Treatment Of Caco-2 Cells Research Articles

Inhibition of Colorectal Cancer Cell Line CaCo-2 by Essential Oil of Eucalyptus camaldulensis Through Induction of Apoptosis

Treatment of colorectal cancer is one of the important challenges due to the increase of resistance to chemotherapeutic drugs. Isolated natural compounds from medicinal plants and other sources often are used as novel drugs for treatment of various human cancer. The aim of this study was to investigate the antioxidant and anticancer activity of Eucalyptus camaldulensis essential oil on colorectal cancer cell line Caco-2. The antioxidant activity of extracted E. camaldulensis essential oil (1000, 800, 400, 200, 100, 50, 25, 12.5, 6, and 3 μg/mL) was evaluated by free radicals inactivation method. Moreover, MTT assay was used to examine the cytotoxic effects of E. camaldulensis essential oil on the Caco-2 cell line. The mRNA expression of BAX and BCL-2 genes was studied using quantitative Real-Time PCR method, in treated cancer cells compared to untreated cells. We indicated a significant, impressive antioxidant activity in 1000 μg/mL of E. camaldulensis essential oil, in a concentration-dependent manner. In addition, we found that this product exerted a cytotoxic effect on cancer cells when 100 μg/mL concentration was considered as half-maximal inhibitory concentration (IC50). Also, the expression of BAX and BCL-2 genes were significantly upregulated and downregulated, respectively, in the treated Caco-2 cells with E. camaldulensis essential oil. In conclusion, our study showed significant antioxidant and anticancer activity in E. camaldulensis essential oil in a concentration and time-dependent manner, which may be due to the reduction of free radicals and induction of apoptosis process in colorectal cancer cells.

MiR-141-3p ameliorates RIPK1-mediated necroptosis of intestinal epithelial cells in necrotizing enterocolitis.

Aim: To explore the effects of miR-141-3p on intestinal epithelial cells in necrotizing enterocolitis and the underlying mechanism.Results: The expression of miR-141-3p was significantly downregulated in serum samples of patients with NEC and LPS-treated Caco-2 cells. The in vitro assays showed that miR-141-3p mimics inhibited expression of IL-6 and TNF-α and reduced PI positive rate of the LPS-treated Caco-2 cells. Next, receptor interacting protein kinase 1 (RIPK1) was identified as the downstream molecule of miR-141-3p, and RIPK1 overexpression aggravated LPS-induced Caco-2 cell injury, which was ameliorated by miR-141-3p mimics. Finally, we found miR-141-3p mimics inhibited upregulation of necroptosis-related molecules and interaction of RIPK1 and RIPK3 in LPS-treated Caco-2 cells.Conclusion: Our research indicated that miR-141-3p protected intestinal epithelial cells from LPS damage by suppressing RIPK1-mediated inflammation and necroptosis, providing an alternative perspective to explore the pathogenesis of NEC.Methods: Quantitative real time-polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-141-3p in serum samples of participants and lipopolysaccharide (LPS)-treated Caco-2 cells. Cell Counting Kit-8 (CCK-8) assay, Propidium Iodide (PI) staining and detection of inflammatory cytokines were performed to evaluate the role of miR-141-3p in LPS-treated Caco-2 cells. TargetScanHuman database and luciferase reporter gene assay were utilized to confirm the direct downstream molecule of miR-141-3p. Western blot analysis was used to explore the mechanism.

