Colonic Epithelial Cell Line Research Articles

Glutathione catabolism by Enterobacteriaceae species to hydrogen sulphide adversely affects the viability of host systems in the presence of 5'fluorodeoxyuridine.

Reduced glutathione (GSH) plays an essential role in relieving oxidative insult from the generation of free radicals via normal physiological processes. However, GSH can be exploited by bacteria as a signalling molecule for the regulation of virulence. We describe findings arising from a serendipitous observation that when GSH and Escherichia coli were incubated with 5′fluorodeoxyuridine (FUdR)‐synchronised populations of Caenorhabditis elegans, the nematodes underwent rapid death. Death was mediated by the production of hydrogen sulphide mainly through the action of tnaA, a tryptophanase‐encoding gene in E. coli. Other Enterobacteriaceae species possess similar cysteine desulfhydrases that can catabolise l‐cysteine‐containing compounds to hydrogen sulphide and mediate nematode killing when worms had been pre‐treated with FUdR. When colonic epithelial cell lines were infected, hydrogen sulphide produced by these bacteria in the presence of GSH was also able to inhibit ATP synthesis in these cells particularly when cells had been treated with FUdR. Therefore, bacterial production of hydrogen sulphide could act in concert with a commonly used genotoxic cancer drug to exert host cell impairment. Hydrogen sulphide also increases bacterial adhesion to the intestinal cells. These findings could have implications for patients undergoing chemotherapy using FUdR analogues that could result in intestinal damage.

Interleukin-27 Regulates the Function of the Gastrointestinal Epithelial Barrier in a Human Tissue-Derived Organoid Model.

Simple SummaryThe gut is lined by a single layer of epithelial cells, creating a barrier between the contents of the gut and the underlying tissue. This barrier also controls permeability (to determine if gut contents move to the tissue) and produces proteins that help with immune responses. These epithelial cells are damaged and do not function properly in inflammatory bowel diseases (IBD). IBD is common, and current treatments are not always effective and can have side effects. New treatments are needed. An alternative treatment target is the epithelial gut lining to promote healing. We used an experimental model of human gut tissue epithelial cells (called organoids) and aimed to define the role of the protein interleukin-27 (IL-27) in epithelial barrier function. We show that IL-27 restored permeability associated with changes in several proteins involved in this process, led to changes in genes involved in the immune response and reaction to bugs in the gut and led to enhanced healing and repair of an injury wound in the barrier. In summary, this study demonstrates a new function of IL-27 in the gut to aid epithelial barrier repair. This is a potential new treatment strategy for IBD.A treatment with direct healing effects on the gastrointestinal epithelial barrier is desirable for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an immunoregulatory cytokine, and oral delivery is an effective treatment in murine models of IBD. We aimed to define IL-27 effects on the human gastrointestinal epithelial barrier. We characterised gene and protein expression of permeability mediators in a human colon-derived organoid model. Functional permeability was determined in an organoid-derived 2D monolayer by transepithelial electrical resistance. IL-27 effects on epithelial innate immune responses were assessed through expression of cytokines, anti-microbial peptides and MUC genes. IL-27 effects on wound healing and proliferation were determined in human colon epithelial cell lines. IL-27 led to restoration of permeability regulation following inflammatory cytokine insult (p = 0.001), associated with differential expression of tight junction mediators with decrease in claudin 2 (p = 0.024) and increase in claudin 4 (p < 0.001), E-cadherin (p < 0.001) and zona occludens (p = 0.0014). IL-27 evoked differential gene expression of epithelial-derived innate immune responses (reduced IL1B and IL18, and increased IL33, HBD1, MUC1 and MUC2; p < 0.012). IL-27 induced epithelial barrier wound healing through restitution (p < 0.001), and increased proliferation (p < 0.001) following injury. Overall, IL-27 provokes mucosal healing of the human gastrointestinal epithelial barrier.

Biological Activity of an Epilobium angustifolium L. (Fireweed) Infusion after In Vitro Digestion.

