Normal Human Colonic Cells Research Articles

Streptomyces nigra sp. nov. Is a Novel Actinobacterium Isolated From Mangrove Soil and Exerts a Potent Antitumor Activity in Vitro

A new bacterial strain, designated 452T, was isolated from the rhizosphere soil of the mangrove Avicennia marina in China. As determined, its cell wall peptidoglycan contained LL-diaminopimelic acid; MK-9(H8) and MK-9(H6) were the major isoprenoid quinones; and iso-C16:0 (31.3%), anteiso-C15:0 (16.9%), and iso-C15:0 (12.5%) were the major cellular fatty acids (>10.0%). Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain 452T formed a distinct lineage in the clade of the genus Streptomyces, and was closely related to S. coerulescens DSM 40146T (99.6% sequence identity), S. bellus DSM 40185T (99.5%), and S. coeruleorubidus DSM 41172T (99.3%). The DNA-DNA relatedness between strain 452T and these type strains ranged between 29.3 and 42.3%. Based on the phenotypic, chemotaxonomic, and phylogenetic features, the strain 452T is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces nigra sp. nov. is proposed. The type strain is 452T (=KCTC 39960T = MCCC 1K03346T). Further, strain 452T extracts exhibited a pronounced antitumor activity against human cancer cell lines A549, HCT-116, and HepG2, but not against normal human colon cells CCD-18Co. Active substances in the fermentation broth of strain 452T were isolated by bioassay-guided analysis, and then purified using a macroporous resin, silica gel, sephadex LX-20 column, and semi-preparative high-performance liquid chromatography (HPLC). Eight proline-containing diketopiperazines, namely, cyclo(Pro-Ala), cyclo(Pro-Gly), cyclo(Pro-Phe), cyclo(Pro-Met), cyclo(Pro-Val), cyclo(Pro-Leu), cyclo(Pro-Tyr), and cyclo(L-Leu-trans-4-hydroxy-L-Pro), were identified by electrospray ionization mass spectrometry (MS) and nuclear magnetic resonance (NMR). The compounds displayed different levels of cytotoxicity. The highest cytotoxicity was exhibited by cyclo(Pro-Ala) and cyclo(Pro-Met) against A549 cells, and cyclo(Phe-Pro) and cyclo(Pro-Ala) against HCT-116 cells, with average IC50 values equal to 18.5, 27.3, 32.3, and 47.6 μg/mL, respectively. The diversity of diketopiperazines and other chemicals produced by 452T was further investigated using gas chromatography (GC)-MS and liquid chromatography (LC)-MS. The analysis revealed 16 types of metabolites with antitumor activity and 16 other types of diketopiperazines. Hence, extracts of the newly identified strain may be used a starting material for the development of antitumor agents.

Increased Expression of the YPEL3 Gene in Human Colonic Adenocarcinoma Tissue and the Effects on Proliferation, Migration, and Invasion of Colonic Adenocarcinoma Cells In Vitro via the Wnt/b-Catenin Signaling Pathway.

BackgroundThe aim of this study was to investigate the effects of the expression of the YPEL3 gene in colonic adenocarcinoma cells grown in vitro and in colonic adenocarcinoma tissue from patients treated by surgical resection.Material/MethodsThe study included 108 patients diagnosed with primary colon cancer (Stages I to IV). The expression of the YPEL3 gene in colonic adenocarcinoma tissue and adjacent normal colonic tissue was detected by real-time quantitative PCR (qRT-PCR). The normal human colonic cell line CCD-1Co and colorectal adenocarcinoma cell lines HT-29 and HCT-8 were induced to overexpress the YPEL3 gene, and the effects on cell proliferation, migration, and invasion of colonic adenocarcinoma cells were investigated by the Cell Counting Kit-8 (CCK-8) assay, a transwell migration assay, and a transwell invasion assay, respectively. The effects of YPEL3 gene overexpression on the Wnt/β-catenin signaling pathway were detected by Western blot.ResultsIncreased expression levels of the YPEL3 gene were present in colon adenocarcinoma tissue compared with adjacent normal colonic tissue in 98 of 108 patients. Overexpression of the YPEL3 gene inhibited the proliferation, migration, and invasion of the HT-29 and HCT-8 colonic adenocarcinoma cells, and inactivated the Wnt/β-catenin signaling pathway; treatment with the Wnt agonist, CAS 853220-52-7, reduced the inhibitory effects of YPEL3 overexpression on proliferation, migration, and invasion in vitro.ConclusionsExpression of the YPEL3 gene was upregulated in human colonic adenocarcinoma tissue, and also inhibited the proliferation, migration, and invasion of colonic adenocarcinoma cells in vitro by inactivating the Wnt/β-catenin signaling pathway.

