Human Epithelial Colorectal Adenocarcinoma Caco-2 Research Articles

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

Roles of Alternative Sigma Factors in Invasion and Growth Characteristics of Listeria monocytogenes 10403S Into Human Epithelial Colorectal Adenocarcinoma Caco-2 Cell.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth.

Correlations between molecular structure and biological activity in "logical series" of dietary chromone derivatives

The research was conducted in the “logical series” of seven ligands: chromone, flavone, 3-hydroxyflavone, 3,7-dihydroxyflavone, galangin, kaempferol and quercetin. Each subsequent ligand differs from the previous one, among others by an additional hydroxyl group. The studied chromone derivatives are plant secondary metabolites which play an important role in growth, reproduction, and resistance to pathogens. They are important food ingredients with valuable pro-health properties. The studies of the relationships between their molecular structure and biological activity facilitate searching for new chemical compounds with important biological properties not by trial and error, but concerning the impact of specific changes in their structure on the compound properties. Therefore several pectroscopic methods (FT-IR, FT-Raman, 1H and 13C NMR) were applied to study the molecular structure of the compounds in the series. Moreover the quantum-chemical calculations at B3LYP/6-311++G** were performed to obtained the theoretical NMR spectra, NBO atomic charge, global reactivity descriptors and thermodynamic parameters. The antioxidant activity of the compounds was tested in the DPPH and FRAP assays and the mechanism of antioxidant activity was discussed based on the results on theoretical calculations. The cytotoxicity of the ligands toward human epithelial colorectal adenocarcinoma Caco2 cells was estimated and correlated with the lipophilicity of the compounds. The principal component analyses (PCA) and hierarchical cluster analysis were used to study the dependency between the molecular structure of ligands and their biological activity. The experimental data were related to the theoretical ones. The found regular changes in physicochemical properties correlated well with the systematic changes in antioxidant and biological properties.

Characterization of Riboflavin-Producing Strains of Lactobacillus plantarum as Potential Probiotic Candidate through in vitro Assessment and Principal Component Analysis.

Lactic acid bacteria (LAB) are known for their probiotic properties, but only a few strains produce riboflavin. We evaluated the probiotic properties of four riboflavin-producing strains of Lactobacillus plantarum (BBC33, BBC32A, BIF43, and BBC32B) by using in vitro assessment and carried out multivariate principal component analysis (PCA) to select the best strain. Safety, antioxidant, and exopolysaccharide-producing properties were also studied. Lact. plantarum BBC33 showed better probiotic potential, followed by strain BIF43. Lact. plantarum BBC32A degraded mucin and excluded as a potential probiotic candidate. Lact. plantarum BIF43, BBC33, and BBC32A tolerated simulated gastrointestinal conditions and their overnight cell-free culture supernatants (CFSs, pH4.0-4.3) inhibited the growth of Escherichia coli AF10, Salmonella Typhi MTCC98, Bacillus cereus NCDC250, and Pseudomonas aeruginosa NCDC105. Lact. plantarum BIF43 and BBC33 did not degrade mucin, adhered to human epithelial colorectal adenocarcinoma Caco-2 cells (22-25%), and aggregated with indicators (30-50%). Moreover, both were non-hemolytic and sensitive to most antibiotics tested. Of the two selected strains, BIF43 showed better exopolysaccharides (EPS) producing phenotype. The CFSs of all strains showed high (85-93%) 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity. PCA confirmed the results obtained from in vitro probiotic experiments and supported the selection of Lact. plantarum BIF33 and BBC43, as potential probiotics.

Addition of lemon before boiling chlorinated tap water: A strategy to control halogenated disinfection byproducts