The biological activity of an in vitro digested infusion of Epilobium angustifolium (fireweed) was examined in a model system of intestinal epithelial and colon cancer tissues. The content of selected phenolic compounds in the digested aqueous extract of fireweed was determined using HPLC-ESI-QTOF-MS/MS. Biological activity was examined using the human colon adenocarcinoma cell lines HT-29 and CaCo-2 and the human colon epithelial cell line CCD 841 CoTr. Cytotoxicity was assessed by an MTT assay, a Neutral Red uptake assay, May-Grünwald-Giemsa staining, and a label-free Electric Cell-Substrate Impedance Sensing cytotoxicity assay. The effect of the infusion on the growth of selected intestinal bacteria was also examined. The extract inhibited the growth of intestinal cancer cells HT-29. This effect can be attributed to the activity of quercetin and kaempferol, which were the most abundant phenolic compounds found in the extract after in vitro digestion. The cytotoxicity of the fireweed infusion was dose-dependent. The highest decrease in proliferation (by almost 80%) compared to the control was observed in HT-29 line treated with the extract at a concentration of 250 μg/mL. The fireweed infusion did not affect the growth of beneficial intestinal bacteria, but it did significantly inhibit E. coli. The cytotoxic effect of the fireweed extract indicates that it does not lose its biological activity after in vitro digestion. It can be concluded that the fireweed infusion has the potential to be used as a supporting agent in colon cancer therapy.

MicroRNA-133b Regulates Cell Proliferation and Cell Cycle Progression via Targeting HuR in Colorectal Cancer

MicroRNAs (miRNAs/miRs) have been identified to serve a key role in the development of tumors. However, the role of miR-133b in colorectal cancer (CRC) remains largely unclear. This study will investigate the role and mechanism of miR-133b in CRC. Reverse transcription-quantitative polymerase chain reaction analysis was performed to detect the level of miR-133b in CRC cell lines. Bioinformatics software TargetScan predicted the potential target genes of miR-133b, and a dual luciferase reporter assay was used to confirm this. To investigate the role of miR-133b in CRC cells, miR-133b was upregulated or downregulated in CRC cell lines (SW620 and HT-29) by transfecting with a miR-133b mimic or inhibitor, respectively. Subsequently, cell viability was analyzed using MTT assay, whereas cell apoptosis and the cell cycle distribution were analyzed by flow cytometry. In addition, the associated protein levels were detected using western blot analysis. The results demonstrated that miR-133b was significantly downregulated in CRC cell lines when compared with the normal colonic epithelial NCM-460 cell line. Human antigen R (HuR; also termed ELAVL1) was demonstrated to be a direct target of miR-133b and was negatively regulated by miR-133b. HuR was also notably upregulated in the CRC cell lines when compared with the normal control. Transfection of SW620 and HT-29 cells with the miR-133b mimic significantly inhibited cell viability, and induced cell apoptosis and G1 phase arrest, while upregulation of HuR demonstrated the opposite effects. Furthermore, the present data demonstrated that the miR-133b mimic significantly enhanced the protein levels of p21 and p27, and downregulated cyclin D1 and cyclin A levels in SW620 and HT-29 cells; the opposite effects were observed following treatment with the miR-133b inhibitor. In conclusion, the data indicate that miR-133b suppressed CRC cell growth by targeting HuR.

The High Ratio of the Plasma miR-96/miR-99b Correlated With Poor Prognosis in Patients With Metastatic Colorectal Cancer.

Object: This study aims to clarify the expression of plasma miRNA in CRC patients, and to clarify the potential use of these miRNAs in diagnosis and prognosis, and to establish a prognostic model to initially explore its clinical value. Methods: We detected the expression of 6 miRNAs in normal colon epithelial cell lines and colorectal cancer cell lines by qRT-PCR and they were validated in the tissues of three subtypes: 20 healthy subjects, 41 pCRC and 49 mCRC patients. COX regression and ROC analyses use to evaluate the diagnostic and prognostic efficacy of candidate miRNAs. Subsequently, we initially established a nomogram prognostic model. MiRNA is also used to construct miRNA-mRNA interaction network and PPI network modules. Results: Five miRNAs showed significant differential expression in pCRC, mCRC patients and normal groups. ROC analysis showed that CEA, miR-96, miR-99b and miR-96/miR-99b are distinguishable from pCRC and mCRC patients, with AUC ranging from 0.65 to 0.91; among them, the ratio of miR-96/miR-99b is stronger than any diagnostic indicators, such as CEA and CA125. Multivariate survival analysis identified miR-96, miR-99b, N stage, M stage and clinical stage as independent prognostic indicators of mCRC. The nomogram based on these 5 characteristics has satisfactory prognostic values. Conclusion: Our data indicate that plasma miR-96/miR-99b can be used as a promising biomarker for early detection of mCRC patients; our nomogram has a promising evaluation value.

