Recent studies have demonstrated that cancer stem cells (CSCs) can initiate and sustain tumor growth and exhibit resistance to clinical cytotoxic therapies. Therefore, CSCs represent the main target of anticancer therapy. Interleukin-6 (IL-6) promotes cellular proliferation and drug resistance in colorectal cancer, and its serum levels correlate with patient survival. Therefore, IL-6 and its downstream signaling molecule the signal transducer and activator of transcription-3 (STAT3) represent potential molecular targets. In the present study, we investigated the effects of IL-6 and its downstream signaling components on stem cell biology, particularly the chemoresistance of CSCs, to explore potential molecular targets for cancer therapy. The colon cancer cell line WiDr was cultured in serum-free, non-adherent, and three-dimensional spheroid-forming conditions to enrich the stem cell-like population. Spheroid-forming cells slowly proliferated and expressed high levels of Oct-4, Klf4, Bmi-1, Lgr5, IL-6, and Notch 3 compared with adherent cells. Treatment with an anti-human IL-6 receptor monoclonal antibody reduced spheroid formation, stem cell-related gene expression, and 5-fluorouracil (5-FU) resistance. In addition, IL-6 treatment enhanced the levels of p-STAT3 (Tyr705), the expression of Oct-4, Klf4, Lgr5, and Notch 3, and chemoresistance to 5-FU. siRNA targeting Notch 3 suppressed spheroid formation, Oct-4 and Lgr5 expression, and 5-FU chemoresistance, whereas STAT3 inhibition enhanced Oct-4, Klf4, Lgr5, and Notch 3 expression and 5-FU chemoresistance along with reduced spheroid growth. Taken together, these results indicate that IL-6 functions in dichotomous pathways involving Notch 3 induction and STAT3 activation. The former pathway is involved in cancer stem-like cell biology and enhanced chemoresistance, and the latter pathway leads to accelerated proliferation and reduced chemoresistance. Thus, an anti-human IL-6 receptor monoclonal antibody or Notch 3 inhibition may be superior to STAT3 inhibition for CSC-targeting therapies concomitant with anticancer drugs.

The emergence of chemoresistance is a major limitation of current cancer therapies, and checkpoint kinase (Chk1) 1 positively correlates with resistance to chemo‑ or radio‑therapy. Cancer cells lacking p53 pathways are completely dependent on the S and G2/M checkpoints via Chk1; therefore, Chk1 inhibition enhances the cytotoxicity of DNA‑damaging agents only in p53‑deficient cells. However, little is known about the synergistic effect of Chk1 inhibition with 5‑FU, the most frequently used antimetabolite, in chemoresistant colorectal cells. In this study, we found that 5‑FU induced S‑phase arrest only in p53‑deficient colorectal cancer cells. 5‑FU treatment induced DNA damage and activation of ataxia telangiectasia mutated (ATM) and Chk1, leading to S‑phase arrest, and Chk1 inhibition using SB218078 reduced S‑phase arrest and increased apoptosis in the presence of 5‑FU. In contrast, in p53‑deficient, 5‑FU‑resistant (5FUR) colon cancer cells that we developed, 5‑FU enhanced DNA damage but did not induce Chk1/ATM activation or cell cycle arrest. SB218078 in combination with 5‑FU did not induce apoptosis. These results indicate that 5‑FU‑resistance abrogated the anticancer effect amplified by Chk1 inhibition, even in p53‑deficient cancer cells.

