Colon Adenocarcinoma Cell Line Caco-2 Research Articles

Избирательное изменение экспрессии α-субъединиц интегринов в клетках кишечного эпителия Caco-2 при гипоксии в условиях микроциркуляции

Intestinal epithelial cells are constantly exposed to physiologically hypoxic environment. The further reduction of tissue oxygen delivery may result in the intestinal epithelial cells function impairment, being a sign of active inflammation. The cultivation conditions are important when performing in vitro studies, since those may affect the cells’ properties. The study was aimed to assess the integrin receptor expression in the human colon adenocarcinoma Caco-2 cell line when simulating both hypoxic condition using the cobalt chloride and microcirculation. Transcriptome analysis revealed the significantly increased expression of the integrin receptors ITGA2 and ITGA5 α2- and α5-subunit genes under hypoxic conditions, as well as the reduction of ITGA5 during incubation in the microfluidic chip. The expression of β-subunits did not change. Analysis of microRNA transcriptomes revealed the decreased expression of hsa-miR-766-3p and hsa-miR-23b-5p microRNA. One of the validated targets for both microRNAs is mRNA of gene ITGA5. It has been shown that microcirculation makes it possible to bring the intestinal epithelial cells cultivation conditions closer to physiological conditions. The possible biological significance of the detected integrin expression profile alterations and the role of microcirculation have been discussed.

Cytotoxic and DNA-Damaging Effects of Aronia melanocarpa, Cornus mas, and Chaenomeles superba Leaf Extracts on the Human Colon Adenocarcinoma Cell Line Caco-2.

Aronia melanocarpa, Cornus mas, and Chaenomeles superba leaf extracts contain large amounts of bioactive compounds—mainly polyphenols, which possess many health benefits including anti-cancer properties. Here, we investigate the biological effects of A. melanocarpa, C. mas, and C. superba leaf extracts on the human colon adenocarcinoma cell line Caco-2. The antiproliferative activity of the extracts was measured using the MTT assay. The most cytotoxic extract was C. mas (IC50 = 0.60%). The extracts caused morphological changes in the Caco-2 cells, including partial detachment of cells, necrotic cells, chromatin condensation, cytoplasmic vacuolization, cell nuclei lysis, and nucleus fragmentation. The DNA damage in the Caco-2 cells after exposure to the leaf extracts was measured using the alkaline comet assay. The extracts increased DNA damage in a concentration dependent manner. However, at lower non-cyto- and non-genotoxic (IC0) concentrations the extracts induced DNA repair in Caco-2 cells after exposure to hydrogen peroxide. In conclusion, the results of these studies suggest that A. melanocarpa, C. mas and C. superba leaf extracts can show anticancer activity. However, further research is required on the mechanisms of anti-cancer activity by these extracts, with the application of more advanced and wide-ranging techniques including in vivo experiments.

Systematic evaluation of potential pathogenicity of Salmonella Indiana

Salmonella Indiana has emerged in recent years as an important zoonotic pathogen, but its pathogenicity has not been fully elucidated. In this study, using in vivo and in vitro animal and cellular experimental model systems, we evaluated the pathogenicity of Salmonella Indiana (S. Indiana) compared with three other serotypes of Salmonella, S. Enteritidis, S. Typhimurium and S. Thompson. The animal experiments included observations of clinical symptoms, pathological changes and determination of median lethal dose in mice. The adhesion and invasiveness and intracellular proliferative capacity of Salmonella in vitro were measured with the murine macrophage-like cell line RAW264.7 cells and the human colon adenocarcinoma cell line Caco-2 cells. The results of animal experiments showed that S. Indiana, S. Enteritidis, S. Typhimurium and S. Thompson caused histopathological changes in most organs to varying degrees, primarily in the liver and intestine of mice. The gross lesions included white necrotic foci on the liver surface with different levels. The histopathological changes of monocyte/macrophage infiltration and coagulative necrosis were observed in the liver. Intestinal villi became short and were sloughed off, and lymphocyte infiltration was found in the submucosa. Compared with the other serotypes, the pathological changes caused by S. Indiana were slighter and had a relatively high median lethal dose in mice. The results of adhesion and invasion tests showed that the intracellular growth trend of most Salmonella strains was positively correlated with the number of pathogens adhering to and invading cells. Compared with the strains of the other three serotypes, most S. Indiana strains exhibited significantly lower adhesion and invasiveness to RAW264.7 and Caco-2 cells within 30 min. Most S. Indiana strains displayed twice to four times lower intracellular proliferation within 24 h in RAW264.7 cells. In conclusion, S. Indiana was pathogenic, but its pathogenicity was lower than that of S. Typhimurium and S. Enteritidis, and was similar to that of S. Thompson.