Mechanism underlying the significant role of the miR-4262/SIRT1 axis in children with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a term used to describe chronic and recurrent gastrointestinal disease. In total, >2 million individuals worldwide have been diagnosed with IBD, including ulcerative colitis (UC), Crohn's disease (CD) and indeterminate colitis. There is accumulating evidence to indicate that microRNAs (miRNAs or miRs) are involved in the development and progression of IBD. miR-4262, an underlying promoter in tumor diseases, has been reported to regulate inflammatory responses. However, the potential mechanisms underlying the role of miR-4262 in IBD remain unknown. The present study attempted to explore the role and mechanisms of miR-4262 in IBD. Firstly, reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of miR-4262 in 30 IBD colonic mucosa tissues, 30 normal tissues, 2% dextran sulfate sodium (DSS)-treated Caco-2 cells and normal cells. It was demonstrated that the expression levels of miR-4262 in IBD colonic mucosa tissues and 2% DSS-stimulated Caco-2 cells were markedly higher compared with those in the control groups. Target gene prediction databases and dual-luciferase reporter assays were then used, and sirtuin 1 (SIRT1) was identified as a target gene of miR-4262. Furthermore, the levels of SIRT1 in 2% DSS-stimulated Caco-2 cells and IBD colonic mucosa tissues were suppressed compared with the corresponding control groups. In addition, it was observed that miR-4262 negatively regulated SIRT1 expression in Caco-2 cells. Thereafter, Caco-2 cells were treated with inhibitor control, miR-4262 inhibitor, control-siRNA or SIRT1-siRNA for 48 h, followed by 2% DSS treatment for 4 days. The secretion of inflammatory factors was analyzed via ELISA and RT-qPCR. MTT assay, flow cytometry and western blot analysis were performed to assess cell viability, apoptosis and NF-κB signaling pathway-related protein levels, respectively. The results indicated that DSS enhanced the inflammatory response, suppressed cell viability and promoted cell apoptosis, and this was decreased following transfection with an miR-4262 inhibitor. In addition, 2% DSS upregulated p-p65 expression and enhanced the ratio of p-p65/p65, while the miR-4246 inhibitor exerted an opposite effect. All the effects of miR-4262 inhibitor on Caco-2 cells were eliminated following transfection with SIRT1-siRNA. It was thus concluded that miR-4262 may serve a role in the progression of IBD via targeting SIRT1, and miR-4262/SIRT1 may represent a potential target for the diagnosis and treatment of IBD.

Giardia duodenalis induces extrinsic pathway of apoptosis in intestinal epithelial cells through activation of TNFR1 and K63 de-ubiquitination of RIP1 in vitro

Giardia duodenalis is one of main causative agents of diarrhea that affects the health of millions of people on a global scale per year. It has been clear that attachment of G. duodenalis trophozoites to intestinal epithelium cells (IECs) can induce cell death, while the underlying cellular and molecular mechanisms remain to be explored. It was shown in this study that treatment of Caco-2 cells with Giardia trophozoites could result in reduced cell viability. RNA sequencing analysis demonstrated that expressions of many apoptosis-related genes and some deubiquitinase genes displayed marked changes in trophozoite-treated cells. Trophozoites activated the death-signaling receptor TNFR1 on the IEC surface and caspase-3/8 (CASP3/8) signaling pathways in Caco-2 cells. K63 ubiquitination level of RIP1 was reduced upon stimulation with trophozoites, in parallel, the expressions of deubiquitinases CYLD and A20 were increased. The caspase inhibitor Q-VD-OPH could rescue trophozoite-induced cell apoptosis. Likewise, TNFR1, CYLD, and A20 silencing decreased the levels of cleaved CASP3/8 in trophozoite-treated cells and reversed the pro-apoptosis induction effect of trophozoites. These data suggest that Giardia trophozoite stimulation can activate CASP3/8 signaling pathways via activation of TNFR1 and K63 de-ubiquitination of RIP1 caused by up-regulated expressions of CYLD and A20, and promote Caco-2 cell apoptosis. The present study deepens our understanding of the mechanism of interaction between Giardia and IECs.

Selective Cytotoxic Activity of Prodigiosin@halloysite Nanoformulation.

Prodigiosin, a bioactive secondary metabolite produced by Serratia marcescens, is an effective proapoptotic agent against various cancer cell lines, with little or no toxicity toward normal cells. The hydrophobicity of prodigiosin limits its use for medical and biotechnological applications, these limitations, however, can be overcome by using nanoscale drug carriers, resulting in promising formulations for target delivery systems with great potential for anticancer therapy. Here we report on prodigiosin-loaded halloysite-based nanoformulation and its effects on viability of malignant and non-malignant cells. We have found that prodigiosin-loaded halloysite nanotubes inhibit human epithelial colorectal adenocarcinoma (Caco-2) and human colon carcinoma (HCT116) cells proliferative activity. After treatment of Caco-2 cells with prodigiosin-loaded halloysite nanotubes, we have observed a disorganization of the F-actin structure. Comparison of this effects on malignant (Caco-2, HCT116) and non-malignant (MSC, HSF) cells suggests the selective cytotoxic and genotoxic activity of prodigiosin-HNTs nanoformulation.

Assessment of the Immunomodulatory Properties of the Probiotic Strain Lactobacillus paracasei K5 in vitro and In Vivo.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1α (IL-1α), ΙL-1β, IL-6, tumor necrosis factor-alpha (TNF-α), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character.