Objective MicroRNAs (miRNAs) act as tumour suppressor genes or oncogenes in the regulation of multiple carcinogenic processes. Aberrant miRNA expression is reported in Helicobacter pylori (H pylori)-related gastritis and gastric cancer. The cytotoxin-associated gene A (CagA) of H pylori has a pathophysiologically important role in gastric carcinogenesis. A study was undertaken to evaluate the effect of CagA on miRNA expression and its regulatory mechanism. Methods The effect of CagA on miRNA expression was assessed by comprehensive miRNA microarray. The mechanisms of the in vitro and in vivo effects of CagA on histone modification and DNA methylation and the involvement of CagA-dysregulated signal transduction on let-7, an important representative miRNA in gastric carcinogenesis, were investigated. Results In in vitro experiments, CagA significantly attenuated let-7 expression leading to Ras pathway activation. CagA enhanced c-myc, DNA methyltransferase 3B (DNMT3B) and Enhancer of Zeste homologue 2 (EZH2) expression and attenuated miR-26a and miR-101 expression, which resulted in the attenuation of let-7 expression by histone and DNA methylation. Experiments performed in CagA transgenic mice revealed that c-myc, EZH2 and DNMT3B expression were enhanced and let-7 expression was attenuated to induce Ras oncoprotein expression in the stomach, with no associated inflammation. Conclusions H pylori CagA induces aberrant epigenetic silencing of let-7 expression, leading to Ras upregulation.

Objective: Inflammatory bowel disease (IBD) is a chronic inflammatory process in the digestive tract and patients with IBD develop osteopenia. Although vitamins K and D are important for maintaining bone health and inhibiting inflammation, their roles in patients with IBD are not clear. We investigated the roles of vitamins K and Din the bone health and inflammation in patients with IBD.Methods: Bone mineral density (BMD) of patients with IBD (Crohn's disease [CD], n = 47, and ulcerative colitis [UC], n = 40) was measured with dual-energy X-ray absorptiometry. Vitamin K and D levels of patients with IBD and healthy volunteers (n = 41) were evaluated by measuring serum undercarboxylated osteocalcin and 1,25 dihydroxyvitamin D, respectively. Clinical activity index was evaluated in patients with CD and UC.Results: BMD was low in patients with CD and UC. Serum undercarboxylated osteocalcin levels were significantly higher in patients with CD, but not with UC, compared with healthy subjects, indicating that bone vitamin K is insufficient in patients with CD. The levels of undercarboxylated osteocalcin were significantly correlated with the clinical activity index of CD, although they were not correlated with BMD. The levels of 1,25 dihydroxyvitamin D were significantly lower in patients with CD and UC than in healthy subjects. The levels of 1,25 dihydroxyvitamin D were inversely correlated with BMD in patients with UC and were not correlated with the clinical activity index of CD.Conclusion: Vitamins K and D are insufficient in patients with IBD. Insufficiency of vitamin K is suggested to be associated with inflammatory processes of CD. (C) 2011 Elsevier Inc. All rights reserved.

Background.Current models have proposed that part of the DNA damage response (DDR) is dependent on the DNA mismatch repair (MMR) system, but there are no good cellular models that demonstrate that MMR proteins recognize DNA damage to subsequently signal cell-cycle checkpoints or apoptosis machinery.For measurement of DNA MMR activity in a living cell, un-nicked circular heteroduplex plasmid DNA is reported to be useful as a substrate.However, multiple factors significantly affect the yield of heteroduplex plasmid DNA, which voids the ability to demonstrate MMR action for DDR.Aims: To establish a method for efficient preparation of a heteroduplex plasmid containing a DNA damage lesion.Methods.We used 5-fluorodeoxyuracil (5FdU) as a DNA damage lesion, which models 5-fluorouracil incorporated into DNA as we previously demonstrated for In Vitro MMR binding assay.We first compared the efficacy between two methods of 5FdU-containing heteroduplex plasmid (5FdU plasmid) construction; one was constructed by a ligation reaction between linearized double-strand DNA (dsDNA) plasmid and annealed synthetic oligonucleotide containing 5FdU (ligation method), and the other was by primer extension/ ligation reaction by using both T4 DNA polymerase and T4 DNA ligase at one time after annealing a 5FdU-containing phosphorylated oligomer with a pGEM7Zf(+) single-strand DNA (ssDNA) plasmid (primer extension method).Based on the results, we further analyzed determinant factors of each process to purify high quality 5FdU plasmid.Results.With the ligation method, we yielded less than 1.0 μg from 10 μg of parental dsDNA plasmid, whereas with the primer extension method, 7.4-12 μg could be purified from the 10 μg ssDNA plasmid.We found it easier to detect the heteroduplex site by direct sequencing with the primer extension method compared to the ligation method.To improve quality and quantity of 5FdU plasmid preparation, we realized the following determinant factors: 1) It is easier and more efficient to isolate ssDNA plasmid using M13K07 helper phage through generating individual transformed colonies of JM109/template dsDNA plasmid/M13K07 than conventional M13K07 infection; 2) Phosphorylation of 5FdU-containing oligomer improved the closed 5FU plasmid recovery rate; 3) A shorter time for primer extension/ligation reaction had higher yield due to T4 polymerase having delayed 3'-5' exonuclease activity.The purified 5FdU plasmids could be transfected, and enabled us to observe cellular cytotoxicity when compared to positive and the negative control plasmids, which suggests usefulness for further analysis of DDR response.Conclusion.We established efficient production of a heteroduplex plasmid containing DNA damage lesion, which will be useful to analyze a pure DDR effect in live cells.