Roles of N-terminal Annexin A2 phosphorylation sites and miR-206 in colonic adenocarcinoma

AimsAnnexin A2 (ANXA2) is closely associated with tumor malignancy and its N-terminus includes a vital domain for its function. The aims are to explore the correlation between the sites (Tyr23, Ser1, Ser11 and Ser25) in the domain and its roles. Main methodsWe re-expressed ANXA2 with mutated sites in ANXA2-deleted human colonic adenocarcinoma cell line caco2 (ANXA2-/-caco2). A series of analyses were used to determine the correlation of each site with ANXA2 activation, cell malignancy enhancement and motility-associated microstructural development. Bioinformatics and luciferase reporter assays were employed to validate ANXA2-targeted miRNAs. Key findingsThe in vitro results showed that all single and multiple mutations of the ANXA2 N-terminal sites inhibited ANXA2 phosphorylation at different levels and subsequently inhibited the proliferation, motility, and polymerization of F-actin and β-tubulin in caco2 cells. Motility-associated microstructures were significantly remodeled when these sites were mutated. The forced expression of miR-206 significantly suppressed the proliferation, motility and epithelial-mesenchymal transition (EMT) of caco2 cells. The in vivo results showed that all the ANXA2 N-terminal site mutations and forced expression of miR-206 significantly inhibited tumor growth. Overall, this study demonstrated that the sites of the ANXA2 N-terminus, especially Tyr23, play crucial roles in maintaining the high malignancy of colonic adenocarcinoma. Furthermore, miR-206 targets ANXA2 and plays a role as a cancer suppressor in colonic adenocarcinoma. SignificanceOur study provided evidence that further elucidates the molecular mechanism of ANXA2 and its roles in colonic adenocarcinoma and suggested potential targets of ANXA2 for colonic adenocarcinoma therapy by using miR-206 as a novel strategy.

Toxic effects and mechanisms of three commonly used fungicides on the human colon adenocarcinoma cell line Caco-2

Fungicides, usually refer to the chemical agents that can effectively control or kill the pathogenic microorganisms. Here, we revealed the effects of three different fungicides, imazalil (IMZ), chlorothalonil (CTL) and carbendazim (CBZ), which are typical broad-spectrum fungicides that are detected at high levels in the natural environment, on heterogeneous human epithelial colorectal cells (Caco-2 cells). All three fungicides had the potential to induce different degrees of toxicity, cause apoptosis, reactive oxygen species (ROS) and even change the cell cycle in the cells. The half maximal inhibitory concentration (IC50) of CTL is the lowest among these three fungicides, suggesting that it may have the highest exposure risk, followed by IMZ, and CBZ. The results of the real-time PCR, Western blotting, and mitochondrial membrane potential (MMP) assays and the activities of key enzymes suggested that CTL induced apoptosis in Caco-2 cells via a mitochondrial-dependent pathway, as indicated by the upregulation of the expression of the apoptotic p53 and bax genes, the increase of the apoptosis marker cytochrome-c, the decrease of mRNA level of bcl-2 gene, and the decrease in the MMP. Exposure to two other fungicides also upregulated the transcriptional level of bax and the expression of cytochrome-c, but the mRNA level of bcl-2 was increased (IMZ) or unchanged (CBZ), suggesting that other pathways may be involved in the induction of cellular apoptosis by these two fungicides. In addition, all three of the fungicides could induce oxidative stress in Caco-2 cells. Our data showed that the three different kinds of fungicides all caused toxic effects in Caco-2 cells through various pathways.

Absorption Rates and Mechanisms of Avenanthramides in a Caco-2 Cell Model and Their Antioxidant Activity during Absorption.