Chlorine disinfection is required to inactivate pathogens in drinking water, but it inevitably generates potentially toxic halogenated disinfection byproducts (halo-DBPs). A previous study has reported that the addition of ascorbate to tap water before boiling could significantly decrease the concentration of overall halo-DBPs in the boiled water. Since the fruit lemon is rich in vitamin C (i.e., ascorbic acid), adding it to tap water followed by heating and boiling in an effort to decrease levels of halo-DBPs was investigated in this study. We examined three approaches that produce lemon water: (i) adding lemon to tap water at room temperature, termed “Lemon”; (ii) adding lemon to boiled tap water (at 100 °C) and then cooling to room temperature, termed “Boiling + Lemon”; and (iii) adding lemon to tap water then boiling and cooling to room temperature, termed “Lemon + Boiling”. The concentrations of total and individual halo-DBPs in the resultant water samples were quantified with high-performance liquid chromatography-tandem mass spectrometry and the cytotoxicity of DBP mixtures extracted from the water samples was evaluated using human epithelial colorectal adenocarcinoma Caco-2 cells and hepatoma HepG2 cells. Our results show that the “Lemon + Boiling” approach substantially decreased the concentrations of halo-DBPs and the cytotoxicity of tap water. This strategy could be applied to control halo-DBPs, as well as to lower the adverse health effects of halo-DBPs on humans through tap water ingestion.

Ferrate(VI) pretreatment before disinfection: An effective approach to controlling unsaturated and aromatic halo-disinfection byproducts in chlorinated and chloraminated drinking waters

Disinfection is an essential process of drinking water treatment to eliminate harmful pathogens, but it generates potentially toxic disinfection byproducts (DBPs). Ferrate (FeO42-, Fe(VI)) was used to pre-oxidize natural organic matter (NOM, the precursor of DBPs) in source water to control DBP formation in subsequent chlorine or chloramine disinfection. Currently, it is unclear how Fe(VI) changes the structure of NOM, and no information details the effect of Fe(VI) pretreatment on the aromatic DBPs or the speciation of overall DBPs generated in subsequent disinfection of drinking water. In the present paper, Fe(VI) was applied to pretreat simulated source water samples at a Fe(VI) to dissolved organic carbon mole ratio of 1:1 at pH 8.0. 13C nuclear magnetic resonance spectroscopy was newly employed to characterize NOM in simulated source waters with and without Fe(VI) treatment, and it was demonstrated that Fe(VI) converted unsaturated aromatic C functional groups in NOM to saturated aliphatic ones. High-resolution mass spectrometry (HRMS) and high performance liquid chromatography/triple quadrupole MS were applied to analyze the DBPs generated in chlorination and chloramination of the source waters with and without Fe(VI) pretreatment. It was confirmed that Fe(VI) pretreatment followed by chlorination (or chloramination), generated DBPs containing less unsaturated, halogenated, and aromatic moieties than chlorination (or chloramination) without pretreatment by Fe(VI). Finally, the cytotoxicity of disinfected drinking water samples were assessed with the human epithelial colorectal adenocarcinoma Caco-2 cell line (a model of the intestinal barrier for ingested toxicants), and the results show that Fe(VI) pretreatment detoxified the chlorinated and chloraminated drinking waters.

Abundance of Lactobacillus plantarum Strains with Beneficial Attributes in Blackberries (Rubus sp.), Fresh Figs (Ficus carica), and Prickly Pears (Opuntia ficus-indica) Grown and Harvested in Algeria.

This first study performed on traditional fruits consumed in North Africa reveals their richness in microorganisms with beneficial attributes like cholesterol lowering capabilities. Blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) are fruits largely and traditionally consumed in Kabylia, a beautiful northern Algerian region. Here, 85 lactic acid bacteria (LAB)-isolates were isolated and identified by MALDI-TOF mass spectrometry. The identified species belong to Lactobacillus and Leuconostoc genera. These 85 LAB-isolates were then assessed for their capabilities to grow under conditions mimicking the gastrointestinal tract, and the resulting data were statistically treated with principal component analysis (PCA). After which, only 26 LAB-isolates were selected and characterized for their genetic relatedness using random amplified polymorphic DNA (RAPD) method. Following the genetic relatedness assessment, only 10 LAB-strains, among which nine Lactobacillus plantarum and one Lactobacillus paracasei were studied for their pathoproperties and some probiotic features. Interestingly, all of these 10 LAB-strains were devoid of adverse effects, but capable to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells. Of note, these 10 LAB-strains exhibited an important in vitro hypocholesteromia effect, in strain-dependent manner. Moreover, the Lactobacillus strains exhibited a high bile salt hydrolase (BSH) activity which was correlated with expression of bsh2, bsh3 and bsh4 genes.

