Normal Human Colon Research Articles

P025 CYSTATHIONINE-GAMMA-LYASE REGULATES MACROPHAGE INTERLEUKIN-10 (IL-10) PRODUCTION IN COLITIS

Cystathionine-β-synthase (CBS) & cystathionine-γ-lyase (CSE), convert homocysteine into cysteine & produce H2S, an anti-inflammatory mediator. In rodent colitis models, CBS & CSE activity is elevated and inhibition or knockout of CSE worsens colitis & decreases levels of anti-inflammatory cytokine IL-10. IL-10 KO mice develop spontaneous colitis accompanied by homocysteinemia & decreased H2S production, which are reversed by IL-10 administration. However, the cellular source of CSE-dependent IL-10 production is unknown. We hypothesize that macrophage-derived CSE is critical for IL-10 production. Human colitis & normal colon specimens were collected. CSE knockout (CSE KO) mice and wild-type (WT) controls received either 7 days of colitis-inducer DSS (2% in drinking water) or vehicle. CSE & cell-type specific markers were evaluated in human/mouse colons by immunofluorescent microscopy & flow cytometry. Peritoneal macrophages from CSE KO & WT mice (N = 6) were isolated & treated with CSE inhibitor propargyl glycine (PAG), H2S donor NaHS or vehicle. IL-10 & β-actin expression levels were determined by qRT-PCR. Confocal microscopy of colitis & normal human colon tissue localized CSE to cells of the lamina propria expressing CD45, a marker of hematopoietic cells. Colonic tissue from mice receiving either DSS or vehicle also showed CSE staining in the lamina propria co-localized with macrophage marker F4/80. Flow cytometry showed co-expression of CSE with F4/80 but not Treg marker FOXP3. Comparison of macrophages from CSE KO to WT mice revealed a 6-fold reduction of IL-10 mRNA. Further, treatment of macrophages from WT mice with PAG inhibited IL-10 expression compared to controls. Addition of NaHS did not alter CSE KO macrophage expression of IL-10. We demonstrate that in macrophages CSE & IL-10 expression are linked in an H2S-independent manner. This suggests that CSE regulates inflammation through both H2S & IL-10.

Preliminary Report: Multiple Clusters of Proliferating Cells in Non-dysplastic Corrupted Colonic Crypts Underneath Conventional Adenomas.

The finding was recently reported of clusters of colonic crypts lined with indigenous normal epithelium displaying irregular shapes underneath the adenomatous glands of conventional (tubular or villous) adenomas. These abnormal crypts were named non-dysplastic corrupted colonic crypts (NDCs). This study explored the characteristics of cell proliferation in NDCs present in a cohort of conventional adenomas. Sections from six conventional adenomas were challenged with the proliferation marker Ki-67 (MIB1). MIB+ proliferating clusters were regarded as those exhibiting two or more adjoining MIB+ cells. A total of 46 (range=1-18) NDCs were found underneath the six conventional adenomas. Out of the 46 NDCs, two exhibited only one proliferative cluster/crypt, 14 NDCs two clusters/crypt, 14 three clusters/crypt and the remaining 16 NDCs more than four distinct clusters/crypt. This preliminary study showed, evidently for the first time, that multiple, apparently haphazardly distributed clusters of proliferating cells are present in NDCs. Since the Ki-67 proliferation marker only labels progenitor daughter cells generated by stem cells, each MIB+ cluster in each NDC must have been produced by a single stem cell. Consequently, individual NDCs may harbor several stem cells, a deduction that is in concert with recent studies showing that in the normal human colon, the number of stem cells per crypt is of the order of five to six, or about 5% of the cell population of a single crypt.