Avenanthramides (AVNs) are a unique kind of polyphenols that were only detected in the oats and have been demonstrated to exhibit strong antioxidant activities but low bioavailability. The purpose of the present research was to evaluate the absorption rates and mechanisms of AVNs (AVN 2c, AVN 2f, and AVN 2p) using a human colon adenocarcinoma cell line (Caco-2) cell model and clarify the influence of the absorption process on the antioxidant capacities of AVNs. Furthermore, the absorption rates and antioxidant activities of ferulic acid and caffeic acid were compared with those of AVNs. Results showed that the apparent absorption rates (Papp) of AVN 2c, AVN 2f, and AVN 2p were 0.65 ± 0.05 × 10-6, 1.18 ± 0.16 × 10-6, and 1.44 ± 0.09 × 10-6 cm/s, respectively, which were significantly lower than those of caffeic acid (3.76 ± 0.31 × 10-6 cm/s) and ferulic acid (1.69 ± 0.13 × 10-5 cm/s). Moreover, the metabolites (caffeic acid, ferulic acid, and AVN 2f) of AVNs after absorption were detected and quantified by high-performance liquid chromatography-mass spectrometry. Before absorption, although the antioxidant capacities of AVNs were significantly stronger than those of ferulic acid and caffeic acid, there was an opposite result after absorption. In addition, AVNs transported the Caco-2 monolayer by paracellular diffusion and were affected by monoamine oxidase and efflux transporters (P-gp, MRP2) during absorption. The co-administration of quercetin could significantly improve the absorption rates of AVNs.

Characteristics and bioactive properties of mannooligosaccharides derived from agro-waste mannans

Mannooligosaccharides (MOS) were derived using Aspergillus oryzae β-mannanase (ManAo) from different mannan-rich agro-wastes, palm kernel cake (PKC), guar gum and copra meal (CM). Guar gum (GG) released higher amount of MOS (56.31% w/w) from which purification of mannobiose (0.68 mg) and mannotriose (1.26 mg) was demonstrated using size-exclusion chromatography. FTIR analysis of mannan hydrolysates showed characteristic peaks in 1200–900 cm−1 region indicating the presence of MOS. 1H &13C NMR spectra showed presence of anomeric sugar forms of MOS in different mannan hydrolysates. MOS from locust bean gum and guar gum had both α- and β-anomers while PKC and CM had only α-anomer. Growth promotional activities of different MOS were demonstrated using two probiotic Lactobacilli. Besides, enzymatically derived MOS also showed metal (Fe2+) chelating and anti-oxidant activities, wherein best anti-glycating agent was evaluated as MOS from PKC. PKC derived MOS showed highest cytotoxicity (74.19%) against human colon adenocarcinoma cell line (Caco-2). This study demonstrated the prebiotic potential of agro-waste derived MOS and possibility of their utilization as a functional food ingredient.

Acrylamide Decreases Cell Viability, and Provides Oxidative Stress, DNA Damage, and Apoptosis in Human Colon Adenocarcinoma Cell Line Caco-2.

Acrylamide (AA) toxicity remains an interesting subject in toxicological research. The aim of the research performed in this paper was to determine mechanisms of cyto- and genotoxic effects of AA on the human colon adenocarcinoma cell line Caco-2, to estimate the inhibitory concentration (IC)50 values in cell viability assays, to measure the basal and oxidative DNA damage as well as the oxidative stress leading to apoptosis, and to assess the morphological changes in cells using microscopic methods. It has been proven that AA induces cytotoxic and genotoxic effects on Caco-2 cells. Higher cytotoxic activity was gained in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay compared with the PrestoBlue assay, with IC50 values of 5.9 and 8.9 mM after 24 h exposure, respectively. In the single-cell gel electrophoresis assay, the greatest DNA damage was caused by the highest concentration of acrylamide equal to 12.5 mM (89.1% ± 0.9%). AA also induced oxidative DNA damage and generated reactive oxygen species (ROS), which was concentration dependent and correlated with the depletion of mitochondrial membrane potential and apoptosis induction. In the microscopic staining of cells, AA in the dosage close to the IC50 induced morphological changes typical for apoptosis. Taken together, these results demonstrate that AA has a pro-oxidative effect on Caco-2 cells, leading to apoptotic cell death.

Assessment of drugs belonging to inhibitors and inductors of p-glycoprotein in vitro

The article describes modern approaches for testing of drugs belonging to substrates and inhibitors of P-glycoprotein (Pgp, ABCB1-protein, MDR1-protein) according to the recommendations of Food and Drug Administration (United States) and European Medicines Agency. In vitro methods on cell lines with hyperexpression of the transporter are presented. The same analysis was done on human colon adenocarcinoma cell line (Caco-2).

