Colon Carcinoma Caco-2 Cells Research Articles

Protective effects of culture extracts (CB08035-SCA and CB08035-SYP) from Marinobacter hydrocarbonoclasticus (strain CB08035) against oxidant-induced stress in human colon carcinoma Caco-2 cells

The present study investigated the effect of culture extracts (CB08035-SCA and CB08035-SYP) from Marinobacter hydrocarbonoclasticus (strain CB08035) on cell viability and the potential protective effects attributed to molecular mechanisms underlying antioxidant response to survive oxidative stress injuries. Caco-2 cells were submitted to oxidative stress by treatment with tert-butyl hydroperoxide (t-BOOH). Both extracts prevented cell damage and enhanced activity of antioxidant defenses (NQO1 and GST activities and GSH levels) reduced by treatment with t-BOOH. Increased ROS and caspase 3/7 activity induced by t-BOOH were dose-dependently prevented when cells were treated with the extracts. CB08035-SCA caused up-regulation of Nrf2, AKT1 and Bcl-2 gene expressions. Moreover, CB08035-SCA and CB08035-SYP treatments reduced significantly Bax, BNIP3, APAF1, ERK1, JNK1, MAPK1, NFκB1, TNFα, IL-6, IL-1β and HO-1 gene expressions of apoptosis, proinflammation and oxidative stress induced by t-BOOH. CB08035-SCA and CB08035-SYP CPE extracts confer a significant protection against oxidative insults to cells. Our results show that culture extracts CB08035-SCA and CB08035-SYP from M. hydrocarbonoclasticus (strain CB08035) appeared to have antioxidant potential, based on their ability to protect antioxidant enzymes and mRNA gene expressions linked to apoptosis/oxidative pathways. These results suggest that culture extracts CB08035-SCA and CB08035-SYP can be a potential ingredient in the pharmaceutical and cosmeceutical industries.

Novel phenethylimidazolium based ionic liquids: Design, microwave synthesis, in-silico, modeling and biological evaluation studies

An eco-friendly preparation method for the novel bioactive imidazolium ionic liquids halides (ILs) was developed under microwave-assisted conditions. Synthesized ILs were characterized by spectroscopic techniques. Selected ILs were investigated for their antimicrobial activity against highly resistant Gram-positive and Gram-negative bacterial strains. Overall, 3-(2-chlorobenzyl)-1-phenethyl-1H-imidazol-3-iumchloride (4) showed high antimicrobial activity against the Staphylococcus aureus strain in the inhibition zone tests and displayed low MIC and MBC levels against almost all tested bacteria. Furthermore, the ILs were screened in vitro against human hepatocellular carcinoma (HepG-2), human breast adenocarcinoma (MCF-7), and human colon carcinoma (Caco-2) cell lines. The screening results showed excellent to moderate anticancer activity across the ILs. Among the synthesized ILs, Overall, 3-(2-chlorobenzyl)-1-phenethyl-1H-imidazol-3-ium chloride (4) and 1-phenethyl-3-(3-phenoxypropyl)-1H-imidazol-3-ium bromide (7) were found to exhibit the most promising ant proliferative effects and had the lowest IC50 values. The docking study suggested strong interaction of ILs with DNA with binding energy ranging from −4.9 to −4.1 kcal/mol. ILs 4 and 7 were most strongly bonded with −4.9 and −4.8 kcal/mol binding energy; confirming in vitro anticancer results.

