Human Cell Line Caco-2 Research Articles

Nuclear expression of claudin-3 in human colorectal adenocarcinoma cell lines and tissues.

Claudins are members of a large family of transmembrane proteins, which are essential for the formation of tight junctions and have a significant effect on the biological behavior of tumor progression. Previous studies have demonstrated that several claudins show aberrant expression patterns in numerous types of cancer. The present study investigated the expression and localization of claudin-3 and claudin-7 in human colorectal adenocarcinoma cell lines and tissues. The protein expression levels of claudin-3 and claudin-7 were determined using immunocytochemical and immunohistochemical staining. Claudin-3, but not claudin-7, exhibited nuclear localization in the human colorectal adenocarcinoma Caco-2 and SW620 cell lines. Surgically resected colorectal adenocarcinoma tissue specimens were obtained, and the associations between the expression of claudin-3 or claudin-7 and various clinicopathological parameters were analyzed. The membranous expression rates of claudin-3 and claudin-7 were 58.0 and 50.0%, while their nuclear expression rates were 22.0 and 2.0%, respectively. The membranous expression of claudin-3 and claudin-7 was not associated with any clinicopathological factors, whereas the nuclear expression of claudin-3 was associated with histological type and was significantly increased in colorectal mucinous adenocarcinomas compared with that in well- to moderately-differentiated colorectal adenocarcinomas (P<0.01). However, no associations were observed between the nuclear expression of claudin-7 and any clinicopathological parameter. In conclusion, the nuclear expression of claudin-3 in colorectal mucinous adenocarcinoma may be involved in the biological transformation of tumors. The results from the present study indicated that claudin-3 is an important protein associated with histological type and has potential as a prognostic marker. Although the mechanisms underlying the nuclear localization of claudin-3 in tumorigenesis have not yet been elucidated in detail, the present results indicated the potential of claudin-3 as a histopathological biomarker for colorectal adenocarcinomas.

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28.

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faecium BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto-aggregation phenotypes. Among them, En. faecium BGGO9-28 showed the strongest auto-aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen-binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, agg, gelE, esp, hylN, ace, efaAfs, and efaAfm) showed that BGGO9-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature.

Knockdown of long non-coding RNA prostate cancer-associated ncRNA transcript 1 inhibits multidrug resistance and c-Myc-dependent aggressiveness in colorectal cancer Caco-2 and HT-29 cells.

The long non-coding RNA (lncRNA) prostate cancer-associated ncRNA transcript 1 (PCAT-1) has been shown to promote prostate cancer cell proliferation through c-Myc and is associated with the poor prognosis of CRC patients. In the current study, it was hypothesized that the effect of PCAT-1 on the aggressiveness of CRC cells was dependent on the function of c-Myc. Human CRC cell lines Caco-2 and HT-29 were transfected with specific PCAT-1 shRNAs, and cell migration, invasiveness, and resistance to 5-fluorouracil were measured. To elucidate the role of c-Myc in PCAT-1 function, c-Myc was overexpressed in PCAT-1-silenced CRC cells and the effect of c-Myc overexpression on the aggressiveness of PCAT-1-silenced cells was detected. The results showed that knockdown of PCAT-1 in CRC cells suppressed cell motility and invasiveness, and sensitized the cells to 5-fluorouracil, as evidenced by the reduced viability and induced apoptosis in PCAT-1-silenced cells compared to the parental cells in response to 5-fluorouracil treatment. The expression of c-Myc in PCAT-1-silenced CRC cells was down-regulated, and forced expression of c-Myc partially restored the invasiveness in PCAT-1-silenced cells. In summary, the findings outlined in the current study suggest that PCAT-1 regulates the invasiveness and drug resistance in CRC cells and that PCAT-1 may promote CRC cell invasion by modulating the expression of c-Myc.