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.

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.

Polyphenols-rich extract from Pleurotus eryngii with growth inhibitory of HCT116 colon cancer cells and anti-inflammatory function in RAW264.7 cells.

Edible mushrooms are rich sources of bioactive components. In this study, a polyphenol-rich extract, designated as PPEP, was isolated from an edible mushroom, Pleurotus eryngii. Using ultra high performance liquid chromatograph combined with triple time-of-flight mass spectrometry (UPLC-TOF/MS/MS), gallic acid monohydrate, 3-(3,4-dihydroxyphenyl)-propionic acid, methyl gallate, syringic acid, ellagic acid and catechin were identified in PPEP. This phenolic-rich extract PPEP exhibited anti-inflammatory effect in lipopolysaccharide-stimulated RAW264.7 macrophages by inhibiting the overproduction of pro-inflammatory mediators including nitric oxide (NO) and reactive oxygen species (ROS). It was demonstrated that the anti-inflammatory effects of PPEP were associated with the inhibition of iNOS expression, suppression of p-IκB protein expression and inhibition of NF-κB and IκB mRNA expression. Next, the inhibitory effect of PPEP against human colon cancer cells was also determined. PPEP suppressed cell proliferation of human colon cancer HCT116 cells in a dose- and time-dependent fashion, while it showed no inhibitory effect on normal human colonic myofibroblasts CCD-18Co cells at the same tested concentrations (0-200 μg mL-1). Moreover, PPEP induced cell cycle arrest and led to extensive cellular apoptosis in human colon cancer cells, which was associated with the downregulation of cell cycle-related signaling protein, e.g. cyclin B and cyclin E, and the upregulation of apoptosis-related signaling protein caspase-3 and cleaved caspase-3. Overall, our results provided a basis for using PPEP as a promising preventive agent against inflammatory disease and colon cancer.

Characterization of methylthioadenosin phosphorylase (MTAP) expression in colorectal cancer

Purpose: Tumour seriously affects people's quality of life. Colorectal cancer is a refractory tumour in digestive tract tumors. In colorectal cancer, gene expression abnormalities is the main reason for its incidence, we mainly focus on the molecular mechanism of MTAP in the development of colorectal cancer.Methods: The tumour tissue and its adjacent tissue samples of 50 patients with colorectal cancer were screened from July 2011 to February 2015, and the expression of MTAP was detected. Cell lines that overexpress MTAP and low expression of MTAP were constructed in colorectal cancer cell lines. The cell proliferation, invasion and migration was detected in the cells with different expression levels of MTAP. Immunohistochemistry was used to detect the expression of MTAP in liver metastasis and to investigate its clinical significance. And statistics of clinical significance.Results: Q-PCR results showed that the expression of MTAP in colorectal cancer cell lines were significantly higher than that normal human colonic myofibroblasts cell line. Cell proliferation test results showed that cell proliferation was accelerated when MTAP was overexpression, cell invasion and migration were simultaneously accelerated. The expression of MTAP in primary liver was positively correlated with metastatic disease in patients with liver metastatic colorectal cancer via EMT.Conclusions: MTAP accelerates the growth and metastasis of colorectal cancer through EMT.

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.

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.

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.

Anti-glycation and anti-oxidative effects of a phenolic-enriched maple syrup extract and its protective effects on normal human colon cells.

Oxidative stress and free radical generation accelerate the formation of advanced glycation endproducts (AGEs) which are linked to several chronic diseases. Published data suggest that phenolic-rich plant foods, show promise as natural anti-AGEs agents due to their anti-oxidation capacities. A phenolic-enriched maple syrup extract (MSX) has previously been reported to show anti-inflammatory and neuroprotective effects but its anti-AGE effects remain unknown. Therefore, herein, we investigated the anti-glycation and anti-oxidation effects of MSX using biochemical and biophysical methods. MSX (500 μg mL-1) reduced the formation of AGEs by 40% in the bovine serum albumin (BSA)-fructose assay and by 30% in the BSA-methylglyoxal (MGO) assay. MSX also inhibited the formation of crosslinks typically seen in the late stage of glycation. Circular dichroism and differential scanning calorimeter analyses demonstrated that MSX maintained the structure of BSA during glycation. In the anti-oxidant assays, MSX (61.7 μg mL-1) scavenged 50% of free radicals (DPPH assay) and reduced free radical generation by 20% during the glycation process (electron paramagnetic resonance time scan). In addition, the intracellular levels of hydrogen peroxide induced reactive oxygen species were reduced by 27-58% with MSX (50-200 μg mL-1) in normal/non-tumorigenic human colon CCD-18Co cells. Moreover, in AGEs and MGO challenged CCD-18Co cells, higher cellular viabilities and rapid extracellular signal-regulated kinase (ERK) phosphorylation were observed in MSX treated cells, indicating its protective effects against AGEs-induced cytotoxicity. Overall, this study supports the biological effects of MSX, and warrants further investigation of its potential as a dietary agent against diseases mediated by oxidative stress and inflammation.

Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line.

AIMTo determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.METHODSNormal human colon epithelial cells (ATCC® CRL.1790™) were stimulated with either heat killed E. coli or heat killed murine cecal contents (HKC) and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production, mitochondrial autophagy and oxidant status. TNFα, IL-1β and IL-8 protein concentrations were measured within the supernatants. Fluorescent microscopy was performed to quantify the production of reactive oxygen species (ROS) using an oxidation responsive fluorogenic probe. Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria. Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells. Autophagy was detected by staining for the presence of autophagic vesicles. Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine. Mitochondrial morphology, ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software (ImageJ).RESULTSExposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress. Heat killed cecal contents treatment induced a 10-12 fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h (P < 0.001). Heat killed E. coli stimulation resulted in a 4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point (P < 0.001). Both heat killed E. coli and HKC stimulated robust ROS production at 6 (P < 0.001), and 12 h (P < 0.01). Mitochondrial morphologic abnormalities were detected at 6 and 12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential, P < 0.01. Microbial stimulation also induced significant autophagy at 6 and 12 h, P < 0.01. Lastly, blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.CONCLUSIONThe findings from this study suggest that CRL.1790 cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

Refractive index mapping of single cells with a graphene-based optical sensor

Graphene-based optical sensors exhibit good performance in cell refractive index (RI) measurements, with high resolution and large detection depths. In this manuscript, we report an RI mapping method based on the polarization-sensitive light absorption of graphene, in focused scanning mode. Several RI mapping images of normal and cancerous human colonic cells have been obtained. A high-numerical-aperture microscope objective is used to minimize the focusing spot size. Under optimized conditions, the experimental spatial resolution reaches 2.34μm. A ring-shaped perfect optical vortex beam with a tunable diameter is generated to control the incident angles, and the experimental RI resolution is 9.3×10−4 refractive index units. The large longitudinal penetration depth of the graphene evanescent field allows the recognition of RI difference between two individual cancer cells. The RI distribution and morphology of the cells can be determined, and different carcinogenesis stages can also be concluded from the image. This method has potential in biomedical sciences, especially in cell imaging, differentiation, and sorting.

Anti-inflammatory Effects of Herbal Preparations STW5 and STW5-II in Cytokine-Challenged Normal Human Colon Cells.

Inflammatory bowel diseases (IBD) are chronic relapsing intestinal disorders characterized by up-regulation of pro-inflammatory cytokines followed by invasion of immune cells to the intestinal lamina propria. Standard therapies consist of anti-inflammatory or immunosuppressive drugs. Since clinical efficiency is not satisfactory and the established drugs have massive side effects, new strategies to treat IBD are required. Herein, we investigate the protective effect of the fixed combination herbal preparations STW5 and STW5-II and the contribution of the corresponding single components in an in vitro inflammation model. The normal human colon epithelial cell line, NCM460, was treated with STW5, STW5-II or their single components for 4 h followed by experimental conditions comparable to induction of colitis. A pro-inflammatory cytokine cocktail consisting of TNF-α, IL-β, and IFN-γ was used to simulate inflammatory stimuli normally caused by immune cells. The effects on NCM460 cells were investigated by enzyme-linked immunoassay and Proteome Profiler®. Levels of IP-10, MCP-1, I-TAC, Groα, and IL-8 were elevated in chemokine-treated cells compared to untreated cells, but significantly reduced upon pretreatment with STW5 or STW5-II. However, the single compounds revealed only little effects on protein expression. Furthermore, we investigated the effect of both combination preparations on pro-inflammatory transcription factors of the STAT family using Western blot. In addition, we tested the effects on upstream MAPK p38. Both, STW5 and STW5-II did not show any effect on MAPK p38, but were effective in reducing phosphorylated levels of STAT1. In conclusion, both combination preparations act in an anti-inflammatory manner by influencing cytokine secretion via reduced activity of the JAK/STAT1 pathway. Relevant differences between STW5 and STW5-II were not found indicating similar efficacies.

Lactonic Sophorolipids Increase Tumor Burden in Apcmin+/- Mice.

Sophorolipids (SL) are amphiphilic biosurfactant molecules consisting of a disaccharide sophorose with one fatty acid at the C1 position and optional acetylation at the C6’and C6” positions. They exist in a closed ring lactonic (LSL) or open acidic (ASL) structure Sophorolipids are produced in crude mixtures in economically viable amounts by the yeast Starmerella bombicola and used in a variety of consumer products. Varying levels of anti- proliferative and anti-cancer activity of crude sophorolipid mixtures are described in a number of tumor cell lines in vitro. However, significant inter-study variation exists in the composition of sophorolipid species as well as other biologically active compounds in these mixtures, which makes interpretation of in vitro and in vivo studies difficult. We produced a 96% pure C18:1 lactonic sophorolipid that dose-dependently reduces the viability of colorectal cancer, as well as normal human colonic and lung cell lines in vitro. Oral administration of vehicle-only; or lactonic sophorolipids (50 mg/kg for 70 days), to Apcmin+/- mice resulted in an increase in the number (55.5 ± 3.3 vs 70.50 ± 7.8: p < 0.05) and size (modal size 2mm vs 4mm) of intestinal polyps. Lactonic administration resulted in a systematic effect via reduced hematocrit (49.5 ± 1.0 vs 28.2 ± 2.0 vs: p<0.03) and splenomegaly (0.56 ± 0.03g vs 0.71 ± 0.04g; p<0.01) confirming exacerbation of disease progression in this model.

Synergystic Interaction of Common Bean (Phaseolus vulgaris L.) Peptides and Oxaliplatin on Proliferation of HCT116 Colorectal Cancer Cells through Mitochondrial Depolarization and DNA Damage

The objectives of this study were to evaluate the antiproliferative effect of common bean (Phaseolus vulgaris L.) peptides on HCT116 human colorectal cancer cells and their interaction with oxaliplatin; furthermore, to determine their potential mechanisms of action. Pure peptides GLTSK, LSGNK, GEGSGA, MTEEY and MPACGS and the peptides in the non‐digestible fraction from common bean cultivars Azufrado Higuera (AH) and Bayo Madero (BM) were tested for cytotoxic effects on HCT116, and also on CCD‐33Co human normal colon cells. None of the peptides were toxic to normal cells at concentrations up to 1 mM. GLTSK (IC50=134.6 μM). On the contrary, according to MTS assay, GEGSGA (IC50=156.7 μM) exerted antiproliferative effects on HCT116 cells.. Based on the combination indexes (γ) of the isobolographic analysis, GLTSK (γ=0.64) and GEGSGA (γ=0.78) interacted synergistically (p &lt; 0.05) with oxaliplatin inhibiting HCT116 cells. Using flow cytometry, it was determined that GLTSK caused loss of mitochondrial membrane potential (Δψm) (16% of cells), increased (p &lt; 0.05) in intracellular reactive oxygen species (ROS) (12.1‐fold compared with untreated cells), cell cycle arrest in G2 (54% of cells) and induced apoptosis (33% of cells). GEGSGA had a limited effect on Δψm and intracellular ROS, but caused arrest in G1 phase (64% of cell population) and promoted increase (p &lt; 0.05) in p53 (290%) and cleavage of PARP (184%) compared to untreated cells, promoting DNA damage. The different mechanisms of action were further established by confocal microscopy. An increase (p &lt; 0.05) in the expression and activation of nuclear p53 (145% of fluorescence intensity/μm2) was observed in response to GEGSGA treatment compared to untreated cells in a time‐frame of 3 to 8 h. Moreover, GLTSK depolarized the cells (38% decrease of fluorescence intensity/μm2) and increased (p &lt; 0.05) intracellular ROS (180% of fluorescence intensity/μm2). In addition, peptide fractions from bean cultivar Azufrado Higuera interacted synergistically with oxaliplatin inducing apoptosis (49% of cells), loss of Δψm (21.7% of cells) and increase of intracellular ROS (13.1‐fold compared to untreated cells). This is the first report demonstrating that dietary peptides from common bean interact synergistically with oxaliplatin inhibiting HCT116 human cancer cells. These results support the hypothesis that physicochemical properties of peptides would determine their mechanism of action.Support or Funding InformationBasic Science, National Council for Science and Technology, Mexico CONACyT‐grant number 220924

Various Acylglycerols from Common Oils Exert Different Antitumor Activities on Colorectal Cancer Cells

