| Background:Renal cell carcinoma (RCC) is a kidney cancer that takes place in the lining of the proximal convoluted tubule. It is the most common type of kidney cancer in adults in approximately 80-90% of cases. RCC is resistant to radiation therapy and chemotherapy. Surgery treatment is the first choice for the earlier or intermediate stage. The 5-year survival rate is 65-90% in cases where the cancer has not metastasized, but lower if the cancer has spread.About 25%-30% in the early diagnosis of renal cell carcinoma, local invasion or distant metastasis.Kidney cancer was early detected no transfer or transfer were less, the survival rate is higher.,Kidney cancer was low sensitivity for chemotherapy for early treatment, surgical local excision is first selection.But for late transfer of kidney cancer, After nephrectomy, systemic treatment is still needed, such as targeted therapy and immunotherapy.Kidney cancer have multiple drug resistance, so for kidney cancer chemotherapy curative effect is poor.Kidney cancer resistance may be due to the existence of a high concentration of multiple drug resistance gene product MDR-1 p glycoprotein, It can actively to pump chemotherapy drugs out of the cancer cells.Studies have shown that chemotherapy combined with immunotherapy can achieve good effect.In recent years the emergence of molecular targeted therapeutic drugs has brought new hope for the treatment of patients with advanced kidney cancer,.In recent years as the gene epigenetics that is not to change the genetic base sequence change only in-depth study of its modification, DNA methylation in academia epigenetics development plays an important role on the incidence of tumor, the generation of tumor associated with DNA methylation disorders and imbalances, compared with normal cells, cancer cells in a wide range of low methylation and CpGs island high methylation, which is the key factor for tumor progression and metastasis.The demethylating reagent 5-aza-2’-deoxycitidine (DAC) blocks DNA methyl-transferases and makes a changeover of DNA methylation. It has been verified that DAC also can inhibit the growth of multiple kinds of cancer cells. A phase 1 study using DAC treating hematopoietic malignancies indicated the effectiveness of DAC against the tumor cells. One report also showed that DAC had clinical activity against metastatic lung carcinoma. Several studies are also being implemented to test the synergistic effects of DAC and other chemotherapeutic agents against tumor cells in vitro. A previous report suggested DAC increased the cytotoxicity of Cisplatin (CDDP) and made lung cancer cells sensitive to CDDP treatment. These phenomena proposed that DAC combined with CDDP is a promising therapy for tumor treatment. However, the mechanisms underlying its anticancer activity and other biological effects are not fully understood.Cell apoptosis is under the gene regulation of programmed cell death, apoptosis and the occurrence of tumor is thought to be unbalanced, genes that promote cell apoptosis decreased, inhibiting apoptosis genes are expressed increment.Apoptosis protease activating factor-1 (APAF-1) is a p53 pathway-related gene that functions in caspase activation, which is associated with mitochondrial-mediated apoptosis. Also, studies have shown that its decreased expression presents in various tumors. Briefly, p53 triggers the mitochondrial apoptotic pathway by inducing the expression of specific apoptotic genes, such as APAF-1, Bax, PUMA, Noxa, etc. As a result, studying the effect of APAF-1 over-expression on the the synergism of DAC with the CDDP-induced apoptosis may contribute to exploring the anti-apoptotic pathway of tumor cells and may also provide guidance for treatment.In this research, to discover the potential anti-tumor effect of DAC, we evaluated the DNA demethylation by DAC in human renal carcinoma cells and assessed the synergistic action of the demethylation with the toxicity of CDDP, which is a commonly used anti-tumor agent for renal carcinoma. We found that DAC promoted a significant global genomic demethylation and improved APAF-1 expression at both mRNA and protein levels. Also, the DAC treatment decreased the CDDP-induced viability of Caki or ACHN cells and synergized the CDDP-induced apoptosis in the ACHN cells. The treatment using DAC combined with CDDP promoted a significantly higher level of renal carcinoma cell apoptosis than DAC or CDDP treatment alone. The knockdown of APAF-1 significantly inhibited the synergism of DAC with the CDDP-induced apoptosis in ACHN cells. For exploring the antiapoptotic approach and treatment of renal cancer cells, the study can be used in subsequent experiments, to improve the treatment of renal cell carcinoma of the curative effect, improve the patients prognosis is of great importance