Yuichiro Miyaki

Increased Polyamine Intake Inhibits Age-Associated Alteration in Global DNA Methylation and 1,2-Dimethylhydrazine-Induced Tumorigenesis

Polyamines (spermine and spermidine) play many important roles in cellular function and are supplied from the intestinal lumen. We have shown that continuous high polyamine intake inhibits age-associated pathologies in mice. The mechanism by which polyamines elicit these effects was examined. Twenty-four week old Jc1:ICR male mice were fed one of three experimental chows containing different polyamine concentrations. Lifetime intake of high polyamine chow, which had a polyamine content approximately three times higher than regular chow, elevated polyamine concentrations in whole blood, suppressed age-associated increases in pro-inflammatory status, decreased age-associated pathological changes, inhibited age-associated global alteration in DNA methylation status and reduced the mortality in aged mice. Exogenous spermine augmented DNA methyltransferase activity in Jurkat and HT-29 cells and inhibited polyamine deficiency-induced global alteration in DNA methylation status in vitro. In addition, increased polyamine intake was associated with a decreased incidence of colon tumors in BALB/c mice after 1,2-demethylhydrazine administration; 12 mice (60%) in the low polyamine group developed tumors, compared with only 5 mice (25%) in the high polyamine group (Fishers exact probability = 0.027, p = 0.025). However, increased polyamine intake accelerated the growth of established tumors; maximal tumor diameter in the Low and High groups was 3.85±0.90 mm and 5.50±1.93 mm, respectively (Mann-Whitney test, p = 0.039). Spermine seems to play important roles in inhibiting age-associated and polyamine-deficient induced abnormal gene methylation as well as pathological changes including tumorigenesis.

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability

BackgroundRecent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.MethodsWith an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.ResultsUnsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3′ end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).ConclusionsSomatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Array-based identification of common DNA methylation alterations in ulcerative colitis.

Patients with long-standing ulcerative colitis (UC) have higher risk of developing colorectal cancer. Albeit the causes remain to be understood, epigenetic alterations have been suggested to play a role in the long-term cancer risk of these patients. In this work, we developed a novel microarray platform based on methylation-sensitive amplified fragment length polymorphism (MS-AFLP) DNA fingerprinting. The over 10,000 NotI sites of the human genome were used to generate synthetic primers covering these loci that are equally distributed into CpG rich regions (promoters and CpG islands) and outside the CpG islands, providing a panoramic view of the methylation alterations in the genome. The arrays were first tested using the colon cancer cell line CW-2 showing the reproducibility and sensitivity of the approach. We next investigated DNA methylation alterations in the colonic mucosa of 14 UC patients. We identified epigenetic alterations affecting genes putatively involved in UC disease, and in susceptibility to develop colorectal cancer. There was a strong concordance of methylation alterations (both hypermethylation and hypomethylation) shared by the cancer cells of the CW-2 cell line and the non-cancer UC samples. To the best of our knowledge, this work defines the first high-throughput aberrant DNA methylation profiles of the colonic mucosa of UC patients. These epigenetic profiles provide novel and relevant knowledge on the molecular alterations associated to the UC pathology. Some of the detected alterations could be exploited as cancer risk predictors underlying a field defect for cancerization in UC-associated carcinogenesis.

The accumulation of DNA demethylation in Sat α in normal gastric tissues with Helicobacter pylori infection renders susceptibility to gastric cancer in some individuals

Helicobacter pylori (HP) infection is widely recognized as a risk factor for gastric cancer, but only a minority of infected individuals develop gastric cancer. The aim of this study was to determine whether DNA demethylation in non-cancerous gastric mucosa (NGM) significantly enhances susceptibility to gastric cancer. A total of 165 healthy volunteers, including 83 HP-positive and 82-negative individuals, as well as 83 patients with single and 18 with synchronous double gastric cancer (GC) were enrolled in this study. The relative demethylation levels (RDLs) of repetitive sequences, including Alu, LINE-1 and Sat α, were quantified by real-time methylation-specific polymerase chain reaction. The Alu RDL did not exhibit any differences within each respective group, whereas LINE-1 RDL was significantly elevated in cancer tissues compared with the NGM in the other groups (P<0.001). Our results indicated that a gradual increase in Sat α RDL correlated with HP infection and cancer development. Sat α RDL was significantly elevated in the NGM in HP-positive compared with HP-negative (P<0.001), and significantly elevated in cancer tissues (P<0.001). Although the Sat α RDL of the NGM in the total population increased in an age-dependent manner, it was significantly increased in a fraction of younger GC patients (<45 years) compared with all of the others (45 years or older, P=0.0391). In addition, double GC exhibited a significantly higher Sat α RDL in the NGM compared with single GC (P=0.0014). In these two fractions, Sat α RDL in the NGM exhibited an inverse correlation with age. In conclusion, the present study demonstrated that the accumulation of DNA demethylation in Sat α RDL in the NGM with HP infection potentially renders susceptibility to gastric cancer in a fraction of GC patients younger than 45 years or in patients with multiple cancers.