P096 RADIATION AND IDO1-SELECTIVE INHIBITOR EPACADOSTAT EXHIBIT THERAPEUTIC SYNERGY IN COLITIS ASSOCIATED CANCER

We have previously demonstrated that gene deletion of the immunosuppressive enzyme IDO1 (indoleamine-2,3-dioxygenase 1) reduces but does not eliminate tumor burden in a mouse model of colitis associated cancer (CAC). In the current study, we test the hypothesis that a new highly specific IDO1 inhibitor epacadostat (Incyte Corp.) will synergize with cytotoxic radiation therapy (RT) as a novel treatment strategy for CAC. CAC was modeled in vivo and in vitro using IDO1 expressing colorectal cancer (CRC) cell lines from mice (CT26, MC38) and humans (HT29, HCT116, and DLD1) cell lines, as well as normal human primary epithelial colonoids and normal colon and small intestinal (SI) cell lines (FHC and IEC6, respectively). Standard molecular biology assays were used to assay gene expression, proliferation (WST), cell death (LDH) and apoptosis (TUNEL/Caspase). CAC was modeled with heterotopic implants with treatment groups of control, epacadostat (300mg/kg/day), focal RT, and epacadostat+ focal RT (n=7/group). Tumor infiltrating lymphocytes (TIL) were examined by flow cytometry and cytokine multiplex assays. In Vitro and in vivo: RT enhanced IDO1 gene expression in CRC cells, but not in normal colon or SI cells. The combination of epacadostat and RT decreased CRC proliferation and colony formation (p<0.01) more than either monotherapy in vitro. In vivo, the combinationalso reduced the tumor growth and size than either treatment alone (Figure 1a, b). Impressively, unilateral RT also enhanced anti-tumor effect of epacadostat in the contralateral unirradiated tumor indicating an enhancement of the recently described abscopal effect. Consistent with this, TILs and cytokine profiles demonstrated enhanced anti-tumor immune environment. These data identify a therapeutic synergy of IDO1 inhibition and RT in CAC/CRC. A Phase 1 clinical study examining this combination strategy is being initiated.

Reconstruction of the Human Colon Epithelium InVivo.

Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs invivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium invivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer anLGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling invivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs invivo, with potential therapeutic applications in regenerative medicine.

Biphasic regulation of spindle assembly checkpoint by low and high concentrations of resveratrol leads to the opposite effect on chromosomal instability

Resveratrol (RSV) is a naturally occurring polyphenolic phytoalexin possessing numerous health-promoting effects. Chromosomal instability (CIN), usually results from defective spindle assembly checkpoint (SAC), is a major contributor to many diseases. While it’s recently recognized that RSV exhibits a nonlinear dose response for disease prevention, whether it’s the case for its role in CIN remains unknown. Here, we investigated the potential of a broad range of RSV concentrations (0.01–100μM) on CIN and the underlying mechanisms in human normal colon epithelial NCM460 cells. CIN was measured by cytokinesis-block micronucleus assay; mitotic fidelity was determined by aberrant mitosis analysis; SAC activity was assessed by nocodazole-challenge assay, and the expression of SAC genes was examined by RT-qPCR. We found that 0.1μM RSV significantly reduced CIN (P<0.01), while 100μM RSV significantly induced it (P<0.05). Mitotic infidelity was significantly prevented by 0.1μM RSV but promoted by 100μM RSV (P<0.05 for both). Moreover, the function of SAC was sustained and impaired by 0.1μM and 100μM RSV, respectively. Several SAC genes, including Aurora-B, Aurora-C, Plk-1 and CENP-E, were significantly up-regulated and down-regulated by 0.1μM and 100μM RSV, respectively (P<0.05). In conclusion, RSV exhibited a biphasic dose-dependent effect on CIN that was exerted via the regulation of mitotic fidelity through the SAC network. The health implications of these findings were summarized.

Supercritical carbon dioxide-developed silk fibroin nanoplatform for smart colon cancer therapy.