EZH2 inhibition promotes ANGPTL4/CREB1 to suppress the progression of ulcerative colitis

AimsEnhancer of zeste homolog 2 (EZH2) is associated with ulcerative colitis development. However, the mechanism of EZH2 in ulcerative colitis progression remains unclear. Main methodsLipopolysaccharide (LPS)-treated Caco-2 cells and dextran sodium sulfate (DSS)-treated mice were used as model of ulcerative colitis. The levels of EZH2, angiopoietin-like 4 (ANGPTL4) and cyclic adenosine monophosphate response element-binding protein 1 (CREB1) were tested via quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell viability and apoptosis was measured via 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide or flow cytometry. The abundances of inflammatory cytokines were examined via qRT-PCR and enzyme-linked immunosorbent assay. The association between EZH2 and ANGPTL4 was explored via chromatin immunoprecipitation. The colon damage in DSS-treated mice was investigated by colon length, histological analysis, inflammatory response and apoptosis. Key findingsLPS induced viability inhibition, inflammatory response and apoptosis in Caco-2 cells. EZH2 expression was increased but ANGPTL4 and CREB1 levels were decreased in LPS-challenged Caco-2 cells. Overexpression of ANGPTL4 or CREB1 suppressed LPS-induced damage in Caco-2 cells. EZH2 could target ANGPTL4 to mediate CREB1 expression. Inhibition of EZH2 suppressed LPS-caused injury. Moreover, knockdown of ANNGPTL4 or CREB1 attenuated the role of EZH2 inhibition. DSS caused the reduced colon length and increased inflammatory response as well as apoptosis. EZH2 expression was up-regulated but ANGPTL4 and CREB1 expression were down-regulated in DSS-treated mice. SignificanceInhibition of EZH2 declined LPS-induced injury in Caco-2 cells by mediating ANGPTL4 and CREB1, indicating the potential of EZH2 in treatment of ulcerative colitis.

Synthesis and characterisation of isothiocyanate functionalised silicon nanoparticles and their uptake in cultured colonic cells.

The functionalisation of silicon nanoparticles with a terminal thiocyanate group, producing isothiocyanate-capped silicon nanoparticles (ITC-capped SiNPs) has been successfully attained. The procedure for the synthesis is a two-step process that occurs via thermally induced hydrosilylation of hydrogen terminated silicon nanoparticles (H-SiNPs) and further reaction with potassium thiocyanate (KSCN). The synthesis was confirmed by Fourier transform infrared (FTIR) spectroscopy and X-Ray photoelectron spectroscopy (XPS). At the same time, the internalisation and the cytotoxicity of the ITC-capped SiNPs in vitro were assessed in two cell lines: Caco-2, human colorectal cancer cells and CCD-841, human colon "normal" cells. The results showed that above concentrations of 15 µg ml-1, the cell viability of both cell lines was depleted significantly when treated with ITC SiNPs, particularly over a 48 hour period, to approximately 20% cell viability at the highest treatment concentration (70 µg ml-1). Flow cytometry was employed to determine cellular uptake in Caco-2 cells treated with ITC SiNPs. It was observed that at lower SiNP concentrations, uptake efficiency was significantly improved for time periods under 12 hours; overall it was noted that cellular uptake was positively dependent on the period of incubation and the temperature of incubation. As such, it was concluded that the mechanism of uptake of ITC SiNPs was through endocytosis. Synchrotron FTIR spectroscopy, by means of line spectral analysis and IR imaging, provided further evidence to suggest the internalisation of ITC SiNPs displays a strong localisation, with an affinity for the nucleus of treated Caco-2 cells.

RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin.

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation.

Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. All-trans retinoic acid (10 μM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1β and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

Protective effects of Bee pollen extract on the Caco-2 intestinal barrier dysfunctions induced by dextran sulfate sodium

Bee pollen (BP) is a natural medicine from the hive with various potential health-promoting benefits, but until now there is no study to determine its protective roles in inflammatory bowel disease (IBD). The aim of this study was to reveal the in vitro gastrointestinal protective effects of BP against IBD using molecular and metabolic methods. Dextran sulfate sodium (DSS) challenged Caco-2 cell monolayers were applied to mimic intestinal epithelial cell dysfunctions and metabolic disorders. The pretreatment with BP extract rich in polyphenols ameliorated DSS-induced cell viability losses. It also exerted protective effects against intestinal barrier impairment by strengthening epithelial integrity and tight junction losses induced by DSS. BP up-regulated anti-oxidant (NQO1, Txnrd1, Nrf2) and down-regulated inflammatory (TNF-α and IL-6) mRNA expressions, in accompany with MAPK signaling inhibition. Furthermore, metabolomics analysis based on UPLC-Q-TOF/MS revealed that BP, and DSS treated Caco-2 cells have different metabolomic profiles, with significant changes on key metabolites involved in glycerophospholipid metabolism. Our results showed that BP has great therapeutic potential throughout the early stages of DSS-induced colitis.

