Intestinal Cancer Cells Research Articles

Investigating the synergistic effects of gold nanoparticles and electroporation in sensitization of human intestinal colon cancer HT-29 cells to 6MV photon beam

Introduction: Radiation therapy (RT) is the gold standard treatment for more than half of known tumors. There is increasing evidence that combining radiation therapy with a radiosensitizer can enhance the efficiency of this treatment modality. A radiosensitizer preferably enhances dose at the site of tumor and increases discrimination between tumor and normal surrounding tissues. Gold nanoparticles (GNPs) and electroporation (EP) have shown good potential as radiosensitizers. Therefore, the present study assessed the sensitizing effects of EP, GNPs, and combined EP-GNPs on the dose enhancement factor(DEF) for 6 MV photon energy. Materials and Methods: In this in-vitro study, intestinal colon cancer (HT-29) cells treated with four different protocols: ionizing radiation alone (IR, control group), EP+IR, GNPs+IR and GNPs+EP+IR. Radiosensitization was assessed by colony formation assay at 6 MV photon energy. The survival curve was estimated using MATLAB software and DEF for each combination treatment group was calculated by dividing of LD50 (50% lethal dose) of control group with combination treatment group. Results: when the cells were treated with EP prior to IR the DEF of 1.36 was achieved. Treatment of cells with GNPs immediately before irradiation resulted in DEF of 1.17. In this group, GNPs did not significantly enhance the effect of IR due to having not enough time for GNPs to enter and accumulate in the target cells. However, when EP was added to the protocol of GNPs+IR group, DEF of 2.61 was observed. The observed difference between DEF values of GNPs+IR and GNPs+EP+IR groups can be attributed to the act of EP as a GNPs delivery system in GNPs+EP+IR group. Conclusion: Combined GNPs-EP protocol could induce synergistic effects in HT-29 cells and it can be beneficially used for the treatment of intrinsically less radiosensitive tumors.

Metabonomics of mice intestine in Codonopsis foetens induced apoptosis of intestine cancer cells

Intestinal cancer is a disease with high morbidity and high mortality in China. Previous studies have shown that Codonopsis foetens can inhibit cellular autophagy and promote the apoptosis of intestine cancer cells. Based on metabolomics method coupled with liquid chromatography-mass spectrometry (LC-MS) technology, we aimed to analyze intestinal small molecule metabolites in the intestinal cancer model group and the Codonopsis foetens treated group. Principal component analysis (PCA) and Partial Least Squares (PLS-DA) were used to identify the pattern of the data. And the metabolic characteristics of the cancer model group were explored based on the metabolic differences between the groups. Multivariate statistical analysis revealed that metabolites presented with differences included: Acetamide, Phosphoric acid, Hydrogen sulfite, Pyruvic acid, Cytosine, 2-Hydroxypyridine, Phosphoric acid, Uracil, Gamma-Aminobutyric acid, Glycerol alpha-monochlorohydrin, Thiosulfic acid, L-Valine, Cysteamine, Taurine, Creatine, Homocysteine, Hypoxanthine, Se-Methylselenocysteine, 5-Hydroxymethyluracil, Oxoglutaric acid, LysoPC(20:0), LysoPC(22:4(7Z,10Z,13Z,16Z)), LysoPC(18:2(9Z,12Z)), LysoPC(16:1(9Z)), LysoPE(0:0/16:0), LysoPE(0:0/18:2(9Z,12Z)), LysoPE(18:0/0:0), LysoPE(20:1(11Z)/0:0), etc. Combined with metabolic pathway analysis, pathways presented with differences included: Citrate cycle (TCA cycle), ABC transporters, 2-Oxocarboxylic acid metabolism, Taurine and hypotaurine metabolism, Butanoate metabolism), Phenylalanine, tyrosine and tryptophan biosynthesis, Biosynthesis of amino acids, Protein digestion and absorption, Aminoacyl-tRNA biosynthesis, C5-Branched dibasic acid metabolism, GABAergic synapse, Proximal tubule bicarbonate reclamation, Mineral absorption, Phenylalanine metabolism. The results showed that the proliferation of intestinal cancer cells caused cell metabolism disorders, manifesting as changes in metabolic pathways and resulting in changes in metabolites.