141), and asymptomatic age-matched controls (n=202).Genotyping was performed on known tagging SNPs in highly conserved parts of the 5'regulatory region of the VDR gene.Radioactive gel shift assay was used to investigate GATA binding affinity.In 25 of the genotyped BE patients, VDR mRNA levels were determined in esophageal biopsies.Results: Dose-risk allele analysis showed that subjects carrying the 1633G/1453T haplotype were less susceptible to EE (OR 0.48; 95%CI 0.28-0.81),BE (OR 0.46; 95%CI 0.26-0.80),and EAC (OR 0.50; 95%CI 0.27-0.96).For BE, this observation was replicated in an independent cohort of 150 patients (OR 0.44; 95%CI 0.23-0.85).Super shift analysis showed that the 1453C→T mutation increased binding of the transcription factor GATA-1.Moreover, in BE patients carrying two GT-alleles VDR mRNA levels in esophageal biopsy samples were 3fold lower than in patients lacking .Conclusion: This study shows that GT-haplotype near the 1c region of the VDR gene is associated with a decreased susceptibility to EE, BE, and EAC.It causes an increase in GATA-1 binding to this regulatory region, thereby inhibiting transcription of the VDR gene.

of H. pylori negative and positive volunteers were assessed by western blot and immunohistochemistry for angiotensin II receptors (AT1R, AT2R) and other RAS components (angiotensinogen, renin, angiotensin converting enzyme and neprilysin).Mucosa levels of myeloperoxidase (MPO) served as protein markers of neutrophil infiltration.Results: The AT1R protein expression was significantly higher in both antral and corporal specimens from H. pylori positive subjects compared to specimens from H. pylori negatives.The gastric mucosal AT1R protein expression correlated with mucosa levels of myeloperoxidase.No significant differences or correlations were found with regard to expression of the other investigated RAS components.Confocal microscopy showed that AT1R was highly expressed by a subpopulation of antral endocrine cells.Most other epithelial cells, as well as all leukocytes infiltrating the H. pylori-infected mucosa, also expressed AT1R and AT2R.Angiotensinogen and renin were expressed by resident mesenchymal cells in the lamina propria and all investigated RAS components were found in vascular endothelial cells.Conclusions: The results suggest that AT1R in the H. pylori infected gastric mucosa might influence neutrophil ROS generation and hormonal release from gastric endocrine cells.These mechanisms might be important for the development of H pylori induced peptic ulcers and gastric adenocarcinoma.This study also demonstrates local expression of several other RAS components in the human gastric mucosa that all might serve as pharmacological targets for modulation of RAS in the H. pylori infected gastric mucosa.

