XRCC1 downregulated through promoter hypermethylation is involved in human gastric carcinogenesis

OBJECTIVE: To analyze the expression and aberrant methylation of X-ray repair cross-complementing gene 1 (XRCC1) in gastric carcinogenesis, and identify the molecular mechanism of gastric carcinogenesis. METHODS: The method based on methyl binding domain protein (MBD) immuno-precipitation and promoter microarray was employed to screen the gastric cancer-related methylation-sensitive gene. An immunohistochemistry assay was applied to detect the protein expression of XRCC1 in the multistep progression of gastric carcinogenesis. The mRNA expression of XRCC1 was determined by real-time PCR in tumor tissues and their corresponding non-tumorous tissues. The methylation status and Arg194Trp and Arg399Gln polymorphisms of XRCC1 in gastric cancer and gastritis tissues were analyzed by methylation-specific PCR, bisulfite genomic sequencing and direct DNA sequencing, respectively. RESULTS: Promoter microarray screening and identification suggested that XRCC1 was a methylation-sensitive gene. Immunochemistry results showed that XRCC1 protein expression gradually decreased with progression of gastric mucosal lesions (P < 0.05). The positive rate of XRCC1 in patients with well/moderately differentiated gastric cancer was significantly higher than patients with poorly differentiated gastric cancer (P < 0.05). The mRNA expression of XRCC1 in gastric cancer tissues was significantly lower than that in the non-tumorous tissues (P < 0.05). Meanwhile, XRCC1 methylation in gastric cancer tissues was more frequent than that in the gastritis tissues (P < 0.05), and the downregulation of XRCC1 expression was relevant to methylation (P < 0.05). CONCLUSION: The expression of XRCC1 is downregulated in gastric carcinogenesis, and promoter hypermethylation may be one of the mechanisms contributing to its downregulation.     

Impact of MTHFR gene C677T polymorphism on Bcl-2 gene methylation and protein expression in colorectal cancer

Abstract

Objective. To investigate the impact of MTHFR C677T polymorphism on Bcl-2 gene promoter CpG island (CGI) methylation and Bcl-2 protein expression. Material and methods. MTHFR polymorphisms of 86 sporadic colorectal cancer (CRC) patients and 100 healthy volunteers were analyzed by PCR-based restriction fragment length polymorphism, and Bcl-2 promoter CGI methylation in 86 CRC tissues and 86 paired nonneoplastic adjacent tissues was determined by methylation-specific PCR. Bcl-2 oncoprotein expression in 70 CRC tissues and paired nonneoplastic adjacent tissues was detected by immunohistochemistry. Results. The frequency of MTHFR 677 T allele and combined variant genotypes (677CT + TT) in CRC patients was significantly higher than that in healthy controls (p = 0.023 and p = 0.035, respectively), and there is a significant association between 677TT or 677(CT + TT) genotypes and CRC (OR = 2.534, p = 0.045 and OR = 1.888, p = 0.035, respectively). The frequency of methylated Bcl-2 promoter CGI in tumor tissues was significantly lower than that in nonneoplastic adjacent tissues (p = 0.014). The frequency of methylated Bcl-2 promoter CGI in CRC tissues of the individuals with CC genotype was significantly higher than that of those with CT/TT genotypes (p = 0.018), there was significant distribution difference of C and T alleles between individuals with methylated and unmethylated Bcl-2 promoter CGI in colorectal cancer tissues (p = 0.023). Bcl-2 promoter hypomethylation was significantly correlated with Bcl-2 oncoprotein expression in colorectal cancer tissues (r = 0.558, p < 0.001). Conclusion. Bcl-2 promoter is hypomethylated in colorectal cancer tissue, and there is a significant correlation between MTHFR 677 TT or CT/TT genotypes and CRC or Bcl-2 promoter CGI methylation/oncoprotein expression in CRC.     

KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients

AIM:To examine the effect of aberrant methylation of the KISS1 promoter on the development of colorectal cancer(CRC)and to investigate reversing aberrant methylation of the KISS1 promoter as a potential therapeutic target.METHODS:KISS1 promoter methylation,mRNA expression and protein expression were detected by methylation-specific polymerase chain reaction(PCR),real-time quantitative PCR and Western blotting,respectively,in 126 CRC tissues and 142 normal colorectal tissues.Human CRC cells with KISS1 promoter hypermethylation and poor KISS1 expression were treated in vitro with 5-aza-2’-deoxycytidine(5-Aza-CdR).After treatment,KISS1 promoter methylation,KISS1 mRNA and protein expression and cell migration and invasion were evaluated.RESULTS:Hypermethylation of KISS1 occurred frequently in CRC samples(83.1%,105/126),but was infrequent in normal colorectal tissues(6.34%,9/142).Moreover,KISS1 methylation was associated with tumor differentiation,the depth of invasion,lymph node metastasis and distant metastasis(P<0.001).KISS1methylation was also associated with low KISS1 expression(P<0.001).Furthermore,we observed re-expression of the KISS1 gene and decreased cell migration after 5-Aza-CdR treatment in a CRC cell line.CONCLUSION:These data suggest that KISS1 is down-regulated in cancer tissues via promoter hypermethylation and therefore may represent a candidate target for treating metastatic CRC.     

5-aza—dC和丁酸钠对人胃癌细胞株MKN28细胞周期、凋亡以及抑癌基因表达的影响

背景：表遗传修饰的主要方式DNA甲基化和组蛋白乙酰化是胃癌发生机制研究中的热点内容。目的：探讨表遗传学调控对人胃癌细胞株MKN28细胞周期、凋广以及抑癌基因Runx3、p21^WAF1表达的影响。方法：培养人胃癌细胞株MKN28,并分为5-氮-2’-脱氧胞苷（5-aza—dC）组、丁酸钠组、5-aza—dC＋丁酸钠组和对照组。以Annexin V—FITC／PI双染法检测细胞周期和凋亡,以逆转录聚合艏链反应（RT—PCR）检测Runx3、p21^WAF1 mRNA表达,以甲基化特异性PCR（MSP）检测Runx3基因启动于区甲基化状态。结果：与对照组相比,5-aza—dC对细胞周期无明显影响,丁酸钠可使细胞周期阻滞于G0／G1期（P〈0．05）;5-aza—dC组和丁酸钠组细胞凋亡率均显著增高（8．3％±1．3％、20．8％±2．4％对2．0％±0．5％,P〈0．05）5-aza—dC可诱导Runx3mRNA重新表达（P〈0．05）,但对p21^WAF1 mRNA表达无影响。丁酸钠可增强p21^WAF1 mRNA表达（P〈0．05）,但不能诱导Runx3 mRNA表达。联合5-aza—dC和丁酸钠可显著绣导细胞凋亡和增强抑癌基因Runx3、p21^WAF1Ⅲ mRNA表达（P〈0．05）．5-azgt—dC十预后,Runx3基因启动子区呈去甲基化状态：结论：去甲基化制剂5-aza-dC或组生白去乙酰化酶抑制剂丁酸钠通过重新表达Runx3或增强p21^WAF1表达而诱导胃癌MKN28细胞凋亡,从而发挥抗肿瘤的作用。     