Cadmium induces iron deficiency anemia through the suppression of iron transport in the duodenum

Cadmium (Cd) is an environmental contaminant that triggers toxic effects in various tissues such as the kidney, liver, and lung. Cd can also cause abnormal iron metabolism, leading to anemia. Iron homeostasis is regulated by intestinal absorption. However, whether Cd affects the iron absorption pathway is unclear. We aimed to elucidate the relationship between the intestinal iron transporter system and Cd-induced iron deficiency anemia. C57BL/6J female and male mice, 129/Sv female mice, and DBA/2 female mice were given a single oral dose of CdCl2 by gavage. After 3 or 24 h, Cd decreased serum iron concentrations and inhibited the expression of iron transport-related genes in the duodenum. In particular, Cd decreased the levels of divalent metal transporter 1 and ferroportin 1 in the duodenum. In addition, human colon carcinoma Caco-2 cells were treated with CdCl2. After 72 h, Cd decreased the expression of iron transport-related factors in Caco-2 cells with a pattern similar to that seen in the murine duodenum. These findings suggest that Cd inhibits iron absorption through direct suppression of iron transport in duodenal enterocytes and contributes to abnormal iron metabolism.

Cytotoxicity and genotoxicity assays of halobenzoquinones disinfection byproducts using different human cell lines.

Recently, halobenzoquinones (HBQs) disinfection byproducts, including 2,6-dichloro-1, 4-benzoquinone (DCBQ), 2,6-dichloro-3-methyl-1, 4-benzoquinone (DCMBQ), 2,3,6-trichloro-1, 4-benzoquinone (TCBQ), and 2,6-dibromobenzoquinone (DBBQ), have been of increasing concern due to their reported ability to induce oxidative damage, and thus genotoxicity. However, data on the risk of genotoxicity due to chromosomal damage by HBQs are still scarce. Here, the cytotoxicity and genotoxicity of the four HBQs were assessed using human cell lines (bladder cancer 5637 cells, colon carcinoma Caco-2 cells, and gastric MGC-803 cells). The four HBQs exhibited significant concentration-response relationships in all the three cell lines. Cytotoxicity of DCBQ, DCMBQ, TCBQ, and DBBQ, represented by the 50% concentration of inhibition (IC50 ) values, were 80.8-99.5, 41.0-57.6, 122.1-146.6, and 86.9-93.8 μM, respectively. The lowest effective concentrations for cellular micronuclei induction in the cell lines by DCBQ, DCMBQ, TCBQ, and DBBQ were 50-75, 20-41.5, 87.4-100, and 50 μM, respectively. 5637 and Caco-2 cells were more sensitive to the cytotoxic and genotoxic effects of HBQs than MGC-803 cells. These results show that HBQs can induce chromosomal damage; DCMBQ induced the highest cytotoxicity and genotoxicity in all the cell lines, and TCBQ caused the least toxicity.

Recombinant TcpA of Vibrio cholerae induces inflammation and bacterial colonization in vitro

Vibrio cholerae colonizes intestinal epithelial cells (IEC) through expression of adhesins in a CEACAM1-dependent manner. Interestingly, it has been proposed that inflammation in the IEC enhances the expression of CEACAM1 and provokes tissue colonization. In this study, we amplified the tcpA gene and cloned it into pET-28a vector; then, the recombinant protein was purified. Next, the effect of various concentrations of recombinant TcpA (1, 5, 10 and 50 mg/L) was evaluated from expression of ceacam1, IL-1β and TNFα genes in a co-culture model of a human colon carcinoma (Caco-2) cell line and rabbit peripheral blood mononuclear cells. Our findings found a significant dose-dependent increase in ceacam1, IL-1β and TNFα gene expression in the IEC. TcpA stimulated the TNF-α expression level, inducing ceacam1 overexpression in Caco-2 cells; hence provoking V. cholerae attachment and subsequent colonization. We concluded that TcpA affects inflammation and bacterial colonization in vitro; however, more investigations are required to illustrate the effects of bacterial adhesins on the intracellular pathways of IEC.

Anticancer Activity of Alkynylgold(I) with P(NMe2)3 Phosphane in Mouse Colon Tumors and Human Colon Carcinoma Caco-2 Cell Line.

