Dynamic changes in DNA methylation during multistep rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are typical genotoxic carcinogens that can induce tumors in a variety of human and rodent tissues. However, the epigenetic mechanisms underlying their tumorigenesis are unclear. In this study we used a MCA/DEN-induced multistep lung carcinogenesis rat model to study the evolution of alterations in DNA methylation. Rats were treated with a single dose of MCA and DEN in iodized oil by left intra-bronchial instillation. The animals were killed on days 15, 35, 55, 65 and 75 and samples of various pathological phases during carcinogenesis were obtained on these days. The status of global methylation was analyzed for each sample using a monoclonal antibody specific for 5-methycytosine (5-mC) and quantified by image analysis software. We found that the degree of global methylation was, in general, higher in basal cells compared to luminal cells of normal, precancerous and tumor tissues. The combined 5-mC scores of different types of tissues decreased gradually during the progression of carcinogenesis. We also used methylation-sensitive arbitrarily primed PCR (MS-AP-PCR) to screen a total of eight differentially methylated DNA fragments in both precancerous and tumor tissues isolated using laser capture microdissection (LCM), and observed that both unique hypomethylation and hypermethylation fragments coexist after exposure to genotoxic carcinogens. Remarkably, epigenetic alterations in p16 (CDKN2A), but not in p15 (CDKN2B), were observed, and these correlated with the presence of pathologic lung lesions and loss of p16 protein expression. Moreover, defective expression of p16 in methylated primary tumor cell lines recovered markedly after treated with 5-aza-2′-deoxycytidine (5-aza-dC). These results suggest that DNA methylation alterations are an early event in tumorigenesis and play an important role during MCA/DEN-induced multistep rat lung carcinogenesis.     

Analysis of aberrant methylation in DNA repair genes during malignant transformation of human bronchial epithelial cells induced by cadmium

Cadmium (Cd) and its compounds are well-known human carcinogens, but the mechanisms underlying the carcinogenesis are not entirely understood yet. Aberrant methylation was investigated in order to obtain insight into the DNA repair-related epigenetic mechanisms underlying CdCl(2)-induced malignant transformation of human bronchial epithelial cells (16HBE). Gene expression and DNA methylation were assessed in untreated control cells; 5th, 15th, and 35th passage of CdCl2-treated cells and tumorigenic cells (TCs) from nude mice by using high-performance liquid chromatography, real-time PCR, Western blot analysis, and methylation-specific PCR assay. During Cd-induced malignant transformation, global DNA methylation progressively increased and was associated with the overexpression of the DNA methyltransferase genes DNMT1 and DNMT3a but not DNMT3b. Expression of both the messenger RNA and proteins of the DNA repair genes (hMSH2, ERCC1, XRCC1, and hOGG1) progressively reduced and DNA damage increased with Cd-induced transformation. The promoter regions of hMSH2, ERCC1, XRCC1, and hOGG1 were heavily methylated in the 35th passage transformed cells and the TCs. The DNA demethylating agent 5-aza-2'-deoxycytidine could reverse the Cd-induced global DNA hypermethylation, DNMT hyperactivity, and the silencing of hMSH2, ERCC1, XRCC1, and hOGG1 in a time-dependent manner. The results indicate that DNMT1 and DNMT3a overexpression can result in global DNA hypermethylation and silencing of the hMSH2, ERCC1, XRCC1, and hOGG1 genes. They may partly explain the epigenetic mechanisms underlying the carcinogenesis due to Cd.     

DNMT1-mediated PTEN hypermethylation confers hepatic stellate cell activation and liver fibrogenesis in rats

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a negative regulator of this process. PTEN promoter hypermethylation is a major epigenetic silencing mechanism in tumors. The present study aimed to investigate whether PTEN promoter methylation was involved in HSC activation and liver fibrosis. Treatment of activated HSCs with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) decreased aberrant hypermethylation of the PTEN gene promoter and prevented the loss of PTEN expression that occurred during HSC activation. Silencing DNA methyltransferase 1 (DNMT1) gene also decreased the PTEN gene promoter methylation and upregulated the PTEN gene expression in activated HSC-T6 cells. In addition, knockdown of DNMT1 inhibited the activation of both ERK and AKT pathways in HSC-T6 cells. These results suggest that DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the activation of the PI3K/AKT and ERK pathways, resulting in HSC activation.     

