Interactions of clinical and environmental Aeromonas isolates with Caco-2 and HT29 intestinal epithelial cells

AIM: Evaluation of adherence and invasion of Aeromonas spp. to human colon carcinoma cell lines Caco-2 and HT29 and assessment of cytotoxic activity. METHODS AND RESULTS: A number of 27 strains of Aeromonas caviae and 23 strains of Aeromonas hydrophila was analysed. All strains were capable to adhere to sub-confluent monolayers of Caco-2 and HT29 cell types, presenting aggregative and diffuse adherence patterns cells, respectively. In the cytotoxic assays all strains showed cytopathic and/or cytotoxic activities to Vero cells. The evaluation of the tetrazolium salt (MTT test) reduction capability was carried out in Vero, Caco-2, and HT29 cells. MTT test showed that Vero cell line was the most sensitive cell type. In the invasion test, 13 strains were analysed on Caco-2 and HT29 monolayers. Only two (15%) of the 13 strains, A. hydrophila and A. caviae species, both isolated from vegetables were invasive to Caco-2 cells. No strains were able to invade the HT29 cells. CONCLUSIONS: A. hydrophila and A. caviae isolated from human diarrhoeic faeces, vegetables, and water, were able to adhere to and produce cytotoxic/cytopathic effects in intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Aeromonas spp. in food and water samples expressing virulence factors suggest that these sources may act as dissemination vehicles of human pathogen with implication in the public health.     

Molecular characterization and intracellular regulation of the human serotonin transporter in Caco-2 cells.

The serotonin transporter (SERT) has shown itself to be an effective pharmacological target in the treatment of mood disorders and some kinds of gastrointestinal syndromes. Most of the molecular studies of SERT in humans have been carried out using heterologous models. In this work, we have investigated the human enterocyte-like Caco-2 cell line as a potential "in vitro" model to study the human SERT. The results show that these cells express a SERT mRNA identical to the human brain SERT, and a 70 kDa protein immunodetected using a specific antibody. The SERT activity levels in Caco-2 cells increased in correlation with the onset and maintenance of the morphological and functional differentiation of the cells. Caco-2 SERT was also shown to be a high affinity (Kt=0.216 microM) saturable, Na(+) -dependent transporter that was inhibited by fluoxetine (IC(50)=17.6 nM). In addition, SERT activity was inhibited by the intracellular modulators protein kinase C and cAMP, either after short or long-term treatment. In short, the expression and molecular characteristics of the human SERT in Caco-2 cells indicate that this cell line may be an ideal tool to study in vitro the physiology and pharmacology of human SERT.     

Vitamin A metabolism in the human intestinal Caco-2 cell line

The human intestinal Caco-2 cell line, described as enterocyte-like in a number of studies, was examined for its ability to carry out the metabolism of vitamin A normally required in the absorptive process. Caco-2 cells contained cellular retinol-binding protein II, a protein which is abundant in human villus-associated enterocytes and may play an important role in the absorption of vitamin A. Microsomal preparations from Caco-2 cells contained retinal reductase, acyl-CoA-retinol acyltransferase (ARAT), and lecithin-retinol acyltransferase (LRAT) activities, which have previously been proposed to be involved in the metabolism of dietary vitamin A in the enterocyte. When intact Caco-2 cells were provided with beta-carotene, retinyl acetate, or retinol, synthesis of retinyl palmitoleate, oleate, palmitate, and small amounts of stearate resulted. However, exogenous retinyl palmitate or stearate was not used by Caco-2 cells as a source of retinol for ester synthesis. While there was a disproportionate synthesis of monoenoic fatty acid esters of retinol in Caco-2 cells compared to the retinyl esters typically found in human chylomicrons or the esters normally synthesized in rat intestine, the pattern was consistent with the substantial amount of unsaturated fatty acids, particularly 18:1 and 16:1, found in the sn-1 position of Caco-2 microsomal phosphatidylcholine, the fatty acyl donor for LRAT. Both ARAT and LRAT have been proposed to be responsible for retinyl ester synthesis in the enterocyte.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclization of a cell‐penetrating peptide via click‐chemistry increases proteolytic resistance and improves drug delivery