New alkynylgold(I) with P(NMe2)3 (HMPT) phosphane complexes, [Au(C≡C-R)(HMPT)] (R= 4-Ph, 4-MePh, 4-OMe, 4-Br, 4-Cl, 2-py, and 3-py) have been synthesized and characterized, including X-ray studies of complexes with R= 4-OMe and 4-Br; additionally, their physicochemical properties and anticancer activity have been tested. Due to the great water solubility of the HMPT phosphane, all the complexes exhibit an optimal balance of hydrophilicity/lipophilicity. Also, all of these complexes are quite stable in physiological conditions and interact well enough with the transport protein BSA. All complexes exhibit a higher anticancer activity against Caco-2 cells than cisplatin, and some of them do not present cytotoxic activity against enterocyte-like differentiated cells. The selective complexes are proapoptotic drugs by the exposure of phosphatidylserine, results that are also confirmed in primary cultures from mouse colon tumors. Complexes with a halogen unit also arrest the cell cycle in G2/M phase. It is thought that maybe these apoptosis processes are promoted by the observed oxidative damage in the membrane lipids, as a consequence of the inhibition of the thioredoxin reductase enzyme. Based on our results, we conclude that five of our complexes are good candidates to be used in chemotherapy.

Molecular signature of interleukin-22 in colon carcinoma cells and organoid models

Interleukin (IL)-22 activates STAT (signal transducer and activator of transcription) 3 and antiapoptotic and proproliferative pathways; but beyond this, the molecular mechanisms by which IL-22 promotes carcinogenesis are poorly understood. Characterizing the molecular signature of IL-22 in human DLD-1 colon carcinoma cells, we observed increased expression of 26 genes, including NNMT (nicotinamide N-methyltransferase, ≤10-fold) and CEA (carcinoembryonic antigen, ≤7-fold), both known to promote intestinal carcinogenesis. ERP27 (endoplasmic reticulum protein-27, function unknown, ≤5-fold) and the proinflammatory ICAM1 (intercellular adhesion molecule-1, ≤4-fold) were also increased. The effect on CEA was partly STAT3-mediated, as STAT3-silencing reduced IL-22-induced CEA by ≤56%. Silencing of CEA or NNMT inhibited IL-22-induced proliferation/migration of DLD-1, Caco-2, and SW480 colon carcinoma cells. To validate these results in primary tissues, we assessed IL-22-induced gene expression in organoids from human healthy colon and colon cancer patients, and from normal mouse small intestine and colon. Gene regulation by IL-22 was similar in DLD-1 cells and human and mouse healthy organoids. CEA was an exception with no induction by IL-22 in organoids, indicating the 3-dimensional organization of the tissue may produce signals absent in 2D cell culture. Importantly, augmentation of NNMT was 5-14-fold greater in human cancerous compared to normal organoids, supporting a role for NNMT in IL-22-mediated colon carcinogenesis. Thus, NNMT and CEA emerge as mediators of the tumor-promoting effects of IL-22 in the intestine. These data advance our understanding of the multifaceted role of IL-22 in the gut and suggest the IL-22 pathway may represent a therapeutic target in colon cancer.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Polyphenols associated to pectic polysaccharides account for most of the antiproliferative and antioxidant activities in olive extracts

• Olive extracts rich in polysaccharides associated with polyphenols were obtained. • The extracts showed strong antioxidant and antiproliferative activities. • These activities were reduced when the phenols were removed by sodium chlorite. • Bleached extracts had a similar antiproliferative activity as citrus modified pectin. Extracts rich in pectic polysaccharides associated to polyphenols were obtained from alperujo, the by-product of olive oil extraction, by different thermal treatments and subsequent chemical treatments. The resulting “Pectoliv” extracts exhibited strong antioxidant activities and a high antiproliferative capacities in vitro against colon carcinoma Caco-2 and leukemia monocytic THP-1 cell lines. Bleaching of the extracts with sodium chlorite removed the phenolic compounds and significantly reduced antiproliferative and antioxidant activities, confirming that these were due mainly to the associated polyphenols present in Pectoliv. However, even after bleaching, these activities remained very similar to that of a commercial citrus modified pectin obtained by chemical treatment from citrus pectin, which has been implicated in cancer treatment and prevention. Thus Pectolivs have a high potential as antiproliferative or antioxidant agents.