丝裂原激活蛋白激酶阻断剂与DNA甲基化酶抑制剂对人结肠癌细胞的协同影响

目的研究细胞外信号渊节激酶-丝裂原激活蛋白激酶途径（ERKMAPK）与DNA甲基化问的关系及对结肠癌细胞生物学行为的协同影响。方法培养人结肠癌细胞SW1116，分别以PBS、二甲基亚砜（DMSO）为对照组，PD 9805950μmol／L、5氮脱氧胞苷（5-aza—dC）5μmol／L、PD9805950μmol／L＋5-aza-dC5μmol／L进行药物干预．以定量RT-PCR检测DNA甲基化酶（Dnmt）1、3a和3b基因转录水平；流式细胞仪分析细胞周期；MTT测定细胞活力；光学显微镜下观察细胞形态学变化。结果ERK—MAPK途径阻断剂PD98059下调Dnmt 1和Dnmt 3b．Dnmt抑制剂5-aza-dC下调Dnmt 1、Dnmt 3a和Dnmt 3b，且5-aza-dC联合PD98059对Dnmt1及Dnmt 3a mRNA的表达下调更为显著。5-azo-dC明显降低G0／G1期细胞百分比（P〈0．05），G2／M期细胞百分比明显增加（P〈0．05）；PD98059使G0／G1期细胞百分比降低（P（0．05）．G2／M期增加（P〈0．05）。PD98059明显抑制细胞生长。PD98059促进细胞分化，呈上皮样改变，细胞变狭长，胞质减少，细胞排列开始出现相对整齐；5-azm-dC干预组细胞大小不一，出现较多多倍体细胞（多个核分裂相）。结论ERK-MAPK途径阻断剂及Dnmt抑制剂均能抑制结肠癌SW1116细胞分裂、增殖，并诱导细胞分化；两者有协同作用；ERK—MAPK信号转导途径能调控DNA甲基化水平。     

Novel methylation gene panel in adjacent normal tissues predicts poor prognosis of colorectal cancer in Taiwan

BACKGROUND It is evident that current clinical criteria are suboptimal to accurately estimate patient prognosis.Studies have identified epigenetic aberrant changes as novel prognostic factors for colorectal cancer(CRC).AIM To estimate whether a methylation gene panel in different clinical stages can reflect a different prognosis.METHODS We enrolled 120 CRC patients from Tri-Service General Hospital in Taiwan and used the candidate gene approach to select six genes involved in carcinogenesis pathways.Patients were divided into two groups based on the methylation status of the six evaluated genes,namely,the<3 aberrancy group and≥3 aberrancy group.Various tumor stages were divided into two subgroups(local and advanced stages)on the basis of the pathological type of the following tissues:Tumor and adjacent normal tissues(matched normal).We assessed DNA methylation in tumors and adjacent normal tissues from CRC patients and analyzed the association between DNA methylation with different cancer stages and the prognostic outcome including time to progression(TTP)and overall survival.RESULTS We observed a significantly increasing trend of hazard ratio as the number of hypermethylated genes increased both in normal tissue and tumor tissue.The 5-year TTP survival curves showed a significant difference between the≥3 aberrancy group and the<3 aberrancy group.Compared with the<3 aberrancy group,a significantly shorter TTP was observed in the≥3 aberrancy group.We further analyzed the interaction between CRC prognosis and different cancer stages(local and advanced)according to the methylation status of the selected genes in both types of tissues.There was a significantly shorter 5-year TTP for tumors at advanced stages with the promoter methylation status of selected genes than for those with local stages.We found an interaction between cancer stages and the promoter methylation status of selected genes in both types of tissues.CONCLUSION Our data provide a significant association between the methylation markers in normal tissues with advanced stage and prognosis of CRC.We recommend using these novel markers to assist in clinical decision-making.     

Hypermethylation of <Emphasis Type="Italic">SFRP2</Emphasis> as a Potential Marker for Stool-Based Detection of Colorectal Cancer and Precancerous Lesions

DNA methylation is a key mechanism of colorectal carcinogenesis. Analysis of aberrantly methylation in stool DNA might provide a novel strategy for noninvasive detection of colorectal cancer (CRC). To explore the feasibility of this approach, we have assessed the methylation status of secreted frizzled-related protein gene 2 (SFRP2) in stool samples from patients with CRC with respect to a series of healthy individuals and patients with benign colorectal diseases, using methylation-specific polymerase chain reaction. Methylated SFRP2 occurs in 94.2%, 52.4%, 37.5%, and 16.7% of patients with CRC, adenomas, hyperplstic polyps, and ulcerative colitis, respectively. Of the 24 normal individuals, only 1 revealed methylated DNA. The pilot study revealed that aberrant methylated SFRP2 could be detected frequently in stools from patients with CRC and precancerous lesions. Methylation testing of fecal DNA may be a simple, promising, and noninvasive screening tool for colorectal neoplasia.     