Lathyrus sativus diamine oxidase counteracts histamine-induced cell proliferation, migration and pro-angiogenic mediators release in human colon adenocarcinoma cell line Caco-2.

Because histamine is a modulator of cancer cell proliferation and invasiveness, this study aimed at investigating the effect of Lathyrus sativus-derived diamine oxidase (LSAO) and its mechanism of action on Caco-2 cell line, considering that LSAO catalizes the oxidative deamination of histamine to the corresponding aldehyde, NH3 and H2 O2 . Histamine (0.01-1μM) caused a proliferative effect on Caco-2 cells promoting cell migration, invasion and nitric oxide and vascular endothelial growth factor release. Histamine (1μM) stimulus also down regulated occludin expression, favouring up regulation of pro-proliferative nuclear protein Ki67. Incubation with LSAO (0.004-0.4μM) resulted in a significant inhibition of histamine-induced effects. LSAO rescued occludin expression and down regulated Ki67, and it inhibited histamine-induced increase of both MMP-2 and 9 expression. Histamine effects were mediated by RhoA-GTP down regulation and inversely related to phospho-p38MAPK/p50/65 up regulation. These effects were counteracted by LSAO incubation. Histamine catabolism by LSAO accounts for a significant down regulation of proliferation and invasiveness of Caco-2 cells. This study highlights the importance to control histamine levels in contrasting pro-angiogenic and metastatization capability of colon cancer cells and expands the knowledge about the diamine oxidase from L.sativus seeding as a phytotherapeutic approach for colon cancer.

Role of tropomyosin 4 gene misregulation in colon epithelial cell tumorigenesis.

e15184 Background: Ordered migration of colon epithelial cells from the bottom to the top of the crypt is required for their differentiation. Because aberrant colon cell migration may contribute to early stages of colon tumorigenesis, elucidation of how this migration is regulated provides insight into how colon cancer can be prevented and treated before malignancy. Certain actin-binding tropomyosins, including tropomyosin 4 (TPM4), regulate cytoskeletal contractility and migration in many different cell types. Methods: To determine whether aberrant TPM4 regulation may be an early event in colon cell tumorigenesis, we measured TPM4 RNA expression in uninvolved, grossly normal colon tissue from 10 human familial adenomatous polyposis (FAP) patients genetically predisposed to colon cancer versus 7 patients not genetically predisposed (normal). We also examined TPM4 expression in a colon adenocarcinoma cell line (Caco2). These are highly proliferative, undifferentiated cells but can be induced to growth arrest and differentiate, thus acquiring many characteristics of normal absorptive colon epithelial cells. Reporter assays were used to identify sequences that regulate TPM4 gene expression in Caco2 cells. Results: Average TPM4 RNA expression is 2.59 fold higher in the FAP versus normal tissue. TPM4 RNA and protein are highly expressed in proliferating, undifferentiated Caco2 cells but are severely reduced in growth arrested, differentiated cells. Reporter assays have identified genetic regions, near the TPM4 transcriptional start site, that may regulate TPM4 gene expression in proliferating Caco2 cells. Conclusions: This data suggests that aberrantly upregulated TPM4 may be an early event in colon tumorigenesis and that normal TPM4 expression, by fostering ordered colon epithelial cell migration, may promote normal cell differentiation and mitigate adoption of a premalignant state.

Daunorubicin oral bioavailability enhancement by surface coated natural biodegradable macromolecule chitosan based polymeric nanoparticles.

Daunorubicin hydrochloride (DAUN·HCl), due to low oral bioavailability poses the hindrance to be marketed as an oral formulation. To develop a natural biodegradable macromolecule i.e. Chitosan (CS)-coated-DAUN-PLGA-poly(lactic-co-glycolic acid)-Nanoparticles (NPs) with an aim to improve oral-DAUN bioavailability and to develop as well as validate UHPLC-MS/MS (ESI/Q-TOF) method for plasma quantification and pharmacokinetic analysis (PK) of DAUN. A particle size (198.3 ± 9.21 nm), drug content (47.06 ± 1.16 mg/mg) and zeta potential (11.3 ± 0.98 mV), consisting of smooth and spherical shape was observed for developed formulation. Cytotoxicity studies for CS-DAUN-PLGA-NPs revealed; a comparative superiority over free DAUN-S (i.v.) in human breast adenocarcinoma cell lines (MCF-7) and a higher permeability i.e. 3.89 folds across rat ileum, as compared to DAUN-PLGA-NPs (p < 0.01) inhuman colon adenocarcinoma cell line (Caco-2). For PK, CS-DAUN-PLGA-NPs as compared to DAUN-S, exhibited a 10.0 fold higher bioavailability in Wister rat's plasma due to presence of a natural biodegradable macromolecule i.e. CS coated on the PLGA-NPs. With regard to bioanalytical method, easy as well as a rapid method for DAUN-plasma quantification was developed as; 2.75 min and 528.49/321.54 m/z for DAUN along with 1.94 min and 544.36/397.41 m/z for IS i.e. Doxorubicin, for elution time and transition, respectively. A novel natural biodegradable approach used in the preparation of CS coated DAUN-NPs for oral administration of DAUN is reported in this study which is can be utilized as an alternate for intravenous therapy.

