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.     

Lack of association between genetic polymorphisms in cytokine genes and disease expression in patients with hereditary non-polyposis colorectal cancer

Objective. Hereditary non-polyposis colorectal cancer (HNPCC) is caused by germline mutations in DNA mismatch repair (MMR) genes and is characterized by familial aggregations of early-onset epithelial cancers. Inflammatory cells produce an attractive environment for tumour growth since reactive oxygen and nitrogen species generated by inflammatory cytokine induction can cause damage to DNA and proteins. In this study the objective was to investigate single nucleotide polymorphisms (SNPs) in cytokine genes to assess their impact on disease expression in individuals diagnosed with HNPCC. Material and methods. DNA samples from 220 participants diagnosed with HNPCC were genotyped for SNPs in IL-6, IL-1β, TNF-α, IFN-γ, IL-10, IL-4 and IL-1RN. The association between the polymorphisms and disease characteristics, i.e. affected or unaffected with colorectal cancer (CRC) and age of diagnosis of CRC, was tested with the Pearson χ² test and by Kaplan-Meier survival analysis. Results. There was no significant difference between CRC patients and unaffected MMR gene mutation carriers for any of the SNPs studied and the Kaplan-Meier survival analysis showed no significant difference between age of diagnosis of CRC and genotype. Conclusions. The SNPs selected for this study do not appear to modify disease expression in HNPCC. Given the complexity of the inflammatory response, the limited number of SNPs studied does not rule out the notion that other cytokine polymorphisms could act as disease modifiers of disease expression in HNPCC.     

Association of TET1 expression with colorectal cancer progression

Objective: The ten–eleven translocation (TET) proteins, as methylcytosine dioxygenases, catalyze 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). The altered expression of TET1 disrupts the balance between DNA methylation and demethylation. This alteration has been reported to be associated with carcinogenesis in various malignancies. The aim of the present study was to investigate changes in expression and the role of TET1 in colorectal cancer (CRC).

Material and methods: A total of 109 CRC patients who underwent radical surgical colon resection were enrolled. The QuantiGene Plex Assay was used to detect the expression of TET1 in CRC tissues and matching adjacent normal tissues. We analyzed the associations between TET1 expression levels and various clinicopathologic features of CRC. TET1 overexpression and depletion cells were constructed to investigate its biological role in CRC.

Results: Compared to normal tissues, the expression level of TET1 in CRC was significantly lower. The ratio of TET1 in CRC tissues to that in adjacent normal tissues (C/N-TET1) was an independent overall survival predictive factor. Moreover, in vitro studies showed that TET1 could inhibit cell growth and promote cell metastasis and invasion.

Conclusions: These findings indicated that TET1 played a multifaceted role in the pathogenesis of CRC, and thereby resulting in multiple effects on tumor progression.     

Genome-wide DNA methylation profiling in nonalcoholic fatty liver reveals predictive aberrant methylation in PRKCE and SEC14L3 promoters

BackgroundOptimal non-invasive biomarkers for diagnosis and treatment of nonalcoholic fatty liver disease (NAFLD) remain to be identified.AimsTo identify potential DNA methylation biomarkers for NAFLD.MethodsGenome-wide DNA methylation profiling was performed to identify differentially methylated CpG sites in peripheral blood leukocytes. Differentially methylated regions were validated using the MassCLEAVE assay. The expression levels of candidate genes were explored by Gene Expression Omnibus database.ResultsThe hypomethylation of PRKCE CpG 4.5 and CpG 18.19 was associated with nonalcoholic fatty liver (NAFL), the odds ratio (OR) and 95% confidence interval (CI) were 0.129 (0.026–0.639) and 0.231 (0.069–0.768). The methylation level of CpG 1.2 and average methylation level of SEC14L3 were correlated with NAFL, with OR (95% CI) being 0.283 (0.093–0.865) and 0.264 (0.087–0.799). PRKCE CpG 4.5 and cg17802464 of SEC14L3 were correlated with body mass index, waist circumference, total triglyceride, high-density lipoprotein cholesterol, alanine aminotransferase and aspartate aminotransferase. All selected datasets showed high expression levels of PRKCE and SEC14L3 in patients with NAFLD.ConclusionsOur findings suggest that the hypomethylation of PRKCE and SEC14L3 promoters represent attractive biomarkers for NAFLD. Further studies are warranted to validate these biomarkers as molecular tools for diagnosis of NAFLD and therapeutic targets.     