Short Chain Fatty Acids Enhance Aryl Hydrocarbon (Ah) Responsiveness in Mouse Colonocytes and Caco-2 Human Colon Cancer Cells

Aryl hydrocarbon receptor (AhR) ligands are important for gastrointestinal health and play a role in gut inflammation and the induction of T regulatory cells, and the short chain fatty acids (SCFAs) butyrate, propionate and acetate also induce similar protective responses. Initial studies with butyrate demonstrated that this compound significantly increased expression of Ah-responsive genes such as Cyp1a1/CYP1A1 in YAMC mouse colonocytes and Caco-2 human colon cancer cell lines. Butyrate synergistically enhanced AhR ligand-induced Cyp1a1/CYP1A1 in these cells with comparable enhancement being observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and also microbiota-derived AhR ligands tryptamine, indole and 1,4-dihydroxy-2-naphthoic acid (DHNA). The effects of butyrate on enhancing induction of Cyp1b1/CYP1B1, AhR repressor (Ahrr/AhRR) and TCDD-inducible poly(ADP-ribose)polymerase (Tiparp/TiPARP) by AhR ligands were gene- and cell context-dependent with the Caco-2 cells being the most responsive cell line. Like butyrate and propionate, the prototypical hydroxyamic acid-derived histone deacetylase (HDAC) inhibitors Panobinostat and Vorinostat also enhanced AhR ligand-mediated induction and this was accompanied by enhanced histone acetylation. Acetate also enhanced basal and ligand-inducible Ah responsiveness and histone acetylation, demonstrating that acetate was an HDAC inhibitor. These results demonstrate SCFA-AhR ligand interactions in YAMC and Caco-2 cells where SCFAs synergistically enhance basal and ligand-induced expression of AhR-responsive genes.

MiR-378 suppresses the proliferation, migration and invasion of colon cancer cells by inhibiting SDAD1

BackgroundMicroRNAs (miRNAs) play important roles in the growth and metastasis of colon cancer. It is known that one set of miRNAs are dysregulated in colon cancer cells, but the mechanism of their role in cancer development is still largely unknown. Our study focuses on the role of miR-378 in colon cancer cells.MethodsHuman colon cancer tissues and adjacent non-tumor tissues were collected from patients diagnosed in pathological examinations. In addition, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and a normal colonic mucosa cell line NCM460 were included. Quantitative RT-PCR was used to detect the miR-378 level in the clinical tissues and cell lines. In SW480 and HCT-116, miR-378 was artificially overexpressed or suppressed. Cell viability and proliferation were measured using MTT and colony formation assays, and apoptosis was detected via annexin V-PI staining and flow cytometry analysis. The transwell technique was applied to detect the migration and invasion of the colon cancer cells, and their epithelial–mesenchymal transition (EMT) was evaluated by detecting EMT-associated markers using Western blotting. Bioinformatics methods were used to predict the potential targets of miR-378, and luciferase reporter assays were performed to conform the direct binding between miR-378 and its target mRNA. The activity of the Wnt/β-catenin pathway was evaluated by detecting the key factors through Western blotting.ResultsWe found that miR-378 expression was low in colon cancer tissues and cell lines. Overexpression of miR-378 not only inhibits the proliferation of colon cancer cells in vitro by inducing apoptosis, but also inhibits migration and invasion by inhibiting the EMT of colon cancer cells. SDAD1 is a direct target gene of miR-378, and knockdown of SDAD1 suppresses the proliferation, migration and invasion of colon cancer cells. We also confirmed that miR-378 alleviated the malignant phenotypes of colon cancer cells by inhibiting the Wnt/β-catenin pathway.ConclusionmiR-378 inhibits the proliferation, migration and invasion of colon cancer cells by targeting SDAD1, defining miR-378 as a potential target for the diagnosis and treatment of colon cancer.

Antioxidant potential of buffalo and cow milk Cheddar cheeses to tackle human colon adenocarcinoma (Caco-2) cells.

ObjectiveThe aim of present study was to assess the anti-oxidant potential of water-soluble peptides (WSPs) extract derived from buffalo and cow milk Cheddar cheeses at different stages of ripening.MethodsThe antioxidant potential of WSPs extract was assessed through 2,2’-azinobis-3-ethylbenzothiazoline-6sulfonic acid (ABTS)-radical scavenging activity. In addition, impact of WSPs extract on cell viability and production of reactive oxygen species (ROS) in human colon adenocarcinoma Caco-2 (tert-butylhydroperoxide-induced) cell lines was also evaluated.ResultsThe ABTS-radical scavenging activity increased progressively with ripening period and dose-dependently in both cheeses. However, peptide extract from buffalo milk Cheddar cheese demonstrated relatively higher activity due to higher contents of water-soluble nitrogen. Intracellular ROS production in Caco-2 cells decreased significantly (p<0.05) till 150th day of cheese ripening and remained constant thereafter. Additionally, dose-dependent response of WSPs extract on antioxidant activity was noticed in the Caco-2 cell line.ConclusionOn the basis of current in vitro study, the Cheddar cheese WSPs extract can protect intestinal epithelium against oxidative stress due to their antioxidant activity.