The Effect of Prim-O-Glucosylcimifugin on Tryptase-Induced Intestinal Barrier Dysfunction in Caco-2 Cells.

The intestinal barrier dysfunction is a critical pathological change in irritable bowel syndrome (IBS). The objective of this study was to evaluate the effect of Prim-O-glucosylcimifugin (POG) on intestinal barrier dysfunction and reveal possible molecular mechanisms. Human colon adenocarcinoma cell line (Caco-2) cell monolayers induced by tryptase (TRYP) were used to establish an intestinal barrier dysfunction model. Caco-2 cell monolayers from both functional and dysfunctional samples were treated with POG (30, 60 and 120 µg/mL) for 2, 8, 24, 36, 48 and 72 h. The Caco-2 cell monolayers were assessed by measurement of trans-epithelial electrical resistance (TEER) and percentage of fluorescein permeation (PFP). The expression of Protease Activated Receptor 2 (PAR-2) and myosin light chain kinase (MLCK) mRNA was analyzed by RT-PCR and the level of Zonula Occludens-1 (ZO-1) protein expression was determined by Western blot. In addition, the impact of POG on the distribution of the tight juction protein of Occludin was performed by immunofluorescence. Our results showed that POG elevated the TEER and decreased the PFP of the functional Caco-2 cell monolayers, as well as the dysfunctional Caco-2 cell monolayers. Furthermore, POG inhibited the expression of PAR-2 mRNA and MLCK mRNA and increased the level of ZO-1 protein expression in dysfunctional Caco-2 cells. The distribution of the Occludin proteins was ameliorated simultaneously. This study demonstrates that POG can enhance the intestinal barrier function of Caco-2 cell monolayers by inhibiting the expression of PAR-2 and MLCK and up-regulating the expression of ZO-1 protein, and ameliorated the distribution of Occludin protein.

Olive oil polyphenols reduce oxysterols -induced redox imbalance and pro-inflammatory response in intestinal cells

Dietary habits may strongly influence intestinal homeostasis. Oxysterols, the oxidized products of cholesterol present in cholesterol-containing foodstuffs, have been shown to exert pro-oxidant and pro-inflammatory effects, altering intestinal epithelial layer and thus contributing to the pathogenesis of human inflammatory bowel diseases and colon cancer. Extra virgin olive oil polyphenols possess antioxidant and anti-inflammatory properties, and concentrate in the intestinal lumen, where may help in preventing intestinal diseases. In the present study we evaluated the ability of an extra virgin olive oil phenolic extract to counteract the pro-oxidant and pro-inflammatory action of a representative mixture of dietary oxysterols in the human colon adenocarcinoma cell line (Caco-2) undergoing full differentiation into enterocyte-like cells. Oxysterols treatment significantly altered differentiated Caco-2 cells redox status, leading to oxidant species production and a decrease of GSH levels, after 1 h exposure, followed by an increase of cytokines production, IL-6 and IL-8, after 24 h. Oxysterol cell treatment also induced after 48 h an increase of NO release, due to the induction of iNOS. Pretreatment with the phenolic extract counteracted oxysterols effects, at least in part by modulating one of the main pathways activated in the cellular response to the action of oxysterols, the MAPK-NF-kB pathway. We demonstrated the ability of the phenolic extract to directly modulate p38 and JNK1/2 phosphorylation and activation of NF-kB, following its inhibitor IkB phosphorylation. The phenolic extract also inhibited iNOS induction, keeping NO concentration at the control level. Our results suggest a protective effect at intestinal level of extra virgin olive oil polyphenols, able to prevent or limit redox unbalance and the onset and progression of chronic intestinal inflammation.