Potential anticancer effects of cell wall protein fractions from Lactobacillus paracasei on human intestinal Caco-2 cell line.

Consumption of probiotics has an important role in colorectal cancer prevention. In this study, we aimed to explore that the cell wall protein fractions from Lactobacillus paracasei could induce apoptosis on Caco-2 cell line. The cell wall proteins from L. paracasei were fractionated by gel filtration chromatography (F1, F2 and F3) and characterized by polyacrylamide gel electrophoresis (SDS-PAGE). The anticancer properties were evaluated using MTT assay and Annexin V-FITC/PI staining. Administration of L. paracasei increased a significant concentration- and time-dependent anti-proliferative effect on Caco-2 cell line, determined by cell viability assays. However, a dramatic decrease in cell viability of Caco-2 cells was observed at the concentration of 100µgml-1 of F1 L. paracasei for 72h (58% cell viability, P<0·05) The results showed that F1 L. paracasei could induce apoptosis in Caco-2 cancer cell line by increased in annexin V and propidium iodide staining for 72h (up to 90·6%, P<0·001). These results indicated the importance of the anticancer effects of cell wall protein fractions of L. paracasei in human colon carcinoma Caco-2 cell line. Thus, cell wall protein fractions of L. paracasei can be a potential chemotherapeutic agent against Caco-2 cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: Significance and Impact of the Study: Our findings revealed that the newly identified cell wall protein fractions from probiotic Lactobacillus paracasei inhibit the cell growth of human colon carcinoma cell line (Caco-2), and the results indicated that the cell wall proteins from L. paracasei can be a potential chemotherapeutic agent against Caco-2 cell lines.

Interactive anticancer effect of nanomicellar curcumin and galbanic acid combination therapy with some common chemotherapeutics in colon carcinoma cells.

In the current investigation, we aimed to study the combined cytotoxicity of curcumin, as a nanomicellar formulation, and galbanic acid (Gal), dissolved in DMSO against the murine C26 and human Caco-2 colon carcinoma cells. Further, curcumin potential for cisplatin and doxorubicin (Dox) co-therapy was studied. The combined cytotoxic effect of these phytochemicals at varying dose ratios were examined using the MTT colorimetric assay. Moreover, the time-dependent toxicity of curcumin, cisplatin, Dox, and pegylated liposomal Dox (Doxil) was determined. The interactive anti-proliferative behavior of these compounds was examined using the CompuSyn software. Nanomicellar curcumin showed considerable cytotoxicity in C26 cells 24 hr post-treatment. Co-treatment of cells with curcumin nanomicelles: Gal had a synergistic effect in C26 (at 10:1 molar ratio), and Caco-2 (at 1:5 molar ratio) cell lines in cell cultures. Nanomicellar curcumin showed strong and mild synergistic inhibitory effects in C26 cells when co-administered with Doxil and cisplatin, respectively. Curcumin nanomicelles and Gal had a synergistic effect in C26 and Caco-2 cell lines. It is speculated that nanomicellar curcumin shows synergistic cancer cell killing if administered 24-hr post-injection of Doxil and cisplatin.

Virulence, attachment and invasion of Caco-2 cells by multidrug-resistant bacteria isolated from wild animals

