Effect of 5-Aza-2’-deoxycytidine on immune-associated proteins in exosomes from hepatoma

AIM: To study the effect of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on heat shock protein 70 (HSP70), human leucocyte antigen-Ⅰ (HLA-Ⅰ) and NY-ESO-1 proteins in exosomes produced by hepatoma cells, HepG2 and Hep3B. METHODS: Exosomes derived from HepG2 and Hep3B cells treated with or without 5-aza-CdR were isolated and purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. The number of exosomes was counted under electron microscope. Concentration of proteins in exosomes was measured by bicinchoninic acid protein assay. Expression of HSP70, HLA-Ⅰ and NY-ESO-1 proteins in exosomes was detected by Western blotting and immunoelectron microscopy. mRNA expression of p53 gene was detected by reverse transcription polymerase chain reaction.RESULTS: The mRNA expression of p53 gene was increased in both hepatoma cell lines after treatment with 5-Aza-CdR. The number of exosomes and the concentration of total proteins in exosomes were increased signifi cantly after treatment with 5-aza-CdR (P < 0.05). After treatment with 5-Aza-CdR, immunoelectron microscopy and Western blotting showed that the HSP70, HLA-Ⅰ and NY-ESO-1 proteins were increased in exosomes produced by both hepatoma cell lines. CONCLUSION: 5-aza-CdR, an inhibitor of DNA methyltransferase, can increase exosomes produced by hepatoma cells and immune-associated protein component of exosomes, which may be mediated by p53 gene upregulation and 5-Aza-CdR demethylation.

Inhibitory Effects of 5-Aza-2'-Deoxycytidine and Trichostatin A in Combination with p53-Expressing Adenovirus on Human Laryngocarcinoma Cells

Objective： To investigate the effects of 5-Aza-2＇-deoxycytidine （5-Aza-Cdr） and trichostatin A （TSA） combined with p53-expressing adenovirus （Ad-p53） on Hep-2 cell line in vivo and in vitro, in order to explore its possibility in biological treatment of laryngocarcinoma. Methods： Effects of 5-Aza-Cdr and TSA in combination with Ad-p53 on Hep-2 cell line in vivo were determined by Cell Counting Kit-8 （CCK-8） assay. The effect of drug combination was calculated by Jin＇s formula. Effects on the cell line in vitro were investigated by establishing the nude mice model. Results： 5-Aza-Cdr and TSA showed inhibitory effects on the proliferation of Hep-2 cells in dose- and time-dependent manner. Ad-p53 can inhibit the growth of Hep-2 cells in vivo and in vitro. However, the combination of epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 was less effective than individual use of Ad-p53. 5-Aza-Cdr and Ad-p53 inhibited the growth of transplanted tumors and reduced the volume of tumors, and the tumor volume of Ad-p53 group was significantly smaller than that of the control group （P0.05）. Conclusion： Both epigenetic reagents （5-Aza-Cdr/TSA） and Ad-p53 can suppress cell proliferation on Hep-2 in vivo and in vitro and there may be some antagonistic mechanism between Ad-p53 and epigenetic reagents （5-Aza-Cdr/ TSA）.

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.

Gene Expression Profiling of Human Myeloid Leukemic MV4-11 Cells Treated with 5-Aza-2’-deoxycytidine

The pyrimidine analog, 5-aza-2’-deoxycytidine (5-aza-dC) is a DNA methyltransferase inhibitor that triggers DNA demethylation leading to the reactivation of epigenetically silenced tumor suppressor genes. To understand the shift in gene expression which mediates the beneficial 5-aza-dC effects in leukemia, we have treated human myeloid derived leukemic cells with 5-aza-dC. Target genes were identified first in MV4-11 cells using a genome-wide gene expression profiling assay to detect differences in treated and untreated cells. From this analysis six genes were identified (HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2) as being significantly different expressed after treatment. To validate microarray data, we performed quantitative PCR on these genes from multiple leukemic cells. The results suggest that these genes are epigenetically regulated indicating that dysregulation of HOXA4, HOXD4, HOXA8, HOXD12, CD9 and RGS2 expression may play an important role in establishing the malignant phenotype in AML.

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.

