Increased ENT2 Expression and Its Association With Altered Purine Metabolism in Different Stages of Colorectal Cancer Cell Lines

Background Colorectal cancer (CRC) is one of the most prevalent malignant cancers worldwide. Although the purine metabolism pathway is known to be vital for cancer cells survival mechanism, not much is known on ENT2 role in CRC development and its association with purine metabolites. Hence this study is aimed to determine the level of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine, uric acid (UA), and the activity of xanthine oxidase (XO) and relate the findings with the ENT2 expression level in different CRC stages. Methods and results Normal colon cell line; CCD-841CoN and a panel of human CRC cell lines; SW480, HCT15 and HCT116, representing different CRC stages; Dukes’ B, C, and D respectively, have been used to measure HPRT, hypoxanthine/xanthine, UA levels and the activity of XO by biochemical assays. The level of ENT2 gene expression was also performed by qRT-PCR. The levels of HPRT, hypoxanthine were significantly higher (P< 0.05), while XO and UA were lower (P< 0.05) in all CRC stages as compared to the normal colon cells. Furthermore, ENT2 expression was found to be increased in all CRC stages. Despite having the highest level of HPRT and hypoxanthine, ENT2 level is lower in Dukes' D when compared to Dukes' B and C. Conclusion The rate of salvage pathway is increased in CRC development as indicated by the elevated levels of HPRT and hypoxanthine in different CRC stages. Increase ENT2 expression implies its importance in assisting hypoxanthine uptake. This step is vital in order to increase DNA synthesis via hypoxanthine recycling. Thus, ENT2 may be a potential marker in therapeutic development.

Effects of Sargassum plagiophyllum extract pretreatment on tissue histology of constipated mice

Purpose: To evaluate the toxicity of the dried seaweed, Sargassum plagiophyllum, extract (SPE) pretreatment in constipated mice.Methods: The dried seaweed powder was mixed with distilled water and extracted by autoclave at 121°C. Antioxidant activity of the extract was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Human normal colon cells were pretreated with SPE at 0 - 100 μg/mL for 24 h before challenging them with 100 μM hydrogen peroxide (H2O2). Intracellular reactive oxygen species (ROS) were quantified using 2',7'- dichlorodihydrofluorescein diacetate (H2DCFDA). Male ICR mice were pretreated for 14 consecutive days with SPE at 100, 500 and 1,000 mg/kg or lactulose at 500 mg/kg. Body weight and food intake were recorded daily. Constipation was induced with loperamide on days 12, 13 and 14 and fecal pellets evacuated over a 4-hr period. The ileum, liver, kidney, and spleen were collected for histopathological examination.Results: The IC50 for the radical scavenging capacity of SPE was 343.90 ± 4.21 μg/mL compared to 14.14 ± 0.71 μg/mL for ascorbic acid. Pretreatment with SPE was significantly reduced ROS production in human normal colon cells. Oral administration of all doses of SPE and lactulose for 14 consecutive days had no effect on food intake or body weight when compared to the normal control group. Defecation was significantly more frequent in mice pretreated with SPE at 100 mg/kg than in the constipation control group. Histopathological examination of the ileum, liver, kidney and spleen of pretreated constipated mice revealed no toxic effect from either SPE or lactulose. On the other hand, the loss of mucus-producing cells in the ileum of constipated mice was significantly lower in mice pretreated with SPE.Conclusions: These findings support the safety of SPE supplementation and may broaden itsapplication in clinical fields as an alternative drug or supplement for constipation management.