Wild animals may be considered important reservoirs for bacterial pathogens and, consequently, possible sources of infection for humans. In this study, selected multidrug-resistant bacteria (Acinetobacter spp., Aeromonas salmonicida, Klebsiella pneumoniae, Pseudomonas fluorescens and Shewanella putrefaciens) isolated from wild animals were characterized on their ability to attach and invade/internalize human colonic carcinoma (Caco-2) cells. In addition, the viability of these bacteria to survive under simulated human gastrointestinal tract conditions as well as the production of virulence factors (homoserine lactones signal molecules, gelatinases, proteases, siderophores and biofilm formation) were studied. The results suggests that all the bacteria presented the capacity to attach and internalize into Caco-2 cells. A. salmonicida and P. fluorescens exhibited the highest ability to internalize. These bacteria were also found to be the highest proteases producers. A. salmonicida and K. pneumoniae survived under simulated human gastrointestinal conditions. These were the bacteria with the highest capacity to produce biofilms. K. pneumoniae was the only bacterium producing siderophores. Taken together, the present results reinforce the need for the “One Health” initiative, underscoring the environment and the animals as important reservoirs of infectious determinants.

Effects of arginine-based surface modifications of liposomes for drug delivery in Caco-2 colon carcinoma cells

Liposomal encapsulation of chemotherapeutics improves circulation time and decreases off-target effects through the enhanced permeability and retention (EPR) effect. Improving the efficacy of these drug carriers through surface modification could benefit patients. A library of arginine derivatives was conjugated to liposomes through carbodiimide chemistry. Both unmodified and modified liposomes were loaded with doxorubicin and exposed to Caco-2 colon carcinoma cells to measure the half maximal inhibitory concentration (IC50). Most of the modifications improved the toxicity of doxorubicin. Principal component analysis (PCA) was used to uncover correlations between physicochemical properties (lipophilicity (log P), partition coefficient (log D), number of hydrogen bond donors, number of hydrogen bond acceptors, freely rotating bonds, surface tension, polarization surface area, and isoelectric point) and the IC50 of encapsulated doxorubicin. Generalized rules for improved toxicity were also developed, which stated that improved drug carriers should have at least 4 hydrogen bond donors, between 4 and 6 freely rotating bonds, an isoelectric point above 5.5, and a log P between -2 and -1. Using these relationships along with previously obtained correlations for macrophages, selective targeting and the understanding of how to rationally design such drug carriers can be improved.

Development of a Caco-2 Cell Line Carrying the Human Intestine-Type CES Expression Profile as a Promising Tool for Ester-Containing Drug Permeability Studies.

Carboxylesterase 2 (CES2), which is a member of the serine hydrolase superfamily, is primarily expressed in the human small intestine, where it plays an important role in the metabolism of ester-containing drugs. Therefore, to facilitate continued progress in ester-containing drug development, it is crucial to evaluate how CES2-mediated hydrolysis influences its intestinal permeability characteristics. Human colon carcinoma Caco-2 cells have long been widely used in drug permeability studies as an enterocyte model. However, they are not suitable for ester-containing drug permeability studies due to the fact that Caco-2 cells express CES1 (which is not expressed in human enterocytes) but do not express CES2. To resolve this problem, we created a new Caco-2 cell line carrying the human small intestine-type CES expression profile. We began by introducing short-hairpin RNA for CES1 mRNA knockdown into Caco-2 cells to generate CES1-decifient Caco-2 cells (Caco-2CES1KD cells). Then, we developed Caco-2CES1KD cells that stably express CES2 (CES2/Caco-2CES1KD cells) and their control Mock/Caco-2CES1KD cells. The results of a series of functional expression experiments confirmed that CES2-specific activity, along with CES2 mRNA and protein expression, were clearly detected in our CES2/Caco-2CES1KD cells. Furthermore, we also confirmed that CES2/Caco-2CES1KD cells retained their tight junction formation property as well as their drug efflux transporter functions. Collectively, based on our results clearly showing that CES2/Caco-2CES1KD cells carry the human intestinal-type CES expression profile, while concomitantly retaining their barrier properties, it can be expected that this cell line will provide a promising in vitro model for ester-containing drug permeability studies.

3D Model Replicating the Intestinal Function to Evaluate Drug Permeability.