In this work we report synthesis and biological evaluation of a cell‐penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne‐azide click reaction. Cell viability studies in several cell‐lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF‐7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Microarray analysis revealing common and distinct functions of promyelocytic leukemia protein (PML) and tumor necrosis factor (TNF alpha) signaling in endothelial cells

ABSTRACT: BACKGROUND: Promyelocytic leukemia protein (PML) is a tumor suppressor that is highly expressed in endothelial cells nonetheless its role in endothelial cell biology remains elusive. Tumor necrosis factor alpha TNF-a is an important cytokine associated with many inflammation-related diseases. We have previously demonstrated that TNF-a induces PML protein accumulation. We hypothesized that PML may play a role in TNF-a signaling pathway. To identify potential PML target genes and investigate the putative crosstalk between PML's function and TNF-a signaling in endothelial cells, we carried out a microarray analysis in human primary umbilical endothelial cells (HUVECs). RESULTS: We found that PML and TNF-a regulate common and distinct genes involved in a similar spectrum of biological processes, pathways and human diseases. More importantly, we found that PML is required for fine-tuning of TNF-a-mediated immune and inflammatory responses. Furthermore, our data suggest that PML and TNF-a synergistically regulate cell adhesion by engaging multiple molecular mechanisms. Our biological functional assays exemplified that adhesion of U937 human leukocytes to HUVECs is co-regulated by PML and TNF-a signaling. CONCLUSIONS: Together, our study identified PML as an essential regulator of TNF-a signaling by revealing the crosstalk between PML knockdown-mediated effects and TNF-a-elicited signaling, thereby providing novel insights into TNF-a signaling in endothelial cells.     

hZip1 (hSLC39A1) regulates zinc homoeostasis in gut epithelial cells

Zinc is an essential trace element required for enzyme catalysis, gene regulation and signal transduction. Zinc absorption takes place in the small intestine; however, the mechanisms by which cells accumulate zinc are not entirely clear. Zip1 (SLC39A1) is a predicted transmembrane protein that is postulated, but not conclusively proven to mediate zinc influx in gut cells. The aim of this study was to investigate a role for hZip1 in mediating zinc uptake in human enterocytes. Both hZip1 mRNA and protein were detected in human intestinal tissue. In non-differentiated Caco-2 human gut cells, hZip1 was partially localised to the endoplasmic reticulum. In contrast, in differentiated Caco-2 cells cultured in extracellular matrix, the hZip1 protein was located in proximity to the apical microvilli. Lack of surface antibody binding and internalisation indicated that hZip1 was not present on the plasma membrane. Functional studies to establish a role for hZip1 in cellular zinc accumulation were carried out using ⁶⁵Zn. In Caco-2 cells harbouring an hZip1 overexpression construct, cellular zinc accumulation was enhanced relative to the control. Conversely, Caco-2 cells with an hZip1 siRNA construct showed reduced zinc accumulation. In summary, we show that the Caco-2 cell differentiation endorses targeting of hZip1 to a region near the apical domain. Given the absence of hZip1 at the apical plasma membrane, we propose that hZip1 may act as an intracellular sensor to regulate zinc homoeostasis in human gut cells.     

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.     

Novel bioassay system for evaluating anti-oxidative activities of food items: use of basolateral media from differentiated Caco-2 cells