Genetic variation in the promoter of DNMT3B is associated with the risk of colorectal cancer

Purpose  

DNA methyltransferase-3B (DNMT3B) plays an important role in the generation of aberrant methylation in carcinogenesis. Polymorphisms of the DNMT3B gene may influence DNMT3B enzyme activity on DNA methylation, thereby modulating the susceptibility to colorectal cancer (CRC).     

A genome-wide search identifies epigenetic silencing of somatostatin, tachykinin-1, and 5 other genes in colon cancer

BACKGROUND & AIMS: Gene silencing via promoter hypermethylation is a central event in the pathogenesis of cancers. To identify novel methylation targets in colon cancer, we conducted a genome-wide, microarray-based, in silico, and epigenetic search. METHODS: Complementary DNA microarray experiments were first performed to identify genes down-regulated in primary colon cancers and up-regulated in colon cancer cell lines after global DNA demethylation by 5-aza-2'-deoxycitidine. Candidate methylation targets were then identified by combining these microarray data with in silico genetic and functional searches. Candidate genes recognized by these searches were further investigated for promoter hypermethylation in colon cancer using methylation-specific polymerase chain reaction. RESULTS: We identified 51 novel and 3 known candidate methylation targets. Subsequent epigenetic analysis revealed that primary colon cancers demonstrated frequent methylation of somatostatin (SST, 30 of 34 cases, 88%) and the substance P precursor gene tachykinin-1 (TAC1; 16 of 34 cases, 47%). TAC1 methylation intensity was significantly higher in Dukes A/B than in Dukes C/D cancers (P = .01). SST methylation intensity was significantly higher in low-level microsatellite instability (MSI-L) than in non-MSI-L cancers (P = .02). Methylation was associated with messenger RNA down-regulation for both SST and TAC1. Furthermore, we isolated 5 additional novel promoter methylation targets: NELL1, AKAP12, caveolin-1, endoglin, and MAL. CONCLUSIONS: These data strongly suggest that SST and TAC1 are involved in colon carcinogenesis. Further studies are now indicated to elucidate mechanisms underlying their involvement in colon cancer and their values as clinical biomarkers. NELL1, AKAP12, caveolin-1, endoglin, and MAL are also promising tumor suppressor gene candidates deserving of further study.     

Promoter methylation profile in gallbladder cancer

Background Methylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time. Methods Twenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained. Results All cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied. Conclusions The high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.     

Field Cancerization in Sporadic Colon Cancer

Background/AimsAberrant DNA methylation has a specific role in field cancerization. Certain molecular markers, including secreted frizzled-related protein 2 (SFRP2), tissue factor pathway inhibitor 2 (TFPI2), N-Myc downstream-regulated gene 4 (NDRG4) and bone morphogenic protein 3 (BMP3), have previously been shown to be hypermethylated in colorectal cancer (CRC). We aim to examine field cancerization in CRC based on the presence of aberrant DNA methylation in normal-appearing tissue from CRC patients.MethodsWe investigated promoter methylation in 34 CRC patients and five individuals with normal colonoscopy results. CRC patients were divided into three tissue groups: tumor tissue, adjacent and nonadjacent normal-appearing tissue. The methylation status (positive: methylation level >20%) of SFRP2, TFPI2, NDRG4, and BMP3 promoters was investigated using methylation-specific PCR.ResultsThe methylation frequencies of the SFRP2, TFPI2, NDRG4 and BMP3 promoters in tumor/adjacent/nonadjacent normal-appearing tissue were 79.4%/63.0%/70.4%, 82.4%/53.6%/60.7%, 76.5%/61.5%/69.2%, 41.2%/35.7%/50.0%, respectively. The methylation levels of the SFRP,TFPI2, NDRG4 and BMP3 promoters in tumor tissues were significantly higher than those in normal-appearing tissue (SFRP2, p=0.013; TFPI2, p<0.001; NDRG4, p=0.003; BMP3, p=0.001). No significant correlation was observed between the methylation levels of the promoters and the clinicopathological variables.ConclusionsThe field effect is present in CRC and affects both the adjacent and nonadjacent normal-appearing mucosa.     