Assessing the effects of silver nanoparticles on monolayers of differentiated Caco-2 cells, as a model of intestinal barrier

Since ingestion is one of the main routes of entry of nanoparticles (NPs) in our organism, simple and fast in vitro models of the intestinal barrier can be helpful to evaluate NPs risk. The human colon adenocarcinoma Caco-2 cell line has been extensively used due to its ability to differentiate, forming a well-structured cell monolayer. In this study, we have used these differentiated cells as a model of intestinal barrier to evaluate a wide set of effects caused by exposure to silver nanoparticles (AgNPs) with an average size of 7.74 nm. Different parameters such as toxicity, monolayer integrity and permeability (assessed by changes in cells' morphology and gene expression pattern), internalization (uptake), translocation, and induction of DNA damage (DNA breaks and oxidative DNA damage) were evaluated. No significant effects were observed on the monolayer's integrity/permeability after exposure to silver nanoparticles, although cellular uptake was demonstrated by using confocal microscopy. Despite the observed uptake, no translocation of AgNPs to the basolateral chamber was demonstrated with any of the different experimental approaches used. The genotoxic effects evaluated using the comet assay indicate that, although AgNPs were not able to induce direct DNA breaks, its exposure induced a significant increase in the oxidative DNA damage levels, at non-toxic concentrations.

Ellagitannins from Rubus idaeus L. Exert Geno- and Cytotoxic Effects against Human Colon Adenocarcinoma Cell Line Caco-2.

Ellagitannins possess several biological activities, including anticancer properties. The goal of the present study was to investigate the cyto- and genotoxic activities of a red raspberry ellagitannin preparation (REP) in the concentration range of 2.5-160 μg/mL, as well as that of the main individual raspberry ellagitannins, sanguiin H-6 (SH-6, 12.8-256 μM) and lambertianin C (LC, 9.3-378 μM), against human colon adenocarcinoma cell line Caco-2. The ellagitannin concentrations used in the study correspond to those found in foodstuffs containing raspberry fruit. REP, SH-6, and LC exhibited strong concentration-dependent genotoxic properties, inducing DNA damage ranging from 7.3 ± 1.3 to 56.8 ± 4.3%, causing double-strand breaks and oxidation of DNA bases. At IC50 (124 μg/mL) the REP affected the nuclear morphology and induced the apoptosis of Caco-2 cells. Because the REP has been found to possess chemopreventive activity, it can be used as a natural food additive to enhance the health benefits of foodstuffs.

Human rotavirus strain Wa downregulates NHE1 and NHE6 expressions in rotavirus-infected Caco-2 cells.

Rotavirus (RV) is the most common cause of severe gastroenteritis and fatal dehydration in human infants and neonates of different species. However, the pathogenesis of rotavirus-induced diarrhea is poorly understood. Secretory diarrhea caused by rotavirus may lead to a combination of excessive secretion of fluid and electrolytes into the intestinal lumen. Fluid absorption in the small intestine is driven by Na+-coupled transport mechanisms at the luminal membrane, including Na+/H+ exchanger (NHE). Here, we performed qRT-PCR to detect the transcription of NHEs. Western blotting was employed for protein detection. Furthermore, immunocytochemistry was used to validate the NHE's protein expression. Finally, intracellular Ca2+ concentration was detected by confocal laser scanning microscopy. The results demonstrated that the NHE6 mRNA and protein expressed in the human colon adenocarcinoma cell line (Caco-2). Furthermore, RV-Wa induced decreased expression of the NHE1 and NHE6 in Caco-2 cell in a time-dependent manner. In addition, intracellular Ca2+ concentration in RV-Wa-infected Caco-2 cells was higher than that in the mock-infected cells. Furthermore, RV-Wa also can downregulate the expression of calmodulin (CaM) and calmodulin kinase II (CaMKII) in Caco-2 cells. These findings provides important insights into the mechanisms of rotavirus-induced diarrhea. Further studies on the underlying pathophysiological mechanisms that downregulate NHEs in RV-induced diarrhea are required.