Synthesis, structural characterization, QSAR and docking studies of a new binuclear nickel (II) complex based on the flexible tetradentate N-donor ligand as a potent antibacterial and anticancer agent

A new nickel (II)complex namely [Ni2(Lt)Cl4] derived from the NiCl2.6H2O and 1,1,3,3-tetrakis(3,5-dimethyl-1-pyrazolyl)propane (Lt) has been synthesized and fully characterized by the single crystal X-ray diffraction, elemental analysis, FT-IR, UV–vis, density functional theory (DFT) calculations, antibacterial and anticancer activities. In the title complex, each of the Ni(II) atoms is tetrahedrally coordinated by two N atoms from one of the chelating bidentate bis(3,5-dimethylpyrazolyl)methane units of the Lt ligand and two Cl as terminal ligands. The neighboring [Ni2(Lt)Cl4] molecules are linked together by the intermolecular CH⋯Cl hydrogen bonds to generate a 1D chain structure. The chains are further stabilized by the intermolecular CH⋯π interactions to form a two-dimensional non-covalent bonded structure. The antibacterial activity of the free Lt ligand and its Ni (II) complex shows that the ability of these compounds to inhibit growth of the tested bacteria increase from the Lt to binuclear nickel (II) complex. Scanning probe microscopy (SPM) study of the treated B. subtilis and E. coli bacteria was implemented to understand the structural changes caused by the interactions between the nickel (II) complex and the target bacteria. The cytotoxicity test of the Lt ligand and its complex was evaluated against the human carcinoma cell line (Caco-2) using the MTT assay. The results indicate that the Lt ligand and its complex display cytotoxicity against Caco-2 with the IC50 values of 36.29μM and 12.97μM, respectively. Therefore, the complex can be nominated as a potential anticancer agent. Molecular docking investigations on the five standard antibiotic, five standard anticancer drugs, free Lt ligand, title complex and twelve receptors were performed by Autodock vina function. The results of docking and DFT calculations are in line with the in vitro data obtained via the antibacterial and anticancer activity of Lt ligand and its made-complex.

Characterization of Candidate probionts isolated from human breast milk.

This study was designed to isolate and identify the potential probionts present in 32 healthy mothers' breast milk. Microbial culture media and 16SrRNA sequencing were used to isolate and identify the bacteria and all isolates were analyzed for their antagonistic potential, resistance to acidic pH, bile salts and survival under simulated gastric and intestinal conditions. The colonization potential was further assessed based on adherence to human enterocyte-like Caco-2 cell lines. The breast milk samples harbored significant numbers of Gram positive and catalase negative (85%) bacteria. Based on 16SrRNA sequencing, these isolates were identified as Lactobacillus casei, L.gasseri, L.fermentum, L.plantarum, Pediococcus acidilactici, and Enterococcus facieum. Among the isolates, P. acidilactici was the most frequent species (71%) present in these samples. Few Gram and catalase positive isolates, Staphylococcus aureus and S.hominiis were also observed. The isolates were viable and unviable in pH 3 and 1.5, respectively, while all isolates survived in 1.0% bile salt. As putative probionts, P.acidilactici 1C showed a significantly higher percentage of adhesion to Caco-2 cells (p&lt; 0.05)than the other two isolates L.plantarum 7A and E.facieum 2C. Bacterial strains isolated from human breast milk were shown to have probiotic properties including anti-infective protection and may be considered as future therapeutics for infants.

Antigiardial Activity of Podophyllotoxin-Type Lignans from Bursera fagaroides var. fagaroides.