The Effect of Flubendazole on Adhesion and Migration in SW480 and SW620 Colon Cancer Cells.

Colon cancer is the most common type of gastrointestinal cancer. Despite advances during the last two decades, the efficacy of colorectal cancer treatment is still insufficient and new anticancer agents are necessary. In our study, colon cancer cells derived from a primary tumor (SW480) and lymph node metastasis (SW620) from the same patient were used and compared. The effect of flubendazole (FLU) on cell adhesion and migration was monitored using the x-CELLigence Real-Time Cell Analysis system. Expressions of molecules involved in adhesion and migration were analyzed using RT-PCR and western blot. Furthermore, RNA silencing of nuclear factor-κB in SW620 cells was used to determine the involvement of the NF-κB p65 regulation pathway in FLU action. FLU significantly suppressed the adhesion of SW480 cells and reduced the expression of adhesion markers (ICAM-1, αE-catenin; β-catenin; integrin α5 and β1). Moreover, a significant anti-migratory potential of FLU was manifested in the SW620 cells. In addition, FLU suppressed the phosphorylation of NF-κB p65 and potentiated the suppression of several metastatic markers (ICAM-1, EpCAM, integrin α5, β1, α-tubulin) caused by NF-κB p65 silencing. FLU has a significant anti-migratory effect in intestinal cancer cell SW480 and its lymph node metastatic cells SW620. FLU decreases the expression of some proteins involved in metastatic processes and inhibits activation of NF-κB p65.

A Research on the Mechanism of TH-302 on Hypoxic Condition and TH-302 Combined with Radiation Applied in Gastrointestinal Tumors

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. TH-302 mainly acts on hypoxic condition instead of normal oxygen condition. There have been some phase II clinical trials of TH-302 in soft tissue sarcoma and pancreatic cancer, it shows that TH-302 has good effect on these tumors. Radiation is less sensitive to tumor cells on hypoxic conditon. This study is to investigate the effect of TH-302 combined with radiation in gastrointestinal tumors and the mechanism of TH-302 on hypoxic condition. HCT116 cells, HT29 cells (both are intestinal cancer cells) and MKN28 cells (gastric cancer cells) are chosen for the following experiments. Firstly, 48h and 72h IC50s of TH-302 both on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) are obtained through drug cytotoxicity tests. Then tumor cells on hypoxic condition are dealt with TH-302 and radiation, CCK8 of the cells are tested. What’s more, cells are treated with TH302 (24h) on normal oxygen condition (O2%=21%) and hypoxic condition (O2%=1.3%) respectively. Cell apoptosis is examined using Flow Cytometry. At last, expression of HIF-1α protein related to hypoxia mechanism is examined through Western Blot and expression of HIF-1α gene is examined through qPCR. TH-302 is sensitive to tumor cells on hypoxic condition and has lower IC50.The 48h IC50 of TH-302 in HCT116 cells on normal oxygen and hypoxic condition is 2.8*10ˆ8nmol/L and 2,074nmol/L respectively.In HT29 and MKN28 cells the data is 118,400 nmol/L vs 40,790 nmol/L and 29,143 nmol/L vs 1,473 nmol/L.TH-302 combined with radiation on hypoxic condition has more effect than radiation and TH-302 alone. The figure will be shown in the final paper. Tumor cells dealt with TH-302 have more apoptosis on hypoxic condition than normal oxygen condition. In HCT116 +/+, HCT116 -/-, MKN28 cells, the apoptosis ratio is 11.3% vs 0.9%, 17.0% vs 4.3% and 73.8% vs 16.5%. There is not much difference of HIF-1 gene expression between hypoxia and normal oxygen condition. ON hypoxic condition, tumor cells have higher HIF-1 protein expression level. TH-302 is sensitive to tumor cells on hypoxic condition. TH-302 can be used as a radiosensitizer in gastrointestinal tumors. The mechanism of how TH-302 works on hypoxic condition has relationship with apoptosis.

Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells.

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

Abstract 3466: Growth inhibitory MEK-ERK signaling in the intestinal epithelium

Abstract Increasing evidence points to members of the PKC family of serine/threonine kinases as important tumor suppressors; however, the signaling pathways involved remain poorly defined. Our previous studies have determined that PKCα, which acts as a tumor suppressor in the epithelium of the intestine/colon, is activated coincident with growth arrest in intestinal crypts in vivo. PKCα activity induces cell cycle withdrawal in intestinal epithelial cells, a process that involves downregulation of cyclin D1 and Id1, upregulation of the cyclin dependent kinase inhibitors p21cip1 and p27kip1, and characteristic changes in pocket proteins and DNA licensing factors. Pharmacological inhibitors of the ERK pathway revealed that these changes are dependent on activation of MEK-ERK signaling, thus identifying a growth inhibitory ERK-mediated signaling axis in the intestinal epithelium. Given the interest in inhibitors of the ERK pathway for treatment of colon and other cancers, characterization of this tumor suppressive pathway will be essential for rational development of this treatment modality and to avoid and/or manage potential adverse effects of ERK inhibition in patients. To this end, the mechanistic basis for the growth suppressive effects of the PKCα→ERK module on cyclin D1, Id1 and p21cip1 are being investigated in non-transformed intestinal epithelial cells and colon cancer cells. Use of selective inhibitors and siRNA-mediated knockdown has determined that PKCα activates multiple components of the ERK pathway, including RAS, RAF, MEK and ERK. Notably, tumor suppressive effects are seen in colon cancer cells that harbor activating mutations in KRAS, indicating that growth inhibitory ERK signaling can override oncogenic signals arising from RAS mutation. PKCα signaling is associated with activation of ARAF, BRAF and CRAF and with induction of RAF heterodimers. A requirement for RAF activity was confirmed by the ability of LY3009120 or sorafenib to block growth inhibitory effects of PKCα. However, knockdown of individual RAF proteins fails to block these effects of PKCα, indicating some redundancy between the RAF proteins in mediating growth inhibitory signals. While the scaffolding protein KSR1 has been shown to be required for oncogenic RAS-ERK signaling, PKCα was still able to elicit growth inhibitory effects in KSR1 knockdown HCT-116 colon cancer cells. These data indicate that differential use of scaffolding proteins may explain both the growth inhibitory effects of PKCα-ERK signaling and their ability to override oncogenic RAS signals. A better understanding of these mechanisms may serve to guide the use of ERK inhibitors for colon cancer therapy. Supported by NIH grants DK60632, CA54807, CA157774 and CA191894. Citation Format: Navneet Kaur, Robert Lewis, Adrian Black, Jennifer Black. Growth inhibitory MEK-ERK signaling in the intestinal epithelium [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 3466.

P-14 - Effect of ginger extract on membrane potential changes and AKT activation on a peroxide-induced oxidative stress cell model

Zingiber officinale is a type of Ginger with antioxidant activity. Its use is described in cellular models such as pancreatic and intestinal cancer cell lines. However, the biochemical bases of antitumor, anti-inflammatory and antioxidant activities have not yet been fully elucidated. The aim of this study was to evaluate in H2O2-induced oxidative stress the effect of ginger extract (GE) on HT1080 cell viability, ROS production, AKT activation, and mitochondrial membrane potential. Results revealed a significant cell viability decrease at increasing GE concentrations; at 200 and 400 µg/ml GE resulted in decreased ROS production; at 200 µg/ml produced a significant increase in mitochondrial membrane potential, but no difference was observed with GE at 400 µg/ml. In untreated cells basal AKT activation was not observed. Conversely, treatment with 750 uM H2O2 led to Ser473 phosphorylation. Additionally, treatment with GE at 200 µg/ml decreased AKT activation. Reports describe GE biological activities; none the less, novel approaches to investigate intracellular changes are described in this study. In conclusion, this study characterized GE induced intracellular changes, leading to changes in mitochondrial membrane potential and signaling protein levels as AKT, reducing cell viability

Protective effect of the riboflavin-overproducing strain Lactobacillus plantarum CRL2130 on intestinal mucositis in mice.