PurposeTo deliver insoluble natural compounds into colon cancer cells in a controlled fashion.Materials and methodsCurcumin (CM)–silk fibroin (SF) nanoparticles (NPs) were prepared by solution-enhanced dispersion by supercritical CO2 (SEDS) (20 MPa pressure, 1:2 CM:SF ratio, 1% concentration), and their physicochemical properties, intracellular uptake efficiency, in vitro anticancer effect, toxicity, and mechanisms were evaluated and analyzed.ResultsCM-SF NPs (<100 nm) with controllable particle size were prepared by SEDS. CM-SF NPs had a time-dependent intracellular uptake ability, which led to an improved inhibition effect on colon cancer cells. Interestingly, the anticancer effect of CM-SF NPs was improved, while the side effect on normal human colon mucosal epithelial cells was reduced by a concentration of ~10 μg/mL. The anticancer mechanism involves cell-cycle arrest in the G0/G1 and G2/M phases in association with inducing apoptotic cells.ConclusionThe natural compound-loaded SF nanoplatform prepared by SEDS indicates promising colon cancer-therapy potential.

Perilla frutescens Extracts Protects against Dextran Sulfate Sodium-Induced Murine Colitis: NF-κB, STAT3, and Nrf2 as Putative Targets.

Perilla frutescens is a culinary and medicinal herb which has a strong anti-inflammatory and antioxidative effects. In the present study, we investigated the effects of Perilla frutescens extract (PE) against dextran sulfate sodium (DSS)-induced mouse colitis, an animal model that mimics human inflammatory bowel disease (IBD). Five-week-old male ICR mice were treated with a daily dose of PE (20 or 100 mg/kg, p.o.) for 1 week, followed by administration of 3% DSS in double distilled drinking water and PE by gavage for another week. DSS-induced colitis was characterized by body weight loss, colon length shortening, diarrhea and bloody stool, and these symptoms were significantly ameliorated by PE treatment. PE administration suppressed DSS-induced expression of proinflammatory enzymes, including cyclooxygenase-2 and inducible nitric oxide synthase as well as cyclin D1, in a dose-dependent fashion. Nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription 3 (STAT3) are major transcriptional regulators of inflammatory signaling. PE administration significantly inhibited the activation of both NF-κB and STAT3 induced by DSS, while it elevated the accumulation of Nrf2 and heme oxygenase-1 in the colon. In another experiment, treatment of CCD841CoN human normal colon epithelial cells with PE (10 mg/ml) resulted in the attenuation of the tumor necrosis factor-α-induced expression/activation of mediators of proinflammatory signaling. The above results indicate that PE has a preventive potential for use in the management of IBD.

ToF-SIMS and principal component analysis of lipids and amino acids from inflamed and dysplastic human colonic mucosa.

Here, time of flight secondary ion mass spectrometry (ToF-SIMS) and multivariate analysis were combined to study the role of ulcerative colitis (UC), a type of inflammatory bowel disease (IBD), in the colon cancer progression. ToF-SIMS was used to obtain mass spectra and chemical maps from the mucosal surface of human normal (NC), inflamed (IC), and dysplastic (DC) colon tissues. Chemical mapping with a lateral resolution of ≈1μm allowed to evaluate zonation of fatty acids and amino acids as well asthe morphological condition of the intestinal glands. High mass resolution ToF-SIMS spectra showed chemical differences in lipid and amino acid composition as a function of pathological state. In positive ion mode, mono- (MAG), di- (DAG), and triacylglycerol (TAG) signals were detected in NC tissues, while in IC and DC tissues, the only cholesterol was present as lipid class representative. Signals from fatty acids, collected in negative ion mode, were subjected to principal component analysis (PCA). PCA showed a strict correlation between IC and DC samples, due to an increase of stearic, arachidonic, and linoleic acid. In the same way, differences in the amino acid composition were highlighted through multivariate analysis. PCA revealed that glutamic acid, leucine/isoleucine, and valine fragments are related to IC tissues. On the other hand, tyrosine, methionine, and tryptophan peaks contributed highly to the separation of DC tissues. Finally, a classification of NC, IC, and DC patients was also achieved through hierarchical cluster analysis of amino acid fragments. In this case, human colonic inflammation showed a stronger relationship with normal than dysplastic condition. Graphical Abstract ᅟ.

Mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells: reversal by non-steroidal anti-inflammatory drugs.

Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

Photo-catalytic, anti-bacterial, and anti-cancer properties of phyto-mediated synthesis of silver nanoparticles from Artemisia tournefortiana Rchb extract

Metal nanoparticles have largely been investigated due to their potential medicinal activities. This study demonstrates the biological properties of green-synthesized silver nanoparticles (AgNPs) by using Artemisia tournefortiana Rchb ethanol extract. Instrumentations such as ultraviolet-visible spectra analysis, high-resolution transmission electron microscopy, scanning electron microscopy, energy-dispersive X-ray analysis, X-ray diffraction, and Fourier transform infrared spectroscopy were used to reveal the synthesized AgNPs. Microscopic results showed that the particles were mostly spherical in shape, having an average diameter of 22.89±14.82nm. The antibacterial activity of the phyto-fabricated AgNPs was investigated by the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The in vitro cytotoxicity effect was investigated against normal human embryonic kidney (HEK293) cells and human colon adenocarcinoma cancer (HT29) cells. The apoptotic cells were identified by annexin V/PI FITC staining, and morphological assessment. The expressions of Bax and Bcl2 were evaluated by quantitative real time PCR method. The phyto-synthesized AgNPs have shown increased cell apoptosis and demonstrated dose-dependent cytotoxicity in HT29 cancer cells. Moreover, the photocatalytic activity of the phyto-synthesized AgNPs was evaluated by degradation of Coomassie Brilliant Blue G-250 under UV light exposure and these fabricated Ag nanoparticles demonstrated efficacy in degrading the dye within 60min. Overall, the present results highlighted the antibacterial and anticancer properties of fabricated AgNPs, suggesting that phyto-synthesized silver nanoparticles could possess potent anti-pathogenic bacteria and anti-colon cancer activities.

Different molecular organization of two carotenoids, lutein and zeaxanthin, in human colon epithelial cells and colon adenocarcinoma cells

Two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of exogenous carotenoids, either zeaxanthin or lutein. Both carotenoids demonstrated cytotoxicity with respect to cancer cells but not to normal cells. Cells from both the cell lines were analyzed with application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. Both imaging techniques show effective incorporation of carotenoid molecules into growing cells. Comparison of the Raman scattering and fluorescence lifetime characteristics reveals different molecular organization of carotenoids in the carcinoma and normal cells. The main difference consists in a carotenoid aggregation level which is substantially lower in the carcinoma cells as compared to the normal cells. Different molecular organization of carotenoids was interpreted in terms of a different metabolism of normal and carcinoma cells and has been concluded to provide a possibility of cancer diagnosis based on spectroscopic analyses.

Reduction of hydrogen sulfide synthesis enzymes cystathionine-β-synthase and cystathionine-γ-lyase in the colon of patients with Hirschsprungs disease

