Comparative Evaluation of the Effects of 5-Aza-2'-deoxycytidine and Trichostatin A on Reactivation of hMLH1 in COC1/DDP Ovarian Cancer Cell Line

Objective： hMLH1 protein serves to detect the DNA damage caused by cisplatin （DDP） and destroys the cell. The absence of hMLH1 expression has been correlated with acquired resistance of ovarian cancer cells to platinum. The aim of this study was to determine the possible role of DNA methylation and histone H3 lysine 9 （H3-K9） acetylation on the loss of hMLH1 expression, and to evaluate the reversal effects of 5-Aza-2＇-deoxycytidine （5-Aza-dC） and Trichostatin A （TSA） on DDP-resistance in ovarian cancer cell lines. Methods： Two human ovarian cancer cell lines, COC1 and its DDP-resistant subline, COCI/DDP were cultured. The two cancer cells were treated with 5-Aza-dC or TSA. Using COC1 cells as a control, we used methylation-specific PCR （MSP） to analyze DNA methylation at hMLHI gene promoter, hMLH1 mRNA and protein expressions were analyzed by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot. Chromatin immunoprecipitation assay （CHIP） was used to test the levels of histone H3-K9 acetylation at hMLH1 gene promoter. Results： In COC1 cells, there was no DNA methylation at hMLH1 gene promoter, while there were hMLH1 mRNA and protein expression. In COC1/DDP cells, there was DNA hypermethylation at hMLH1 gene promoter, while there was no hMLH1 mRNA or protein expression. The treatment with 5-Aza-dC resulted in DNA demethylation at the promoter region, as well as restoration of hMLH1 expression in COC1/DDP cells. The treatment with TSA had no effects on DNA demethylation or restoration of hMLH1 expression in COC1/DDP cells. Conclusion： Hypermethylation of DNA at the promoter is related to the silencing of hMLH1 in COC1/DDP ovarian cancer cells. DNA methylation at hMLH1 promoter could play a significant role in determining the sensitivity of ovarian cancer to DDP. The drug resistance mediated by methylation of hMLH1 could be overcome by 5-Aza-dC.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury

Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.

Effects of 5-Aza-CdR on Cell Proliferation of Breast Cancer Cell Line MDA-MB-435S and Expression of maspin Gene

The effects of 5-Aza-2＇-deoxycytidine （5-Aza-CdR） on the proliferation of MDA-MB-435S cells and the expression of tumor suppressor gene maspin were investigated. Human breast cancer cell line MDA-MB-435S was treated with 5 μmol/L 5-Aza-CdR, a specific demethylating agent for 0 to 8 days. The growth of MDA-MB-435S cells was observed by MTT assay before and after 5-Aza-CdR treatment, respectively. The expression of maspin mRNA was detected by reverse transcfiption-polymerase chain reaction （RT-PCR）. The cell cycle of MDA-MB-435S cells was analyzed by flow cytometry. The results showed that the growth of MDA-MB-435S cells treated with 5-Aza-CdR for 8 days was significantly suppressed as compared with the control groups, and the inhibition rate increased sharply from 5 day to 8 day （35.42% to 71.29%）. Flow cytometry showed that 5 μmol/L 5-Aza-CdR could induce G2/M cell cycle arrest and decrease the percentage of mitosis cell number in this cell line. Maspin mRNA was expressed in MDA-MB-435S cells after 5-Aza-CdR treatment, but it was weakly detectable before the treatment. It was concluded that Maspin gene might be transcriptional silencing by hypermethylation and the re-expression of maspin gene by 5-Aza-CdR can inhibit the proliferation and induce the G2/M arrest of MDA-MB-435S breast cancer cells.

Gene induction and apoptosis in human hepatocellular carcinoma cells SMMC-7721 exposed to 5-aza-2′-deoxycytidine

Background Aberrant DNA methylation plays a key role in human carcinogenesis. 5-aza-2＇-deoxycytidine inhibits DNA methylation and induces the expression of genes putatively silenced by promoter methylation in vitro. There are few studies of the biological and clinical significance of 5-aza-2＇-deoxycytidine in human hepatocellular carcinoma. This study explored the mechanism of 5-aza-2＇-deoxy.cytidine targeting transcriptional repressor complexes affecting global gene expression in hepatocellular carcinoma cell line. Methods High density oligonucleotide gene expression microarrays were used to examine the effects of 5-aza-2＇-deoxycytidine treatments on human hepatocellular carcinoma cell line SMMC-7721. The 5＇ ends of the genes upregulated or downregulated in this manner were compared with BLAST database to determine whether they might have promoter CpG islands. Flow cytometry was used to detect stages of the cell cycle and apoptosis of SMMC-7721 after being treated with 5-aza-2＇-deoxycytidine. Results Data obtained 3 days after 4 days of treatment with 5-aza-2＇-deoxycytidine showed that more genes were induced in tumorigenic cells including genes that function in cell proliferation, differentiation, regulation of transcription, and cytokine signalling. Approximately 30% of induced genes did not have CpG islands within their 5＇ regions, suggesting that some genes activated by 5-aza-2＇-deoxycytidine may not result from the direct inhibition of promoter methylation. This phenomenon may contribute to a number of upregulated genes involving regulation of transcription in the treated cell. Results showed that 100 lumol/L 5-aza-2＇-deoxycytidine blocked cell cycle at S/G2-M phase increasing rate of apoptosis. Notably, we found differential expression of molecular action in the methylation although DNA methyltransferases did not show significant difference in the treated cell line. Conclusion 5-aza-2＇-deoxycytidine could restore some silenced genes expression independently of DNA rnethylation inhibition and expression of DNA methyltransferases.