Aberrant methylation of PSD disturbs Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis in ulcerative colitis-associated carcinogenesis

We previously reported that the Pleckstrin and Sec7 domain-containing (PSD) gene is preferentially methylated in patients with ulcerative colitis (UC) who developed colorectal cancer (CRC), and is implicated in UC-associated carcinogenesis through its inhibition of apoptosis. This study aimed to determine the potential effect of PSD methylation on its downstream molecule, Ras-related C3 botulinum toxin substrate 1 (Rac1), which governs neutrophil chemotaxis and apoptosis signaling. PSD was knocked down in a normal human fibroblast cell line (HNDF) and a neutrophil-like cell line (HL-60). Both NHDF and HL-60 cells exhibited numerous filamentous-actin (F-actin) rich membrane extensions, resulting in the activation of Rac1; this activation was hampered by PSD silencing. Lipopolysaccharide, a reactive oxygen species (ROS) inducer, stimulated NHDF cells to release ROS and activated caspase-3/7 in the presence of neutrophils, which was inhibited by PSD knockdown. Migration assays demonstrated that chemotaxis of HL-60 cells was affected by PSD silencing in NHDF cells. Tissue sections from 6 UC patients with CRC and 15 UC patients without CRC were examined. To verify Rac1-mediated chemotaxis in tissue sections, we evaluated the grade of neutrophil infiltration by histological assessment and assessed F-actin and PSD expression by immunohistochemistry. Neutrophil infiltration, F-actin and PSD expression were significantly decreased in specimens from UC patients with PSD methylation compared with those without. Decreased levels of F-actin expression were observed in colorectal mucosa, as well as in infiltrating cells with PSD methylation. PSD expression was preferentially inhibited in colorectal mucosa by PSD methylation, whereas PSD expression was rarely observed in infiltrating cells, regardless of PSD methylation status. These data indicate that aberrant methylation of PSD occurs in UC-associated colorectal mucosa, enabling circumvention of Rac1-mediated immune responses governing neutrophil chemotaxis and apoptosis, and thus plays a pivotal role in the mechanisms underlying UC-associated carcinogenesis.

Aberrant methylation of the Pleckstrin and Sec7 domain-containing gene is implicated in ulcerative colitis-associated carcinogenesis through its inhibitory effect on apoptosis.

The Pleckstrin and Sec7 domain-containing (PSD) gene, which regulates skeletal rearrangements, has been found to be more frequently methylated both in ulcerative colitis (UC)-associated colorectal cancer tissues (5 of 7; 71.4%) and matched normal epithelia (4 of 7; 57.1%) compared to non-neoplastic UC epithelia (6 of 22; 27.3%) and sporadic colorectal cancer tissues (6 of 32; 18.8%). The levels of PSD mRNA were positively correlated with the methylation status of PSD, as shown by both MSP and bisulfite sequencing. To determine the potential role of PSD silencing in the mechanisms underlying UC-associated carcinogenesis, the levels of senescence, proliferation and apoptosis were evaluated in a normal human fibroblast cell line (NHDF) in which 93% of PSD expression was knocked down by a small-interfering RNA (si-RNA). Although there were no significant differences in the levels of senescence and proliferation caused by PSD knockdown, the level of apoptosis was significantly decreased by PSD knockdown (5.3% in siControl-treated cells vs. 0.67% in siPSD-treated cells, p=0.0001). In addition, reactive oxygen species inducers accelerated apoptosis in NHDF and a neutrophil-like cell line, which was significantly reduced by PSD knockdown. To verify the effect of PSD methylation in tissue sections including 21 samples from UC patients with or without tumors, we elucidated PSD promoting accumulation of filamentous-actin (F-actin) and apoptosis by immunohistochemistry and TUNEL assay, respectively. Both levels of accumulation of F-actin and apoptosis were significantly decreased in specimens from UC patients with PSD methylation compared to those without PSD methylation (F-actin: 0.69±0.86 with vs. 1.57±0.51 without, p=0.0031, apoptotic index: 0.31±0.63 with vs. 1.0±0.88 without, p=0.0277). In conclusion, our results indicate that PSD methylation plays a significant role in the mechanisms underlying UC-associated carcinogenesis through its inhibitory effect on apoptosis in the interaction between colorectal mucosa and neutrophils.

Laparoscopic splenectomy for a wandering spleen complicating gastric varices: report of a case

Wandering spleen is a rare clinical entity, and its chronic torsion of the vascular pedicle result in splenic vein occlusion leading to gastric varices. Here, we present a case of wandering spleen complicating gastric varices in a 40-year-old female. Three-dimensional CT (3D-CT) clearly showed the disruption of the splenic vein at the origin of the vascular pedicle and collateral development of the gastric varices. The patient was electively treated with laparoscopic splenectomy. Difficulty of prediction of the splenic vein recanalization to improve the varices was the reason for the use of splenectomy versus splenopexy. The varices were successfully diminished 3 months after the surgery. After review of cases of complicating gastric varices in the literatures, splenectomy is still a secure way to treat an adult patient with wandering spleen with complicating gastric varices.

Asynchronous abdomino-parasacral resection of a giant pelvic lipoma protruding to the left buttock

INTRODUCTION Few reports detail adequate surgical management of giant pelvic tumors that traverse the sciatic foramen. PRESENTATION OF CASE We present a case of a giant retroperitoneal pelvic lipoma that presented with a dumbbell shape on imaging, occupying the entire lesser pelvis and protruding to the gluteus through the sciatic foramen. Surgery was performed for en bloc resection of the tumor. DISCUSSION A parasacral approach with the patient in the prone position was necessary to dissect the tumor in the buttock, manipulate around the sciatic foramen and preserve collateral blood flow for the gluteal muscle. An abdominal approach was also essential to ligate the internal iliac vessels involved in the tumor. Accordingly changings the position of the patient during the operation were required. Division of the sacrotuberous and sacrospinous ligaments and packing of the soft tumor into a plastic bag were useful to pass the buttock portion through the foramen without the tumor breaking off. CONCLUSION The asynchronous abdomino-parasacral approach with several turnings of the patients body and plastic bag packing of the tumor were advantageous to manage en bloc resection of the giant pelvic lipoma presented in this case study.