荷卵巢癌裸鼠肿瘤E-cad启动子去甲基化的研究

目的：观察DNA甲基化转移酶（DNA methyltransferase,DNMT）抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-2'-deoxycytidine,5-Aza-CdR）联合组蛋白去乙酰化酶（histone deacetylase,HDAC）抑制剂苯丁酸钠（Sodium 4-Phenylbutyrate,SPB）对荷卵巢癌细胞系SKOV3裸鼠上皮性钙黏素（epithelial cadherin,E-cad）表达的影响。 方法：在已建立的卵巢癌裸鼠腹腔移植瘤模型上,分别应用或二者联合应用5-Aza-CdR、SPB,免疫组化法检测腹腔移植瘤中DNMT1、HDAC1以及E-cad的表达变化情况;检测各组移植瘤E-cad基因启动子区5’CpG岛甲基化状况及各组移植瘤E-cad mRNA和蛋白的表达变化情况。 结果：①在移植瘤组织中,DNMT1和HDAC1呈高表达,5-Aza-CdR能抑制DNMT1的表达,SPB能抑制HDAC1的表达,与对照组相比,差异显著（P<0.05）。②5-Aza-CdR能够诱导甲基化的E-cad基因去甲基化,从而mRNA及E-cad蛋白恢复表达或者表达增强,联合应用5-Aza-CdR和SPB可更大程度上诱导甲基化的E-cad基因去甲基化而恢复mRNA和E-cad蛋白的表达。 结论：5-Aza-CdR能够降低E-cad基因启动子区的甲基化,可恢复其表达;联合应用5-Aza-CdR和SPB可产生协同效应,显著增强甲基化的E-cad基因的表达,对卵巢癌的治疗有重要作用。     

Mithramycin A inhibits DNA methyltransferase and metastasis potential of lung cancer cells

Abnormal CpG island hypermethylation of multiple tumor-suppressor genes (TSGs) can lead to the initiation and progression of human cancer. The cytosine of the CpG island on the promoter region is methylated by 5'-cytosine-methyltransferases (DNMTs). Pharmacologic inhibitors of CpG island methylation provide a rational approach to reactivate the TSGs in tumor cells and to restore the critical cellular pathways in cancer cells. Mithramycin A (MMA) is known to be a GC- and CG-rich DNA-binding agent. We sought to determine whether MMA could inhibit CpG island methylation and DNMT expression in lung cancer cells. We found that MMA reduced the CpG island methylation of antimetastasis TSGs, including SLIT2 and TIMP-3 genes, and was associated with the prevention of metastasis. When highly metastatic CL1-5 lung cancer cells were treated with low doses (10 nmol/l) of MMA for 14 days, they reexpressed mRNA levels for these genes. MMA also inhibited the invasion phenotypes of CL1-5 cells as indicated by its inhibition of cancer cell migration using wound-healing and transwell assays. Molecular docking of MMA onto the DNMT1 catalytic domain revealed that MMA might interact with the catalytic pocket of DNMT1. Western blots showed that DNMT1 protein levels were depleted after MMA. These data support the idea that MMA has demethylation and antimetastasis effects on lung cancer cells. This mechanism might be mediated by the interaction of MMA and DNMT1, leading to the depletion of the DNMT1 protein and the reversal of the metastasis phenotype in lung cancer cells.     

Cyclophosphamide perturbs cytosine methylation in Jurkat-T cells through LSD1-mediated stabilization of DNMT1 protein

Aberrant cytosine methylation is known to be associated with cancer development. Here, we assessed how common cancer chemotherapeutic agents perturb cytosine methylation in Jurkat-T acute lymphoblastic leukemia cells. We tested six antitumor agents and found that cyclophosphamide induced the most pronounced increase in global DNA cytosine methylation after a 24-h treatment. Long-term treatment with cyclophosphamide led to a time-dependent increase in cytosine methylation level with up to 4 days of treatment, and the extent of cytosine methylation returned to normal level after 8 days. The trend of change in DNA methylation level paralleled that of the expression level of DNMT1 protein, whereas no significant increase in DNMT1 mRNA level was observed. Previous studies showed that the stability of endogenous DNMT1 protein is regulated by lysine methylation through histone lysine methyltransferase Set7 and lysine-specific demethylase 1 (LSD1), with the methylated DNMT1 being the target for proteasomal degradation. We observed that the elevated expression of DNMT1 protein at 4 days of treatment was correlated with the increased expression of LSD1 protein and with the decreased frequency of K142 methylation in DNMT1. Taken together, our results showed that cyclophosphamide perturbed temporarily global cytosine methylation in Jurkat-T cells via regulation of the lysine methylation level in DNMT1.     