The effect of dietary vitamin D supplementation on sodium-dependent phosphate uptake and expression of NaPi-IIb in the small intestine of weanling pigs.

Two experiments were conducted to investigate the effects of 1,25(OH)2D3 to stimulate Na+-dependent phosphate uptake in Caco-2 cells, and the effects of dietary vitamin D supplementation to vitamin D-deficient nursery pigs on Na+-dependent nutrient uptake and mRNA expression of NaPi-IIb cotransporter and calbindin D(9k) in the jejunum. In Exp. 1, 250,000 Caco-2 cells were seeded on Costar 12 mm Snapwell inserts with a 0.40 µm polycarbonate filter and a seeding density of 0.25 × 106 and studied at 15 d postconfluence. Cells were treated with 10 nM of either 1,25(OH)2D3 or vehicle for 48 h and then mounted in modified Ussing chambers for transepithelial measurements. In Exp. 2, pigs (n=32) were removed from sows at 3 d of age, placed on a vitamin D-deficient milk replacer diet and housed in a room devoid of sunlight and UV light in the range of 280 to 300nm. On d 28, serum 25(OH)D3 concentrations were measured to verify low vitamin D status. Pigs (BW 10.10 ± 0.38 kg) were then individually housed d 28 post-weaning and allotted to 1 of 2 dietary treatments. Dietary treatments consisted of corn-soybean based diets with vitamin D supplementations of 0 or 1,500 IU/kg diet for 12 d. Blood samples were taken from the brachiocephalic vein on the initial (d 0) and final day (d 10,11, or 12) of the study for analysis of serum 25(OH)D3 , P, and Ca. Pigs were euthanized and jejunal segments were harvested and used in modified Ussing chambers and for RNA isolation and subsequent quantitative RT-PCR analysis. In Exp. 1, treating Caco-2 cells with 10 nM 1,25(OH)2D3 resulted in a 52% increase (P < 0.005) in Na+-dependent phosphate uptake compared with cells treated with a vehicle. In Exp. 2, Na+-dependent phosphate and glucose transport did not differ (P > 0.10) among treatment groups. Additionally, NaPi-IIb and calbindin D(9k) mRNA expression were not different (P > 0.10) between treatment groups. No differences (P > 0.10) were detected in final serum P or 25(OH)D3 concentrations between treatments. However, serum Ca linearly increased (P < 0.05) as the concentration of supplemental vitamin D increased in the diet. Overall, while 1,25(OH)2D3 stimulated Na+-dependent phosphate uptake in Caco-2 cells, supplementing diets with 1,500 IU/kg Vitamin D3 from cholecalciferol did not increase jejunal Na+-dependent phosphate uptake or NaPi-IIb mRNA expression over that of pigs fed diets with no supplemental cholecalciferol.

Intestinal epithelial tight junction barrier regulation by autophagy-related protein ATG6/beclin 1.

A defective tight junction (TJ) barrier is a key pathogenic factor for inflammatory bowel disease. Previously, we have shown that autophagy, a cell survival mechanism, enhances intestinal epithelial TJ barrier function. Autophagy-related protein-6 (ATG6/beclin 1), a key protein in the autophagy pathway, also plays a role in the endocytic pathway. The constitutive role of beclin 1 in the intestinal TJ barrier is not known. In Caco-2 cells, beclin 1 was found to be coimmunoprecipitated with the TJ protein occludin and colocalized with occludin on the membrane. Treatment of Caco-2 cells with beclin 1 peptide [transactivating regulatory protein (Tat)-beclin 1] reduced TJ barrier function. Activation of beclin 1 increased occludin endocytosis and reduced total occludin protein level. In contrast, beclin 1 siRNA transfection enhanced Caco-2 TJ barrier function. In pharmacologic and genetic autophagy inhibition studies, the constitutive function of beclin 1 in the TJ barrier was found to be autophagy independent. However, de novo induction of autophagy with starvation or rapamycin prevented Tat-beclin 1-induced increase in TJ permeability and reduction in occludin level. Induction of autophagy also resulted in reduced beclin 1-occludin association. In mouse colon, beclin 1 colocalized with occludin on the epithelial membrane. Perfusion of mouse colon with beclin 1 peptide caused an increase in colonic TJ permeability that was prevented by in vivo induction of autophagy. These findings show that beclin 1 plays a constitutive, autophagy-independent role in the regulation of intestinal TJ barrier function via endocytosis of occludin. Autophagy terminates constitutive beclin 1 function in the TJ barrier and enhances the TJ barrier.

Study on Sucrosomial® Iron Endocytosis-Mediated Uptake and Enterocytes Release

Introduction: iron homeostasis is maintained by regulating the iron levels in plasma which is maintained by four coordinated processes: duodenal iron absorption, macrophage iron recycling, hepatic iron storage and erythropoiesis. Iron in the Fe2+ form is transported across the apical duodenal membrane by DMT1 and subsequently transferred to the blood via the iron exporter, Ferroportin the only know cell membrane iron exporter. Due to the presence of two check points at cellular levels, iron absorption and release are mainly regulated, because of this iron containing oral formulations are poorly absorbed and bioavailable. To overcome cellular barriers and increasing the bioavailability of supplemented iron forms, there is a need for new carriers that work protecting the iron as well as enhancing its intestinal absorption and release into the blood stream. Moreover thus reducing dosage and side effects. Sucrosomial® Iron (SI) represents an innovative oral iron-containing carrier in which ferric pyrophosphate is protected by a phospholipid bilayer membrane plus a sucrester matrix. To date, in vitro studies have shown that SI is mostly absorbed as vesicle-like structure, bypassing the conventional iron absorption pathway. Due to its behaviour at the gastrointestinal tract, SI is well tolerated and highly bioavailable compared to conventional iron salts. To deeply understand involvement of endocytosis in SI absorption and release, in vitro experiments using endocytosis and ferroportin inhibitors were carried outAim: to study Sucrosomial® Iron uptake and release in different in vitro systems.Materials and Methods: CACO-2 and THP1 cells were used to investigate the role of FPN in Sucorsomial Iron release from cells. For release study, CACO-2 cells were exposed for 18h to quercetin (150mmol/L) in order to downregulate FPN expression. CACO-2 quercetin pre-treated cells were co-cultured with TPH1 cells, and SI or FAC were added. However, prior to measure cell Ferritin content, the incubation medium was discarded and cells were washed to remove quercetin. Iron uptake-release analysis was performed using co-culture transwell system between CACO-2 cells and TPH1. To investigate the cellular fate of cellular iron in quercetin treated CACO-2 and TPH1 cells we measured cell ferritin content. To inhibit endocytosis absorption pathway, CACO-2, THP1 and HepG2 cells were pre-treated with PitStop2 and Dyngo 4a inhibitors and then treated with SI, or Ferrous Sulfate (FS) or ferric ammonium citrate (FAC). Cellular Ferritin content was measured.Results: in order to understand the effect of quercetin on iron storage, we used CACO2 and TPH1 cells pre-treated with quercetin and then treated with SI, FAC or nothing (control). Quercetin-SI treated CACO-2 cells showed no differences in Ferritin expression compared to control cells (3,94 ngFTL/mg proteins Vs 4,56 ngFTL/mg proteins) while in quercetin-FAC treated cells ferritin expression was decreased compare to control cells (16,3 ngFTL/mg proteins Vs 27,55 ngFTL/mg proteins). In a similar manner, quercetin-SI treated TPH1 cells didn't show increase in Ferritin expression compared to control cells (20 ngFTL/mg proteins Vs 15,15 ngFTL/mg proteins), only in quercetin-FAC treated cells we observed a Ferritin expression increase compared to control untreated cells (16 ngFTL/mg proteins Vs 24 ngFTL/mg proteins). Results from experiments using endocytosis inhibitors showed that SI absorption in CACO-2 cells is inhibited using Dyngo4a (from 4ngFTL/mg proteins to 0,36 ngFTL/ mg proteisn) while PitStop3 seems to reduce SI absorption in THP1 (from 396 ngFL/mg protein to 199,91 ngFTL/mg proteins) and HepG2 cells (from 26,86 ngFL/mg proteins to 3,93 ngFTL/mg proteins), since ferritin expression significantly decrease only in SI treated cells.Conclusions: endocytosis pathway seems to be involved in SI cellular uptake but this process is regulated in different manner probably due to different cell types. Release experiments showed that cells treated with quercetin could reduce for a negative feedback DMT1 expression as well, affecting iron uptake from cells treated with FAC but not with SI and consequently, if SI is able to bypass commonly iron uptake mechanism, FPN inhibition did not show iron release perturbation from cells treated with SI. DisclosuresBrilli:Pharmanutra s.p.a.: Consultancy. Martinelli:Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy; Abbvie: Consultancy; Novartis: Speakers Bureau; Amgen: Consultancy; Ariad/Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Tarantino:Pharmanutra s.p.a.: Employment.

Lactobacillus paracasei K5 displays adhesion, anti-proliferative activity and apoptotic effects in human colon cancer cells.

Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain, isolated recently from feta-type cheese. Its probiotic potential has been demonstrated in a series of established in vitro tests. Moreover, incorporation of L. paracasei K5 as starter culture offered organoleptic and technological advantages to novel fermented food products. In the present study, further investigation of the potential probiotic activity of L. paracasei K5 was performed and its mechanisms of action were investigated. Employing quantitative analysis and confocal, fluorescent microscopy the adhesion properties of the above strain were studied. L. paracasei K5 displayed efficient adherence capacity to Caco-2 colon cancer cells, similarly to the reference strains Lactobacillus casei ATCC 393 and Lactobacillus rhamnosus GG. Moreover, treatment of Caco-2 cells with L. paracasei K5 inhibited cell proliferation in a time-and dose-dependent manner. The anti-proliferative effects appear to be mediated through induction of apoptosis via modulation of expression of specific Bcl-2 family proteins. These results elucidate the mechanisms of action of L. paracasei K5 and enhance its potential probiotic activity.

Abstract 4438: NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells

Abstract Sodium butyrate (NaBt) is a histone deacetylase inhibitor (HDACi) produced in the colon by commensal microbiota-mediated fermentation of dietary fibers. It is regarded to have tumor suppressive and anti-inflammatory effects. NaBt can regulate gene expression through chromatin remodeling and altered transcription. However, it has not been addressed adequately whether inflammatory genes may also be regulated by NaBt in a post transcriptional manner via AU rich elements (ARE) in their 3'UTR. Overexpression of Cyclooxygenase-2 (COX-2) is observed in chronic inflammatory diseases and colorectal cancer (CRC). The aim of this study was to explore whether NaBt regulates COX-2 expression via post-transcriptional mechanisms. Pre-confluent Caco-2 and HT-29 cells were treated with different concentrations (1-5mM) of NaBt for short (3-6h) and long (24-48h) time points. Gene expression was determined by qPCR or Western blot. mRNA stability was determined with an Actinomycin D (ActD) chase assay. 3'UTR activity was determined in an RPSM30 vector containing the ARE rich and corresponding mutated regions of the 3'UTR from COX-2. NaBt strongly reduced COX-2 mRNA and protein expression with both short and long-term treatments. This reduction was not through NF-κB, since short-term treatment of cells with NaBt could not alter NF-κB activity in a luciferase reporter assay. An Act D chase assay indicated that in the absence of new mRNA synthesis, NaBt treatment induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. However, we did not observe any change in the 3'UTR activity of COX-2 in the presence of NaBt alone, indicating that the presence of a stress factor (such as ActD) was necessary for mRNA stabilization. Treatment of Caco-2 cells with NaBt and ActD together resulted in enhanced activation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of the ARE binding protein HuR. This was also reflected in increased COX-2 mRNA and protein levels. In HT-29 cells, simultaneous (NaBt+ActD) treatment resulted in a decrease in p-MK2, but an increase in p-Chk2 compared to ActD alone. p-Chk2 can preserve Cdk1 in a hyperphosphorylated state where it may promote retention of HuR in the nucleus, facilitating COX2 mRNA degradation and lower protein levels. No change in p-Chk2 was observed in Caco-2 cells in a similar experimental set up. These findings suggest that in the presence of a stress-inducing agent like ActD, NaBt can alter mRNA stability of COX-2 in a contextual manner, dependent on upstream signaling. Thus, while the rate of COX-2 mRNA degradation in HT-29 was enhanced, it slows down the same in Caco-2 cells. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of stress. Citation Format: Sreeparna Banerjee, Shabnam Enayat, Sinem Tuncer, Doğukan H. Ulgen, Aydan Torun. NaBt can regulate the expression of COX-2 post-transcriptionally in the presence of chemically induced stress in colon epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4438.

Abstract 4476: Role of MAP-Kinase suppressor in IBD

Abstract BACKGROUND &amp; AIMS: Endogenous suppressor of Mitogen Activated Kinase Pathway (MAPK) are considered cancer suppressors. In this regard , Sprouty (SPRY) proteins that are endogenous suppressor of receptor tyrosine kinase signaling are reported to be tumor suppressors in various cancers, whereas we discovered that SPRY2 augments Epithelial Mesenchymal Transition (EMT) in colorectal cancer (CRC) [Oncogene, 2016 Jun 16; 35(24): 3151-62]. Significance of SPRY proteins in Inflammatory Bowel Disease (IBD) is not clearly understood. MATERIALS &amp; METHODS: Biopsies from Crohn's Colitis patients were assessed for SPRY, Beta-catenin, Occludin and ZO1 expression. Tight junction profiler was employed to assess expression of integrin proteins in CaCO2 colon cancer cell line. SPRY was suppressed in colon cancer cells by siRNA and CRISPR/Cas9 technology and changes were assessed by immunoblotting, real time PCR, phase-contrast and confocal microscopy. Spry1 and Spry2 floxed mice were utilized to dissect SPRY functions in mesenchymal cells. RESULTS: SPRY, E-cadherin, Beta-catenin and Occludin mRNA transcripts were significantly different in IBD patients (p&amp;lt;0.05). No significant changes in ZO1 mRNA transcripts were noted. Suppression of SPRY significantly decreased expression of several integrins in CaCO2 cells (p&amp;lt;0.05) . Treatment of CaCO2 cells with Dextran Sodium Sulfate (DSS) significantly altered SPRY, Beta-catenin, Occludin and ZO1 expressions that was reversed by SPRY suppression. Spry1-/- and Spry2-/- double mutant murine embryonic fibroblasts (MEFs) demonstrated restoration of several epithelial markers which protected cells from DSS-induced changes. CONCLUSIONS: SPRY expression in colonic epithelium regulates IBD. SPRY has prognostic value in IBD. Citation Format: Sharad Khare, Qiong Zhang. Role of MAP-Kinase suppressor in IBD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4476.

Resveratrol mediated cancer cell apoptosis, and modulation of multidrug resistance proteins and metabolic enzymes

BackgroundThe degree of intracellular drug accumulation by specific membrane transporters, i.e., MDR1, BCRP, and MRP, and the degree of detoxification by intracellular metabolic enzymes, i.e., CYP3A4 and GST, provide control for cancer chemotherapy through diminishing the propensity of cancer cells to undergo apoptosis which in turn modulates the unresolved and complex phenomenon of multidrug resistance (MDR) for the cancer cells. Hypothesis/PurposeThis study dwells into the interaction details involving ABC-transporters, CYP3A4, GST and cytotoxic effects of resveratrol on different cell lines. MethodsResveratrol was evaluated for its ability modulating the expression and efflux functions of P-gp /MDR1, MRP1, and BCRP in the multidrug-resistant human colon carcinoma cell line, Caco-2, and CEM/ADR5000 cells through flow cytometry and RTPCR technique. ResultsThe resveratrol influenced P-gp and MRP1 efflux functions whereby it increased rhodamine 123 with calcein accumulation in concentration-dependent manner (1 – 500 µM) in the Caco-2 cell lines and inhibited the effluxes of both the substrates also as concentration-dependent phenomenon (10 – 100 µM) in the p-gp overexpressing CEM/ADR5000 cells through FACS (full form). The treatment of drug-resistant Caco-2, and CEM/ADR5000 cells with doxorubicin (DOX) along with 20 µM of resveratrol in the mixture. It increased the cell sensitivity DOX towards the DOX and enhanced the cytotoxicity. The resveratrol inhibited both CYP3A4 and GST enzymatic activity in a concentration-dependent way and induced apoptosis in the resistance cell lines because of increased levels of caspase-3, -8,-6/9 and incremental phosphatidyl serine (PS) exposure as detected by flow cytometry. The treatment of Caco-2 cells with resveratrol showed significantly lower p-gp, MRP1, BCRP, CYP3A4, GST, and hPXR mRNA levels in a 48 h observation. ConclusionThe result confirmed resveratrol mediated inhibition of ABC-transporters’ overall efflux functions, and its expression, and apoptosis as well as metabolic enzymes GST and CYP3A4 activity.