Background and aims: Clopidogrel and aspirin (ASA) are wildly used in the prevention of cardiovascular and embolic events. However, these combined medications cause significant risk of peptic ulcers and bleeding complications. This case-control study was designed to evaluate the risk of upper gastrointestinal bleeding (UGIB) in clopidogrel and aspirin users who continue receiving standard dose of proton pump inhibitors (PPI). . Methods: Data for clinical information and endoscopic findings were collected during January 2009 and November 2009 from patients who used clopidogrel and/or ASA and continue receiving standard dose of PPI. Patients with history of prior UGIB or abdominal surgery were excluded. Clopidogrel or ASA user was defined as consumption of clopidogrel or ASA for at least 7 days period preceding the episode of bleeding. The UGIB was defined as overt bleeding (hematemesis, positive nasogastric aspirate, and melena) or fall in baseline hematocrit ≥ 5 points within 24 hours of admission. Ulcer was defined at endoscope by breaking mucosa > 3mm in diameter. Results:A total of 175 patients (82 men and 93 women, mean age of 65.3 years) were evaluated in this study including 54 patients (30.9%) with UGIB and 121 patients (69.1%) with dyspeptic symptoms. Male were significantly more common than female patients in bleeding group (61.1 % vs 38.9%: P=0.01). However, the underlying diseases of the patients including cardiovascular diseases, rheumatological diseases and diabetes mellitus were not different between these 2 groups. UGIB was significantly higher in current ASA (325mg) plus clopidogrel user than non-user (16.7% vs 5.8%; P=0.02). The multivariable model suggested that the probability of UGIB event increased with current ASA (325mg) plus clopidogrel use (OR= 2.3, 95%CI =1.2-3.9) in the patients receiving concomitant PPI. Summary: Risk of UGIB events still occur in ASA and clopidogrel users in spite of receiving concomitant PPI. Lower dose of ASA, concomitant higher dose PPI and carefully monitoring of UGIB should be considered in combined ASA and clopidogrel user patients.

Background and aims: Immunoglobulin (Ig) G carries N-linked oligosaccharides at the Cgamma2 domain.We have reported that agalactosyl fraction in the fucosylated IgG oligosaccharides was increased in inflammatory bowel disease (IBD) and that agalactosyl IgG is an effective serological marker in IBD, especially in Crohns disease (CD) (Am J Gastroenterol. 2008). The mRNA level and activity of beta-1,4-galactosyltransferase I (Beta4GalT I), which is indispensable for attaching galactose to IgG oligosaccharides, were significantly lower in B cells and plasma cells of patients with CD than in those of ulcerative colitis patients. To clarify the pathophysiology of agalactosyl IgG elevation in IBD, we investigated the role of oligosaccharide alterations in colitis by using Beta4GalT I deficient mice. Methods: Beta4GalT I hetero-type mice (HT) and littermate wild-type mice (WT) were used because Beta4GalT I null mice were semi-lethal and Beta4GalT I activity in HT was reported to be half in WT. Both HT and WT were administered with 3% dextran sodium sulfate (DSS; MW 3500050000) ad libitum for 7 days. Solution was replaced to normal water for 3 days and mice were sacrificed at day 10. The severity of colitis was evaluated by the colon length, the body weight loss and the histological examination. CD4+, CD11b+ and CD19+ cells were separated from mononuclear cells from the spleen, mesenteric lymph nodes and lamina propria by MACS system. Cytokine productions in the culture supernatants of these cells were measured by ELISA. Results: Agalactosyl oligosaccharide structures of serum IgG were increased in Beta4GalT I HT compared to WT littermates and the IgG oligosaccharides in HT showed similar pattern as CD patients in terms of agalactosylation. When DSS colitis was induced in HT and WT, HT showed significantly less shortening in the colon length and less body weight loss than WT. Furthermore, histological score was significantly lower in HT than in WT. Thus, DSS-induced colitis was significantly ameliorated in HT compared to WT. Production of IL-10 in splenocytes of HT, especially in CD11b+ macrophages, was increased compared to that in WT. IgG oligosaccharide structures were not altered by DSS administration. Conclusions: Decrease in Beta4GalT I activity ameliorated murine DSS colitis. Agalactosyl IgG might not be the cause of colitis and is suggested to play a protective role for the colonic inflammation.