Antiparasitic properties of miltefosine-based nanoformulations against protozoan pathogen, Acanthamoeba castellanii

Background: Acanthamoeba castellanii genotype T4 is the causative agent of the progressively increasing sight-threatening Acanthamoeba keratitis and central nervous system infections. Because of the increased prevalence and the ineffectiveness of the current antiamoebic drugs, we synthesized miltefosine poly(lactic-co-glycolic acid) nanoparticles (miltefosine PLGA NP) as a potential potent and biocompatible antiamoebic drug. The advantage to use PLGA NP is to preserve the cells from the toxic effect of miltefosine drug. In particular, miltefosine PLGA nanoformulation offers a better cellular uptake and a sustained drug release compared with the free drug that presents potent cytotoxicity at high concentrations against human colon cancer cell lines. Methods: The miltefosine NP were synthesized using a double emulsion-solvent evaporation method, characterized, and then assessed for their antiamoebic activity against A. castellanii belonging to the T4 genotype. Blank PLGA NP and miltefosine were used as controls. Results: Amoebicidal assays revealed that at 25 and 50 µM, unmodified miltefosine eradicated 83% and 93% of amoebae, respectively. At these same concentrations of 25 and 50 µM, the amount of miltefosine released form PLGA NP formulation was limited to 22.6%. However, it killed 36% and 56% of the protozoa, respectively. Thus, the efficacy of PLGA NP formulation was similar to that of the unmodified miltefosine. Both miltefosine and its PLGA NP significantly inhibited the pretreated amoebae (minimum inhibitory concentration 50% = 37.23 and 55.26 µM, respectively, compared with 147.2 µM of the blank NP; P < 0.05) and reduced amoebae-mediated host cell death. The blank NP and miltefosine NP exhibited minimal cytotoxicity against colon epithelial cell lines. In contrast, the unmodified miltefosine caused 37%, 71%, and 88% of cytotoxicity at 10, 25, and 50 µM, respectively. Conclusion: Overall, these findings suggest that controlling the release of miltefosine from PLGA NP for a short time was almost as effective as miltefosine alone against A. castellanii genotype T4 while reducing host cell toxicity. Hence, this study demonstrates the feasibility of using PLGA NP for the treatment of Acanthamoebic infections.

The dietary fibre rhamnogalacturonan improves intestinal epithelial barrier function in a microbiota-independent manner.

Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function. Monolayers of colonic epithelial cell lines (Caco-2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR-4 and PKC isoforms. Apically applied RGal (1000 μg ml-1 ) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)-dextran flux in Caco-2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco-2 cells in a calcium switch assay. RGal also reversed the barrier-damaging effects of inflammatory cytokines on FITC-dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4-expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ. RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.

Inflammatory activation of surface molecule shedding by upregulation of the pseudoprotease iRhom2 in colon epithelial cells

The metalloproteinase ADAM17 contributes to inflammatory and proliferative responses by shedding of cell-surface molecules. By this ADAM17 is implicated in inflammation, regeneration, and permeability regulation of epithelial cells in the colon. ADAM17 maturation and surface expression requires the adapter proteins iRhom1 or iRhom2. Here we report that expression of iRhom2 but not iRhom1 is upregulated in intestinal tissue of mice with acute colitis. Our analysis of public databases indicates elevated iRhom2 expression in mucosal tissue and epithelial cells from patients with inflammatory bowel disease (IBD). Consistently, expression of iRhom2 but not iRhom1 is upregulated in colon or intestinal epithelial cell lines after co-stimulation with tumor necrosis factor (TNF) and interferon gamma (IFNgamma). This upregulation can be reduced by inhibition of Janus kinases or transcription factors NF-kappaB or AP-1. Upregulation of iRhom2 can be mimicked by iRhom2 overexpression and is associated with enhanced maturation and surface expression of ADAM17 which then results in increased cleavage of transforming growth factor (TGF) alpha and junctional adhesion molecule (JAM)-A. Finally, the induction of these responses is suppressed by inhibition of iRhom2 transcription. Thus, inflammatory induction of iRhom2 may contribute to upregulated ADAM17-dependent mediator and adhesion molecule release in IBD. The development of iRhom2-dependent inhibitors may allow selective targeting of inflammatory ADAM17 activities.

Evaluation of the antimicrobial and anticancer properties of the fruits of Synsepalum dulcificum (Sapotaceae)

Purpose: To evaluate the antimicrobial and anticancer activities of the fruit of Synsepalum dulcificum (Sapotaceae) against six bacterial strains and on two different colorectal cancer cell lines, respectively.Methods: The dried fruit of the plant was extracted in a Soxhlet apparatus successively with petroleum ether and ethanol, and concentrated in a rotary evaporator to obtain petroleum ether and ethanol extract, respectively. Phytochemical screening was done on the two extracts. The antimicrobial effects of the extracts against Bacillus subtilis, Staphylococcus aureus, Streptococcus pyogene, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris were evaluated using agar well diffusiontechnique while the anticancer effect of the ethanol extract was assessed on HCT-116 and primary colon epithelial (PCE) cell lines by MTT assay.Results: The results indicate that the petroleum ether extract of Synsepalum dulcificum fruits exerted stronger antimicrobial activity than the ethanol extract. The ethanol extract also showed significant anticancer activity (p &lt; 0.05). The calculated half-maximal concentration (IC50) of the extract on HCT- 116 cells at 24, 48, and 72 h are 14.99, 8.97, and 8.54 μg/mL, respectively, while the IC50 of the extract on PCE cell lines at 24, 48, and 72 h are 236.25, 206.09, and 196.72 μg/mL, respectively. The extract was more toxic to cancer cells than to normal cells.Conclusion: The results of this study lend some justification for the use of the fruits of Synsepalum dulcificum as an antibacterial and anticancer agent.

3,4,5-O-tricaffeoylquinic acid alleviates ionizing radiation-induced injury in vitro and in vivo through regulating ROS/JNK/p38 signaling.

Ionizing radiation (IR) brings many health problems to humans, causing damage to the digestive system, hematopoietic system, and immune system. Natural compounds derived from plants have attracted widespread attention due to their low toxicity. Here, we found that 3,4,5-O-tricaffeoylquinic acid (tCQA) extracted from natural plant Azolla imbricata could significantly alleviate the systemic damage in mice caused by IR. In order to further explore the molecular mechanism of the radioprotective effect of tCQA, in vitro experiments confirmed that tCQA could attenuate the cytotoxic effect of IR on the colonic epithelial cell line NCM460 and alleviate the IR-induced mitochondrial dysfunction characterized by the decrease of mitochondrial transmembrane potential, ROS production, and caspase-dependent apoptosis. In addition, the generation of ROS induced by H2 O2 could also be reversed by tCQA. Then, Western blot demonstrated that tCQA could reverse the MAPK signaling pathway activated by IR. However, the inhibitory effect of tCQA on JNK and P38 levels activated by the JNK agonist anisomycin is not obvious; meanwhile, tCQA could inhibit the activation of JNK/P38 induced by H2 O2 , which suggests that tCQA might inhibit the JNK/P38 signaling pathway by reducing ROS. In short, tCQA inhibits the generation of ROS caused by IR, and then regulates the activity of caspase in the mitochondrial pathway by inhibiting the JNK/P38 signaling pathway, thereby alleviating the apoptosis of NCM460. This research provides an experimental basis for the development of new types of radioprotective agents for medical diagnosis and radiotherapy.

A Novel Prognostic Prediction Model for Colorectal Cancer Based on Nine Autophagy-Related Long Noncoding RNAs.