Animal models are essential in drug development but present many concerns in the practical and ethical sense. To avoid the unnecessary use of animals other models are used in the beginning of any scientific discovery, the in vitro models. The relevance of in vitro cell based culture models for studying intestinal drug absorption and transcytosis during early stages of drug development is undeniable. Several in vitro co-culture models have been described for this purpose, however excluding the integration of the complex intestinal architecture and neglecting different physiological mechanisms involved in the drug transport. 2-D cell cultures are the current standard, but despite their widely use, they no longer are considered the most trustworthy in vitro models since they do not mimic many aspects that happen in vivo. The simulation of a complete microenvironment capable of mimicking the intestinal mucosa requires therefore further investigation, particularly focused in addressing the abovementioned unmet needs. 3D models came as bridge between the in vitro and in vivo models. These models are proven to be influential of the drug effect in cells, being the most adequate to mimic the live tissue especially in drug development. Supported by the great amount of studies using simple and reductionist co-culture monolayers, and pushing forward an innovative model previously reported by our group, the present study aims to describe a sophisticated and highly reproducible in vitro 3D co-culture intestinal model. Here, the components are assembled in a multistage process into Transwell filters by co-culturing human colon carcinoma Caco-2 and mucus-producing HT29-MTX cells over a layer of collagen embedding intestinal myofibroblasts (CCD-18Co). The 3D co-culture intestinal model described herein represents a particularly powerful and versatile tool that recapitulates the intestinal functioning regarding mucus production, tightness of the different cell types, and the 3D architecture, bridging the gap between simple monolayer cultures of epithelial cells and the complex in vivo physiological conditions. Importantly, it shows tremendous potential in predicting intestinal absorption of orally administered drugs when delivered alone, or encapsulated into micro- and nanosystems, the current leading force of pharmaceutical technology research.

N-3 Polyunsaturated Fatty Acids Stimulate Bile Acid Detoxification in Human Cell Models.

Cholestasis is characterized by the accumulation of toxic bile acids (BAs) in liver cells. The present study aimed to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, on BA homeostasis and toxicity in human cell models. The effects of EPA and/or DHA on the expression of genes involved in the maintenance of BA homeostasis were analyzed in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, as well as in primary culture of human intestinal (InEpC) and renal (RPTEC) cells. Extracellular BA species were quantified in culture media using LC-MS/MS. BA-induced toxicity was evaluated using caspase-3 and flow cytometry assays. Gene expression analyses of HepG2 cells reveal that n-3 PUFAs reduce the expression of genes involved in BA synthesis (CYP7A1, CYP27A1) and uptake (NTCP), while activating genes encoding metabolic enzymes (SULT2A1) and excretion transporters (MRP2, MRP3). N-3 PUFAs also generate a less toxic BA pool and prevent the BA-dependent activation of apoptosis in HepG2 cells. Conclusion. The present study reveals that n-3 PUFAs stimulate BA detoxification.

In Vitro Bioavailability Study of an Antiviral Compound Enisamium Iodide.

An investigation into the biopharmaceutics classification and a study of the in vitro bioavailability (permeability and solubility) of the antiviral compound enisamium iodide (4-(benzylcarbamoyl)-1-methylpyridinium iodide) were carried out. The solubility of enisamium iodide was determined in four different buffers. Apparent intestinal permeability (Papp) of enisamium iodide was assessed using human colon carcinoma (Caco-2) cells at three concentrations. The solubility of enisamium iodide in four buffer solutions from pH 1.2 to 7.5 is about 60 mg/mL at 25 °C, and ranges from 130 to 150 mg/mL at 37 °C, depending on the pH. Based on these results, enisamium iodide can be classified as highly soluble. Enisamium iodide demonstrated low permeability in Caco-2 experiments in all tested concentrations of 10–100 μM with permeability coefficients between 0.2 × 10−6 cm s−1 and 0.3 × 10−6 cm s−1. These results indicate that enisamium iodide belongs to class III of the Biopharmaceutics Classification System (BCS) due to its high solubility and low permeability. The bioavailability of enisamium iodide needs to be confirmed in animal and human studies.