Intestinal mucositis (IM) is a local inflammatory response that causes alterations of the intestinal structure that in turn affect nutrient absorption and a side effect that is commonly associated with cancer treatments. Lactobacillus plantarum CRL2130 is a riboflavin-overproducing strain that has previously been shown to provide antiinflammatory properties. The objective of this study was to evaluate the effects of this riboflavin-producing strain in a chemically induced murine mucositis model. Mucositis was induced by daily injections of 5-fluororacil (5-FU) after which mice were either given L. plantarum CRL2130, CRL725 (strain from which CRL2130 was derived that does not overproduce riboflavin), or commercial riboflavin twice daily during 6 d of chemotherapy agent injections. The effect of the strains and riboflavin was also evaluated in vitro using Caco-2 intestinal cancer cell cultures to determine if they interfere with 5-FU's anticancer activity. The administration of L. plantarum CRL2130 significantly attenuated the pathologic changes induced by 5-FU in mice such as body weight loss, diarrhea, shortening of villus height, increases in proinflammatory cytokine concentrations, and elevated production of interleukin 10. In vitro assays using Caco-2 cells showed that the effectiveness of 5-FU was not affected by L. plantarum CRL2130 and that this strain exerted an inhibitory mechanism against oxidative stress. These results indicate that the riboflavin-overproducing strain L. plantarum CRL2130 could be useful to prevent mucositis during cancer treatments and would not affect the primary treatment.

Metabolomics assisted fingerprint of Hypericum perforatum chemotypes and assessment of their cytotoxic activity

Hypericum perforatum is known as an important medicinal plant, used for the treatment of several diseases, while its pharmacological properties are attributed to the presence of a wide range of secondary metabolites. Due to the great chemotypic variability of Hypericum species in the nature, and the demand for standardized herbal products, a detailed phytochemical investigation was carried out on different parts (herba, leaf, flowers) from wild collected and cultivated populations, using advanced chromatographic tools. Liquid Chromatographic analysis (LC-MS/MS MRM) revealed significant variability in the secondary metabolites content of the examined methanolic extracts. The most common derivatives belong to 9 groups i.e. benzoic acids, phenylpropanoids, coumarins, flavones, flavonols, flavan-3-ols, anthocyanins, phloroglucinols and naphtodianthrones. The main polyphenolic compounds were catechin, epicatechin, quercetin, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, neochlorogenic acid, proanthocyanidins (A and B series) and cyanidin-3-O-glucoside. In addition, the content of the characteristic compounds hypericin and hyperforin in herba crude extracts ranged between 0.5 and 1.7 mg/g and 0.6–3.3 mg/g respectively. The cytotoxic activity of the crude extracts was assessed at concentrations ranged between 0.01 and 100 μg/mL, on Caco-2 intestinal cancer cell cultures, and a cytotoxic behavior was shown only at the highest concentration of 100 μg/mL.

The estrogenic hormone effect in gastric cancer.

