Tomoo Yoshie

A Novel Serum Metabolomics-Based Diagnostic Approach for Colorectal Cancer

Background To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. Methodology/Principal Findings We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0–2 colorectal cancer (82.8%). Conclusions/Significance Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.

Serum metabolomics as a novel diagnostic approach for gastrointestinal cancer.

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and L-serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3-hydroxypropionic acid and pyruvic acid; and L-alanine, glucuronoic lactone and L-glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer.

A Role of the Aryl Hydrocarbon Receptor in Attenuation of Colitis

Background and AimsThe aryl hydrocarbon receptor (AhR), which is a member of the basic helix-loop-helix/Per-Arnt-Sim homology superfamily, plays an important role in multiple biological functions, and AhR knockout (AhR KO) animals suffer from a variety of organ disorders including a decline in the efficacy of their immune system. In addition, AhR activation is known to aid the maintenance of homeostasis in vivo. In this study, we investigated whether AhR is functionally associated with intestinal immunity.Methods and ResultsIn in vivo experiments, it was found that dextran sodium sulfate (DSS)-evoked colitis was more severe in AhR KO mice than in C57BL/6J wild type mice. It was also revealed that the administration of DSS increased the expression levels of AhR and CYP1A1 mRNA in the colon epithelium. In addition, oral administration of β-naphthoflavone (βNF), a non-toxic agonist of AhR, suppressed the pathogenesis of DSS-induced colitis. βNF also attenuated DSS-induced colitis. In cell culture experiments, downregulation of AhR in human colon carcinoma SW480 cells enhanced the inflammatory responses evoked by lipopolysaccharide (LPS), and furthermore, AhR activation attenuated LPS-induced inflammatory responses, suggesting that AhR expressing intestinal epithelial cells are involved in the prevention of colitis.ConclusionsOur findings about the potential role of AhR activators in epithelial immune regulation aid our understanding of mucosal homeostasis and inflammatory bowl disease (IBD) and suggest that AhR activation has therapeutic value for the treatment of IBD.

GCMS‐based metabolomic study in mice with colitis induced by dextran sulfate sodium

Background: Metabolomics provides data about all the metabolic processes of a cell or organism. So far, the changes that occur in the levels of metabolites during the development of colitis have not been fully elucidated. Here we examined the changes of metabolite levels in the serum and colon tissue of colitis mice using gas chromatography mass spectrometry (GC/MS) with the aim of achieving a detailed understanding of the pathogenesis of inflammatory bowel disease (IBD). Methods: To induce colitis, C57BL/6J mice were administered 3.0% dextran sulfate sodium (DSS) in their drinking water for 5 days and were subsequently given drinking water alone. Results: A total of 77 and 92 metabolites were detected in serum and colon tissue, respectively, and among the metabolites the compositions of TCA cycle intermediates and amino acids changed depending on the degree of colitis. Then, partial least square discriminant analysis (PLS‐DA), a multiple classification analysis, showed distinct clustering and clear separation of the groups according to the degree of colitis. Furthermore, PLS‐DA loadings plots revealed that succinic acid, indole‐3‐acetic acid, glutamic acid, and glutamine were the main contributors to the separation of each stage of colitis. In addition, it was revealed that supplementation with glutamine, the level of which was significantly decreased in the acute phase of colonic inflammation, attenuated colitis induced by DSS. Conclusions: Our results suggest that metabolomics is capable of representing the various degrees of colitis, and our findings will aid in the discovery of therapeutic agents for IBD and other inflammatory disorders by metabolomic approaches. (Inflamm Bowel Dis 2011;)

Serum fatty acid profiling of colorectal cancer by gas chromatography/mass spectrometry

AIMS Several screening methods have been applied for the early diagnosis of colorectal cancer, but most colorectal cancer patients are not diagnosed at a localized stage. In order to find novel biomarkers for the diagnosis of colorectal cancer, profiling of the serum levels of fatty acids, which are the main components of fats and are important factors for human metabolism, was performed using the sera of colorectal cancer patients. MATERIALS & METHODS A total of 42 colorectal cancer patients and eight healthy volunteers participated in this study. The serum levels of fatty acids, including free fatty acids and esterified fatty acids, were evaluated by gas chromatography/mass spectrometry. Then, partial least squares discriminant analysis was performed on the basis of the serum fatty acids detected by gas chromatography/mass spectrometry. RESULTS The serum levels of the nine fatty acids exhibited distinct differences between the colorectal cancer patients and healthy volunteers: the levels of four fatty acids were higher in the colorectal cancer patients than the healthy volunteers, and those of the other five fatty acids were lower. These changes were also observed at a very early clinical stage. Furthermore, the levels of very-long-chain fatty acids had a tendency to be increased in the sera of the colorectal cancer patients. CONCLUSIONS The pathogenesis of colorectal cancer leads to changes in the composition of serum fatty acids including free fatty acids and esterified fatty acids. These results suggest that serum fatty acid profiling may be used as a novel diagnostic tool for early-stage colorectal cancer.