Reactive oxygen and nitrogen species, including superoxide and nitric oxide (NO), are known to be mediators of oxidative stress and play pivotal roles in the onset of numerous life style-related diseases. While a number of studies have shown that naturally occurring anti-oxidants may be applicable for prevention and therapy for those diseases, most in vitro anti-oxidation tests reported have not provided significant insight into the absorption efficiency or metabolism of dietary anti-oxidants in the gastrointestinal tract. In the present study, we established a novel assay system by focusing on the bioconversion of food constituents using differentiated Caco-2 cells as a model of human intestinal epithelial cells. Various fresh food preparations [ginger, garlic, shimeji (Hypsizigus marmoreus), onion, carrot] were added to the apical side of differentiated Caco-2 monolayers. After incubation, the medium was recovered and tested for its inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced generation in differentiated HL-60 cells, and on combined lipopolysaccharide (LPS)- and interferon (IFN)-gamma -induced NO generation in RAW 264.7 macrophages. The garlic preparation (25% v/v) basolateral medium abolished generation without any cytotoxicity toward HL-60 cells, though it was cytotoxic to Caco-2 cells. In the NO generation tests, all of the food preparations showed notable inhibitory activity, while the garlic preparation (5% v/v) basolateral medium inhibited NO generation with substantial cytotoxicity toward RAW 264.7 cells. Interestingly, the carrot preparation (1% v/v) basolateral medium inhibited NO generation in both a concentration- and time-dependent manner without any cytotoxicity toward RAW 264.7 or Caco-2 cells, and its activities were higher than those of the carrot preparation alone (1% v/v). Our results indicate that the present assay system is appropriate and reliable for determination of the anti-oxidative efficacy of dietary phytochemicals in vivo.     

In vitro anti-proliferative activities of ellagic acid

The potential cytotoxic and anti-proliferative activities of ellagic acid (a naturally occurring bioactive compound in berries, grapes, and nuts) was evaluated using human umbilical vein endothelial cells (HUVEC), normal human lung fibroblast cells HEL 299, Caco-2 colon, MCF-7 breast, Hs 578T breast, and DU 145 human prostatic cancer cells. Ellagic acid at concentration in the range 10-100 micromol/L did not affect the viability of normal fibroblast cells during a 24-hour incubation. An increase in adenosine triphosphate (ATP) bioluminescence of approximately 18-21% was observed in normal cells incubated with ellagic acid. In contrast, ellagic acid at 1-100 micromol/L dose-dependently inhibited HUVEC tube formation and proliferation on a reconstituted extracellular matrix and showed strong anti-proliferative activity against the colon, breast, and prostatic cancer cell lines investigated. The most sensitive cells were the Caco-2, and the most resistant were the breast cancer cells. Ellagic acid induced cancer cell death by apoptosis as shown by the microscopic examination of cell gross morphology. Ellagic acid induced reduced cancer cell viability as shown by decreased ATP levels of the cancer cells. After 24 hours incubation of 100 micromol/L of ellagic acid with Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells, ellagic acid suppressed fetal bovine serum (FBS) stimulation of cell migration. The apoptosis induction was accompanied by a decreased in the levels of pro-matrix metalloproteinase-2 (pro-MMP-2 or gelatinase A), pro-matrix metalloproteinase-9 (pro-MMP-9 or gelatinase B), and vascular endothelial growth factor (VEGF(165)) in conditioned media. The results suggest that ellagic acid expressed a selective cytotoxicity and anti-proliferative activity, and induced apoptosis in Caco-2, MCF-7, Hs 578T, and DU 145 cancer cells without any toxic effect on the viability of normal human lung fibroblast cells. It was also observed that the mechanism of apoptosis induction in ellagic acid-treated cancer cells was associated with decreased ATP production, which is crucial for the viability of cancer cells.     

Study of benzo[a]phenazine 7,12-dioxide as selective hypoxic cytotoxin-scaffold. Identification of aerobic-antitumoral activity through DNA fragmentation

Phenazine 5,10-dioxides are prodrugs for antitumor therapy that undergo hypoxic-selective bioreduction to form cytotoxic species. Here we investigate the expanded system benzo[a]phenazine 7,12-dioxides as selective hypoxic cytotoxin-scaffold. The clonogenic survival of V79 cells on aerobic and anaerobic conditions, conduct us to study antiproliferative activity on Caco-2 tumoral cells in normoxia. Electrochemical, DNA-interaction and DNA-damage studies were performed to establish the mode of action. The results demonstrated the potential biological properties of the studied scaffold being derivatives 6–10 structural hits for further chemical-modifications to become into therapeutics for solid tumors. Compounds 6 and 8 with cytotoxicity against V79 cells in both conditions (aerobia and anaerobia) were also cytotoxic against Caco-2 tumoral cells in aerobiosis.     

