Butyrate supplementation regulates expression of chromosome segregation 1­like protein to reverse the genetic distortion caused by p53 mutations in colorectal cancer.

The chromosome segregation 1­like (CSE1L) protein, which regulates cellular mitosis and apoptosis, was previously found to be overexpressed in colorectal cancer (CRC) cells harboring mutations. Therefore, regulating CSE1L expression may confer chemotherapeutic effects against CRC. The gut microflora can regulate gene expression in colonic cells. In particular, metabolites produced by the gut microflora, including the short­chain fatty acid butyrate, have been shown to reduce CRC risk. Butyrates may exert antioncogenic potential in CRC cells by modulating p53 expression. The present study evaluated the association between CSE1L expression and butyrate treatment from two non­transformed colon cell lines (CCD­18Co and FHC) and six CRC cell lines (LS 174T, HCT116 p53+/+, HCT116 p53­/­, Caco­2, SW480 and SW620). Lentiviral knockdown of CSE1L and p53, reverse transcription­quantitative PCR (CSE1L, c­Myc and p53), western blotting [CSE1L, p53, cyclin (CCN) A2, CCNB2 and CCND1], wound healing assay (cell migration), flow cytometry (cell cycle analysis) and immunofluorescence staining (CSE1L and tubulin) were adopted to verify the effects of butyrate on CSE1L­expressing CRC cells. The butyrate­producing gut bacteria Butyricicoccus pullicaecorum was administered to mice with 1,2­dimethylhydrazine­induced colon tumors before the measurement of CSE1L expression. The effects of B. pullicaecorum on CSE1L expression were then assessed by immunohistochemical staining for CSE1L and p53 in tissues from CRC­bearing mice. Non­cancerous colon cells with the R273H p53 mutation or CRC cells haboring p53 mutations were found to exhibit significantly higher CSE1L expression levels. CSE1L knockdown in HCT116 p53­/­ cells resulted in G1­and G2/M­phase cell cycle arrest. Furthermore, in HCT116 p53­/­ cells, CSE1L expression was already high at interphase, increased at prophase, peaked during metaphase before declining at cytokinesis but remained relatively high compared with that in HCT116 expressing wild­type p53. Significantly decreased expression levels of CSE1L were also observed in HCT116 p53­/­ cells that were treated with butyrate for 24 h. In addition, the migration of HCT116 p53­/­ cells was significantly decreased after CSE1L knockdown or butyrate treatment. Tumors with more intense nuclear p53 staining and weaker CSE1L staining were found in mice bearing DMH/DSS­induced CRC that were administered with B. pullicaecorum. Taken together, the results indicated that butyrate can impair CSE1L­induced tumorigenic potential. In conclusion, butyrate­producing microbes, such as B. pullicaecorum, may reverse the genetic distortion caused by p53 mutations in CRC by regulating CSE1L expression levels.

Expression pattern of placenta specific 8 and keratin 20 in different types of gastrointestinal cancer.

The aim of the present study was to investigate the expression of keratin 20 (KRT20) and placenta specific 8 (PLAC8) in gastrointestinal (GI) cancer with various differentiation phenotypes. The present study retrospectively investigated archived formalin­fixed paraffin­embedded tissue samples from 12 patients at different stages of GI cancer [four with gastric cancer, four with pancreatic cancer and four with colorectal cancer (CRC)]. The stages were pre­determined, according to differentiation phenotypes, by a pathologist of the Department of Pathology at Sijhih Cathay General Hospital. KRT20 and PLAC8 expression levels were assessed using immunohistochemistry. The CRC cell lines SW620 and Caco­2 were used to assess interactions between KRT20 and PLAC8 via reverse transcription­quantitative PCR. PLAC8 and KRT20 expression was observed consistently only in the well­differentiated CRC tissue samples. Low KRT20 expression levels were observed in the PLAC8 knockdown SW620 cells. In addition, there was a positive association between PLAC8 and KRT20 expression in the differentiated Caco­2 cells. According to the results of the present study, the differentiation status of GI cancer influenced KRT20 expression, particularly in CRC, which may explain why patients with well­differentiated CRC display better clinical outcomes. Therefore, the prognostic significance of KRT20 and PLAC8 may be particularly crucial for patients with CRC displaying a well­differentiated phenotype.

Discovery of genes from feces correlated with colorectal cancer progression.

Colorectal cancer (CRC) is considered to develop slowly via a progressive accumulation of genetic mutations. Markers of CRC may serve to provide the basis for decision-making, and may assist in cancer prevention, detection and prognostic prediction. DNA and messenger (m)RNA molecules that are present in human feces faithfully represent CRC manifestations. In the present study, exogenous mouse cells verified the feasibility of total fecal RNA as a marker of CRC. Furthermore, five significant genes encoding solute carrier family 15, member 4 (SLC15A4), cluster of differentiation (CD)44, 3-oxoacid CoA-transferase 1 (OXCT1), placenta-specific 8 (PLAC8) and growth arrest-specific 2 (GAS2), which are differentially expressed in the feces of CRC patients, were verified in different CRC cell lines using quantitative polymerase chain reaction. The present study demonstrated that the mRNA level of SLC15A4 was increased in the majority of CRC cell lines evaluated (SW1116, LS123, Caco-2 and T84). An increased level of CD44 mRNA was only detected in an early-stage CRC cell line, SW1116, whereas OXCT1 was expressed at higher levels in the metastatic CRC cell line CC-M3. In addition, two genes, PLAC8 and GAS2, were highly expressed in the recurrent CRC cell line SW620. Genes identified in the feces of CRC patients differed according to their clinical characteristics, and this differential expression was also detected in the corresponding CRC cell lines. In conclusion, feces represent a good marker of CRC and can be interpreted through the appropriate CRC cell lines.