Demethylation-mediated upregulation of melanoma-associated antigen-A11 correlates with malignant progression of esophageal squamous cell carcinoma

BackgroundThe expression and methylation status of oncogenes are closely related to the onset and progression of cancer.AimsTo explore the role and methylation status of melanoma-associated antigen-A11 in the pathogenesis of esophageal squamous cell carcinoma.Methods116 esophageal squamous cell carcinoma patients with tumor tissues and corresponding adjacent normal tissues were obtained. The expression level and methylation status of melanoma-associated antigen-A11 in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues were determined respectively.ResultsSignificant up-regulation of melanoma-associated antigen-A11 was detected in esophageal cancer cell lines and esophageal squamous cell carcinoma tissues. Up-regulation of melanoma-associated antigen-A11 contributed to proliferation and invasion in cancer cells. Hypomethylation of the CpG site was associated with pathological differentiation, clinical stage, tumor size, lymph node metastasis and distant metastasis. Esophageal squamous cell carcinoma patients in stage III and IV, with high expression of melanoma-associated antigen-A11 or hypomethylation of the CpG site within the promoter demonstrated poor survival.ConclusionMelanoma-associated antigen-A11 is up-regulated in esophageal squamous cell carcinoma at least partly by hypomethylation of the CpG site within the promoter and this hypomethylation may affect the prognosis of esophageal squamous cell carcinoma patients.     

High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80-100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.     

ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS

Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.     

The risk of developing a mismatch repair deficient colorectal cancer after undergoing cholecystectomy

Objectives: Mismatch repair deficient (dMMR) colorectal cancer (CRC) is caused by inactivation of the MMR DNA repair system, most commonly via epigenetic inactivation of the MLH1 gene, and these tumors occur most frequently in the right colon. The objective was to determine whether cholecystectomy (CCY) increases the risk of a dMMR CRC by comparing CCY incidence in patients with dMMR CRC and proficient MMR (pMMR) CRC to unaffected controls.

Materials and methods: All patients diagnosed with CRC in Iceland from 2000 to 2009 (n?=?1171) were included. They had previously been screened for dMMR by immunohistochemistry (n?=?129 were dMMR). Unaffected age- and sex-matched controls (n?=?17,460) were obtained from large Icelandic cohort studies. Subjects were cross-referenced with all pathology databases in Iceland to establish who had undergone CCY. Odds ratios were calculated using unconditional logistic regression.

Results: Eighteen (13.7%) dMMR CRC cases and 90 (8.7%) pMMR CRC cases had undergone CCY compared to 1532 (8.8%) controls. CCY-related odds ratios (OR) were 1.06 (95% CI 0.90–1.26, p?=?.577) for all CRC, 1.16 (95% CI 0.66–2.05 p?=?.602) for dMMR CRCand 1.04 (95% CI 0.83–1.29, p?=?.744) for pMMR CRC. Furthermore, OR for dMMR CRC was 0.51 (95% CI 0.16–1.67, p?=?.266), 2.04 (95% CI 0.92–4.50, p?=?.080) and 1.08 (95% CI 0.40–2.89, p?=?.875)?<10 years, 10–20 years and?>20 years after a CCY, respectively.

Conclusions: There was no evidence of increased risk of developing dMMR CRC after CCY although a borderline significantly increased 2-fold risk was observed 10–20 years after CCY. Larger studies are warranted to examine this further.     

Epigenetic silencing of LRRC3B in colorectal cancer

Objective. Tumor suppressor gene silencing via promoter hypermethylation is an important event in the pathogenesis of colorectal cancer (CRC). Some aberrant DNA hypermethylation has high tumor specificity, so it may contribute to early diagnosis of CRC. The objective of this study was to establish novel therapeutic and diagnostic strategies against CRC by identifying the novel methylation-related genes. Material andmethods. Two microarray-based approaches were used to identify novel methylation-related genes in CRC. We identified methylation-sensitive genes in colon cancer cell line SW1116 by comparing differential expression genes after treatment with the methylation inhibiting drug, 5-aza-2'-deoxycitidine (5-aza-dC) using gene expression microarray. Promoter microarray analysis was performed to identify cancer-specific, methylation-related genes in two patients with CRC. Gene promoter methylation was identified by methylation-specific polymerase chain reaction (PCR) (MSP) in primary CRC. Gene expression level was assessed using real-time PCR analysis. Results. By using gene expression microarray, up-regulation of 253 genes was detected in the CRC cell line, SW1116, after treatment with 5-aza-dC. Of the 253 genes identified by gene expression microarray analysis, LRRC3B (leucine-rich repeat containing 3B) was isolated as a potential methylation-specific gene by promoter microarray analysis. MSP analysis showed frequent methylation of LRRC3B in primary CRC (24/31 cases, 77%). In addition, the LRRC3B methylation intensity was significantly higher in cancer tissues than in the corresponding non-cancerous tissues. Decreased LRRC3B expression (17/31, 55%) was observed in the cancer tissues by real-time PCR. Conclusions.LRRC3B may be a novel methylation-sensitive tumor suppressor gene in CRC. LRRC3B methylation has significant tumor specificity and may be a biomarker of CRC.     