New cytotoxic lanostanoid triterpenes from Ganoderma lingzhi

Further chemical investigation of the metabolites in the fruiting bodies of Ganoderma lingzhi resulted in isolation of eight triterpenes; two of them are new triterpene acid ethyl esters. Their structures were established based on spectroscopic studies and comparison with the known related compounds. The anticancer potential of the isolates were tested with an in vitro cytotoxic assay against five human cancer cell lines (MCF-7, HeLa, HCT-116, Caco-2 and HepG2) and two normal human cell lines (TIG-1 and HF19). Results showed that the new compounds have a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. Potent cytotoxic activities of some of the known isolated compounds are reported for the first time.     

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

AIMS: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. METHODS AND RESULTS: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. CONCLUSIONS: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.     

Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer

The human intestinal Caco-2 cell line has been extensively used as a model of the intestinal barrier. However, it is widely reported in literature that culture-related conditions, as well as the different Caco-2 cell lines utilized in different laboratories, often lead to problems of reproducibility making difficult to compare results. We developed a new cell-maintenance protocol in which Caco-2 cells were subcultured at 50% of confluence instead of 80% of confluence, as usually suggested. Using this new protocol, Caco-2 cells retained a higher proliferation potential resulting in a cell population, which, on reaching confluence, was able to differentiate almost synchronously, forming a more homogeneous and polarized cell monolayer, as compared to that obtained using a high cell growing density. This comparison has been done by analyzing the gene expression and the structural characteristics of the 21-days differentiated monolayers by microarrays hybridization and by confocal microscopy. We then investigated if these differences could also modify the effects of toxicants on 21-days-differentiated cells. We analyzed the 2 h-acute toxicity of CuCl(2) in terms of actin depolymerization and metallothionein 2A (MT2A) and heat shock protein 70 (HSPA1A) genes induction. Copper treatment resulted in different levels of actin depolymerization and gene expression induction in relationship with culture protocol, the low-density growing cells showing a more homogeneous and stronger response. Our results suggest that cell growing density could influence a number of morphological and physiological properties of differentiated Caco-2 cells and these effects must be taken in account when these cells are used as intestinal model.     

N-Propargylic β-enaminones in breast cancer cells: Cytotoxicity,apoptosis, and cell cycle analyses

Breast cancer is one of the most common cancers worldwide and the discovery of new cytotoxic agents is needed. Enaminones are regarded to be a significant structural motif that is found in a variety of pharmacologically active compounds however the number of studies investigating the anticancer activities of N-propargylic β-enaminones (NPEs) is limited. Herein we investigated the potential cytotoxic and apoptotic effects of 23 different NPEs (1-23) on human breast cancer cells. Cytotoxicity was evaluated via MTT assay. Apoptotic cell death and cell cycle distributions were investigated by flow cytometry. CM-H2DCFDA dye was used to evaluate cellular ROS levels. Expression levels of Bcl-2, Bax, p21, and Cyclin D1 were measured by quantitative real-time PCR. ADME properties were calculated using the ADMET 2.0 tool. NPEs 4, 9, 16, and 21 showed selective cytotoxic activity against breast cancer cells with SI values >2. NPEs induced apoptosis and caused significant changes in Bcl-2 and Bax mRNA levels. The cell cycle was arrested at the G0/G1 phase and levels of p21 and Cyclin D1 were upregulated in both breast cancer cells. ROS levels were significantly increased by NPEs, suggesting that the cytotoxic and apoptotic effects of NPEs were mediated by ROS. ADME analysis revealed that NPEs showed favorable distributions in both breast cancer cell lines, meaning good lipophilicity values, low unfractionated values, and high bioavailability. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.     