DNA甲基化酶抑制剂5-Aza-CdR对人牙髓细胞增殖活性及矿化能力的影响

目的：研究DNA甲基化酶抑制剂5-氮杂脱氧胞苷（5-Aza-2＇-deoxycytidine,5-Aza-CdR）对人牙髓细胞（human dental pulp cells,hDPCs）增殖活性和矿化能力的影响.方法：以酶消化联合组织块法体外培养人牙髓细胞,取第3代细胞并随机分为常规培养组（对照组）、矿化诱导组、5-Aza-CdR组及5-Aza-CdR联合矿化诱导液组（联合组）；分别于培养不同时间后,采用CCK8法检测各组hDPCs细胞的增殖活性；碱性磷酸酶活性检测法以及茜素红矿化结节染色法观察各组hDPCs的矿化能力.结果：与对照组相比,矿化诱导组、5-Aza-CdR组及联合组在培养第3～7天时,hDPCs的增殖活性明显降低（P＜0.05）；第3～14天时,碱性磷酸酶活性明显升高（P＜0.05）.第14天时,除对照组外各组均有矿化结节形成,其中5-Aza-CdR组矿化结节较少,矿化诱导组及联合组可见大片矿化结节,特别是联合组,矿化结节的密度更大、颜色更深.结论：体外培养条件下,DNA甲基化酶抑制剂5-Aza-CdR可降低hDPCs的增殖活性,增强其矿化能力.

5-杂氮-2′-脱氧胞苷上调CK13表达并抑制人肺鳞癌YTMLC-9细胞增殖

目的：探讨DNA甲基转移酶抑制剂5-杂氮2′-脱氧胞苷（5-aza-2′-deoxycytidine）对人肺鳞癌YTMLC-9细胞株增殖的影响及对细胞角蛋白13（CK13）表达和CK13基因甲基化的影响。方法：采用四甲基偶氮唑蓝（MTT）法检测不同浓度（1、2、5、10，20、40μmol/L）5-aza-2′-deoxycytidine处理1～4 d后的5-aza-2′-deoxycytidine对人肺鳞癌YTMLC-9细胞株生长的影响；采用半定量PCR及Western blot检测5μmol/L的5-aza-2′-deoxycytidine作用YTMLC-91～4 d CK13的表达；采用甲基化PCR检测不同浓度（1、2、5、10μmol/L）5-aza-2′-deoxycytidine 处理48 h后CK13甲基化和非甲基化的变化。结果：5-aza-2′-deoxycytidine对人肺鳞癌YTMLC-9细胞株的生长具有明显地抑制作用，当用药浓度高于20μmol/L后产生明显的细胞毒性；5-aza-2′-deoxycytidine能够上调CK13基因表达，呈时间依赖性。结论：5-aza-2′-deoxycytidine抑制人肺鳞癌YTMLC-9细胞的生长，抑制作用呈浓度、时间依赖性。5-aza-2′-deoxycytidine可以使CK13重新表达。其生物学效应可能与CK13启动子甲基化状态改变有关。

5-Aza-2’-deoxycytidine暴露对新生大鼠精子发生的影响

[目的]探讨暴露于DNA甲基化抑制剂5-Aza-2’-deoxycytidine(5-Aza-CdR)对新生SD大鼠生长发育和成年精子发生的影响。[方法]新生大鼠随机分为3组,每组24只雄鼠。自出生第3天(postnatal day 3,PND 3)开始经口给予5-Aza-CdR 25、250μg/kg,对照组给予等量的溶剂。连续暴露5d,最后一次暴露结束后24h,处死半数雄鼠(幼鼠)。剩余部分继续喂养至12周龄(成鼠),乙醚麻醉。取睾丸组织做病理学检查、精子头计数等,附睾做精子畸形检查。[结果]随着剂量的增加,幼鼠体重出现下降趋势,与对照组比较差异有统计学意义(P<0.05)。组织病理学发现幼鼠睾丸组织中出现空泡变性。检查发现染毒结束后继续喂养至12周的成鼠睾丸组织无明显形态和组织学变化;但每克睾丸组织精子头计数及每日精子生成量随幼年时暴露剂量增加呈现下降趋势(P<0.05),而精子畸形率随剂量增加呈现上升趋势(P<0.05)。[结论]新生大鼠对DNA甲基化抑制制5-Aza-CdR生殖毒性作用敏感,低剂量短时间的暴露即可引起成年期生殖功能的异常。

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.

Telomere Elongation in the Breast Cancer Cell Line 21NT after Treatment with an Epigenetic Modifying Drug

Background: Telomere length dysregulation plays a major role in cancer development and aging. Telomeres are maintained by a group of specialized genes known as shelterin and shelterin-associated proteins. In breast cancer lines it has been shown that shelterin proteins are dysregulated thereby affecting the telomere stability and contributing to the neoplastic conversion of the mammary epithelial cells. Interestingly, the regulation of some of the shelterin genes is thought to be controlled epigenetically. Methods and Results: In this study, we set out to measure the effect of increased shelterin gene expression on telomere length in breast cancer cell line 21NT treated with 5-aza-2-deoxycytidine (5-aza-CdR) using known telomere length assays. We measured telomere lengths using: Telomere Restriction Fragment length (TRF), absolute quantitative-PCR and cytogenetic Interphase Quantitative Fluorescent in situ Hybridization (iQ-FISH). We found that non-cytotoxic levels of 5-aza-CdR affect telomere lengths by causing a significant and stable increase in telomere lengths of the breast cancer cell line. The increase in telomere lengths was consistently observed when various telomere length methods were used. Conclusions: Further investigation is required to understand the underlying mechanism involved, and the significance of telomere length elongation in relation to clinical outcome when epigenetic modifying drugs are utilized.