Electrochemotherapy for Colorectal Cancer with Commonly Used Chemotherapeutic Agents in a Mouse Model

We examined here the usefulness of electrochemotherapy against colorectal cancer (CRC) using a mouse model. Electropermeabilization profoundly increased the sensitivity of murine CRC, Colon 26, and MC38 cells to bleomycin (BLM) but not to 5-fluorouracil (5-FU) or to cisplatin (CDDP) in vitro. In vivo experiments revealed that electrochemotherapy with 5-FU, CDDP, or BLM was much more effective against CRC compared with the treatment of the drug alone. Electrochemotherapy with BLM or CDDP exhibited profound antitumor effects on subcutaneously established CRC in mice, and complete tumor regression was observed in five and four of eight animals, respectively. Electrochemotherapy with 5-FU also had an impact on CRC development, and complete cure was observed in one of eight animals. Subsequent analyses revealed that electropermeabilization significantly increased intratumoral amounts of BLM and CDDP but not 5-FU. These results indicate that electrochemotherapy may be a promising treatment modality against CRC.     

Altered distribution of β-catenin and prognostic roles in colorectal carcinogenesis

Objective. Although β-catenin cytoplasmic stabilization and nuclear translocation play a key role in initiation of colorectal cancer (CRC), the mechanisms are far from clear. The aim of this study was to investigate the relation of expressions of cyclooxygenase (COX)-2 and E-cadherin, and the β-catenin gene exon 3 mutation to the altered distribution of β-catenin, and their roles in CRC progression and prognosis. Material and methods. Expressions of β-catenin, COX-2 and E-cadherin in 96 tissue specimens were detected by immunohistochemistry, and mutation of β-catenin gene exon 3 was screened by polymerase chain reaction (PCR) and denaturing high-performance liquid chromatography (DHPLC). Results. Cytoplasmic/nuclear expression of β-catenin and reduced membranous expression of E-cadherin were associated, respectively, with the earlier and later stages of sequential colorectal carcinogenesis (p<0.05). The altered distribution of β-catenin was significantly associated with both high Dukes’ stages and poor differentiation of CRC (p<0.05). It also had a parallel relationship with COX-2 overexpression (p<0.05, Spearman's rank analysis), but not with reduced E-cadherin expression. Kaplan-Meier analysis showed a significantly worse survival rate for CRC patients with altered expression of β-catenin (p<0.05, log-rank test). Nevertheless, we failed to find any exon 3 mutation of β-catenin gene in all 60 cases of CRC. Conclusions. Altered distribution of β-catenin occurs in the early stage of colorectal carcinogenesis and has a parallel relationship with COX-2 overexpression. It may serve as a potential marker for the progression and prognosis of CRC. The exon 3 mutation did not appear contributive to the abnormal expression of β-catenin in CRCs in a Chinese population.     

Inhibition of autophagy aggravates DNA damage response and gastric tumorigenesis via Rad51 ubiquitination in response to H. pylori infection

ABSTRACT

Helicobacter pylori (H. pylori) infection is the strongest known risk factor for the development of gastric cancer. DNA damage response (DDR) and autophagy play key roles in tumorigenic transformation. However, it remains unclear how H. pylori modulate DDR and autophagy in gastric carcinogenesis. Here we report that H. pylori infection promotes DNA damage via suppression of Rad51 expression through inhibition of autophagy and accumulation of p62 in gastric carcinogenesis. We find that H. pylori activated DNA damage pathway in concert with downregulation of repair protein Rad51 in gastric cells, C57BL/6 mice and Mongolian gerbils. In addition, autophagy was increased early and then decreased gradually during the duration of H. pylori infection in vitro in a CagA-dependent manner. Moreover, loss of autophagy led to promotion of DNA damage in H. pylori-infected cells. Furthermore, knockdown of autophagic substrate p62 upregulated Rad51 expression, and p62 promoted Rad51 ubiquitination via the direct interaction of its UBA domain. Finally, H. pylori infection was associated with elevated levels of p62 in gastric intestinal metaplasia and decreased levels of Rad51 in dysplasia compared to their H. pylori- counterparts. Our findings provide a novel mechanism into the linkage of H. pylori infection, autophagy, DNA damage and gastric tumorigenesis.     