85 Background: Estrogen receptors (ERs) are steroid hormone receptors that regulate cellular activities in many physiological and pathological processes in different tissues. A few of studies have examined the expression of ER in gastric cancer. However, considerable controversy is raised as to the expression level of ER and its prognostic value in gastric cancer. In the present study, the expression profile of ERα, ERβ was determined in gastric cancer cell lines according Lauren classification and evaluate that the treatment effect of selective estrogen receptor modulator. Methods: Using four cell lines established from human gastric carcinomas according Lauren classification, check endogenous ERα, ERβ expression levels with RT-PCR. The SERM treatment effect were detected MTT test. Using immunohistochemical detection, the present study analyzed the clinical relevance of ERα, ERβ expression in tumor cells in 197 patients who underwent curative radical surgery and who were observed on long-term follow-up. Results: Endogenous ERα was high expression not intestinal cancer cell lines but in diffuse cancer cell line. Endogenous ERβ was high expression both type cancer cell line than normal gastrointestinal cell lines. According MTT assay, only raloxifene among SERM was significant treatment effect. In immonohistochemial study of gastric tissue, ERα negative and ERβ positive was associated with good prognosis. Conclusions: ERβ may be partly involved in gastric carcinogenesis and ERβ antagonist might be new therapeutic drug for gastric cancer.

Stem Cells in Intestinal Lining May Predict Cancer Cell Behavior

Stem Cells in Intestinal Lining May Predict Cancer Cell Behavior

Exopolysaccharides from Lactobacillus plantarum NCU116 induce c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in mouse intestinal epithelial cancer cells

Exopolysaccharides (EPS) from lactic acid bacteria (LAB) have been reported to play vital parts in the modulation of cell-cycle and apoptosis in cancer cells. However, the mechanisms by which EPS regulate the proliferation and apoptosis of cancer cells remain incompletely understood. We thus used different cancer cells to evaluate the anticancer ability and to investigate the underlying molecular mechanism of EPS from Lactobacillus plantarum NCU116 (EPS116). Our studies showed that EPS116 inhibited the proliferation of cancer cells in a cell type manner, and remarkably repressed the growth and survival of CT26 through induction of apoptosis. Moreover, EPS116 increased the expression of pro-apoptotic genes, including Fas, Fasl and c-Jun, induced the phosphorylation of c-Jun in CT26 cells. Furthermore, TLR2 (Toll like receptor 2) was upregulated by EPS116, and the CT26 cells with TLR2 knockdown were found to be insensitive to EPS116, suggesting that the anti-cancer activity of EPS116 may be TLR2-dependent. Taken together, the suppressive efficacy of EPS116 on the proliferation of CT26 cells may be mediated via TLR2 and the activation of c-Jun dependent Fas/Fasl-mediated apoptotic pathway. Our study has, for the first time, shown that EPS from LAB induced c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in CT26 cells.

Effect of ginger extract on membrane potential changes and AKT activation on a peroxide-induced oxidative stress cell model

Zingiber officinale is a type of Ginger used for the treatment of reactive oxygen species (ROS) associated diseases. Reports describe its use in cellular models, such as pancreatic and intestinal cancer cell lines. However, the biochemical bases of antitumor, anti-inflammatory and antioxidant activities have not yet been fully elucidated. The aim of this study was to evaluate in H2O2-induced oxidative stress the effect of ginger extract (GE) on HT1080 cell viability, ROS production, AKT activation, and mitochondrial membrane potential (ΔΨm). ROS production was measured by DHE probe. Results revealed a significant cell viability decrease with increasing GE concentrations. In addition, GE at 200μg/ml and 400μg/ml resulted in decreased ROS production compared with controls. Moreover, GE at 200μg/ml concentration produced a significant increase in ΔΨm. In contrast, no difference in ΔΨm was observed compared to controls with GE at 400μg/ml. In untreated HT1080 cells basal AKT activation was not observed. Conversely, treatment with 750μM H2O2 led to Ser473 phosphorylation. Additionally, treatment with GE at 200μg/ml decreased AKT activation. Reports in the literature describe GE biological activities; none the less, novel approaches to investigate intracellular changes resulting from GEs are described in this study. In conclusion, this study characterized GE induced intracellular changes, leading to changes in ΔΨm and signaling protein levels, such as AKT, and reduced cell viability.

The effects of β-caryophyllene oxide and trans-nerolidol on the efficacy of doxorubicin in breast cancer cells and breast tumor-bearing mice

BackgroundOne approach to improve effect of chemotherapy is combination of classical cytostatic drugs with natural compounds, e. g. sesquiterpenes. In our previous study, sesquiterpenes β-caryophyllene oxide (CAO) and trans-nerolidol (NER) improved the anti-proliferative effect of doxorubicin (DOX) in intestinal cancer cell lines. PurposeThe present study was designed to evaluate the effect of CAO and NER on DOX efficacy, focusing on cell proliferation, migration, apoptosis and DOX accumulation in breast cancer cells MDA-MB-231 and MCF7 in vitro and in mice bearing solid Ehrlich tumors (EST) in vivo. MethodsThe impact of cytotoxic effect was assessed by the neutral red uptake test. The ability to migrate was tested using real-time measurement in x-CELLigence system. Expressions of molecules were examined using western blot analysis. The accumulation of DOX inside the cells using time lapse microscopy was observed. The mice with inoculated EST cells were treated repeatedly with DOX and DOX+CAO or DOX+NER and the growth of tumors were monitored. DOX concentrations in plasma and tumor were assayed using HPLC. ResultsIn MDA-MB-231, combination of DOX with CAO enhanced anti-proliferative effect and acted strongly synergistic. NER increased accumulation of DOX inside the cells; moreover combination DOX with NER suppressed migration ability in vitro. In vivo, apoptosis was activated especially in group treated with DOX and CAO. However, none of tested sesquiterpenes was able to improve DOX accumulation in tumors and DOX-mediated inhibition of tumor growth. ConclusionIn conclusion, sesquiterpenes CAO and NER increased the efficacy of DOX in breast cancer cells in vitro, but did not improve its effect in vivo, in Ehrlich solid tumor bearing mice.

Activation of Epithelial Signal Transducer and Activator of Transcription 1 by Interleukin 28 Controls Mucosal Healing in Mice With Colitis and Is Increased in Mucosa of Patients With Inflammatory Bowel Disease

We investigated the roles of interleukin 28A (also called IL28A or interferon λ2) in intestinal epithelial cell (IEC) activation, studying its effects in mouse models of inflammatory bowel diseases (IBD) and intestinal mucosal healing. Colitis was induced in C57BL/6JCrl mice (controls), mice with IEC-specific disruption of Stat1 (Stat1IEC-KO), mice with disruption of the interferon λ receptor 1 gene (Il28ra-/-), and mice with disruption of the interferon regulatory factor 3 gene (Irf3-/-), with or without disruption of Irf7 (Irf7-/-). We used high-resolution mini-endoscopy and invivo imaging methods to assess colitis progression. We used 3-dimensional small intestine and colon organoids, along with RNA-Seq and gene ontology methods, to characterize the effects of IL28 on primary IECs. We studied the effects of IL28 on the human intestinal cancer cell line Caco-2 in a wound-healing assay, and in mice colon wounds. Colonic biopsies and resected tissue from patients with IBD (n= 62) and patients without colon inflammation (controls, n= 23) were analyzed by quantitative polymerase chain rection to measure expression of IL28A, IL28RA, and other related cytokines; biopsy samples were also analyzed by immunofluorescence to identify sources of IL28 production. IECs were isolated from patient tissues and incubated with IL28; signal transducer and activator of transcription 1 (STAT1) phosphorylation was measured by immunoblots and confocal imaging. Lamina propria cells in colon tissues of patients with IBD, and mice with colitis, had increased expression of IL28 compared with controls; levels of IL28R were increased in the colonic epithelium of patients with IBD and mice with colitis. Administration of IL28 induced phosphorylation of STAT1 inprimary human and mouse IECs, increasing with dose. Il28ra-/-, Irf3-/-, Irf3-/-Irf7-/-, as well as Stat1IEC-KO mice, developed more severe colitis after administration of dextran sulfate sodium than control mice, with reduced epithelial restitution. Il28ra-/- and Stat1IEC-KO mice also developed more severe colitis in response to oxazolone than control mice. We found IL28 to induce phosphorylation (activation) of STAT1 in epithelial cells, leading to their proliferation in organoid culture. Administration of IL28 to mice with induced colonic wounds promoted mucosal healing. IL28 controls proliferation of IECs in mice with colitis and accelerates mucosal healing by activating STAT1. IL28 might be developed as a therapeutic agent for patients with IBD.