Grape seed proanthocyanidins reactivate silenced tumor suppressor genes in human skin cancer cells by targeting epigenetic regulators

Grape seed proanthocyanidins (GSPs) have been shown to have anti-skin carcinogenic effects in in vitro and in vivo models. However, the precise epigenetic molecular mechanisms remain unexplored. This study was designed to investigate whether GSPs reactivate silenced tumor suppressor genes following epigenetic modifications in skin cancer cells. For this purpose, A431 and SCC13 human squamous cell carcinoma cell lines were used as in vitro models. The effects of GSPs on DNA methylation, histone modifications and tumor suppressor gene expressions were studied in these cell lines using enzyme activity assays, western blotting, dot-blot analysis and real-time polymerase chain reaction (RT-PCR). We found that treatment of A431 and SCC13 cells with GSPs decreased the levels of: (i) global DNA methylation, (ii) 5-methylcytosine, (iii) DNA methyltransferase (DNMT) activity and (iv) messenger RNA (mRNA) and protein levels of DNMT1, DNMT3a and DNMT3b in these cells. Similar effects were noted when these cancer cells were treated identically with 5-aza-2′-deoxycytidine, an inhibitor of DNA methylation. GSPs decreased histone deacetylase activity, increased levels of acetylated lysines 9 and 14 on histone H3 (H3-Lys 9 and 14) and acetylated lysines 5, 12 and 16 on histone H4, and reduced the levels of methylated H3-Lys 9. Further, GSP treatment resulted in re-expression of the mRNA and proteins of silenced tumor suppressor genes, RASSF1A, p16^INK4a and Cip1/p21. Together, this study provides a new insight into the epigenetic mechanisms of GSPs and may have significant implications for epigenetic therapy in the treatment/prevention of skin cancers in humans.     

Snail、E-cadherin 和N-cadherin 在非小细胞肺癌组织的表达及相关性研究

目的：探讨上皮型钙黏附素（E-cadherin）、神经型钙黏附素（N-cadherin）及Snail在非小细胞肺癌（NSCLC）中的表达及其临床意义。方法采用实时荧光定量PCR测定Snail、E-cadherin和N-cadherin基因在10对非小细胞肺癌及其癌旁正常肺组织中的表达情况,同时采用免疫组化法检测105例NSCLC以及41例癌旁组织中Snail、E-cadherin和N-cadherin蛋白的表达。结果与癌旁正常肺组织相比较,非小细胞肺癌组织中E-cadherin的表达显著减少,N-cadherin和Snail表达明显增加（P〈0.05）。NSCLC组织中,Snail的表达与N-cadherin的表达呈明显的正相关（P〈0.05,r=0.21）,与E-cadherin的表达呈显著负相关（P〈0.05,r=-0.39）,而N-cadherinl的表达与E-cadherin的表达也呈明显负相关（P〈0.05,r=-0.53）。结论非小细胞肺癌组织中, Snail、N-cadherin呈显著高表达,而E-cadherin呈显著低表达,三者可能通过相互作用共同参与NSCLC的发生和发展。     

Dust fall PM2.5-induced lung inflammation in rats is associated with hypermethylation of the IFN-γ gene promoter via the PI3K-Akt-DNMT3b pathway

Inflammation is one of the major adverse effects of fine particulate matter (PM_2.5) on the lung system; however, its mechanisms remain unclear. Rats were exposed to different concentrations of PM_2.5 to investigate the mechanism of short-term exposure-induced lung inflammation. The regulation of PI3K-Akt and DNA methyltransferase 3b (DNMT3b) was assessed by using a PI3K inhibitor and a DNA methyltransferase inhibitor. We found that PM_2.5 could decrease interferon-γ (IFN-γ) levels and increase interleukin 4 (IL-4), IL-5 and IL-13 levels in bronchoalveolar lavage fluid (BALF) to promote eosinophil infiltration and eventually lead to allergic pulmonary inflammation. Moreover, the CpG island methylation rate of the IFN-γ promoter and the protein expression of DNMT3b, PI3K and p-Akt were increased in lung tissues after PM_2.5 exposure. Both inhibitors reversed the CpG island hypermethylation of IFN-γ. In conclusion, in PM_2.5-induced lung injury, the activated PI3K-Akt pathway, via an increase in DNMT3b expression, is involved in CpG hypermethylation of the IFN-γ gene promoter.     