Multi-target stool DNA test in the surveillance of inflammatory bowel disease: a cross-sectional cohort study

Background and aim: Colonoscopic surveillance is recommended in patients with longstanding inflammatory bowel disease (IBD) as they are at increased risk of colorectal cancer (CRC). Non-invasive surveillance may improve compliance and access. Multi-target stool DNA (MT-sDNA) has been validated for screening of sporadic CRC but has not been assessed in IBD. Our aim was to assess the performance of a MT-sDNA test in a real-life surveillance setting of patients with longstanding IBD.

Material and methods: A total of 192 IBD patients enrolled from two prospective cohorts submitted an EDTA buffered stool sample and underwent chromo- or white light colonoscopy. Stools were assayed for methylated BMP3 &; NDRG4, mutant KRAS and β-actin by a laboratory blinded to clinical data.

Results: The multitarget-sDNA panel was positive in 2/2 CRC and 5/15 low-grade dysplasia (LGD)?Conclusion: The MT-sDNA panel detected CRC in IBD. Sensitivity for sub-centimeter colorectal neoplasms in IBD patients appears similar to that observed in the general population. The test may be a valuable tool for detection of malignancy during structured surveillance of long-term IBD in a first line hospital setting.     

A Potential Approach for Electrochemotherapy against Colorectal Carcinoma Using a Clinically Available Alternating Current System with Bipolar Snare in a Mouse Model

Background: Although electrochemotherapy appears promising for the treatment of superficial tumors, its usefulness against internal tumors, such as colorectal carcinoma (CRC), has not been well examined. Furthermore, since direct current electric pulses have been used for electropermeabilization of tumors in all in vivo electrochemotherapy studies, including clinical trials, the usefulness of alternating current systems has not been examined at all. In a mouse model it was examined whether the alternating current system with a bipolar snare, which has been employed already as a clinical endoscopic treatment modality, was useful for electrochemotherapy against CRC. Methods: Murine CRC colon 26 cells were implanted subcutaneously into syngeneic BALB/c mice and electrochemotherapy with bleomycin (BLM) using the alternating current system was performed against established CRC tumors. Results: Electrochemotherapy significantly suppressed the growth of established CRC tumors, resulting in significantly prolonged survival of animals with CRC. Histological examination revealed that electrochemotherapy caused massive necrosis of CRC tumors. Subsequent analysis revealed that the delivery of alternating current electric pulses to CRC tumors profoundly increased intratumoral amounts of BLM. Conclusions: Because the alternating current system using a bipolar snare has been used widely as an endoscopic treatment modality in clinical settings, these results indicate that electrochemotherapy using the alternating current system may be a promising approach for the treatment of CRC.     

The Role of the CpG Island Methylator Phenotype on Survival Outcome in Colon Cancer

Background/AimsCpG island methylator phenotype (CIMP)- high colorectal cancers (CRCs) have distinct clinicopathological features from their CIMP-low/negative CRC counterparts. However, controversy exists regarding the prognosis of CRC according to the CIMP status. Therefore, this study examined the prognosis of Korean patients with colon cancer according to the CIMP status.MethodsAmong a previous cohort population with CRC, a total of 154 patients with colon cancer who had available tissue for DNA extraction were included in the study. CIMP-high was defined as 3/5 methylated markers using the five-marker panel (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1).ResultsCIMP-high and CIMP-low/negative cancers were observed in 27 patients (17.5%) and 127 patients (82.5%), respectively. Multivariate analysis adjusting for age, gender, tumor location, tumor stage and CIMP and microsatellite instability (MSI) statuses indicated that CIMP-high colon cancers were associated with a significant increase in colon cancer-specific mortality (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.20 to 8.69; p=0.02). In microsatellite stable cancers, CIMP-high cancer had a poor survival outcome compared to CIMP-low/negative cancer (HR, 2.91; 95% CI, 1.02 to 8.27; p=0.04).ConclusionsRegardless of the MSI status, CIMP-high cancers had poor survival outcomes in Korean patients.