Upregulation of the growth arrest-specific-2 in recurrent colorectal cancers, and its susceptibility to chemotherapy in a model cell system.

Colorectal cancer (CRC) is one of the most common life-threatening malignances worldwide. CRC relapse markedly decreases the 5-year survival of patients following surgery. Aberrant expression of genes involved in pathways regulating the cell cycle, cell proliferation, or cell death are frequently reported in CRC tumorigenesis. We hypothesized that genes involved in CRC relapse might serve as prognostic indicators. We first evaluated the significance of gene sequences in the feces of patients with CRC relapse by consulting a public database. Tumorigenesis of target tissues was tested through tumor cell growth, cell cycle regulation, and chemotherapeutic efficacy. We found a highly significant correlation between CRC relapse and growth arrest-specific 2 (GAS2) gene expression. Based on cell models, the overexpressed GAS2 was associated with cellular growth rate, cell cycle regulation, and with chemotherapeutic sensitivity. Cell division was impaired by treating cells with 2-[4-(7-chloro-2-quinoxalinyloxy)phenoxy]-propionic acid (XK469), even when the cells were overexpressing GAS2. Thus, downregulation of GAS2 expression might control CRC relapse after curative resection. GAS2 could serve as a noninvasive marker from the feces of patients with prediagnosed CRC. Our findings suggest that GAS2 could have potential clinical applications for predicting early CRC relapse after radical resection, and that XK469 might impair tumor cell division by reducing GAS2 expression or blocking its cellular translocation. This will help in selecting the best therapeutic option, 5-fluorouracil in combination with XK469, for patients overexpressing GAS2 in CRC cells. Thus, GAS2 might act as a prognostic biomolecule and potential therapeutic target in patients with CRC relapse.

Upregulation of fecal cytokeratin 19 is associated with prognosis in older colorectal cancer patients.

The upregulation of fecal cytokeratin 19 (CK19) correlates with age and metastatic status in human colorectal cancer (CRC). To further explore its clinical significance in older patients (>60 years), their fecal CK19 was measured by quantitative reverse transcription-polymerase chain reaction. Differences in CK19 transcripts were compared using the nonparametric Mann-Whitney U test. Clinical significance was assessed with the chi-squared test and a binary logistic regression model. The association between overall survival and expressions of fecal CK19 in combination with other serum markers, carcinoembryonic antigen and carbohydrate antigen 19-9 (CA19-9), was evaluated using the Kaplan-Meier method. In these older groups, CRC patients had significantly higher median fecal CK19 expression (p = 0.006) than controls. The highest risk of CRC (odds ratio, 5.8; 95% confidence interval, 2.3-14.7; p < 0.001) was detected when the cutoff value for fecal CK19 expression was set at the median value of the 28 healthy controls. The lowest overall survival (29.2% ± 21.4%) occurred in patients in whom serum CA19-9 levels were high and fecal CK19 was overexpressed. Our results suggest that fecal CK19 expression is associated with CRC, and together with serum carcinoembryonic antigen or CA19-9, it can predict overall survival in older patients.

Clinical meaning of age-related expression of fecal cytokeratin 19 in colorectal malignancy.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of malignant death worldwide. Because young age of onset is often considered a poor prognostic factor for CRC, it is important to identify the poor outcomes of CRC in a younger population and to consider an aggressive approach by implementing early treatment. Our aim was to specifically quantify the fecal cytokeratin 19 (CK19) transcript from CRC patients and investigate its correlation with clinical stage, tumor malignancy, and age. METHODS: The quantitation of fecal CK19 transcript was determined by a quantitative real-time reverse transcription polymerase chain in 129 CRC patients (45 younger than 60 years at diagnosis) and 85 healthy controls. The levels of CK19 protein were examined both in colonic cell lines and tissues. RESULTS: The analysis of 45 younger CRC patients (age

Correlation of K-ras codon 12 mutations in human feces and ages of patients with colorectal cancer (CRC).

Colorectal cancer (CRC) is the predominant gastrointestinal malignancy and constitutes a major medical and economic burden worldwide. A thorough understanding of the oncogenes or genes related to tumorigenesis is the key to developing successful therapeutic strategies. Molecular analysis of feces constitutes a potentially potent and noninvasive method for detection of CRC. Using nested reverse transcription-polymerase chain reaction (RT-PCR) and amplified restriction fragment length polymorphism analysis, sloughed cells from the entire length of the colon and rectum were analyzed for expression of activating K-ras codon 12 mutants, which are becoming attractive targets for antisense treatment. K-ras codon 12 mutant sequences were detected in feces of 5% (1/20) of healthy controls, in feces of 41% (12/29) of CRC patients, in 10% (3/29) of isolates of tissue complementary DNA (cDNA), and in 14% (4/29) of isolates of genomic DNA. Age of patient was significantly associated with K-ras codon 12 sequences in feces: Patients with wild-type K-ras codon 12 sequences were significantly younger than those with mutated forms of K-ras codon 12. Fecal ribonucleic acid (RNA) analysis was demonstrated to be a useful for diagnosis of CRC. This technique may be suitable for screening and determining the clinical significance of active mutations of the K-ras gene in feces and would possibly be useful for identifying patients that would benefit from antisense therapy.