to the quality of life.The first part:5-nitrogen impurity-2’-deoxy cytidine and cisplatin in kidney cancer cells to the mechanism of methylation1.Objective:To research the influence of the degree of methylation to methylation agent DAC ACHN and Caki cells of human kidney cell line, and compare to methylation agent with CDDP DAC individually or in combination, different drug concentrations in human kidney cell line ACHN and Caki two types of cells to the influence of methylation demethylation with CDDP DAC, To provide a molecular mechanism combination in treatment of renal cell carcinoma, and further verification to methylation agent with CDDP DAC dose combination of kidney cells, provide reference for further animal experimental and clinical pharmacology.2. Methods:Uing different concentrations of single or combined with CDDP DAC ACHN and Caki cultivated human kidney cancer cell line.By methylation specific PCR method for determining the kidney cancer cell lines of different methods cultivated ACHN and Caki methylation level.There were determined by MTT cell activity detection and cell apoptosis.Results used SPSS 13.0 software for statistical processing, the experimental results with two-tailed t-test test.3.Results:1). DAC and ACHN and Caki cell culture, reduced the ACHN and Caki cell DNA methylation level.2). With CDDP DAC synergy reduced the activity of renal cancer cells.3.) The concentration of the DAC and CDDP associated with kidney cancer cell activity.The second part:The research of 5-nitrogen impurity-2’-deoxy cytidine and cisplatin by enhancing apoptosis in renal cancer cells the activity of enzyme activation factor 1 mechanism of apoptosis induced by collaborativel.Objective:Through in vitro from the mRNA and protein level to research to methylation agent DAC ACHN human kidney cell line and Caki APAF two types of cells-1 the influence of the degree of methylation, and to compare methylation agent with CDDP DAC individually or in combination, different drug concentrations in human kidney cell line ACHN and Caki cells influence on APAF-1, as to methylation agent with CDDP DAC provide a molecular mechanism combination in treatment of renal cell carcinoma.2.Methods:1)With the different concentrations of DAC and CDDP ACHN alone or combination to cultivate human kidney cell line and Caki, measured by immune protein imprinting APAF 1 protein levels.2)With the different concentrations of DAC and CDDP alone or combination ACHN and Caki, cultivate human kidney cell line by methylation specific PCR and real-time reverse transcription PCR measuring methylation CPG and APAF the methylation level 1 mRNA.3) With the different concentrations of DAC and CDDP ACHN alone or combination to cultivate human kidney cell line and Caki, detect the activity of the two types of cells, and compare the differences of effects of two types of cell growth.4) With the different concentration dose with CDDP DAC ACHN alone or combination to cultivate human kidney cell line and Caki, detect of two kinds of cells apoptosis rate, and compare the differences of effects of two kinds of fine apoptosis.5) With the concentrations of DAC and CDDP individually or in combination with transfection remove APAF ACHN human kidney cell line 1 of a culture, using LEHDase analysis and the analysis of DEADase cells caspase 9 and the activity of caspase 3, and detection of cell apoptosis, DCA analysis APAF-1 in methylation agent and the mechanism of action of CDDP treatment of renal cell carcinoma.5)A11 data using SPSS 13.0 statistical analysis software, more difference between different groups was compared using analysis of variance between groups, the comparison between the two groups with double tail t test, significance level set to 0.05, when the P value is less than 0.05, the difference is statistically significant.3.Conclusion(1) In Caki and ACHN cells, the DAC treatment reduced DNA methylation level, improved the expression level of APAF-1.(2) In kidney cancer ACHN and the synergy of DAC and CDDP Caki cells induced apoptosis effectively.(3) Inhibiting APAF-1 the expression of retarding the ACHN cells by DAC and CDDP synergy inducing cell apoptosis.conclusion of full text(1) we successfully cultivated the ACHN human kidney cell line and Caki, silent the expression of APAF ACHN cells-1 (for specific siRNA APAF-1 transfection ACHN cells)(2) In Caki and ACHN cells, the DAC treatment reduced DNA methylation level, improve the expression level of APAF-1.(3) The synergy of DAC and CDDP reduced the activity of renal cancer cells(4) The kidney ACHN and the synergy of DAC and CDDP Caki cells induced cell research effectively(5) Inhibited expression of the block APAF- 1 the ACHN cells by DAC and CDDP synergy could induce cell apoptosis. |
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