Recurrent Superenhancer of the Oncogene POU5F1B in Colorectal Cancers

Superenhancer usages in single cancer form such as colorectal cancer (CRC) may provide novel efficient targeting candidates. It is unclear whether CRC contains recurrent superenhancers that confer a predisposition to malignancy. We investigated the superenhancer profile of CRC cell line HCT116 and compared it to that of a healthy sigmoid colon. We found that HCT116 had lost most of the normal colon superenhancer activities but gained a new set of tumor-favoring superenhancers that facilitate tumor proliferation, growth signalling, and hypoxia resistance. Inhibiting the superenhancers by JQ-1 treatment had significantly decreased the colony formation capability of HCT116. Then, by comparing the superenhancer genes and robust CRC upregulated genes, we identified a superenhancer associated with a common CRC upregulated oncogene, POU5f1B. POU5f1B overexpression is related to the worse outcome in CRCs. Via performing ChIP-PCR in 35 clinical samples and investigating CRC anti-H3K27ac ChiP-seq public dataset consisting of 36 samples, we further identified that the superenhancer of oncogene POU5F1B is recurrently activated in CRCs, taking 62 and 72 per cent, respectively. Moreover, JQ-1 treatment successfully inhibited the POU5F1B expression in 5 out of 6 POU5F1B superenhancer-positive samples. Therefore, we concluded that the superenhancer activation of POU5F1B contributes partially to its high expression in CRCs, in addition to the well-known gene amplification aetiology.

Effect of vitamin C and E supplementation on human gastrointestinal tract tissues and cells: Raman spectroscopy and imaging studies

Cancer of gastrointestinal tract, such as colorectal cancer (CRC) and gastric cancer (GC), are common types of cancer globally and their origin can be linked to oxidative stress conditions. Commonly available antioxidants, such as vitamins C and E, are widely considered as potential anti-cancer agents. Raman spectra have great potential in the biochemical characterization of matter based on the fact that each molecule has its own unique vibrational properties. Raman spectroscopy allows to precisely characterized cell substructures (nucleus, mitochondria, cytoplasm, cell membrane) and components (proteins, lipids, nucleic acids). The paper presents the application of the Raman spectroscopy technique for the analysis of tissue samples and cells of the human colon and stomach. The main goal of this study is to show the differences between healthy and cancerous tissues from the human digestive tract and human normal and cancer colon and gastric cell lines. The paper presents the spectroscopic characterization of normal colon cells - CCD-18 Co in physiological and oxidative conditions and effect of oxidative injury of normal colon cells upon supplementation with vitamin C at various concentrations based on Raman spectra. The obtained results were related to the Raman spectra recorded for human colon cancer cells - Caco-2. In addition, the effect of the antioxidant in the form of vitamin E on gastric cancer cells - HTB-135 is presented and compared with normal gastric cells - CRL-7869. All measured gastric samples were biochemically and structurally characterized by means of Raman spectroscopy and imaging. Statistically assisted analysis has shown that normal, ROS injured and cancerous human gastrointestinal cells can be distinguished based on their unique vibrational properties. The conducted research based on Raman spectra proved that antioxidants in the form of vitamin C and E exhibit anti-cancer properties. In consequence, conducted studies proved that label-free Raman spectroscopy may play an important role in clinical diagnostics differentiation of human normal and cancerous gastrointestinal tissues and may be a source of intraoperative information supporting histopathological analysis.

Colorectal cancer is associated with the presence of cancer driver mutations in normal colon

While somatic mutations in colorectal cancer (CRC) are well characterized, little is known about the accumulation of cancer mutations in the normal colon prior to cancer. Here we have developed and applied an ultra-sensitive, single-molecule mutational test based on CRISPR-DS technology, which enables mutation detection at extremely low frequency (<0.001) in normal colon from patients with and without CRC. We found oncogenic KRAS mutations in the normal colon of about one third of patients with CRC but in none of the patients without CRC. Patients with CRC also carried more TP53 mutations than patients without cancer, and these mutations were more pathogenic and formed larger clones, especially in patients with early onset CRC. Most mutations in normal colon were different from the driver mutations in tumors suggesting that the occurrence of independent clones with pathogenic KRAS and TP53 mutations is a common event in the colon of individuals that develop CRC.