Giardiasis, a diarrheal disease, is highly prevalent in developing countries. Several drugs are available for the treatment of this parasitosis; unfortunately, all of them have variable efficacies and adverse effects. Bursera fagaroides has been known for its anti-inflammatory and antidiarrheal properties in Mexican traditional medicine. We investigated the in vitro anti-giardial activities of four podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides, namely, 5′-desmethoxy-β-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), burseranin (BUR), and podophyllotoxin (POD). All lignans affected the Giardia adhesion and electron microscopy images revealed morphological alterations in the caudal region, ventral disk, membrane, and flagella, to different extents. Only 5-DES, APOD, and POD caused growth inhibition. Using the Caco-2 human cell line as a model of the intestinal epithelium, we demonstrated that APOD displayed direct antigiardial killing activity and low toxicity on Caco-2 cells. This finding makes it an attractive potential starting point for new antigiardial drugs.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

BackgroundBifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant.ResultsA putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages.ConclusionsOur results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Design and Green Synthesis of Thieno[2,3-d]pyrimidine Analogues as Potential Antiproliferative Agents.

Despite of significant progress achieved in the chemotherapy of cancer; it is still among the leading cause of morbidity and mortality worldwide. Taking cognizance of the extensive biological potential of reported thieno[2,3-d]pyrimidines and inspired by the clinically available anticancer agents dasatinib and gefitinib, 4-substituted thieno[2,3-d]pyrimidines have been synthesized. The compounds were synthesized via microwave-assisted methods and screened for their cytotoxic activity against liver HepG-2, lung NCI-H522, melanoma A-375, pancreatic MIA PaCa-2 and colon CaCo-2 human cancer cell lines using MTT assay. The antiproliferative potential of most active compounds 20b and 20f (piperidino substituted); and 22d (hybrid analogue of Dasatinib) was further assessed and confirmed by calcein AM and colony formation assay, which revealed the higher potency of hybrid analogue 22d in comparison to piperidino substituted derivative 20f. Flow cytometer based cell cycle perturbation experiments revealed that antiproliferative effects of the most active compound 22d was associated with increased proportion of cells in the G2/M and subG0/G1 phases of the cell cycle. In silico ADME studies also confer the drug like characteristics of the potent compounds.

Investigation of Cytokine Responses and Variations in the Expression of Beta-defensin-3 of Caco-2 Human Colon Epidermal Adenocarsinoma and THP-1 Human Leukemia Monocyte Cell Lines in Response to Toxoplasma gondii Under Various Conditions

Mononuclear phogocytic cells and epithelial cells are effective during the initiation and regulation of the innate immune response. They have an active role in mucosal immune response both mechanically and by interaction with other cells with cytokine release. Defensins are microbicidal peptides that are expressed in various cells and are thought to be effective in the first line defense against pathogens. IL-12 and IL-10, showing proinflamatory and antiinflamatory activities, respectively, are actors of the cellular immunity and limit the infection of the host without causing immunopathology. The aim of this study was to observe the differences in the release of IL-12 and IL-10 and the expression of human beta-defensin-3 (hBD-3) inCaco-2 (human colon epidermal adenocarcinoma cell) and THP-1 (human leukemia monocytic cell) cell lines cultured alone or in co-culture, by the stimulation of Toxoplasma gondii tachyzoites either in direct contact with the cells or separated by an insert filter from the cells. Twenty-four hours after the addition of RH strain tachyzoites to the cells, the supernatants were collected from the experiment wells, and commercial ELISA kits (Invitrogen) were used according to the manufacturers instructions to measure IL-12 and IL-10 levels. HBD-3 expression of cells collected from the experiment wells afterfour and 24 hourswere analyzed by using real time PCR. For this procedure, complementary c-DNA was obtained (Transcriptor High Fidelity cDNA Synthesis Kit, Roche Diagnostics, Germany)after the extraction of RNA with a commercial kit (High pure RNA isolation kit, Roche Diagnostics, Germany). IL-12 was higher than IL-10 in all experiment wells. IL-12 was induced more in the co-culture wells where Caco-2 and THP-1 cells were challenged together, than the wells in which the cells infected with T.gondii tachyzoites alone. No differences in respect to cytokine response were observed between the cells with which tachyzoites were in contact and the cells which were separated from the parasites with an insert. In hBD-3 experiments, Caco-2 and THP-1 cells interacted in co-culture wells when infected with tachyzoites and displayed a higher level of hBD-3 expression than the condition when they were infected alone. This study showed that, IL-12 release and hBD-3 expression, which play a role in innate immunity, are greater when various antigens of T.gondii interacted with stimulated mononuclear cell and epithelial cells.

Bioavailable Citrus sinensis Extract: Polyphenolic Composition and Biological Activity.

Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrus sinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value.

The Influence of Spirulina platensis Filtrates on Caco-2 Proliferative Activity and Expression of Apoptosis-Related microRNAs and mRNA.