Usefulness of plasma epigenetic changes of five major genes involved in the pathogenesis of colorectal cancer

Purpose

The purpose of present study was to investigate the methylation status of the promoter region in five genes (mothers against decapentaplegic homolog 4, fragile histidine triad protein, death-associated protein kinase 1, adenomatous polyposis coli (APC), and E-cadherin), which are known to be involved in the pathogenesis of colorectal cancer (CRC) and its clinicopathological significance.

Methods

The study subjects were 60 CRC patients, 40 patients with adenomatous colorectal polyp and 60 healthy control individuals. We further enrolled a total of 16 patients (two patients with Crohn’s disease, two patients with ulcerative colitis, one patient with serrated adenoma, and 11 patients with colorectal cancer). The methylation states of the five genes were determined in peripheral blood plasma using methylation-specific polymerase chain reaction single-strand conformation polymorphism analysis.

Results

This study showed the most sensitive epigenetic markers, E-cadherin (60 %), followed by APC (57 %), for detecting CRC. E-cadherin and APC had similar specificities and amplified 84 and 86 %, respectively, of CRC patients compared to non-CRC patients. Additionally, APC was the only marker to be significantly increased (OR?=?6.67, 95 % CI?=?1.19–23.4, P?=?0.045) and the most sensitive (57 %) and specific (89 %) marker in stage I CRC. Though we have not examined the paired cancer tissues and plasma, there was relatively high concordant rate (60–80 %) in our limited number of colorectal cancer patients.

Conclusions

Five genes, promoter methylation, in plasma were statistically significant risk factors in CRC patients. In this study, E-cad and APC genes may be particularly useful epigenetic biomarkers in plasma for the detection of CRC. Additionally, APC may able to identify early potential CRC.     

胰腺癌细胞株HOXA7基因表达及其启动子区甲基化状态

目的 检测HOXA7 mRNA在胰腺癌细胞株中的表达及其启动子区的甲基化状态,探讨两者的相关性.方法 采用RT-PCR法检测人胰腺癌细胞株BxPC3、CFPAC1、PANC1和SW1990细胞的HOXA7 mRNA的表达水平.采用重亚硫酸盐测序PCR(bisulfite sequencing PCR,BSP)和结合重亚硫酸盐的限制性内切酶法(combined bisulfite restriction analysis,COBRA)检测启动子区域甲基化状态.应用去甲基化药物5-氮杂-2'-脱氧胞苷(5-aza-2-deoxycytidine,5-aza-dC)处理各细胞株,检测处理前后细胞HOXA7 mRNA表达和甲基化状态的变化.结果 胰腺癌细胞株BxPC3、CFPAC1和SW1990细胞均表达HOXA7 mRNA,而PANC1细胞不表达HOXA7 mRNA.CFPAC1、BxPC3、PANC1和SW1990中HOXA7启动子甲基化率分别为93.16%、90.65%、90.09%、52.30%,SW1990细胞的HOXA7甲基化率较其他三株细胞均明显降低(P值均＜0.01).经5-aza-dC处理后,PANC1细胞的HOXA7 mRNA重新表达,BxPC3的HOXA7 mRNA表达增强;而CFPAC1和SW1990细胞的HOXA7 mRNA在5-aza-dC处理前后无明显变化.结论 胰腺癌细胞株BxPC3和PANC1细胞的HOXA7mRNA表达与启动子区甲基化状态密切相关,而CFPAC 1和SW1990细胞两者无明显相关性.