Gene expression profiles of normal proliferating and differentiating human intestinal epithelial cells: a comparison with the Caco-2 cell model

cDNA microarray technology enables detailed analysis of gene expression throughout complex processes such as differentiation. The aim of this study was to analyze the gene expression profile of normal human intestinal epithelial cells using cell models that recapitulate the crypt-villus axis of intestinal differentiation in comparison with the widely used Caco-2 cell model. cDNA microarrays (19,200 human genes) and a clustering algorithm were used to identify patterns of gene expression in the crypt-like proliferative HIEC and tsFHI cells, and villus epithelial cells as well as Caco-2/15 cells at two distinct stages of differentiation. Unsupervised hierarchical clustering analysis of global gene expression among the cell lines identified two branches: one for the HIEC cells versus a second comprised of two sub-groups: (a) the proliferative Caco-2 cells and (b) the differentiated Caco-2 cells and closely related villus epithelial cells. At the gene level, supervised hierarchical clustering with 272 differentially expressed genes revealed distinct expression patterns specific to each cell phenotype. We identified several upregulated genes that could lead to the identification of new regulatory pathways involved in cell differentiation and carcinogenesis. The combined use of microarray analysis and human intestinal cell models thus provides a powerful tool for establishing detailed gene expression profiles of proliferative to terminally differentiated intestinal cells. Furthermore, the molecular differences between the normal human intestinal cell models and Caco-2 cells clearly point out the strengths and limitations of this widely used experimental model for studying intestinal cell proliferation and differentiation.     

Induction of apoptosis of endothelial cells by Viscum album: a role for anti-tumoral properties of mistletoe lectins

BACKGROUND: Viscum album (VA) preparations consist of aqueous extracts of different types of lectins of VA. Mistletoe lectins have both cytotoxic and immunomodulatory properties that support their study for the development for cancer therapy. However, the mechanisms of the anti- tumoral properties in vivo of mistletoe lectins are not fully understood. Because endothelial cells (EC) play a pivotal role in tumor angiogenesis, we tested the hypothesis that VA extracts induce endothelial cell death and apoptosis. MATERIALS AND METHODS: We investigated the effect of various VA preparations on both human venous endothelial cell (HUVEC) and immortalized human venous endothelial cell line (IVEC) using morphologic assessment of EC, FACScan analysis after propidium iodine and annexin V labeling, and detection of cleavage of poly(A)DP-ribose polymerase (PARP). RESULTS: All tested VA preparations, except Iscador P, were cytotoxic in IVEC. Apoptosis, assessed by morphologic examination, annexin V labeling, and Western blot analysis for PARP cleavage, was involved in HUVEC cell death induced by VA preparations derived from plants that grow on oak trees (VA Qu FrF). CONCLUSIONS: Results from the present study suggest that VA extract-induced endothelial apoptosis may explain the tumor regression associated with the therapeutic use of VA preparations and support further investigations to develop novel anti-angiogenic compounds based on mistletoe compounds.     

Anti-neoplastic activity of two flavone isomers derived from Gnaphalium elegans and Achyrocline bogotensis

Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5-80 μM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.     

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.     

Potential probiotic Kluyveromyces marxianus B0399 modulates the immune response in Caco-2 cells and peripheral blood mononuclear cells and impacts the human gut microbiota in an in vitro colonic model system

Considering the increase in the consumption of yeasts as human probiotics, the aim of this study was to broadly investigate the beneficial properties of the lactic yeast Kluyveromyces marxianus (formerly Kluyveromyces fragilis) B0399. Several potential probiotic traits of K. marxianus B0399 were investigated by using in vitro assays, including adhesion and immune modulation, and the effect of the administration of 10(7) CFU/day of K. marxianus B0399 on the composition and metabolic activity of the human intestinal microbiota was investigated in a 3-stage continuous-culture system simulating the human colon. We demonstrated that this strain was highly adhesive to human enterocyte-like Caco-2 cells and modulated the immune response, inducing proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs). In the presence of inflammatory stimulation with lipopolysaccharide (LPS), K. marxianus B0399 provoked decreases in the levels of production of proinflammatory cytokines in PBMCs and Caco-2 cells, thus ameliorating the inflammatory response. Furthermore, K. marxianus B0399 impacted the colonic microbiota, increasing the bifidobacterial concentration in the stages of the colonic model system simulating the proximal and transverse colon. The amounts of the short-chain fatty acids acetate and propionate also increased following yeast supplementation. Finally, K. marxianus B0399 was found to induce a decrease of the cytotoxic potential of the culture supernatant from the first stage of the colonic model system. The effects of K. marxianus B0399 on adhesion, immune function, and colonic microbiota demonstrate that this strain possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotic.