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1.
Nutrients ; 13(8)2021 Jul 25.
Article in English | MEDLINE | ID: mdl-34444704

ABSTRACT

γ-Aminobutyric acid (GABA) is a potent bioactive amino acid, and several studies have shown that oral administration of GABA induces relaxation, improves sleep, and reduces psychological stress and fatigue. In a recent study, we reported that exosomes derived from GABA-treated intestinal cells serve as signal transducers that mediate brain-gut interactions. Therefore, the purpose of this study was to verify the functionality of GABA-derived exosomes and to examine the possibility of improving memory function following GABA administration. The results showed that exosomes derived from GABA-treated intestinal cells (Caco-2) activated neuronal cells (SH-SY5Y) by regulating genes related to neuronal cell functions. Furthermore, we found that exosomes derived from the serum of GABA-treated mice also activated SH-SY5Y cells, indicating that exosomes, which are capable of activating neuronal cells, circulate in the blood of mice orally administered GABA. Finally, we performed a microarray analysis of mRNA isolated from the hippocampus of mice that were orally administered GABA. The results revealed changes in the expression of genes related to brain function. Gene Set Enrichment Analysis (GSEA) showed that oral administration of GABA affected the expression of genes related to memory function in the hippocampus.


Subject(s)
Exosomes/metabolism , Memory/drug effects , Neurons/metabolism , gamma-Aminobutyric Acid/administration & dosage , Administration, Oral , Animals , Caco-2 Cells/metabolism , Hippocampus/metabolism , Humans , Mice , MicroRNAs/metabolism , Models, Animal
2.
Biol Pharm Bull ; 44(2): 275-278, 2021.
Article in English | MEDLINE | ID: mdl-33518681

ABSTRACT

α-Defensin 5 has a particularly broad antibacterial spectrum; it eliminates pathogenic microorganisms and regulates intestinal flora. Although Caco-2 cells are similar to small intestinal cells, it is unclear whether they secrete α-defensin 5. Therefore, we investigated whether Caco-2 cells secrete α-defensin 5 and determined the secretion mechanism using cells from three cell banks (ATCC, DSMZ, and RIKEN). The Caco-2 cell proliferation rate increased with the number of culture days, irrespective of cell bank origin. On the other hand, the alkaline phosphatase activity, which affects cell differentiation and the mRNA levels of several cytokines, such as interleukin 8 (IL-8), IL-6, IL-1ß, tumor necrosis factor-α (TNF-α), and IL-2, in the Caco-2 cells fluctuated with the number of culture days, and differed for each cell bank. α-Defensin 5 secretion was detected in all three cell bank Caco-2 cells; particularly, the ATCC Caco-2 cells grew linearly depending on the cell culture day as well as the levels of IL-8 and TNF-α mRNA. This suggested that α-defensin 5 secretion in the ATCC Caco-2 cells was associated with fluctuations in the mRNA levels of various cytokines, such as IL-8 and TNF-α. In conclusion, Caco-2 cells may be a simple model for screening health food components and drugs that affect α-defensin 5 secretion.


Subject(s)
Caco-2 Cells/metabolism , alpha-Defensins/metabolism , Biological Specimen Banks , Cell Proliferation , Cytokines/analysis , Cytokines/metabolism , Drug Evaluation, Preclinical/methods , Feasibility Studies , Humans , Reproducibility of Results , alpha-Defensins/analysis
3.
Pak J Biol Sci ; 24(12): 1217-1225, 2021 Jan.
Article in English | MEDLINE | ID: mdl-34989199

ABSTRACT

<b>Background and Objective:</b> Pineapple (<i>Ananas comosus</i> L.) has antioxidant and other pharmacological properties. This study examined how pineapple modified mitochondrial permeability transition and expression of drug-metabolizing enzymes, i.e., CYP1A2, CYP2C9, CYP3A4, UGT1A6, NAT2 and the drug transporter OATP1B1 in human colorectal adenocarcinoma (Caco-2) cells. <b>Materials and Methods:</b> Caco-2 cells (2.5×10<sup>5</sup> cells well<sup>1</sup> in 24-well plates) were incubated with pineapple (125 to 1,000 µg mL<sup>1</sup>) for 48 hrs in a phenol red-free medium. Mitochondrial permeability transition, resazurin cell viability and AST and ALT levels were investigated. The mRNA expression of target genes was determined by RT/qPCR. <b>Results:</b> Pineapple significantly reduced depolarized mitochondria, slightly decreased cell viability and did not change AST and ALT levels. Pineapple did not modify the mRNA expressions of CYP1A2, CYP2C9 and CYP3A4 but markedly induced UGT1A6 expression. The highest tested concentration of pineapple (1,000 µg mL<sup>1</sup>) significantly suppressed NAT2 and OATP1B1 expression. <b>Conclusion:</b> Although pineapple slightly decreased cell viability to ~80% of control, the morphology and functions of the cells were unaffected. Pineapple showed a beneficial effect to reduce depolarized mitochondria, which consequently decreased reactive oxygen species production. Pineapple did not modify the expression of CYPs, whilst it altered the expression of phase 2 metabolizing genes UGT1A6 and NAT2 and the transporter OATP1B1. Therefore, the consumption of large amounts of pineapple is of concern for the risk of drug interaction via alteration of UGT1A6, NAT2 and OATP1B1 expression.


Subject(s)
Ananas/metabolism , Caco-2 Cells/drug effects , Mitochondrial Transmembrane Permeability-Driven Necrosis/physiology , Pharmaceutical Preparations/metabolism , Caco-2 Cells/metabolism , Humans
4.
J Sci Food Agric ; 101(8): 3156-3164, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33211321

ABSTRACT

BACKGROUND: Rubus chingii Hu is a widely cultivated fruit in China and has declared multiple bioactivities including antioxidative activity. Ethyl carbamate (EC), mostly found in fermented food and alcoholic beverages, is a recognized human carcinogen, and researchers have proposed the correlation between oxidative stress and its toxicity. This study acquired the polysaccharide from R. chingii (RP) and explored its effect on EC-induced cytotoxicity using Caco-2 cells as the cell model. RESULTS: Results showed that RP exhibited protection against EC-induced toxicity by repairing redox imbalance as indicative of mitigated mitochondrial membrane potential collapse, attenuated reactive oxygen species overproduction, and impeded glutathione depletion. Moreover, the structural features of RP were characterized and revealed that it was mainly constituted by galacturonic acid and arabinose, with an average molecular weight of 7.039 × 105 g mol-1 . CONCLUSION: Overall, our results provided a new approach dealing with the toxicity caused by EC from the perspective of oxidative stress and described a new potential healthy value of R. chingii Hu, which could contribute to the development of a promising dietary supplement and functional food. © 2020 Society of Chemical Industry.


Subject(s)
Plant Extracts/pharmacology , Polysaccharides/pharmacology , Protective Agents/pharmacology , Rubus/chemistry , Urethane/toxicity , Antioxidants , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Cell Survival/drug effects , Glutathione/metabolism , Humans , Oxidative Stress/drug effects , Plant Extracts/isolation & purification , Polysaccharides/isolation & purification , Protective Agents/isolation & purification , Reactive Oxygen Species/metabolism
5.
Food Funct ; 11(11): 9526-9534, 2020 Nov 18.
Article in English | MEDLINE | ID: mdl-33089841

ABSTRACT

It is well known that consumption of a high-fat diet (HFD) promotes intestinal inflammation despite little being known about causative factors. Recent evidence implicates dietary peroxidized lipids (POLs), which are typically formed from the oxidation of polyunsaturated fatty acid double bonds, as potential contributors due to their enrichment in HFDs, ability to be formed during gastrointestinal transit, and immunogenic and cytotoxic properties. 13-HPODE, the most common dietary POL, demonstrates pro-inflammatory activity in a variety of immune cells, especially Natural Killer (NK) cells whose role in mediating intestinal inflammation remains unclear. Therefore, we set out to investigate how 13-HPODE and other POLs modulate NK-cell activity in the context of intestinal inflammation. We not only found that NK cells fully decompose exogenous 13-HPODE, but that direct treatment stimulates TNF-α and MCP1 expression as well as Granzyme B (GZMB) secretion in a dose-dependent manner. Similar results were observed upon incubation of NK cells with oxidized, but not-unoxidized, low-density lipoproteins. Secretory products from 13-HPODE-treated NK cells were able to induce Caco2 intestinal cell inflammation in the same way as exogenous GZMB with greater sensitivity in undifferentiated compared to differentiated cells. Results were recapitulated in 13-HPODE-fed mice, demonstrating both spatial and temporal patterns of elevated GZMB expression that favored acute treatments in the distal intestinal epithelium. Collectively, our results suggest that that HFD-derived POLs, like 13-HPODE, potentially contribute to intestinal inflammation by stimulating the secretion of pro-inflammatory granzymes by resident NK cells, ultimately revealing a more direct role for diet in modulating gut homeostasis and the immune environment.


Subject(s)
Inflammation/metabolism , Intestinal Diseases/metabolism , Intestinal Mucosa/drug effects , Linoleic Acids/pharmacology , Lipid Peroxides/pharmacology , Animals , Caco-2 Cells/metabolism , Dietary Fats/adverse effects , Granzymes/metabolism , Humans , Inflammation/chemically induced , Intestinal Diseases/chemically induced , Intestinal Mucosa/metabolism , Killer Cells, Natural/metabolism , Male , Mice , Mice, Inbred C57BL
6.
Nutrients ; 12(8)2020 Aug 18.
Article in English | MEDLINE | ID: mdl-32824739

ABSTRACT

Dry heating of cow's milk protein, as applied in the production of "baked milk", facilitates the resolution of cow's milk allergy symptoms upon digestion. The heating and glycation-induced changes of the protein structure can affect both digestibility and immunoreactivity. The immunological consequences may be due to changes in the peptide profile of the digested dry heated milk protein. Therefore, cow's milk protein powder was heated at low temperature (60 °C) and high temperature (130 °C) and applied to simulated infant in vitro digestion. Digestion-derived peptides after 10 min and 60 min in the intestinal phase were measured using LC-MS/MS. Moreover, digests after 10 min intestinal digestion were applied to a Caco-2 cell monolayer. T-cell epitopes were analysed using prediction software, while specific immunoglobin E (sIgE) binding epitopes were identified based on the existing literature. The largest number of sIgE binding epitopes was found in unheated samples, while T-cell epitopes were equally represented in all samples. Transport of glycated peptide indicated a preference for glucosyl lysine and lactosyl-lysine-modified peptides, while transport of peptides containing epitope structures was limited. This showed that the release of immunoreactive peptides can be affected by the applied heating conditions; however, availability of peptides containing epitopes might be limited.


Subject(s)
Caco-2 Cells/metabolism , Digestion/physiology , Food Handling/methods , Hot Temperature , Infant Nutritional Physiological Phenomena/physiology , Intestines/physiology , Milk Hypersensitivity/prevention & control , Milk Proteins/metabolism , Milk , Peptides/metabolism , Protein Transport , Proteolysis , Animals , Epitopes, T-Lymphocyte , Humans , Immunoglobulin E , In Vitro Techniques , Infant , Milk Proteins/chemistry , Milk Proteins/immunology
7.
Viruses ; 12(7)2020 07 17.
Article in English | MEDLINE | ID: mdl-32708879

ABSTRACT

The zika virus (ZIKV) is a neurotropic virus that causes congenital abnormalities in babies when they are infected in utero. Some studies have reported these congenital abnormalities result from ZIKV attacking neural progenitor cells within the brain which differentiate into neurons, oligodendrocytes, and astrocytes. Each of these glial cells play important roles during development of the fetal brain. In addition to ZIKV-induced congenital abnormalities, infected patients experience gastrointestinal complications. There are presently no reports investigating the role of this virus at the proteomic level in gastrointestinal associated cells, so we conducted an in vitro proteomic study of ZIKV-induced changes in Caco-2, a colon-derived human cell line which is known to be permissive to ZIKV infection. We used SomaScan, a new aptamer-based proteomic tool to identify host proteins that are dysregulated during ZIKV infection at 12, 24, and 48 h post-infection. Bioinformatic analyses predicted that dysregulation of differentially-regulated host proteins results in various gastrointestinal diseases. Validation of the clinical relevance of these promising protein targets will add to the existing knowledge of ZIKV biology. These potential proteins may be useful targets towards the development of therapeutic interventions.


Subject(s)
DNA Damage , Gastrointestinal Tract/virology , Proteome/metabolism , Zika Virus Infection/pathology , Caco-2 Cells/metabolism , Caco-2 Cells/virology , Gastrointestinal Tract/metabolism , Gene Expression Regulation, Viral , Humans , Zika Virus Infection/metabolism
8.
Food Funct ; 11(4): 3741-3748, 2020 Apr 30.
Article in English | MEDLINE | ID: mdl-32314770

ABSTRACT

Impairment of the intestinal barrier often occurs in inflammatory bowel diseases, and pro-inflammatory factors play a vital role in the pathogenesis of intestinal diseases. In our study, the potential protective effects of Lycium barbarum polysaccharides (LBP) against intestinal barrier dysfunction evoked by pro-inflammatory factors and its anti-inflammatory effects were investigated. Caco-2 cells were stimulated with or without tumor necrosis factor (TNF)-α in the presence or absence of LBP. Our findings showed that LBP assuaged the increase of paracellular permeability and the decrease of transepithelial electrical resistance (TER) in Caco-2 cells. In addition, LBP also prevented the secretion of pro-inflammatory markers (IL-8, IL-6, ICAM-1 and MCP-1) in TNF-α-challenged Caco-2 cells. Moreover, LBP inhibited the overexpression of tight junction (TJ) proteins (claudin-1, ZO-3, and occludin) and the increase of MLCK, pMLC, p-IκBα and NFκBp65 protein expression evoked by TNF-α was suppressed by LBP pre-incubation. This finding indicated that LBP improve TNF-α-evoked intestinal barrier dysfunction via suppressing the MLCK-MLC signaling pathway mediated by NFκB.


Subject(s)
Caco-2 Cells/drug effects , Drugs, Chinese Herbal/pharmacology , Caco-2 Cells/metabolism , Humans , Intestinal Mucosa/metabolism , Myosin Light Chains/metabolism , Myosin-Light-Chain Kinase/metabolism , Signal Transduction
9.
Mol Pharm ; 17(3): 757-768, 2020 03 02.
Article in English | MEDLINE | ID: mdl-32011888

ABSTRACT

Salmon calcitonin (sCT) is a potent calcium-regulating peptide hormone and widely applied for the treatment of some bone diseases clinically. However, the therapeutic usefulness of sCT is hindered by the frequent injection required, owing to its short plasma half-life and therapeutic need for a high dose. Oral delivery is a popular modality of administration for patients because of its convenience to self-administration and high patient compliance, while orally administered sCT remains a great challenge currently due to the existence of multiple barriers in the gastrointestinal (GI) tract. Here, we introduced an orally targeted delivery system to increase the transport of sCT across the intestine through both the paracellular permeation route and the bile acid pathway. In this system, sCT-based glycol chitosan-taurocholic acid conjugate (GC-T)/dextran sulfate (DS) ternary nanocomplexes (NC-T) were produced by a flash nanocomplexation (FNC) process in a kinetically controlled mode. The optimized NC-T exhibited well-controlled properties with a uniform and sub-60 nm hydrodynamic diameter, high batch-to-batch reproducibility, good physical or chemical stability, as well as sustained drug release behaviors. The studies revealed that NC-T could effectively improve the intestinal uptake and permeability, owing to its surface functionalization with the taurocholic acid ligand. In the rat model, orally administered NC-T showed an obvious hypocalcemia effect and a relative oral bioavailability of 10.9%. An in vivo assay also demonstrated that NC-T induced no observable side effect after long-term oral administration. As a result, the orally targeted nanocomplex might be a promising candidate for improving the oral transport of therapeutic peptides.


Subject(s)
Calcitonin/administration & dosage , Calcium-Regulating Hormones and Agents/administration & dosage , Drug Delivery Systems/methods , Intestinal Absorption/drug effects , Nanocomposites/chemistry , Administration, Oral , Animals , Biological Availability , Biological Transport , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Calcitonin/adverse effects , Calcitonin/blood , Calcitonin/pharmacokinetics , Calcium/blood , Calcium-Regulating Hormones and Agents/adverse effects , Calcium-Regulating Hormones and Agents/blood , Calcium-Regulating Hormones and Agents/pharmacokinetics , Chitosan/chemistry , Dextran Sulfate/chemistry , Drug Liberation , Drug Stability , Half-Life , Humans , Hypocalcemia/chemically induced , Injections, Subcutaneous , Male , Rats , Rats, Sprague-Dawley , Taurocholic Acid/chemistry
10.
J Pharm Pharmacol ; 72(4): 575-582, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31975441

ABSTRACT

OBJECTIVES: Ezrin (Ezr), radixin (Rdx) and moesin (Msn) (ERM) proteins anchor other proteins to the cell membrane, serving to regulate their localization and function. Here, we examined whether ERM proteins functionally regulate breast cancer resistance protein (BCRP) and P-glycoprotein in cell lines derived from lung, intestinal and renal cancers. METHODS: ERM proteins were each silenced with appropriate siRNA. BCRP and P-gp functions were evaluated by means of efflux and uptake assays using 7-ethyl-10-hydroxycamptothecin (SN-38) and rhodamine123 (Rho123) as specific substrates, respectively, in non-small cell lung cancer HCC827 cells, intestinal cancer Caco-2 cells and renal cancer Caki-1 cells. KEY FINDINGS: In HCC827 cells, the efflux rates of SN-38 and Rho123 were significantly decreased by knockdown of Ezr or Msn, but not Rdx. However, BCRP function was unaffected by Ezr or Rdx knockdown in Caco-2 cells, which do not express Msn. In Caki-1 cells, Rdx knockdown increased the intracellular SN-38 concentration, while knockdown of Ezr or Msn had no effect. CONCLUSIONS: Our findings indicate that regulation of BCRP and P-gp functions by ERM proteins is organ-specific. Thus, if the appropriate ERM protein(s) are functionally suppressed, accumulation of BCRP or P-gp substrates in lung, intestine or kidney cancer tissue might be specifically increased.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Cytoskeletal Proteins/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Neoplasm Proteins/metabolism , Caco-2 Cells/metabolism , Carcinoma, Non-Small-Cell Lung , Cell Line, Tumor , Cytoskeletal Proteins/genetics , Humans , Irinotecan/metabolism , Kidney Neoplasms/metabolism , Membrane Proteins/genetics , Microfilament Proteins/genetics , RNA, Messenger/metabolism , RNA, Small Interfering , Rhodamine 123/metabolism
11.
Food Funct ; 11(1): 700-710, 2020 Jan 29.
Article in English | MEDLINE | ID: mdl-31909774

ABSTRACT

Vanillin is a popular flavoring agent in the food, tobacco, and perfume industries. In this paper, we investigated the effect of vanillin on the transport rates of drugs with different levels of permeability (acyclovir, hydrochlorothiazide, propranolol and carbamazepine) through a Caco-2 cell bidirectional transport experiment. We also explored the underlying mechanism using an in silico technique and fluorescence anisotropy measurements. The influence of vanillin on the pharmacokinetics of drugs whose transport rates were affected by vanillin in vitro was then studied in vivo. Results showed that vanillin (100 µM) increased the cumulative amount of passively transported drugs (2.1-fold of hydrochlorothiazide, 1.49-fold of propranolol, 1.35-fold of acyclovir, and 1.34-fold of carbamazepine) in vitro. Molecular dynamics simulations revealed that vanillin disordered the structure of the lipid bilayer and reduced the energy barrier of drugs across the center of the membrane. The anisotropy of TMA-DPH also decreased in Caco-2 cells after treatment with vanillin (25 and 100 µM) and indicated an increase in membrane fluidity, which was dose-dependent. An oral bioavailability study indicated that vanillin (100 mg kg-1) significantly enhanced the Cmax and AUC0-6 of hydrochlorothiazide by 1.42-fold and 1.28-fold, respectively, and slightly elevated the Cmax of propranolol. In conclusion, vanillin can significantly increase the absorption of drugs with moderate oral bioavailability in vitro and in vivo by loosening the membrane. Thus, the concurrent consumption of drugs with food containing vanillin may result in increased drug plasma concentration and pose potential health risks.


Subject(s)
Benzaldehydes/pharmacology , Intestinal Absorption/drug effects , Plant Extracts/pharmacology , Acyclovir/pharmacokinetics , Administration, Oral , Animals , Anti-Arrhythmia Agents/pharmacokinetics , Anticonvulsants/pharmacokinetics , Antiviral Agents/pharmacokinetics , Area Under Curve , Benzaldehydes/administration & dosage , Biological Availability , Biological Transport , Caco-2 Cells/metabolism , Carbamazepine/pharmacokinetics , Diuretics/pharmacokinetics , Humans , Hydrochlorothiazide/pharmacokinetics , In Vitro Techniques , Male , Plant Extracts/administration & dosage , Propranolol/pharmacokinetics , Rats , Rats, Sprague-Dawley
12.
J Bioenerg Biomembr ; 51(6): 403-412, 2019 12.
Article in English | MEDLINE | ID: mdl-31845097

ABSTRACT

Candida albicans causes mucosal diseases and secretes farnesol, a quorum-sensing molecule, which plays a vital role in suppressing the yeast-to-mycelia switch. Farnesol can also regulate immune cell function. However, how farnesol interacts with the intestinal epithelium remains unknown. Herein, we identified that farnesol promotes intestinal barrier function, by promoting transepithelial electrical resistance, reducing paracellular flux, inducing the Zonula Occludens-1 Protein (ZO-1) and occludin expression. Moreover, the JAK/STAT3 signaling pathway was activated after farnesol treatment, and inhibition of STAT3 phosphorylation by stattic remarkably suppressed the expression level of ZO-1. Additionally, chromatin immunoprecipitation assay (Chip) revealed that farnesol facilitated the transcriptional activation of STAT3 to significantly enhance the expression of ZO-1. Taken together, our findings demonstrated that farnesol facilitated intestinal epithelial barrier transcriptional regulation via activating JAK/STAT3 signaling. The involved molecules may be potentially targeted for treatment of Candida albicans invasion.


Subject(s)
Caco-2 Cells/metabolism , Epithelial Cells/metabolism , Farnesol/therapeutic use , Intestinal Mucosa/metabolism , STAT3 Transcription Factor/metabolism , Tight Junctions/metabolism , Cell Differentiation , Farnesol/pharmacology , Humans , Signal Transduction , Transfection
13.
Sci Rep ; 9(1): 16647, 2019 11 12.
Article in English | MEDLINE | ID: mdl-31719636

ABSTRACT

The present state of cancer chemotherapy is unsatisfactory. New anticancer drugs that marginally improve the survival of patients continue to be developed at an unsustainably high cost. The study aimed to elucidate the effects of insulin (INS), an inexpensive drug with a convincing safety profile, on the susceptibility of colon cancer to chemotherapeutic agents: 5-fluorouracil (FU), oxaliplatin (OXA), irinotecan (IRI), cyclophosphamide (CPA) and docetaxel (DOC). To examine the effects of insulin on cell viability and apoptosis, we performed an in vitro analysis on colon cancer cell lines Caco-2 and SW480. To verify the results, we performed in vivo analysis on mice bearing MC38 colon tumors. To assess the underlying mechanism of the therapy, we examined the mRNA expression of pathways related to the signaling downstream of insulin receptors (INSR). Moreover, we performed Western blotting to confirm expression patterns derived from the genetic analysis. For the quantification of circulating tumor cells in the peripheral blood, we used the maintrac method. The results of our study show that insulin-pretreated colon cancer cells are significantly more susceptible to commonly used chemotherapeutics. The apoptosis ratio was also enhanced when INS was administered complementary to the examined drugs. The in vivo study showed that the combination of INS and FU resulted in significant inhibition of tumor growth and reduction of the number of circulating tumor cells. This combination caused a significant downregulation of the key signaling substrates downstream of INSR. The results indicate that the downregulation of PIK3CA (phosphatidylinositol 3-kinase catalytic subunit alpha), which plays a critical role in cell signaling and GRB2 (growth factor receptor-bound protein 2), a regulator of cell proliferation and differentiation may be responsible for the sensitizing effect of INS. These findings were confirmed at protein levels by Western blotting. In conclusion, these results suggest that INS might be potentially applied to clinical use to enhance the therapeutic effectiveness of chemotherapeutic drugs. The findings may become a platform for the future development of new and inexpensive strategies for the clinical chemotherapy of tumors.


Subject(s)
Antineoplastic Agents/therapeutic use , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Colorectal Neoplasms/drug therapy , GRB2 Adaptor Protein/antagonists & inhibitors , Insulin/pharmacology , Animals , Blotting, Western , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Class I Phosphatidylinositol 3-Kinases/metabolism , Colorectal Neoplasms/metabolism , Cyclophosphamide/therapeutic use , Docetaxel/therapeutic use , Down-Regulation/drug effects , Drug Synergism , Female , Fluorouracil/therapeutic use , GRB2 Adaptor Protein/metabolism , Humans , Irinotecan/therapeutic use , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplastic Cells, Circulating/drug effects , Neoplastic Cells, Circulating/metabolism , Oxaliplatin/therapeutic use
14.
Article in English | MEDLINE | ID: mdl-31475120

ABSTRACT

Enterococcus faecium is a clinically important pathogen associated with opportunistic infection and multi-drug resistance. E. faecium has been shown to produce membrane vesicles (MVs), but MV production by E. faecium under antibiotic stress conditions and the pathogenic traits thereof have yet to be determined. This study investigated the production of MVs in E. faecium ATCC 700221 cultured with sub-minimum inhibitory concentrations (MICs) of vancomycin or linezolid and determined their pathologic effects on colon epithelial Caco-2 cells. E. faecium ATCC 700221 cultured with 1/2 MIC of vancomycin or linezolid produced 3.0 and 1.5 times more MV proteins than bacteria cultured without antibiotics, respectively. Totals of 438, 461, and 513 proteins were identified in MVs from E. faecium cultured in brain heart infusion broth (MVs/BHI), BHI broth with 1/2 MIC of vancomycin (MVs/VAN), or BHI broth with 1/2 MIC of linezolid (MVs/LIN), respectively. Intact MVs/BHI induced cytotoxicity and the expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells in a dose-dependent manner, but proteinase K-treated MVs significantly suppressed these pro-inflammatory responses. MVs/LIN were more cytotoxic toward Caco-2 cells than MVs/BHI and MVs/VAN, whereas MVs/VAN stimulated more pro-inflammatory cytokine gene expression in Caco-2 cells than MVs/BHI and MVs/LIN. Overall results indicated that antibiotics modulate the biogenesis and proteomes of MVs in E. faecium at subinhibitory concentrations. MVs produced by E. faecium cultured under antibiotic stress conditions induce strong host cell responses that may contribute to the pathogenesis E. faecium.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enterococcus faecium/drug effects , Enterococcus faecium/metabolism , Epithelial Cells/metabolism , Extracellular Vesicles/metabolism , Gram-Positive Bacterial Infections/immunology , Bacterial Proteins , Caco-2 Cells/immunology , Caco-2 Cells/metabolism , Cell Survival , Chemokines/genetics , Chemokines/metabolism , Drug Resistance, Bacterial , Enterococcus faecium/pathogenicity , Gram-Positive Bacterial Infections/microbiology , Host-Pathogen Interactions , Humans , Microbial Sensitivity Tests , Proteome/drug effects , Proteome/metabolism , Stress, Physiological , Vancomycin/pharmacology , Virulence/drug effects , Virulence Factors/metabolism
15.
Article in English | MEDLINE | ID: mdl-31481446

ABSTRACT

P-glycoprotein (ABCB1), an ATP-binding-cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions (DDIs). ABCB1 has been suggested to interact with many antivirals used to treat HIV and/or chronic hepatitis C virus (HCV) infections. Using bidirectional transport experiments in Caco-2 cells and a recently established ex vivo model of accumulation in precision-cut intestinal slices (PCIS) prepared from rat ileum or human jejunum, we evaluated the potential of anti-HIV and anti-HCV antivirals to inhibit intestinal ABCB1. Lopinavir, ritonavir, saquinavir, atazanavir, maraviroc, ledipasvir, and daclatasvir inhibited the efflux of a model ABCB1 substrate, rhodamine 123 (RHD123), in Caco-2 cells and rat-derived PCIS. Lopinavir, ritonavir, saquinavir, and atazanavir also significantly inhibited RHD123 efflux in human-derived PCIS, while possible interindividual variability was observed in the inhibition of intestinal ABCB1 by maraviroc, ledipasvir, and daclatasvir. Abacavir, zidovudine, tenofovir disoproxil fumarate, etravirine, and rilpivirine did not inhibit intestinal ABCB1. In conclusion, using recently established ex vivo methods for measuring drug accumulation in rat- and human-derived PCIS, we have demonstrated that some antivirals have a high potential for DDIs on intestinal ABCB1. Our data help clarify the molecular mechanisms responsible for reported increases in the bioavailability of ABCB1 substrates, including antivirals and drugs prescribed to treat comorbidity. These results could help guide the selection of combination pharmacotherapies and/or suitable dosing schemes for patients infected with HIV and/or HCV.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Anti-HIV Agents/pharmacology , Antiviral Agents/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Aged , Animals , Atazanavir Sulfate/pharmacology , Benzimidazoles/pharmacology , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Carbamates , Drug Interactions , Female , Fluorenes/pharmacology , HIV Infections/complications , HIV Infections/drug therapy , HIV Infections/virology , Hepatitis C/complications , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Imidazoles/pharmacology , Intestines/drug effects , Lopinavir/pharmacology , Male , Maraviroc/pharmacology , Middle Aged , Pyrrolidines , Rats , Rats, Wistar , Ritonavir/pharmacology , Saquinavir/pharmacology , Valine/analogs & derivatives , Zidovudine/pharmacology
16.
Sci Rep ; 9(1): 10609, 2019 07 23.
Article in English | MEDLINE | ID: mdl-31337851

ABSTRACT

4-thiazolidinones, which are privileged structures in medicinal chemistry, comprise the well-known class of heterocycles and are a source of new drug-like compounds. Undoubtedly, the 5-bulky-substituted-2,4-thiazolidinediones - a class of antihyperglycemic glitazones, which are peroxisome proliferator-activated receptor gamma (PPARγ) agonists, are the most described group among them. As there are various chemically distinct 4-thiazolidinones, different subtypes have been selected for studies; however, their main pharmacological profiles are similar. The aim of this study was to evaluate the anticancer activity of 5Z-(4-fluorobenzylidene)-2-(4-hydroxyphenylamino)-thiazol-4-one (Les-236) in four human cancer cell lines, A549, SCC-15, SH-SY5Y, and CACO-2, and investigate its impact on the production of reactive oxygen species (ROS) and the apoptotic process as well as cytotoxicity and metabolism in these cell lines. The cell lines were exposed to increasing concentrations (1 nM to 100 µM) of the studied compound for 6, 24, and 48 h, and later, ROS production, cell viability, caspase-3 activity, and cell metabolism were examined. The obtained results showed that the studied compound decreased the production of ROS, increased the release of lactate dehydrogenase, and decreased cell metabolism/proliferation in all the five cell lines at micromolar concentrations. Interestingly, over a wide range of concentrations (from 1 nM to 100 µM), Les-236 was able to increase the activity of caspase-3 in BJ (after 6 h of exposure), A549, CACO-2, and SCC-15 (after 48 h of exposure) cell lines which could be an effect of the activation of PPARγ-dependent pathways.


Subject(s)
Antineoplastic Agents/pharmacology , Thiazoles/pharmacology , A549 Cells/drug effects , A549 Cells/metabolism , Apoptosis/drug effects , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Caspase 3/metabolism , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Dose-Response Relationship, Drug , Humans , L-Lactate Dehydrogenase/metabolism , Reactive Oxygen Species/metabolism
17.
J Crohns Colitis ; 13(8): 1067-1080, 2019 Aug 14.
Article in English | MEDLINE | ID: mdl-30722010

ABSTRACT

BACKGROUND AND AIMS: Endogenous H2S regulates multiple physiological and pathological processes in colon epithelial tissues. The current study investigated the role of cystathionine ß-synthase [CBS], a major producer of H2S in colon epithelial cells, in the pathogenesis of ulcerative colitis [UC]-related intestinal barrier injury. The expression and DNA methylation level of CBS were investigated in inflamed and non-inflamed colon tissues collected from UC patients, and the effect of decreased CBS levels on Caco-2 monolayer barrier injury and altered status of tight junctions elicited by tumour necrosis factor/interferon [TNF/IFN] was determined. METHODS: The expression of CBS and the methylation level of the CBS promoter were assessed in non-inflamed and inflamed colon epithelial tissue samples collected from UC patients. Barrier function, status of tight junction proteins and activation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway were further investigated in Caco-2 monolayers. RESULTS: Decreased expression of CBS and elevated methylation levels of the CBS promoter were observed in inflamed sites compared with in non-inflamed sites in the colon epithelial samples from UC patients. In Caco-2 monolayers, decreased expression of CBS exacerbated TNF/IFN-induced barrier injury and altered localization of tight junction proteins. Decreased expression of CBS predisposed Caco-2 monolayers to injury elicited by TNF/IFN via augmentation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway. CONCLUSIONS: Decreased expression of CBS propagates the pathogenesis of UC by exacerbating inflammation-induced intestinal barrier injury. Elevated methylation of the CBS promoter might be one of the mechanisms underlying the decreased expression of CBS in inflamed sites of colon epithelial tissues from UC patients.


Subject(s)
Colitis, Ulcerative , Cystathionine beta-Synthase , DNA Methylation , Intestinal Mucosa , Tight Junctions/physiology , Adult , Caco-2 Cells/metabolism , Cells, Cultured , Colitis, Ulcerative/genetics , Colitis, Ulcerative/metabolism , Colitis, Ulcerative/surgery , Cystathionine beta-Synthase/genetics , Cystathionine beta-Synthase/metabolism , Female , Humans , Interferons/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Signal Transduction , Transcription Factor RelA , Transcriptome , Tumor Necrosis Factor-alpha/metabolism
18.
Microb Pathog ; 120: 79-84, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29715536

ABSTRACT

Listeria monocytogenes expresses various virulence factors enabling the invasion and multiplying in host cells, and together induces cytokines transcription. In order to explore the relationship between virulence factors of L. monocytogenes wild-type EGD-e and cellular response in human colonic epithelial cell line(Caco-2), we constructed mutant strains with in-frame deletions of critical virulence genes of inlA, inlB, hly, actA and virulence regulatory factor prfA from EGD-e, respectively. Compared with EGD-e, mutant strains showed significantly decreased invasion and apoptosis in Caco-2 cells. However, mutant strains were capable to evoke cytokines transcription of interleukin-8 (IL-8), mononuclear chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and CXCL-2 production in Caco-2 cells. Interestingly, EGD-e Δhly-infected Caco-2 cells showed a significant decrease of IL-6, IL-8 and MCP-1 transcription compared with EGD-e at 1 h post-infection. Simultaneously, EGD-e ΔinlB-infected cells showed a decrease in IL-6 transcription, while EGD-e ΔactA-infected cells reflected a decrease in MCP-1 transcription. Virulence genes play a role in inflammatory transcription, but the interaction between pathogenic bacteria and the host cells predominates in inflammatory transcription. Overall, the data showed cellular response of Caco-2 cells infected with EGD-e mutant strains.


Subject(s)
Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Listeria monocytogenes/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Virulence Factors/adverse effects , Apoptosis/drug effects , Bacterial Proteins/adverse effects , Bacterial Toxins/adverse effects , Caco-2 Cells/immunology , Chemokine CCL2/metabolism , Chemokine CXCL2/metabolism , Cytokines/metabolism , Host-Pathogen Interactions/immunology , Host-Pathogen Interactions/physiology , Humans , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Listeria monocytogenes/pathogenicity , Membrane Proteins/adverse effects , Peptide Termination Factors/adverse effects , Tumor Necrosis Factor-alpha/metabolism , Virulence/genetics , Virulence Factors/genetics
19.
J Nat Med ; 72(4): 897-904, 2018 Sep.
Article in English | MEDLINE | ID: mdl-29797179

ABSTRACT

In modern medical care in which Kampo and Western drugs are often combined, it is extremely important to clarify drug-drug interaction (DDI) to ensure safety and efficacy. However, there is little evidence of DDI in Kampo medicines. Therefore, as part of our studies to clarify the DDI risk for Kampo medicines, we evaluated the effects of five Kampo medicines [yokukansan (YKS), rikkunshito (RKT), shakuyakukanzoto (SKT), hangeshashinto (HST), and goshajinkigan (GJG)] that are widely used in Japan, on drug transporter P-glycoprotein (P-gp) using a Caco-2 permeability assay. These Kampo medicines inhibited the P-gp transport of digoxin through a Caco-2 cell monolayer. The IC50 values were 1.94-10.80 mg/ml. Of the five Kampo medicines, YKS showed the strongest inhibition (IC50 = 1.94 mg/ml), which was attributed to Uncariae Uncis Cum Ramulus. Unfortunately, we could not find the active ingredients responsible for its action. Finally, the Igut/IC50 values for the five Kampo medicines were calculated, and the DDI risk was objectively evaluated according to the criteria in the DDI guidance issued by the Japanese Ministry of Health, Labor, and Welfare and the US Food and Drug Administration. The Igut/IC50 values for the five Kampo medicines were ≤3.4. As these values were <10, they were evaluated as having a weak P-gp inhibitory effect that does not require further verification in humans, suggesting that the DDI risk due to P-gp inhibition for these Kampo medicines is low. The results should provide useful clinical information on the safety and efficacy of the combined use of Kampo and Western medicines.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects , Biological Transport/drug effects , Caco-2 Cells/metabolism , Medicine, Kampo/methods , Drug Interactions , Humans , Permeability
20.
Pharm Res ; 35(4): 74, 2018 Feb 26.
Article in English | MEDLINE | ID: mdl-29484506

ABSTRACT

PURPOSE: To identify conditions allowing the use of cell-based models for studies of drug absorption during in vitro lipolysis of lipid-based formulations (LBFs). METHODS: Caco-2 was selected as the cell-based model system. Monolayer integrity was evaluated by measuring mannitol permeability after incubating Caco-2 cells in the presence of components available during lipolysis. Pure excipients and formulations representing the lipid formulation classification system (LFCS) were evaluated before and after digestion. Porcine mucin was evaluated for its capacity to protect the cell monolayer. RESULTS: Most undigested formulations were compatible with the cells (II-LC, IIIB-LC, and IV) although some needed mucin to protect against damaging effects (II-MC, IIIB-MC, I-LC, and IIIA-LC). The pancreatic extract commonly used in digestion studies was incompatible with the cells but the Caco-2 monolayers could withstand immobilized recombinant lipase. Upon digestion, long chain formulations caused more damage to Caco-2 cells than their undigested counterparts whereas medium chain formulations showed better tolerability after digestion. CONCLUSIONS: Most LBFs and components thereof (undigested and digested) are compatible with Caco-2 cells. Pancreatic enzyme is not tolerated by the cells but immobilized lipase can be used in combination with the cell monolayer. Mucin is beneficial for critical formulations and digestion products.


Subject(s)
Caco-2 Cells/drug effects , Drug Evaluation, Preclinical/methods , Drug Liberation , Lipolysis , Pharmaceutical Preparations/metabolism , Administration, Oral , Caco-2 Cells/metabolism , Enzymes, Immobilized/metabolism , Enzymes, Immobilized/toxicity , Excipients/chemistry , Fungal Proteins , Gastrointestinal Absorption , Humans , Lipase/metabolism , Lipase/toxicity , Lipids/chemistry , Mucins/metabolism , Pancrelipase/metabolism , Pancrelipase/toxicity , Recombinant Proteins/metabolism
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