Regulation of the metabolite profile by an APC gene mutation in colorectal cancer

Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography–mass spectrometry‐based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non‐polyp tissues of APCmin/+ mice than in the normal tissues of the control mice and the polyp tissues of APCmin/+ mice. Ribitol, the levels of which were decreased in the polyp lesions of the APCmin/+ mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full‐length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APCmin/+ mice than in their non‐polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 μM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer. (Cancer Sci 2012; 103: 1010–1021)

AP1B plays an important role in intestinal tumorigenesis with the truncating mutation of an APC gene

Recent evidence has suggested that carcinoma is accompanied by the loss of cell polarity. An epithelial cell‐specific form of the AP‐1 clathrin adaptor complex, AP1B, is involved in the polarized transport of membrane proteins to the basolateral surface of epithelial cells. In our study, we investigated whether AP1B is involved in intestinal tumorigenesis. The cellular polarity of intestinal tumor cells was examined using APCMin/+ mice as an in vivo model and SW480 cells with a truncating mutation in the adenomatous polyposis coli (APC) gene as an in vitro model by confocal microscopy. Next, the expression of AP1B in intestinal tumor cells was examined by real‐time polymerase chain reaction (PCR) and Western blotting. The localization of β‐catenin and the expression of AP1B in the tumor tissue of patients with colorectal cancer were evaluated by confocal microscopy and real‐time PCR, respectively, and the relationships among cell polarity, AP1B expression and intestinal tumorigenesis were examined. Cellular polarity was lost in intestinal tumor cells, and the expression of AP1B was downregulated. In addition, the reduction in the expression level of AP1B correlated with the nuclear localization of β‐catenin in human colorectal cancer. Our study indicates the close associations between AP1B, intestinal tumorigenesis and mutations in the APC gene. This is the first report to reveal the relationships among AP1B, cellular polarity and intestinal tumorigenesis, and achieving a detailed understanding of AP1B will hopefully lead to discovery of therapeutic targets and novel biomarkers for intestinal cancer.

Anti-inflammatory Lipid Mediators Derived from ω-6 and ω-3 Polyunsaturated Fatty Acids as a Treatment Option for IBD

Lipoxins and resolvins are endogenous lipid mediators derived from ω-6 and ω-3 polyunsaturated fatty acids (PUFAs), respectively. Lipoxins, such as lipoxin A4 (LXA4) and lipoxin B4 (LXB4), are known as the first proresolving mediators, and their appearance leads to the resolution of inflammation. In addition, resolvins, such as D series resolvins (RvD) and E series resolvins (RvE), play important roles in the resolution of inflammation. So far, the anti-inflammatory effects of lipoxins and resolvins have been revealed in various experimental models of inflammatory disorders, and much attention has been paid to PUFAs and lipid mediators derived from PUFAs as a therapeutic strategy for inflammatory disorders including inflammatory bowl diseases (IBD). Recent studies using animal experimental models demonstrated that lipoxins; aspirin-triggered lipoxins; and their stable analogues, such as LXA4 and aspirin-triggered 15-epi-LXA4, were able to attenuate colitis. Resolvins, such as RvE1, were also demonstrated to protect against colitis. Moreover, it has been proposed that the biological abilities of endogenous anti-inflammatory lipid mediators are induced via their corresponding receptors, for example, FPR2/ALX for LXA4 and ChemR23 for RvE1, and the expression levels of their receptors were reported to be increased in macrophages and intestinal epithelium stimulated with exogenous antigens such as lipopolysaccaride. In this paper, the anti-inflammatory effects of lipid mediators derived from PUFAs, especially LXA4 and RvE1, are outlined, and the possibility of their use as a therapeutic strategy for IBD is discussed.