异硫氰酸苯已酯诱导骨髓瘤U266细胞P16基因去甲基化

目的研究异硫氰酸苯己酯(PHI)对骨髓瘤U266细胞株P16基因的去甲基化作用并探讨其作用机制。方法应用半定量逆转录-聚合酶链反应检测U266细胞经过PHI处理后DNA甲基转移酶DNMT1、DNMT3a和DNMT3b基因的mRNA的表达变化,用甲基化特异性聚合酶链反应检测PHI作用前后U266细胞株P16基因甲基化状态的变化。结果使用不同浓度的PHI处理U266细胞72h后,U266细胞表达DNMT1、DNMT3a、DNMT3bmRNA的水平明显降低并呈浓度依赖性。以不同浓度的PHI处理U266细胞10d后,P16基因的甲基化状态被逐渐逆转。结论 PHI可能通过抑制DNA甲基转移酶的表达水平诱导P16基因产生去甲基化,使失活的抑癌基因重新激活,诱导瘤细胞凋亡。     

SOCS1 hypermethylation mediated by DNMT1 is associated with lipopolysaccharide-induced inflammatory cytokines in macrophages

Macrophages activation which releases the pro-inflammatory cytokines is an essential event in the process of inflammation. SOCS1 has been shown to act as a negative regulator of cytokine signals and plays a key role in the suppression of tissue injury and inflammatory diseases. DNA methylation mediated by specific DNA methyltransferases1 (DNMT1) which contributes to the epigenetic silencing of multiple genes. SOCS1 promoter hypermethylation is by far the best categorized epigenetic change in tumors. Our study with a view to investigate whether the loss of SOCS1 due to SOCS1 promoter methylation was involved in the course of inflammatory cytokines released from lipopolysaccharide (LPS)-stimulated macrophages. Here, we found that treatment of LPS-induced RAW264.7 macrophage cells with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) reduced aberrant promoter hypermethylation of SOCS1 and prevented the loss of the expression of SOCS1 in macrophages which secret inflammatory cytokines. Knockdown of DNMT1 gene not only attenuated the SOCS1 gene promoter methylation but also up-regulated the expression of SOCS1 in activated RAW264.7 cells. Furthermore, silencing of DNMT1 prevented the activation of JAK2/STAT3 pathway in LPS-induced RAW264.7 cells. These studies demonstrated that DNMT1-mediated SOCS1 hypermethylation caused the loss of SOCS1 expression results in negative regulation of activation of the JAK2/STAT3 pathway, and enhanced the release of LPS-induced pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages.     

DNMT1通过下调SOX1表达参与宫颈癌生长和转移的机制研究

目的 探讨DNA甲基转移酶1（DNMT1）通过调节性别决定区Y-框1（SOX1）表达对宫颈癌（CC）生长和转 移的影响。方法 采用亚硫酸氢盐定量PCR测定CC组织和细胞及CC癌旁组织和正常宫颈细胞中SOX1甲基化水 平,Western blot检测DNMT1、SOX1蛋白表达。将HeLa229和SW756细胞分为DNMT1-NC组、DNMT1-siRNA组和空 白组,采用亚硫酸氢盐定量 PCR 测定 SOX1 甲基化比例,Western blot 检测 DNMT1、SOX1、c-Myc、Cyclin D1 及 β- catenin蛋白表达,细胞克隆和裸鼠成瘤实验测定细胞增殖,Transwell实验测定细胞迁移和侵袭。结果 CC组织较癌 旁组织DNMT1表达水平及SOX1甲基化比例增高,SOX1蛋白表达降低（P＜0.05）;宫颈癌HeLa229、ME-180、SiHa、 SW756细胞较宫颈上皮Ect1/E6E7细胞DNMT1表达水平及SOX1甲基化比例增高,SOX1蛋白表达降低（P＜0.05）。 与空白组、DNMT1-NC组比较,DNMT1-siRNA组HeLa229和SW756细胞体外克隆形成数、迁移和侵袭数量减少,裸 鼠内瘤体质量减轻,DNMT1、c-Myc、Cyclin D1、核/质β-catenin蛋白表达及SOX1甲基化比例降低,SOX1蛋白表达增 加（P＜0.05）。结论 DNMT1在CC组织和细胞中高表达,可能通过维持SOX1启动区高甲基化并抑制其蛋白表达, 促进CC生长和转移。     