Spirulina platensis (SP) is a blue-green microalga that has recently raised attention not only as a nutritional component, but also as a source of bioactivities that have therapeutic effects and may find application in medicine, including cancer treatment. In the present study we determined the cytotoxic effect of S. platensis filtrates (SPF) on human colon cancer cell line Caco-2. Three concentrations of SPF were tested—1.25%, 2.5%, and 5% (v/v). We have found that the highest concentration of SPF exerts the strongest anti-proliferative and pro-apoptotic effect on Caco-2 cultures. The SPF negatively affected the morphology of Caco-2 causing colony shrinking and significant inhibition of metabolic and proliferative activity of cells. The wound-healing assay showed that the SPF impaired migratory capabilities of Caco-2. This observation was consistent with lowered mRNA levels for metalloproteinases. Furthermore, SPF decreased the transcript level of pro-survival genes (cyclin D1, surviving, and c-Myc) and reduced the autocrine secretion of Wnt-10b. The cytotoxic effect of SPF involved the modulation of the Bax and Bcl-2 ratio and a decrease of mitochondrial activity, and was related with increased levels of intracellular reactive oxygen species (ROS) and nitric oxide (NO). Moreover, the SPF also caused an increased number of cells in the apoptotic sub-G0 phase and up-regulated expression of mir-145, simultaneously decreasing expression of mir-17 and 146. Obtained results indicate that SPF can be considered as an agent with anti-cancer properties that may be used for colon cancer prevention and treatment.

Polyphenol Composition, Antioxidant Activity and Cytotoxicity of Seeds from Two Underexploited Wild Licania Species: L. rigida and L. tomentosa.

Studies have shown the benefit of antioxidants in the prevention or treatment of human diseases and promoted a growing interest in new sources of plant antioxidants for pharmacological use. This study aimed to add value to two underexploited wild plant species (Licania rigida) and L. tomentosa) from Brazilian flora. Thus, the phenolic compounds profile of their seed ethanol extract and derived fractions were elucidated by HPLC, the antioxidant capacity was assessed by in vitro chemical tests and the cytotoxicity determined using the human carcinoma cell lines MCF-7 and Caco-2. Eleven phenolic compounds were identified in the extracts of each species. The extracts and fractions showed excellent antioxidant activity in the DPPH assay (SC50, ranging from 9.15 to 248.8 µg/mL). The aqueous fraction of L. rigida seeds was most effective in preventing lipid peroxidation under basal conditions (IC50 60.80 µg/mL) whereas, in the presence of stress inducer, the methanolic fraction of L. tomentosa performed best (IC50 8.55 µg/mL). None of the samples showed iron chelating capacity. Ethanolic seed extracts of both species did not reveal any cytotoxicity against MCF-7 and Caco-2 cells. Both plant species showed a promising phenolic profile with potent antioxidant capacity and deserve attention to be sustainably explored.

An examination of the antimicrobial and anticancer properties of Garcinia cambogia fruit pericarp extracts

Background: Garcinia cambogia (synonym Garcinia gummi-gutta) is commonly known as brindleberry and Malabar tamarind. It has received considerable recent interest due to its potential in the prevention and treatment of obesity and obesity related diseases. The fruit pericarp also has traditional uses in the treatment of a wide variety of diseases and medical conditions, yet these have received relatively little investigation to date. Methods: Garcinia cambogia fruit extracts were investigated for their ability to inhibit the growth of a panel of bacteria of medicinal importance. The extracts were also tested for their ability to block the proliferation of the CaCo2 and HeLa human carcinoma cell lines. Results: Garcinia cambogia methanolic, aqueous and ethyl acetate extracts displayed broad spectrum antimicrobial activity, inhibiting the growth of between 16 (64 %; ethyl acetate extract) and 22 (88 % aqueous extract) of the 25 bacterial species tested. Strong inhibitory activity was detected with minimum inhibitory concentration (MIC) values <1000 μg/ml against many bacteria. All extracts were effective against both Gram negative and Gram positive bacteria, although Gram negative bacteria were more sensitive. All extracts displayed anti-proliferative activity against CaCo2 and HeLa carcinoma cells, yet were non-toxic in the Artemia franciscana bioassay, with LC50 values greatly in excess of 1000 μg/ml. Conclusion: The inhibitory bioactivity against a range of microbes, the anti-proliferative activity against CaCo2 and HeLa cells, as well as the lack of toxicity, indicate the potential for G. cambogia in the discovery and development of new natural pharmaceuticals.