PurposeHirschsprung associated enterocolitis (HAEC) is the most common cause of morbidity and mortality in Hirschsprung Disease (HSCR). The pathogenesis of HAEC is poorly understood. In recent years, there is increasing evidence that a compromised intestinal barrier function plays a major role in the pathogenesis of HAEC. Hydrogen sulfide, synthesized from L-cysteine by two key enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lysase (CSE) is reported to play a key role in regulating gastrointestinal motility and promoting resolution of inflammation. We designed this study to test the hypothesis that CBS and CSE expression is altered in the colon of patients with HSCR. MethodsWe investigated CBS and CSE protein expression in both the aganglionic and ganglionic regions of HSCR patients (n=10) versus healthy control colon (n=10). Protein distribution was assessed by using immunofluorescence and confocal microscopy. Gene and protein expression was quantified using quantitative real-time polymerase chain reaction (qPCR), Western blot analysis, and densitometry. Main resultsqPCR and Western blot analysis revealed that CBS and CSE are expressed in the normal human colon. CBS and CSE expression was significantly decreased (p<0.003) in the ganglionic and aganglionic bowel in HSCR compared to controls. Confocal microscopy revealed that CBS and CSE expression in smooth muscles, interstitial cells of Cajal, platelet-derived growth factor-alpha receptor-positive cells, enteric neurons and colonic epithelium was markedly decreased in HSCR specimens compared to controls. ConclusionWe demonstrate for the first time the expression and distribution of CBS/CSE in patients with HSCR. The observed decreased expression of CBS and CSE may affect mucosal integrity and colonic contractility and thus render HSCR patients more susceptible to develop HAEC.

RUFY3 interaction with FOXK1 promotes invasion and metastasis in colorectal cancer

RUFY3 is highly expressed in brain tissue and has a role in neuronal development. Transcriptional factor FOXK1 is involved in cell growth and metabolism. We knew that RUFY3 or FOXK1 has been correlated with the malignant of tumor cells. However, the role of these molecules in colorectal cancer (CRC) progression remains unknown. We investigated the protein expression levels by Western blot, immunofluorescence and immunohistochemistry analyses. The migration and invasive abilities of CRC cells were assessed using shRNA-mediated inhibition in vitro and in vivo. We showed that RUFY3 expression was up-regulated in CRC compared with its expression in a normal human colon cell line (FHC). RUFY3 suppression inhibited anchorage independent cell tumorigenesis. RUFY3 induced elevated expression of eight major oncogenes. Moreover, RUFY3 physically interacts with FOXK1 in CRC. A positive correlation was observed between the expression patterns of RUFY3 and FOXK1. Furthermore, RUFY3 and FOXK1 expression were correlated with tumor progression and represented significant predictors of overall survival in CRC patients. SiRNA-mediated repression of FOXK1 in RUFY3-overexpressing cells reversed the epithelial-mesenchymal transition (EMT) and metastatic phenotypes. In vivo, FOXK1 promoted RUFY3-mediated metastasis via orthotopic implantation. These findings suggest that the RUFY3-FOXK1 axis might promote the development and progression of human CRC.

The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02µM, whereas for NCM460 cells 0.56µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca2+ concentration, but decreased GSH concentration in the cells. The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

Cryptotanshinone induces ROS-dependent autophagy in multidrug-resistant colon cancer cells

The development of novel chemotherapeutic agents is highly desired for colon cancer treatment, in particular for the multidrug-resistant cancer types. Cryptotanshinone (CTS), an active quinoid diterpene isolated from Salvia miltiorrhiza Bunge, was previously reported to induce autophagy in various colon cancer cell lines. However, its mechanisms of action have not been fully understood. The current study aims to explore the mechanisms by which CTS induces autophagy in a multidrug-resistant human colon cancer cell line SW620 Ad300. Using MTT assay, CTS at 10 μM exhibited no significant cytotoxicity on human normal colon fibroblasts CCD-18Co, but induced 45.67% and 48.35% cell death in SW620 and SW620 Ad300 cells, respectively. Further studies revealed that CTS induced weak apoptosis (9.37%) and significant caspase-independent cell death in SW620 Ad300 cells. In the same cell line, CTS also induced significant autophagy, which was found to promote cell death and to mediate the cytotoxicity of CTS in these multidrug-resistant cells. Moreover, activation of ROS-p38 MAPK-NF-κB signaling pathway was involved in autophagic cell death induced by CTS in SW620 Ad300 cells. Interestingly, our results also demonstrated a complementary relationship between CTS-induced apoptosis and autophagic cell death in SW620 Ad300 cells. Taken together, CTS induces autophagic cell death in SW620 Ad300 cells via the ROS-p38 MAPK-NF-κB signaling pathway, and it might be a potential candidate as a chemotherapeutic agent for the treatment of multidrug-resistant colon cancer.