DNA甲基转移酶 凋亡抑制蛋白在肺癌组织中的表达及意义

目的观察DNA甲基转移酶(DNMT)1、DNMT3β和凋亡抑制蛋白-2(IAP2)在肺癌组织中的表达及与肺癌的临床病理特征关系。方法采用组织微阵列和免疫组织化学技术检测DNMT1、DNMT3β在60例肺癌组织及30名健康人正常肺组织中的表达。结果肺癌组织中DNMT1、DNMT3β、IAP2表达的阳性率均高于正常对照组(P<0.01);3种蛋白的表达在性别、吸烟史、年龄、淋巴结转移、肿瘤分期之间差异无统计学意义(P>0.05);DNMT1、DNMT3β表达与IAP-2的表达明显相关(P<0.01)。结论 DNMT1、DNMT3β表达升高是肺癌形成、发展中的普遍事件,可作为诊断的有效指标,DNMT1与DNMT3β的表达可能存在共同调节机制;且可能参与了凋亡抑制蛋白IAP2调节作用。     

吸入氢气对脓毒症小鼠海马组织 DNA甲基化的影响

目的 评价吸入氢气对脓毒症小鼠海马组织的 DNA甲基化状态的影响。方法 54只健康雄性 C57BL/6小鼠采用随机数字表法分为 3组：假手术组（Sham组）、脓毒症组（Sepsis组）和氢气治疗组（Sepsis+H2组）,每组 18只。Sepsis组和 Sepsis+H2组采用盲肠结扎穿孔法（CLP）制备小鼠脓毒症模型,Sepsis+H2组小鼠于手术后 1 h和 6 h吸入用空气混合的 2%氢气 1 h,Sham组只开腹不进行盲肠结扎和穿孔。3组小鼠于假手术或 CLP后 1、3、7 d取小鼠海马组织,比色法测定全基因组 DNA甲基化水平;实时定量 PCR法检测 DNA甲基化转移酶（DNMTs,包括 DNMT1、DNMT3a和 DNMT3b）的 mRNA水平;Western blot法检测 DNMT1、DNMT3a和 DNMT3b蛋白表达水平。结果 与 Sham组比较,Sepsis组小鼠在建模后 1、3、7 d海马组织全基因组甲基化水平明显下降（P＜0.05）,DNMT1和 DNMT3a的 mRNA和蛋白表达水平升高,DNMT3b的 mRNA和蛋白表达水平降低（P＜0.05）;与 Sepsis组比较,Sepsis+H2组全基因组甲基化水平升高（P＜0.05）,DNMT1和 DNMT3a的 mRNA和蛋白表达水平下降,DNMT3b的 mRNA和蛋白表达水平升高（P＜0.05）。结论 吸入氢气可纠正脓毒症小鼠海马组织的 DNA甲基化紊乱状态,改善 DNA甲基化紊乱状态是氢气治疗脓毒症相关性脑病的重要机制之一。     

Heart failure and angiotensin II modulate atrial Pitx2c promotor methylation

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Effect of berberine against cerebral ischemia and reperfusion involving in the methylation of PPARγ promoter

Cerebral ischemia has higher incidence and causes irreversible damage to people. As a traditional drug for anti-inflammation, berberine (BBR) has recently been reported to have protective effect against cerebral ischemia. However, the mechanism has not been explored thoroughly. By employing in vivo and in vitro models for cerebral ischemia and reperfusion, we studied the mechanism of BBR against the ischemia-reperfusion. We found that BBR regulated the expression of peroxisome proliferator-activated receptor (PPARγ) in a specific way upon ischemia-reperfusion injury. BBR enhanced the PPARγ expression during cerebral ischemia-reperfusion. By inhibiting PPARγ activity uisng GW9662, a PPARγ inhibitor, we confirmed that BBR protected the mouse brain against the ischemia in a PPARγ-dependent mechanism. In addition, we found that BBR reduced the overall global methylation, declined the expressions of DNMT1 (DNA methyltransferases 1) and DNMT3a (DNA methyltransferases 3a) in the ischemia-reperfusion and reduced the methylation of PPARγ promoter region. Therefore, our data suggested that PPARγ was one of major targets of BBR, and such BBR-induced PPARγ expression during cerebral ischemia and reperfusion might be correlated to the reduced methylation of PPARγ promoter.