Tumor necrosis factor alpha reduces intestinal vitamin C uptake: a role for NF-κB-mediated signaling.

Sodium-dependent vitamin C transporter-1 (SVCT-1) is the major transporter mediating intestinal vitamin C uptake. Intestinal inflammation and prolonged infection are associated with increased serum and intestinal mucosa levels of tumor necrosis factor-α (TNF-α), which also exerts profound effects on the intestinal absorption process. Elevated levels of TNF-α have been linked to the pathogenesis of inflammatory bowel disease (IBD) and malabsorption of nutrients, and patients with this condition have low levels of vitamin C. To date, little is known about the effect of TNF-α on intestinal absorption of vitamin C. We studied the impact of TNF-α on ascorbic acid (AA) transport using a variety of intestinal preparations. The expression level of human SVCT-1 mRNA is significantly lower in patients with IBD. TNF-α treated Caco-2 cells and mice showed a significant inhibition of intestinal 14C-AA uptake. This inhibition was associated with significant decreases in SVCT-1 protein, mRNA, and heterogeneous nuclear RNA levels in TNF-α treated Caco-2 cells, mouse jejunum, and enteroids. Also, TNF-α caused a significant inhibition in the SLC23A1 promoter activity. Furthermore, treatment of Caco-2 cells with celastrol (NF-κB inhibitor) blocked the inhibitory effect caused by TNF-α on AA uptake, SVCT-1 protein, and mRNA expression, as well as the activity of SLC23A1 promoter. Treatment of TNF-α also led to a significant decrease in the expression of hepatocyte nuclear factor-1-α, which drives the basal activity of SLC23A1 promoter, and this effect was reversed by celastrol. Together, these findings show that TNF-α inhibits intestinal AA uptake, and this effect is mediated, at least in part, at the level of transcription of the SLC23A1 gene via the NF-κB pathway. NEW & NOTEWORTHY Our findings show that tumor necrosis factor-α inhibits intestinal ascorbic acid uptake in both in vitro and in vivo systems, and this inhibitory effect is mediated, at least in part, at the level of transcription of the SLC23A1 (sodium-dependent vitamin C transporter-1) gene via the NF-κB pathway.

Cholera Toxin Decreases SLC26A3 Expression and Function in Intestinal Epithelial Cells

Down‐regulated in adenoma (DRA, SLC26A3), the major Cl−/HCO3− exchanger involved in electroneutral NaCl absorption in the mammalian intestine, has emerged as a novel therapeutic target for diarrhea. Diarrhea commonly involves decreased absorption and/or increased secretion of fluid and electrolytes. We and others have shown decreased expression and function of DRA in infectious diarrhea. In this regard, cholera toxin (CTX) secreted by Vibrio cholerae is one of the most potent agents causing infectious diarrhea. Whether DRA function/expression is altered in CTX‐induced diarrhea is not known. Therefore, the current studies were undertaken to examine the effects of CTX on DRA in vitro in intestinal epithelial Caco‐2 cells. DRA function (Cl−/HCO3− exchange activity) was measured as DIDS sensitive 125I− uptake, mRNA and protein levels were assessed by quantitative real‐time PCR and immunoblotting respectively. Our results showed that treatment of Caco‐2 cells with CTX (1, 5 and 10 ng/ml) for 24 h significantly decreased Cl−/HCO3− exchange activity in a dose dependent manner. Concomitant dose‐dependent decrease in DRA mRNA levels was observed in response to CTX [1 ng/ml (46.3 ± 8.8 % p&lt; 0.0001), 5 ng/ml (69.5 ± 3.1 % P &lt; 0.004) and 10 ng/ml (76.5 ± 2.1%, P&lt; 0.02)] compared to untreated control. Similarly, DRA protein levels were also significantly decreased [1 ng/ml (39 ± 15.7 5% p&lt; 0.0001), 5 ng/ml (60.8 ± 6.3 % P &lt; 0.0001) and 10 ng/ml (56.7 ± 12.6%, P&lt; 0.0001)]. In conclusion, our data suggest that CTX‐induced decrease in DRA expression and function could contribute to the pathophysiology of CTX‐induced diarrhea.Support or Funding Information(Supported by NIDDK/Department of Veteran Affairs)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.