ABSTRACTColorectal cancer is one of the leading causes of death in Western countries; therefore, the implementation of healthy dietary habits in order to prevent its occurrence is a desirable action. We show here that both free fatty acids (FFAs) and some acylglycerols induce antitumoral actions in the colorectal cancer cell line HT-29. We tested several C18 polyunsaturated fatty acid-enriched oils (e.g., sunflower and Echium) as well as other oils, such as arachidonic acid-enriched (Arasco®) and docosahexaenoic acid-enriched (Marinol® and cod liver oil), in addition to coconut and olive oils. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test indicated inhibitory effects on HT-29 cells viability for FFAs, and monoacylglycerol and diacylglycerol (DAG) species, while the lactate dehydrogenase test proved that FFAs were the more effective species to induce membrane injury. Conversely, all species did not exhibit actions on CCD-18 normal human colon cells viability.Furthermore, transmission electron microscopy showed the presence of necrosis and apoptosis, while the monoacylglycerol lipase (MAGL) inhibition test demonstrated high activity for 2-monoacylglycerols derived from Arasco and sunflower oils. However, different monoacylglycerols and DAGs have also the potential for MAGL inhibition. Therefore, checking for activity on colon cancer cells of specifically designed acylglycerol-derivative species would be a suitable way to design functional foods destined to avoid colorectal cancer initiation.

Bifidobacterially produced, C18:3 and C18:4 conjugated fatty acids exhibit in vitro anti‐carcinogenic and anti‐microbial activity

Conjugated fatty acid (CFA)s have shown a range of in vitro and in vivo bioactivities prompting increased endeavours both to identify novel sources and to characterize their properties. Strains of bifidobacteria possess the capacity to produce a range of CFAs including conjugated α‐linolenic acid (CLNA), conjugated γ‐linolenic acid (CGLA), and conjugated stearidonic acid (CSA), and in the current study these CFA were assessed for anti‐proliferative activity using the SW480 colon cancer cell line. Additionally, these fatty acids were also assessed for inhibitory activity against select gastro‐intestinal pathogens and potential probiotic CFA producers. The results demonstrated that the CFA displayed greater inhibitory activity against the SW480 cells than a normal fetal human colonic cell line (FHC), suggesting selective anti‐proliferative potential in vitro. Studies into the mechanism behind this inhibitory activity indicated potential roles for increased cellular lipid peroxidation, altered cellular phospholipid composition, and reductions in the cellular content of Bcl‐2. Microbiological studies also demonstrated that the CFA displayed inhibitory activity against selected pathogens, while CFA producing bifodobacteria remained unaffected. CFAs were particularly effective in the inhibition of the methicillin resistant strain Staphylococcus aureus ATCC 43300.Practical applications: We have demonstrated that bifidobacterially produced C18:3 and C18:4 CFAs may have potential in the control of colon cancer. Additionally, their inhibitory properties against common gastrointestinal pathogens (particularly the methicillin resistant strain S. aureus ATCC 43300) and not potential probiotic CFA producing bifidobacteria may indicate further therapeutic potential or a metabolic process that gives CFA producing microbes a competitive advantage in the complex environment that is the gastrointestinal tract.Strains of bifidobacteria such as Bifidobacterium breve DPC 6330 possess the capacity to bioconvert α‐linolenic, γ‐linolenic, and stearidonic acids to their conjugated derivatives under fermentative conditions. In vitro bioassays have indicated that these conjugated derivatives possess anti‐microbial activity, particularly against microbial pathogens including Staphyloccus aureus, and anti‐carcinogenic properties against colonic cancer cell lines such as SW480.

Selective mechanism of action of dietary peptides from common bean on HCT116 human colorectal cancer cells through loss of mitochondrial membrane potential and DNA damage

The inhibitory effect of pure peptides from common bean on human colorectal cancer cells, their ability to enhance oxaliplatin effects, and mechanisms of action were evaluated. Peptides GLTSK, LSGNK, GEGSGA, MTEEY, and MPACGSS were tested for cytotoxic effects on HCT116 and CCD-33Co human normal colon cells; no peptide was toxic to normal cells. GLTSK (IC50 = 134.6 µM) and GEGSGA (IC50 = 156.7 µM) exerted anti-proliferative effects on HCT116 cells; LSGNK, MTEEY, and MPACGSS were less potent. GLTSK (γ = 0.64) and GEGSGA (γ = 0.78) interacted synergistically with oxaliplatin inhibiting HCT116 cells. GLTSK caused loss of mitochondrial potential (Δψm) (15.8%) and increased intracellular ROS (12.1-fold) suggesting mitochondrial membrane disruption. GEGSGA caused arrest in G1 phase (63.6% of cell population) and promoted cleavage of PARP (183.7%) suggesting DNA damage. GEGSGA and oxaliplatin caused activation and nuclear translocation of p53. Thus, peptides from common beans selectively induced apoptosis through loss of Δψm and DNA damage on human colorectal cancer cells.