IntroductionColorectal cancer (CRC) is the most common gastrointestinal cancer and has a low overall survival rate. Tumor–node–metastasis staging alone is insufficient to predict patient prognosis. Autophagy and long noncoding RNAs play important roles in regulating the biological behavior of CRC. Therefore, establishing an autophagy-related lncRNA (ARlncRNA)-based bioinformatics model is important for predicting survival and facilitating clinical treatment.MethodsCRC data were retrieved from The Cancer Genome Atlas. The database was randomly divided into train set and validation set; then, univariate and multivariate Cox regression analyses were performed to screen prognosis-related ARlncRNAs for prediction model construction. Interactive network and Sankey diagrams of ARlncRNAs and messenger RNAs were plotted. We analyzed the survival rate of high- and low-risk patients and plotted survival curves and determined whether the risk score was an independent predictor of CRC. Receiver operating characteristic curves were used to evaluate model sensitivity and specificity. Then, the expression level of lncRNA was detected by quantitative real-time polymerase chain reaction, and the location of lncRNA was observed by fluorescence in situ hybridization. Additionally, the protein expression was detected by Western blot.ResultsA prognostic prediction model of CRC was built based on nine ARlncRNAs (NKILA, LINC00174, AC008760.1, LINC02041, PCAT6, AC156455.1, LINC01503, LINC00957, and CD27-AS1). The 5-year overall survival rate was significantly lower in the high-risk group than in the low-risk group among train set, validation set, and all patients (all p < 0.001). The model had high sensitivity and accuracy in predicting the 1-year overall survival rate (area under the curve = 0.717). The prediction model risk score was an independent predictor of CRC. LINC00174 and NKILA were expressed in the nucleus and cytoplasm of normal colonic epithelial cell line NCM460 and colorectal cancer cell lines HT29. Additionally, LINC00174 and NKILA were overexpressed in HT29 compared with NCM460. After autophagy activation, LINCC00174 expression was significantly downregulated both in NCM460 and HT29, while NKILA expression was significantly increased.ConclusionThe new ARlncRNA-based model predicts CRC patient prognosis and provides new research ideas regarding potential mechanisms regulating the biological behavior of CRC. ARlncRNAs may play important roles in personalized cancer treatment.

Anti-proliferative activity of yerba mate (Ilex paraguariensis) aqueous extracts on human colorectal cancer cell lines

Background: Yerba mate, a popular, tea-like beverage prepared from the dried leaves of Ilex paraguariensis, is widely consumed, and has several reported health benefits. Compared with other herbal teas, the effect of yerba mate on human cells in the context of cancer has not been extensively studied. The method of extraction of bioactive compounds from the yerba mate leaves plays an important role in its effect on cancer cells. Methods: In this study we assessed the viability, anti-proliferative, and apoptotic effect of the aqueous yerba mate extract, prepared using the same conditions employed for consumption, on different human colorectal cancer cell lines (Caco-2, HT-29, and HCT116) and on the non-tumorigenic human colon epithelial cell line (NCM460).Results: Cytotoxicity of aqueous yerba mate extract was studied and a dose-dependent decrease in viability was observed in all the tested cell lines. At 24 hrs., viability decreased to 19.7% with Caco-2 cells, 2.7% with HCT116, and 8.4% with HT-29 cells at a concentration of 4.8 mg/mL of yerba mate extract. The effect was less prominent on the NCM460 cell line where the viability of cells at the same concentration was 65.2%. Yerba mate extract also showed concentration-dependent anti-proliferative effects as determined by the WST-1 proliferation kit. IC50 values ranged between 0.22-0.69 mg/mL at 24 hr for cell lines tested. To study whether cell death was due to apoptosis, Caco-2 cells were stained with Annexin V-FITC assay and an increase in the percentage of late apoptotic Caco-2 cells was observed with yerba mate extract at 0.6-4.8 mg/mL. Cell cycle analysis using DNA content by flow cytometry showed an increase in the percentage of Caco-2 cells in the subG0/G1 phase and the G0/G1 phase after treatment with 2.4 mg/mL extract. Collectively, our data suggest that yerba mate aqueous extract exhibits an anti-proliferative effect on tested cell lines by inducing apoptosis. Conclusions: Yerba mate aqueous extract exhibits a strong anti-proliferative activity against adenocarcinoma cell lines studied and constitutes a promising functional food adjuvant to anti-cancer therapy. Further work is needed to identify active components and mechanisms of action. Keywords: Aqueous extract, yerba mate, anti-proliferative activity, adenocarcinoma cell lines

Suppression of Transmembrane Tumor Necrosis Factor Alpha Processing by a Specific Antibody Protects Against Colitis-Associated Cancer.

Soluble tumor necrosis factor-α (sTNF-α) plays an important role in colitis-associated cancer (CAC); however, little is known about transmembrane TNF-α (tmTNF-α). Here, we observed an increase in sTNF-α mainly in colitis tissues from an azoxymethane/dextran sodium sulfate (DSS)-induced CAC mouse model whereas tmTNF-α levels were chiefly increased on epithelial cells at the tumor stage. The ratio of intracolonic tmTNF-α/sTNF-α was negatively correlated with the levels of pro-inflammatory mediators (IL-1β, IL-6, and NO) and M1 macrophages but positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and the level of the anti-inflammatory cytokine IL-10, suggesting an anti-inflammatory effect of tmTNF-α. This effect of tmTNF-α was confirmed again by the induction of resistance to LPS in colonic epithelial cell lines NCM460 and HCoEpiC through the addition of exogenous tmTNF-α or transfection of the tmTNF-α leading sequence that lacks the extracellular segment but retains the intracellular domain of tmTNF-α. A tmTNF-α antibody was used to block tmTNF-α shedding after the first or second round of inflammation induction by DSS drinking to shift the time window of tmTNF-α expression ahead to the inflammation stage. Antibody treatment significantly alleviated inflammation and suppressed subsequent adenoma formation, accompanied by increased apoptosis. An antitumor effect was also observed when the antibody was administered at the malignant phase of CAC. Our results reveal tmTNF-α as a novel molecular marker for malignant transformation in CAC and provide a new insight into blocking the pathological process by targeting tmTNF-α processing.

Protease-activated receptor-2 activation enhances epithelial wound healing via epidermal growth factor receptor

ABSTRACT The intestinal barrier function relies on the presence of a single layer of epithelial cells. Barrier dysfunction is associated with the inflammatory bowel diseases (IBD). Understanding the mechanisms involved in intestinal wound healing in order to sustain the barrier function has a great therapeutic potential. Activation of protease-activated receptor-2 (PAR2) induces COX-2 expression in intestinal epithelial cells via EGFR transactivation. COX-2 is well known for its protective effects in the gastrointestinal tract. Therefore, we hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through COX-2-derived lipid mediators and EGFR transactivation. Immunofluorescence and calcium assay were used to characterize CMT-93 mouse colonic epithelial cell line for PAR2 expression and its activity, respectively. Treatment with PAR2 activating peptide 2-furoyl-LIGRLO-NH2 (2fLI), but not by its inactive reverse-sequence peptide (2fO) enhanced wound closure in scratch wounded monolayers. The EGFR tyrosine kinase inhibitor (PD153035), broad-spectrum matrix metalloproteinase inhibitor (GM6001) and Src tyrosine kinase inhibitor (PP2) inhibited PAR2-induced wound healing. However, PAR2 activation did not induce COX-2 expression in CMT-93 cells and inhibition of COX-2 by COX-2 selective inhibitor (NS-398) did not alter PAR2-induced wound healing. In conclusion, PAR2 activation drives wound healing in CMT-93 cells via EGFR transactivation. Matrix metalloproteinases and Src tyrosine kinase activity may involve in EGFR transactivation and PAR2-induced wound healing is independent of COX-2 activity. These findings provide a mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases.

Biogenically engineered silver nanoparticles using bael leaf extract and evaluation of its therapeutic potential

ABSTRACT The current study aims to explore the antimicrobial and anticancer activity of biogenic silver nanoparticles (B-AgNPs) synthesized using Bael leaf extract. AgNPs nanoparticles characterized using FESEM, TEM, XRD, and UV-Spectrophotometry. TEM analysis revealed that B-AgNPs were predominantly spheroidal with particle size distribution in the range of 7-10 nm. In MTT assay, when B-AgNPs were applied in colon cancer (HCT-116) cells and human colon epithelial cell line (FHC) cells. B-AgNPs exhibited specific cytotoxicity to HCT-116 as minimal effect observed in normal FHC cells. Moreover, the fluorescence microscopic examination after nucleus staining as well as DNA fragmentation also confirmed AgNPs spectific cytotoxicity towards HCT-116. Further, AgNPs demonstrated excellent antibiofilm and antibacterial activity in both Gram-Positive and Gram-negative bacteria. Hence, overall, we categorically concluded that the biogenic silver nanoparticles synthesized using Bael leaf extract possess multiple biological activities and might be possibly used as multipurpose therapeutic material with no or little systemic toxicity.