In silico-in vitro discovery of untargeted kinase–inhibitor interactions from kinase-targeted therapies: A case study on the cancer MAPK signaling pathway

Protein kinase inhibitors have been widely used as therapeutic agents to treat a variety of diseases, but many of them may cause off-target effects by unexpectedly targeting other noncognate kinases due to high conversion across the protein kinase family. The mitogen-activated protein kinase (MAPK) signaling pathway plays an essential role in tumorigenesis, which has been recognized as a high priority in the druggable target candidates of anticancer therapy. Here, we attempt to investigate the untargeted kinase–inhibitor interactions (UKIIs) of kinase-targeted therapies for the cancer MAPK signaling cascade via an integration of biomolecular modeling, cell viability assay and kinase inhibition analysis. A systematic kinase–inhibitor interaction profile is created for 28 FDA-approved kinase inhibitor drugs across 9 caner-related MAPK kinases. The created profile is analyzed at structural, energetic and dynamic levels and, consequently, totally 18 promising UKII pairs with high theoretical affinity are derived, from which the noncognate inhibitors Cabozantinib, Regorafenib and Crizotinib are selected to test their cytotoxic effects on human epithelial colorectal adenocarcinoma Caco-2 cell line and inhibition activity against the recombinant protein of human p38α kinase domain. The obtained results are compared with two cognate MAPK inhibitors JNK-IN-8 and BIRB796. As might be expected, the Regorafenib, Crizotinib and Cabozantinib exhibit high, moderate and low cytotoxicities, respectively. In addition, the Regorafenib is determined to have a potent p38α-inhibitory activity. This is basically in line with the test results of positive controls JNK-IN-8 and BIRB796 and can be well confirmed by computational modeling.

Anti-pathogenic and probiotic attributes of Lactobacillus salivarius and Lactobacillus plantarum strains isolated from feces of Algerian infants and adults

Sixty-seven (67) lactic acid bacteria (LAB) isolates belonging to Lactobacillus genus were isolated from human feces and tested for their auto-aggregation and cell surface hydrophobicity in order to establish their adhesion capabilities, a prerequisite for probiotic selection. Strains with the upmost auto-aggregation and cell surface hydrophobicity scores were identified by MALDI-TOF spectrometry and 16S rDNA sequencing as Lactobacillus plantarum (p25lb1 and p98lb1) and Lactobacillus salivarius (p85lb1 and p104lb1). These strains were also able to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells, with percentages ranging from 4.68 to 9.59%. They displayed good survival under conditions mimicking the gastrointestinal environment and remarkably impeded adhesion and invasion of human Caco-2 by Listeria monocytogenes and Enteropathogenic Escherichia coli. It should also be noted that Lb. plantarum p98lb1 was able to reduce in vitro cholesterol concentration by about 32%, offering an additional health attribute.

An Eco-Friendly Ultrasound-Assisted Synthesis of Novel Fluorinated Pyridinium Salts-Based Hydrazones and Antimicrobial and Antitumor Screening.

The present work reports an efficient synthesis of fluorinated pyridinium salts-based hydrazones under both conventional and eco-friendly ultrasound procedures. The synthetic approach first involves the preparation of halogenated pyridinium salts through the condensation of isonicotinic acid hydrazide (1) with p-fluorobenzaldehyde (2) followed by the nucleophilic alkylation of the resulting N-(4-fluorobenzylidene)isonicotinohydrazide (3) with a different alkyl iodide. The iodide counteranion of 5–10 was subjected to an anion exchange metathesis reaction in the presence of an excess of the appropriate metal salts to afford a new series of fluorinated pyridinium salts tethering a hydrazone linkage 11–40. Ultrasound irradiation led to higher yields in considerably less time than the conventional methods. The newly synthesized ILs were well-characterized with FT-IR, 1H NMR, 13C NMR, 11B, 19F, 31P and mass spectral analyses. The ILs were also screened for their antimicrobial and antitumor activities. Within the series, the salts tethering fluorinated counter anions 11–13, 21–23, 31–33 and 36–38 were found to be more potent against all bacterial and fungal strains at MIC 4–8 µg/mL. The in vitro antiproliferative activity was also investigated against four tumor cell lines (human ductal breast epithelial tumor T47D, human breast adenocarcinoma MCF-7, human epithelial carcinoma HeLa and human epithelial colorectal adenocarcinoma Caco-2) using the MTT assay, which revealed that promising antitumor activity was exhibited by compounds 5, 12 and 14.