Absorption characteristics of 5 components of Xinshao extract in Caco-2 cells

To study the absorption characteristics of Xinshao extracts in Caco-2 cells. In this paper, human colon adenocarcinoma cell line Caco-2 cell model was established, and UPLC-MS method was applied to determinate the contents of five components of Xinshao extracts(albiflorin, gallic acid, caffeic acid, scutellarin and apigenin-7-O-glucronide) in cell lysates. This model was also used to study the effect of different drug concentrations, pH, time and temperature on the absorption of five components, investigate the transport of the five components of Xinshao extracts under the conditions with or without P-glycoprotein inhibitors, and predict the absorption mechanism of these five components in Caco-2 cells. The experimental results showed that the absorption of five components of Xinshao extracts in Caco-2 cells was time-dependent at 37 ℃, and concentration-dependent in the range of 0.5-12.5 g•L⁻¹, with a passive diffusion mechanism. At the pH of 4-7.4, the absorption of caffeic acid, scutellarin and apigenin-7-O-glucronide was significantly declined with the increase of pH(P<0.05). At the temperature of 4 to 37 ℃, the absorption of caffeic acid was declined with the increase of temperature, while the absorption of other four components was increased with the increase of temperature. Compared with the control group, caffeic acid and scutellarin cell absorption was significantly higher(P<0.05) after treatment with P-glycoprotein inhibitors(verapamil and cyclosporine A). The results indicated that, the absorption mechanism of five components in Xinshao extracts may be of passive diffusion, and the caffeic acid and scutellarin may be the substrates of P-glycoprotein.

Palmitoylethanolamide Exerts Antiproliferative Effect and Downregulates VEGF Signaling in Caco-2 Human Colon Carcinoma Cell Line Through a Selective PPAR-α-Dependent Inhibition of Akt/mTOR Pathway.

Palmitoylethanolamide (PEA) is a nutraceutical compound that has been demonstrated to improve intestinal inflammation. We aimed at evaluating its antiproliferative and antiangiogenic effects in human colon adenocarcinoma Caco-2 cell line. Caco-2 cells were treated with increasing concentrations of PEA (0.001, 0.01 and 0.1 μM) in the presence of peroxisome proliferator-activated receptor-a (PPAR-α) or PPAR-γ antagonists. Cell proliferation was evaluated by performing a MTT assay. Vascular endothelial growth factor (VEGF) release was estimated by ELISA, while the expression of VEGF receptor and the activation of the Akt/mammalian target of rapamycin (mTOR) pathway were evaluated by western blot analysis. PEA caused a significant and concentration-dependent decrease of Caco-2 cell proliferation at 48 h. PEA administration significantly reduced in a concentration-dependent manner VEGF secretion and VEGF receptor expression. Inhibition of Akt phosphorylation and a downstream decrease of phospho-mTOR and of p-p70S6K were observed as compared with untreated cells. PPAR-α, but not PPAR-γ antagonist, reverted all effects of PEA. PEA is able to decrease cell proliferation and angiogenesis. The antiangiogenic effect of PEA depends on the specific inhibition of the AkT/mTOR axis, through the activation of PPAR-α pathway. If supported by in vivo models, our data pave the way to PEA co-administration to the current chemotherapeutic regimens for colon carcinoma. Copyright © 2016 John Wiley & Sons, Ltd.

Radioactive Permeability Studies of Doxycycline Hyclate from Microemulsion and Solution

Doxycycline Hyclate (DOX) is an antibacterial drug which is member of the second tetracycline group and the absorption of DOX is reduced about 20% in the presence of skimmed milk. Therefore new drug delivery system can be advisable for DOX to reduce the food and drug interaction and improve the bioavailability. The aim of the present study is to prepare a microemulsion system of DOX which could result in reducing in food interaction and an improvement of oral bioavailability by increasing the drug's permeability. For this purposes, DOX was radiolabeled with 99m Tc. Radiochemical purity was determined with radioactive thin layer chromatography (RTLC) studies. Permeability of DOX from 99m Tc-DOX solution ( 99m Tc-DOX-S) and 99m Tc-DOX loaded microemulsion ( 99m Tc-DOX-M) was investigated with in vitro cell culture studies by using human colonic adenocarcinoma cell line (Caco-2). The radioactivity of 99m Tc-DOX-M for the apical to basolateral direction (P app (A→B)) and basolateral to apical direction (P app (B→A)) were found higher than 99m Tc-DOX-S. Based on the in vitro cell culture studies, this dosage form is a promising formulation as an alternative for oral drug delivery of DOX.