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.

Human and mouse microarrays-guided expression analysis of membrane protein trafficking-related genes in MDCK cells, a canine epithelial model for apical and basolateral differential protein targeting

MDCK cells are widely used to study the differential targeting of membrane transporters to apical and basolateral membrane but its canine origin limited the commercial tools available for the analysis of protein trafficking machinery. Because apical and basolateral membranes are only found in differentiated epithelial cells, genes critical for differential targeting may be specifically up-regulated upon MDCK cell differentiation. To search for these genes, a cross-species screening strategy was used. We first analyzed the human microarray data for protein trafficking-related genes that were up-regulated in colon carcinoma Caco2 cells upon differentiation. The results of mouse 44K gene expression microarray analysis were then used to extract additional candidate genes that showed higher expression in normal colon epithelium compared to primary embryonic fibroblasts. Finally, NCBI genomic sequence information was used to design RT-PCR primers for 13 candidate and 10 negative control genes and used to analyze MDCK cells at 2, 13 and 17 days after seeding. To determine whether the gene up-regulation was specific in epithelial differentiation, we also performed RT-PCR on rat non-differentiating intestinal IEC-6 cells and mouse C2C12 cells, a differentiating myoblast model. Of the 13 candidate genes, 3 genes, SDCBP2, KIF12, KIF27, met all criteria of specific up-regulation in differentiated MDCK cells. In addition, KIF13A showed up-regulation in differentiated MDCK and C2C12 cells but not in IEC-6 cells cultured for the same duration. The functions of these genes need to be analyzed in the future. This cross-species screening strategy may be useful for other non-human, non-rodent cell models.

Response surface method optimization of a novel Hypericin formulation in P123 micelles for colorectal cancer and antimicrobial photodynamic therapy

The photodynamic properties of Hypericin (Hyp) may be used as an alternative treatment for malignancies of the lower gastrointestinal tract and for the prevention of surgical-site infection; however, its use in photodynamic therapy has been limited because of its poor hydrosolubility. Therefore, in order to improve its water solubility and its photodynamic effect, Hyp was encapsulated in Pluronic P123 (P123) and the photodynamic effects against intestinal and epidermal bacteria and against two lineages of intestinal colon carcinoma cells were investigated. Two response surface methods (RSM) were used to achieve the best in vitro photodynamic activity against Enterococcus faecalis, Escherichia coli and Staphylococcus aureus: in the first (full 23 RSM), Hyp concentration (HC*), incubation time (IT*) and LED-light time (LT*) were considered as the independent variables and E. faecalis inhibition as the dependent variable. In the second (full 32 RSM), Hyp concentration (HC*) and P123 concentration (CC*) were considered as independent variables and E. faecalis, E. coli and S. aureus inhibition as dependent variables. The optimized experimental conditions achieved were: Hyp concentration=37.5μmol/L; P123 concentration=21.5 μmol/L and 6.3J/cm2, which resulted in 2.86±0.12 and 2.30±0.31CFU log-reductions of E. faecalis and S. aureus. No effect was seen against E. coli. The cytotoxic effects of Hyp/P123 were also investigated for Caco-2 and HT-29 intestinal colon carcinoma cells at Hyp/P123 concentrations of 1, 0.5, 0.25 and 0.1μmol/L for Caco-2 cells and 4, 3, 2 and 1μmol/L for HT-29 cells. The cytotoxic concentrations for 50% (CC50) and 90% (CC90) of Hyp/P123 were 0.443 and 0.870μmol/L for Caco-2 cells and 1.4 and 2.84μmol/L for HT-29 cells. The P123 nanocarrier played a significant role in the permeation of Hyp through the cell membrane leading to significant cell death, and showed itself to be a promising photosensitizer for PDT that could be suitable for the treatment of colonic diseases since it is effective against positive Gram bacteria and intestinal colon carcinoma cells.