Evaluation of anticancer activity of water and juice extracts of young Hordeum vulgare in human cancer cell lines HT-29 and A549.

Introduction and objective. Barley (Hordeum vulgare L.) is known as a rich source of different bioactive compounds. At present, considerable attention of researchers is focused on young barley grass. It can be a good source of dietary minerals, vitamins, carbohydrates, amino acids, phenolic compounds and proteins. It is possible that the composition of chemical ingredients beneficial for health may induce an anticancer potential of young barley in human colon adenocarcinoma (HT-29) and human lung adenocarcinoma (A549) cell lines. Materials and method. Hordeum vulgare water extract (HWE) and Hordeum vulgare juice extract (HJE) were prepared. Cell proliferation and viability were examined with the use of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and NR (neutral red) methods. Induction of necrosis was assessed by propidium iodide/Hoechst staining. Progress of the cell cycle involved in cell proliferation, apoptosis, and regulation of transcription was estimated using flow cytometry analysis. Additionally, the capability of free radical scavenging was evaluated with the DPPH assay. Results. The study revealed that extracts inhibited the proliferation of cancer cells. The NR study confirmed the low cytotoxic activity of the tested extracts to normal human colon epithelial cells (CCD 841 CoTr) and human skin fibroblasts (HSF). Furthermore, a dose-dependent cytotoxicity against HT-29 cells, but not A549 cells, has been reported. The free radical scavenging activity was observed in the case of the HWE but not the HJE. Conclusions. The obtained results indicate a cancer chemopreventive potential of young barley as a safe dietary agent in colon carcinoma.

DNA hypomethylation of CBS promoter induced by folate deficiency is a potential noninvasive circulating biomarker for colorectal adenocarcinomas.

Aberrant DNA methylation patterns, which induced by folate deficiency, play important roles in tumorigenesis of colorectal cancer (CRC). Some DNA methylation alterations can also be detected in cell-free DNA (cfDNA) of patients’ plasma, making cfDNA an ideal noninvasive circulating biomarker. However, exact DNA methylation alterations induced by folate deficiency in tumorigenesis of CRC and exact potential circulating cfDNA methylation biomarker are still unclear. Therefore, DNA methylation patterns of the normal human colon mucosal epithelial cell line (NCM460), cultured with normal or low folate content, were screened and the DNA hypomethylation of cystathionine-beta-synthase (CBS) promoter was further validated in vitro and vivo. Then, the correlation analysis between folate level, DNA methylation alteration in promoter and expression of CBS was carried out in vitro and vivo. Further, the methylation patterns of CBS promoter in plasma cfDNA were detected and statistically correlated with pathological parameters and clinical outcome. Our study showed that DNA hypomethylation in CBS promoter, induced by folate deficiency, would lead to up-regulation of CBS both in vitro and vivo. Patients with cfDNA hypomethylation of CBS promoter in plasma were correlated with high tumor stage and poor clinical outcome. In addition, cfDNA hypomethylation of CBS promoter in plasma was shown to be an independent prognostic factor for recurrence and cancer-related death in CRC. Our results indicated that DNA hypomethylation of CBS promoter induced by folate deficiency could serve as a potential noninvasive circulating biomarker and may be helpful in developing more effective prognostic markers for CRC.

Streptomyces colonosanans sp. nov., A Novel Actinobacterium Isolated from Malaysia Mangrove Soil Exhibiting Antioxidative Activity and Cytotoxic Potential against Human Colon Cancer Cell Lines.

Streptomyces colonosanans MUSC 93JT, a novel strain isolated from mangrove forest soil located at Sarawak, Malaysia. The bacterium was noted to be Gram-positive and to form light yellow aerial and vivid yellow substrate mycelium on ISP 2 agar. The polyphasic approach was used to determine the taxonomy of strain MUSC 93JT and the strain showed a range of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus Streptomyces. Phylogenetic and 16S rRNA gene sequence analysis indicated that closely related strains include Streptomyces malachitofuscus NBRC 13059T (99.2% sequence similarity), Streptomyces misionensis NBRC 13063T (99.1%), and Streptomyces phaeoluteichromatogenes NRRL 5799T (99.1%). The DNA–DNA relatedness values between MUSC 93JT and closely related type strains ranged from 14.4 ± 0.1 to 46.2 ± 0.4%. The comparison of BOX-PCR fingerprints indicated MUSC 93JT exhibits a unique DNA profile. The genome of MUSC 93JT consists of 7,015,076 bp. The DNA G + C content was determined to be 69.90 mol%. The extract of strain MUSC 93JT was demonstrated to exhibit potent antioxidant activity via ABTS, metal chelating, and SOD assays. This extract also exhibited anticancer activity against human colon cancer cell lines without significant cytotoxic effect against human normal colon cells. Furthermore, the chemical analysis of the extract further emphasizes the strain is producing chemo-preventive related metabolites. Based on this polyphasic study of MUSC 93JT, it is concluded that this strain represents a novel species, for which the name Streptomyces colonosanans sp. nov. is proposed. The type strain is MUSC 93JT (= DSM 102042T = MCCC 1K02298T).

Transcriptomic Analysis of Calcium Remodeling in Colorectal Cancer.

Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca2+ homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca2+ remodeling includes critical changes in store-operated Ca2+ entry (SOCE) and Ca2+ store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca2+ transport, a comprehensive view of Ca2+ remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca2+ transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca2+ channels and store-operated Ca2+ entry players, transient receptor potential (TRP) channels, Ca2+ release channels, Ca2+ pumps, Na+/Ca2+ exchanger isoforms and genes involved in mitochondrial Ca2+ transport. These data provide the first comprehensive transcriptomic analysis of Ca2+ remodeling in CRC.

FAM83D knockdown regulates proliferation, migration and invasion of colorectal cancer through inhibiting FBXW7/Notch-1 signalling pathway

Colorectal cancer is one of the most common malignant tumors in the digestive system, and in China its incidence is rising in recent years. The FAM83D family with sequence similarity 83, member D is associated with the occurrence and development of various cancers. However, in human colorectal cancer, the biological function of FAM83D and its molecular mechanism are still little known. In our study, we found that FAM83D mRNA expression level was markedly up-regulated in colorectal cancerous tissues when compared with that of adjacent normal colon tissues. We also found that the protein and mRNA expression levels of FAM83D are dramatically increased in human colorectal cancer cell lines, including Caco-2, RKO, DLD-1, HT-29, LoVo, SW480, and HCT116, especially in SW480 and HCT116 cells, when compared with that of human normal colon epithelial cell line (NCM460). Next, in HCT116 and SW480 cells, the biological function of FAM83D was examined. FAM83D-knockdown notably inhibited cell proliferation and colony formation. Cell apoptosis was promoted by FAM83D knockdown. In addition, FAM83D siRNA decreased cell migration and invasion. Moreover, FAM83D knockdown up-regulated the protein expression level of F-box and WD repeat domain-containing 7 (FBXW7), but diminished the Notch1 protein expression level. It also found that FBXW7 siRNA reverses the suppressive effect of FAM83D knockdown on Notch1 protein expression. Notch1 overexpression reversed the effect of FAM83D knockdown on colorectal cancer cell proliferation, cell migration and invasion. In conclusion, FAM83D knockdown promoted colorectal cancer cell apoptosis, inhibited cell proliferation, cell migration and invasion, which might be associated with inhibiting the FBXW7/Notch1 signal pathway. Our findings indicated that FAM83D is a promising molecular target for colorectal cancer treatment.