Ginsenoside Rh2 Suppresses Metastasis and Growth of Colon Cancer via miR-491

Ginsenoside Rh2 is considered as a new direction for future cancer treatment because of its excellent anticancer effect. However, due to its low bioavailability, it cannot exert its significant anticancer effect when applied directly to the human body. Chitosan (CS), a nanomaterial, has been verified to be able to enhance drug efficacy via its coating for drugs. Thus, we designed this study to investigate the impact of CS-coated ginsenoside Rh2 on the metastasis and growth of colon cancer (CC). First, ginsenoside Rh2 chitosan tripolyphosphate (CS-Rh2-TPP) nanoparticles (NPs) were constructed, and MTT, transwell, scratch adhesion, and flow cytometry assays were carried out for determining the impact of CS-Rh2-TPP at various concentrations on growth, metastasis, and apoptosis of colon cancer cells (CCCs). qRT-PCR was used to detect the expression of mircoRNA-491 (miR-491) in CCCs. According to TEM-based image analysis, CS-Rh2-TPP NPs were spherical or spheroidal in even distribution, with a particle size of about 220 mm and a zeta potential of −44.58 ± 2.84 mV. Additionally, CCCs presented lower miR-491 than normal colon cells, and its relative expression in CCCs showed a stronger increase after intervention of CS-Rh2-TPP than that after intervention of ginsenoside Rh2. Moreover, CS-Rh2-TPP suppressed the activity, invasion, as well as migration of CCCs and accelerated their apoptosis more significantly than ginsenoside Rh2. According to these results, CS-Rh2-TPP is able to upregulate miR-491 in CCCs, thus suppressing the metastasis and growth of CC.

The Colon Transcriptome Explorer (CoTrEx) 2.0: a Reference Web-Based Resource for Exploring Population-Based Normal Colon Gene Expression

Gene expression data is key for the functional annotation of single nucleotide polymorphisms (SNPs) identified in genome-wide association studies (GWAS). Expression and splicing quantitative trait loci (e/sQTLs) in normal colon tissue, such as those from the University of Barcelona and University of Virginia RNA sequencing project (BarcUVa-Seq) and the Genotype-Tissue Expression project (GTEx), are required to gain biological insight of colon-related diseases risk loci. Moreover, transcriptome-wide association studies (TWAS) rely on reference gene expression imputation panels in the tissue of interest to nominate susceptibility genes. Also, it is of high interest to study the relationships between genes in a network framework. For facilitating these analyses, we have updated and expanded the scope of the Colon Transcriptome Explorer (CoTrEx) to the version 2.0. This web-based resource provides exhaustive visualization and analysis of transcriptome-wide gene expression profiles of normal colon tissue from BarcUVa-Seq and GTEx. In addition to the integration of new datasets, CoTrEx 2.0 provides additional e/sQTLs sets, as well as gene expression prediction models and regulatory and co-expression networks. It is freely available at https://barcuvaseq.org/cotrex/. Overall, it is of high interest for researchers aiming to investigate the genetic susceptibility to colon-related complex traits and diseases.

The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer

OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated. METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM. RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P < 0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(–) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P < 0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells. CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.

Is histological remission of ulcerative colitis achievable?

Current conception of deep remission in patients with ulcerative colitis (UC) consists of clinical remission, endoscopic mucosal healing and normalization of laboratory markers. Histological remission should not be used as a primary end point for therapeutic efficacy, but instead should be considered as a marker of deep remission. The main goal of UC treatment should be focused on endoscopic healing of colon mucosa, decrease of inflammation activity, prolonged remission, absence of disease recurrence, and also histologic remission. Nevertheless, the term histologic remission has not yet been fully validated and no histologic indexes have been standardized. We need single unified definition for remission, based on multicentral studies analysis. One of important challenge is restoration of normal colon mucosal and results of multiple studies showed contradictory tests for assessing histologic remission, thus remaining an issue for further discussion.