Cytotoxicity analysis of pre- and post-hurricane harvey soil samples collected from greater houston bayous

Rapid urbanization, anthropogenic pollution and frequent flooding events are affecting the soil and water quality along the streams and bayous of Houston. Soil acts as sink and reservoir of heavy metals and nutrients affecting human and animal health. The objectives of the study are 1) to analyze the effects of the metal and nutrient concentration of bayou flood plain surface soil samples on the gut cell cytotoxicity and 2) to evaluate the spatial and temporal difference in soil contamination on cell viability of colon cancer (HT-29) and normal colon epithelial (CCD 841 CoN) cell lines. To evaluate soil contamination between pre- and post-hurricane (Summer and Fall) conditions in six Bayous (Brays, Buffalo, Halls, Hunting, Greens and White Oak Bayous) of Harris County, Texas, in vitro bioassay analysis was applied to soil extracts. The MTT assay determined that, with increase in concentration of Bayou soil from 12.5% to 100%, the viability of CCD 841 CoN and HT-29 cells decreased significantly, across all sampling locations during both summer and fall seasons. Among all the bayous, the viability of CCD 841 CoN cells in summer and fall followed the pattern of White Oak > Greens > Halls > Brays Bayou, where the viability of cells exposed to White Oak soils was 3–4 times higher than cells exposed to Brays Bayou soil at 100% soil concentration. The viability of HT-29 cells in both seasons followed the pattern of Greens > White Oak > Halls > Brays Bayou, where the viability of cells exposed to Greens Bayou soil was more than 3–4 times higher than the cells exposed to Brays Bayou soil at 100% concentration. The higher concentration of metals and nutrients such as P, Zn, Cd, and Cu might have contributed to the significant cell lethality in Brays Bayou samples compared to other locations.

Synthesized Two New Water‐Soluble Fluorescents Calix[4]arene 4‐sulfo‐1,8‐naphthalimide Derivatives Inhibit Proliferation of Human Colorectal Carcinoma Cells

AbstractSynthesis of 1,8‐naphthalimide derivatives and biochemical studies using them as anti‐cancer and cellular imaging agents have attracted significant attention in recent years. This study aimed to synthesize two new fluorescent narrow rim‐functionalized 4‐sulfo‐1,8‐naphthalimide derivatives of calix[4]arenes (4 and 7) and examination of their cytotoxic properties for cancerous cells. For this purpose, p‐tert‐butylcalix[4]arene bearing dihydrazide (3) or diaminopropyl (6) units on its narrow rim were reacted with 4‐sulfo‐1,8‐naphthalic anhydrate. The structure of these synthesized compounds has been characterized using 1H‐NMR, 13C‐NMR, and FTIR techniques. To investigate the anti‐cancer properties, compounds 4, 7, and N,N‐dimethylaminoethyl‐1,8‐naphthalimide (8) were tested on the human colorectal cancer cell line (DLD‐1) as well as healthy colon epithelial cell line (CCD‐18Co). The IC50 values of 4 and 7 were determined as 12.95 μM and 16.13 μM, respectively, on DLD‐1 cells. Furthermore, following the treatment of CCD‐18Co cells with 4 and 7, the IC50 values were determined as 508 μM and 269 μM, respectively. However, it was observed that compound 8 had no cytotoxic effect on both DLD‐1 cells and CCD‐18Co cells. Furthermore, the fluorescence imaging of human colorectal cancer cells treated with the synthesized compounds (4, 7, and 8) was performed on living cells.

Hsa_circ_0136666 activates Treg-mediated immune escape of colorectal cancer via miR-497/PD-L1 pathway

PurposeIn the rankings of cancer mortality and incidence worldwide, colorectal cancer ranks fourth and the third, respectively. Circular RNA hsa_circ_0136666 (hsa_circ_0136666) is reported to participate in the growth of colorectal cancer. However, the mechanism by which hsa_circ_0136666 regulates the tumorigenesis of colorectal cancer needs to be further explored. In this study, we report here the role of hsa_circ_0136666 in the aberrant activation of Treg cells and immune evasion of tumor cells, providing a new strategy for the treatment of colorectal cancer. MethodsWestern blotting assay and qRT-PCR assay were used to determine protein and mRNA expression levels. Dual-luciferase reporter assay was used to evaluate the targeted regulatory relationship. RNA immunoprecipitation was used to detect RNA binding. Colony formation assay was utilized to measure the cell proliferation. Flow cytometry was used to assess cell apoptosis. Xenograft model was setup to evaluate tumor growth. ResultsThe results showed that hsa_circ_0136666 and PD-L1 was increased in colorectal cancer cells while miR-497 was decreased in colorectal cancer cells when compared with normal colon epithelial cell line. Hsa_circ_0136666 was demonstrated to directly target miR-497, which also regulated PD-L1 by binding to its 3′UTR. Further mechanistic studies identified that hsa_circ_0136666 controlled cell proliferation and apoptosis via targeting miR-497 and regulating PD-L1 expression. Of note, hsa_circ_0136666 stimulated Treg cells mediated by miR-497/PD-L1 axis and its downstream signal pathway in Treg cells. Finally, hsa_circ_0136666 was found to accelerate the tumor growth in vivo. ConclusionsOur findings demonstrated that hsa_circ_0136666 promoted the expression of PD-L1 by inhibiting miR-497 level in colorectal cancer, thus inducing the activation of Treg cells and leading to the immune escape of tumor, providing a novel mechanistic insight into the pathogenesis of colorectal cancer.

Assessing the Toxicological Relevance of Nanomaterial Agglomerates and Aggregates Using Realistic Exposure In Vitro.

Low dose repeated exposures are considered more relevant/realistic in assessing the health risks of nanomaterials (NM), as human exposure such as in workplace occurs in low doses and in a repeated manner. Thus, in a three-week study, we assessed the biological effects (cell viability, cell proliferation, oxidative stress, pro-inflammatory response, and DNA damage) of titanium-di-oxide nanoparticle (TiO2 NP) agglomerates and synthetic amorphous silica (SAS) aggregates of different sizes in human bronchial epithelial (HBE), colon epithelial (Caco2), and human monocytic (THP-1) cell lines repeatedly exposed to a non-cytotoxic dose (0.76 µg/cm2). We noticed that neither of the two TiO2 NPs nor their agglomeration states induced any effects (compared to control) in any of the cell lines tested while SAS aggregates induced some significant effects only in HBE cell cultures. In a second set of experiments, HBE cell cultures were exposed repeatedly to different SAS suspensions for two weeks (first and second exposure cycle) and allowed to recover (without SAS exposure, recovery period) for a week. We observed that SAS aggregates of larger sizes (size ~2.5 µm) significantly affected the cell proliferation, IL-6, IL-8, and total glutathione at the end of both exposure cycle while their nanosized counterparts (size less than 100 nm) induced more pronounced effects only at the end of the first exposure cycle. As noticed in our previous short-term (24 h) exposure study, large aggregates of SAS did appear to be similarly potent as nano sized aggregates. This study also suggests that aggregates of SAS of size greater than 100 nm are toxicologically relevant and should be considered in risk assessment.

Abstract 2840: HK1 induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer

Abstract Hexokinase 1 (HK1) has been implicated in tumorigenesis. However, its role in tumor metastasis and progression has not been evaluated. The aim of this study is to evaluate the role of HK1 in the metastasis in CRC. Methods: The expression level of HK1 was detected using quantitative RT-PCR and western blot in 9 CRC cell lines and 1 human colon epithelial cell line NCM460 (Figure 1A). HK1 was overexpressed via lentiviral vector system and silenced using specific small interference RNA (si-HK1) in DLD1 and HCT8. Then wound-healing, migration assay and invasion assay were carried out to investigate the function of HK1 in CRC cells. RT-PCR and Western blot was performed to detect the change of Snail2 following the up-regulated expression of HK1. Results: HK1 expression was overexpressed by HK1-cDNA and was inhibited by si-HK1 in DLD1 and HCT8 (Figure 1B and 1C). Ectopic expression of HK1 promoted cell migration and invasion in CRC cells and promoted lung metastasis in mouse models (Figure 1D). Furthermore, HK1 induced migration and invasion in CRC cells was found to activate expression of Snail2 and in turn, induce epithelial-mesenchymal transition (EMT) and cancer progression (Figure 1E). Conclusions: High expression of HK1 could activate expression of Snail2 and in turn, induce EMT. Our findings suggest that HK1 may be a potential prognostic biomarker and therapeutic target for CRC invasion and metastasis. Keywords: Colorectal Cancer, HK1, Migration, Invasion Figure 1. A. The expression level of HK1 in 10 cell lines. B, C. HK1 expression was overexpressed by HK1-cDNA and was inhibited by si-HK1 in DLD1 and HCT8. *P&amp;lt; 0.05. D. Overexpression of HK1 in HCT8 increased the capacity of cell migration and invasion in vitro and in vivo. E. Expression levels of Snail2 and EMT were induced after HK1 overexpression. Citation Format: Xiaobin Zheng, Huashan Liu, Xiaoli Zhong, Xuanhui Liu, Zerong Cai, Yufeng Chen, Xiaosheng He, Ping Lan, Xiaojian Wu. HK1 induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2840.