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.

DNA Methyltransferase Inhibitors Induce Cerebral Dopamine Neurotrophic Factor Expression in C6 Glioma Cells

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are involved in neuroprotection and mitigating endoplasmic reticulum (ER) stress in the brain and peripheral organs. In earlier work, an increase in histone acetylation, following treatment with an epigenetic modulator, valproic acid, was associated with induction of CDNF and MANF in cultured cells and rat brain. These findings prompted an investigation of the effects of DNA methyltransferase (DNMT) inhibitors, which can alter epigenetic function, on the expression of CDNF and MANF. Rat C6 glioma cells were treated with a micromolar range of DNMT inhibitors: 5-aza-2’-deoxycytidine (DAC or decitabine), 5-azacytidine (AZA) or zebularine (ZEB) for 24 h. Subsequently, qPCR analysis was used to examine the mRNA expression of DNMT1, ten-eleven translocation methylcytosine dioxygenase 2 (TET-2), CDNF and MANF. A significant dose-dependent decrease in DNMT1 mRNA levels, together with a significant increase in TET-2 expression, was observed following treatment with AZA or DAC. Importantly, DAC, AZA and ZEB caused a significant dose-dependent increase in CDNF mRNA levels. In contrast, MANF mRNA expression decreased following treatment with AZA, with no significant effects observed with DAC or ZEB. Western analysis revealed no significant changes in CDNF protein levels following treatment with DAC for 24 h. The significant increase in CDNF expression, following treatment with DNMT1 inhibitors, suggests that DNA methylation is involved in the regulation of this neurotrophic factor. Clarification of the epigenetic or other mechanisms underlying the regulation of CDNF may provide novel therapeutic approaches in neurodegenerative and ER stress-related disorders.

徐淮山羊 Sox2 基因启动子的克隆及其活性的初步分析

本研究旨在确定徐淮山羊Sox2基因启动子区域,找出该基因启动子的核心调控区,初步探讨Sox2基因的表达调控机制。根据GenBank已公布的绵羊Sox2基因的启动子序列,设计特异性PCR引物扩增Sox2基因的一系列启动子缺失片段,经酶切、测序及生物信息学分析,构建包含Sox2基因5′侧翼区一系列启动子缺失片段的pGL3-Sox2双荧光素酶表达载体,转染COS-7和GC1细胞,并进行5-aza-2′-deoxycytidine诱导,检测不同片段的启动子活性。结果表明:徐淮山羊Sox2基因5′侧翼区-1249—+49 bp区域的启动子活性最强(COS-7细胞),-1792—+49 bp区域活性最强(GC1细胞),-224—+49 bp区域为Sox2基因启动子基本活性区域。进一步研究发现,-484―-109 bp区域存在正调控元件,-755—-484 bp区域存在负调控元件。本实验通过构建包含Sox2基因启动子不同片段的重组报告基因载体并比较其转录活性,确定了Sox2基因启动子的核心区域。另外,5-aza-2′-deoxycytidine可以显著增强Sox2启动子的活性。为进一步研究Sox2基因的表达调控机制奠定了基础。

Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor

Background： DNA hypomethylation of long interspersed nuclear elements- 1 （LINEs- 1 ） occurs during carcinogenesis, whereas intbmaation addressing LINE-1 methylation in Wilms tumor （WT） is limited. The main purpose of our study was to quantity, LINE-1 methylation levels and evaluate their relationship with relative telomere length （TL） in WT. Methods： We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction （PCR） pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line. Significant changes were analyzed by t-tests. Results： LINE-1 methylation levels were significantly lower （P 〈 0.05） and relative TLs were sigmificantly shorter （P 〈 0.05） in WT compared with normal kidney. There was a significant positive relationship between LINE- 1 methylation and relative TL in WT （r = 0.671, P = 0.001 ）. LINE- 1 Methylation levels were significantly associated with global DNA methylation （r = 0.332, P 〈 0.01 ）. In addition, relative TL was shortened and LINE- 1 methylation was decreased in a WT cell line treated with the hypomethylating agent 5-aza-2＇-deoxycytidine compared with untreated WT cell line. Conclusion： These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT.