5,12.5μm o 1/L) of 5-aza-dc in 24h after T24 cell apoptosis rate changes. Use the methylation specific PCR (MSP) to detect drug treatment before and after death-associated protein kinase (DAPK) the methylation status.Results1,The proliferation of T24 cell was inhibited significantly by 5-aza-dc from 0.1 to 12.5μmol/L accordingly.Compared with control group,there is significant difference in experimental groups(P<0.01).2,It was found by MTT assay that the growth inhibitory action of 5-aza-dc at different levels (0.1,0.5,2.5,12.5umol/l) on T24 cell was dose-dependent at the same action time. The inhibitory action of the same concentration group on T24 cells increased with time within 24 hours (P<0.01), however, at the level of 12.5 umol/L, the change of inhibitory rate became not so distinct beyond 24 hours. The test showed that 5-aza-dc had the most significant inhibitory action on the growth of T24 cells at 24 hours with the concentration of 12.5umol/L.3,Distinct apoptosis of bladder cancer T24 cells 24 hours after 5-aza-dc at different concentrations (0.1,0.5,2.5 andl2.5umol/L) was given was observed by Flow cytometry assay (Annexin V/PI double stain). The apoptosis rate in administration group increased with the increase of drug concentration compared to that in control group and the apoptosis rate was (24.12±1.4)% at the concentration of 12.5umol/L. Above results indicated that the cellular apoptosis rates at different levels was of significant difference (p<0.01) and the apoptosis rate of T24 cells was dose-dependent.4,With MSP assay, the result of the methylation status of the promoter of DARK gene in T24 cell under normal cultivation conditions showed that the amplification band of methylated primer was positive and the amplification band of non-methylated primer was negative. Twenty four hours after 5-aza-da (12.5umol/L) was given, the amplification band of methylated primer in T24 cell was negative and the amplification band of non-methylated primer was positive. That suggested that the promoter of DARK gene in bladder cancer T24 cells was highly methylated and such high-methylation state of DARK promoter could be inverted into its non-methylation state by the demethylation effect of 5-aza-da.Conclusion1,This study evidenced that 5-aza-da had significant effects of proliferation inhibition and apoptosis induction on human bladder cancer T24 cell line in concentration and time dependent manner; the apoptosis rate distinctly increased with the increase of concentration and the elongation of cultivation time.2,The action mechanism of the drug might be inducing the apoptosis by re-activating tumor suppressor gene, which had been inactivated by methylation. The induction of apoptosis by 5-aza-da might be applied to the clinical treatment for tumors and offer a new idea for further studies on the gene therapy for bladder tumor, which was worthy of further exploration.
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