Bifunctional Succinylated ε-Polylysine-Coated Mesoporous Silica Nanoparticles for pH-Responsive and Intracellular Drug Delivery Targeting the Colon.

Conventional oral drug formulations for colonic diseases require the administration of high doses of drug to achieve effective drug concentrations at the target site. However, this exposes patients to serious systemic toxicity in order to achieve efficacy. To overcome this problem, an oral drug delivery system was developed by loading a large amount (ca. 34% w/w) of prednisolone into 3-aminopropyl-functionalized mesoporous silica nanoparticles (MCM-NH2) and targeting prednisolone release to the colon by coating the nanoparticle with succinylated ε-polylysine (SPL). We demonstrate for the first time the pH-responsive ability of SPL as a "nanogate" to selectively release prednisolone in the pH conditions of the colon (pH 5.5-7.4) but not in the more acidic conditions of the stomach (pH 1.9) or small intestine (pH 5.0). In addition to targeting drug delivery to the colon, we explored whether the nanoparticles could deliver cargo intracellularly to immune cells (RAW 264.7 macrophages) and intestinal epithelial cells (LS 174T and Caco-2 adenocarcinoma cell lines). To trace uptake, MCM-NH2 were loaded with a cell membrane-impermeable dye, sulforhodamine B. The SPL-coated nanoparticles were able to deliver the dye intracellularly to RAW 264.7 macrophages and the intestinal epithelial cancer cells, which offers a highly promising and novel drug delivery system for diseases of the colon such as inflammatory bowel disease and colorectal cancer.

Gold nanoparticles and electroporation impose both separate and synergistic radiosensitizing effects in HT-29 tumor cells: an in vitro study.

Background and objectiveRadiation therapy (RT) is the gold standard treatment for more than half of known tumors. Despite recent improvements in RT efficiency, the side effects of ionizing radiation (IR) in normal tissues are a dose-limiting factor that restricts higher doses in tumor treatment. One approach to enhance the efficiency of RT is the application of radiosensitizers to selectively increase the dose at the tumor site. Gold nanoparticles (GNPs) and electroporation (EP) have shown good potential as radiosensitizers for RT. This study aims to investigate the sensitizing effects of EP, GNPs, and combined GNPs-EP on the dose enhancement factor (DEF) for 6 MV photon energy.MethodsRadiosensitizing effects of EP, GNPs, and combinations of GNPs-EP were comparatively investigated in vitro for intestinal colon cancer (HT-29) and Chinese hamster ovary (CHO) cell lines by MTT assay and colony formation assay at 6 MV photon energy in six groups: IR (control group), GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR.ResultsTreatment of both cell lines with EP, GNPs, and combined GNPs-EP significantly enhanced the response of cells to irradiation. However, the HT-29 showed higher DEF values for all groups. In addition, the DEF value for HT-29 cells for GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR was, respectively, 1.17, 1.47, 1.36, 2.61, and 2.89, indicating synergistic radiosensitizing effect for the GNPs (24 h)+EP+IR group. Furthermore, the synergistic effect was observed just for HT-29 tumor cell lines.ConclusionCombined GNPs-EP protocols induced synergistic radiosensitizing effect in HT-29 cells, and the effect is also tumor specific. This combined therapy can be beneficially used for the treatment of intrinsically less radiosensitive tumors.

TFF3-dependent resistance of human colorectal adenocarcinoma cells HT-29/B6 to apoptosis is mediated by miR-491-5p regulation of lncRNA PRINS

Tumour necrosis factor-α (TNF-α) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-α. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-α/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue repair but is also involved in intestinal tumour progression and scattering. We hypothesised that TFF3 rescues intestinal epithelial cancer cells from TNF-α-induced apoptosis by involving regulatory RNA networks. In silico-based expression analysis revealed TFF3-mediated regulation of selected microRNAs as well as long non-coding RNAs (lncRNAs), whereas miR-491-5p was identified to target the lncRNA ‘psoriasis susceptibility-related RNA gene induced by stress’ (PRINS). RNA interference-based gain- and loss-of-function experiments examined miR-491-PRINS axis to exert the TFF3-mediated phenotype. Chemical inhibition of selected pathways showed that phosphatidylinositol 3-kinase/AKT accounts for TFF3-mediated downregulation of miR-491-5p and accumulation of PRINS. Moreover, we showed that PRINS colocalises with PMAIP1 (NOXA) in nuclei of HT-29/B6 possessing inhibitory effects. Immunoprecipitation experiments proved molecular interaction of PMAIP1 with PRINS. Our study provides an insight into RNA regulatory networks that determine resistance of colorectal cancer cells to apoptosis.

Paradoxical Roles of Elongation Factor-2 Kinase in Stem Cell Survival

Protein synthesis inhibition is an immediate response during stress to switch the composition of protein pool in order to adapt to the new environment. It was reported that this response could be either protective or deleterious. However, how cells choose to live or die upon protein synthesis inhibition is largely unknown. Previously, we have shown that elongation factor-2 kinase (eEF2K), a protein kinase that suppresses protein synthesis during elongation phase, is a positive regulator of apoptosis both in vivo and in vitro Consistently, here we report that knock-out of eEF2K protects mice from a lethal dose of whole-body ionizing radiation at 8 Gy by reducing apoptosis levels in both bone marrow and gastrointestinal tracts. Surprisingly, similar to the loss of p53, eEF2K deficiency results in more severe damage to the gastrointestinal tract at 20 Gy with the increased mitotic cell death in small intestinal stem cells. Furthermore, using epithelial cell lines, we showed that eEF2K is required for G2/M arrest induced by radiation to prevent mitotic catastrophe in a p53-independent manner. Specifically, we observed the elevation of Akt/ERK activity as well as the reduction of p21 expression in Eef2k(-/-) cells. Therefore, eEF2K also provides a protective strategy to maintain genomic integrity by arresting cell cycle in response to stress. Our results suggest that protective versus pro-apoptotic roles of eEF2K depend on the type of cells: eEF2K is protective in highly proliferative cells, such as small intestinal stem cells and cancer cells, which are more susceptible to mitotic catastrophe.

Interference with mutagenic aflatoxin B1-induced checkpoints through antagonistic action of ochratoxin A in intestinal cancer cells: a molecular explanation on potential risk of crosstalk between carcinogens.

Foodborne aflatoxin B1 (AFB1) and ochratoxin A (OTA) cause genotoxic injury and subsequent tumor formation. As a biomarker of oncogenic stimulation by genotoxic mycotoxins, p53-triggered Mdm2 was assessed in intestinal cancer cells. AFB1 increased Mdm2 reporter expression in a dose-dependent manner. However, this was strongly antagonized by OTA treatment. As a positive transcription factor of Mdm2 expression, p53 levels were also increased by AFB1 alone and reduced by OTA. With marginal cell death responses, AFB1 induced p53-mediated S phase arrest and cell cycle-regulating target genes, which was completely suppressed by OTA. Although enterocyte-dominant CYP3A5 counteracted AFB1-induced DNA damage, expression of CYP3A5 was decreased by OTA or AFB1. Instead, OTA enhanced expression of another metabolic inactivating enzyme CYP3A4, attenuation of formation of AFB1-DNA adduct and p53-mediated cell cycle checking responses to the mutagens. Finally, the growth of intestinal cancer cells exposed to the mycotoxin mixture significantly exceeded the expected growth calculated from that of cells treated with each mycotoxin. Although AFB1-induced mutagen formation was decreased by OTA, interference with checkpoints through antagonistic action of OTA may contribute to the survival of tumor cells with deleterious mutations by genotoxic mycotoxins, potently increasing the risk of carcinogenesis.