Transepithelial transport efficiency of bovine collagen hydrolysates in a human Caco-2 cell line model.

Collagen was extracted from raw bovine hide and hydrolyzed by one of three enzymes - Alcalase, Flavourzyme, or trypsin - or by using a combination of two or three of these enzymes. The Alcalase-containing enzymatic hydrolysis treatments generated a greater proportion of hydrolysates with molecular weight (MW) <2kDa (79.8-82.7%). Flavourzyme-containing hydrolysis treatments exhibited the greatest proportion of free amino acids (686-740nmol/mg). The hydrolysates were then subjected to a simulated gastrointestinal (GI) digestion, and transport studies were conducted using a Caco-2 cell model. Due to the lower MW profile, the hydrolyzed collagen showed greater resistance to GI digestion and greater transport efficiency than the unhydrolyzed collagen control. Hydrolysates from a dual enzyme mixture - the Alcalase/Flavourzyme combination - generated the greatest transport efficiency across Caco-2 cell monolayers (21.4%), two-fold more than that of the collagen control.

Ketogenesis contributes to intestinal cell differentiation.

The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body β-hydroxybutyrate (βHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21Waf1). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/βHB signaling in intestinal cells. In contrast, treatment of intestinal cells with βHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/βHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis.

Synthesis and factorial design applied to a novel chitosan/sodium polyphosphate nanoparticles via ionotropic gelation as an RGD delivery system

Chitosan nanoparticles have been extensively studied for both drug and protein/peptide delivery. The aim of this study was to develop an optimized chitosan nanoparticle, by ionotropic gelation method, using 32 full factorial design with a novel polyanion, sodium polyphosphate, well known under the trade name Graham salt. The effects of these parameters on the particle size, zeta potential, and morphology and association efficiency were investigated. The optimized nanoparticles showed an estimated size of 166.20±1.95nm, a zeta potential of 38.7±1.2mV and an efficacy of association of 97.0±2.4%. The Atomic Force Microscopy (AFM) and Scanning Electronic Microscopy (SEM) revealed spherical nanoparticles with uniform size. Molecular interactions among the components of the nanoparticles and peptide were evaluated by Fourier Transform Infrared Spectra (FTIR) and Differential Scanning Calorimetry (DSC). The obtained results indicated that, the developed nanoparticles demonstrated high biocompatible, revealing no or low toxicity in the human cancer cell line (Caco-2). In conclusion, this work provides parameters that contribute to production of chitosan nanoparticles and sodium polyphosphate with desirable size, biocompatible and enabling successful use for protein/peptides delivery.

English

Several investigations have demonstrated in vitro antibacterial activity of plant extracts. Kalanchoe pinnata is globally used in folk medicine for the treatment of various diseases, such as diarrhea, infections, tuberculosis and fever. Among the enteropathogenic bacteria, enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea in the world. EAEC is also capable of forming biofilms, conformation that provides antibiotic resistance and a high degree of dispersion and reinfection, which may represent up to 80% of causes of human microbial infections. Thus, it becomes necessary to search for new antimicrobial agents with activity against biofilms. The aim of this study was to evaluate the cytotoxic, antibacterial and antibiofilm in EAEC from aqueous extracts of leaves and the flavonoids quercetin and rutin, occurring in K. pinnata. Leaf aqueous extracts were obtained and evaluated phytochemically. The cytotoxic activity was evaluated in human carcinoma cell lines HEp-2, Caco-2 and T84. The antibacterial activity was evaluated by the macrodilution method and the evaluation of antibiofilm activity was performed in Escherichia coli enteroaggregative (EAEC 042). Aqueous extracts of K pinnata do not show toxicity to Hep-2, but all other cell lines were sensitive to this extract. Instead, the flavonoids quercetin and rutin showed no cytotoxicity with any of the tested cell lines. Quercetin is capable of inhibiting bacterial growth of all tested strains. The aqueous leaf extract and quercetin were able to inhibit EAEC 042 biofilm formation above 50%. The results indicate the potential use of the species in treatment of bacterial infections. Key words: Antimicrobials, EAEC 042, biofilms, quercetin, Crassulaceae.