Colon Carcinoma Cell Line HT-29 Research Articles

Attenuating Colorectal Cancer Using Nine Cultivars of Australian Lupin Seeds: Apoptosis Induction Triggered by Mitochondrial Reactive Oxygen Species Generation and Caspases-3/7 Activation.

As Australian lupin cultivars are rich sources of polyphenols, dietary fibers, high-quality proteins, and abundant bioactive compounds with significant antioxidant, antidiabetic, and anticancer activities, this research work is aimed at investigating the colon cancer alleviation activity of nine cultivars of lupin seeds on HCT116 and HT29 colon carcinoma cell lines through anti-proliferation assay, measurement of apoptosis, and identification of the mechanism of apoptosis. Nine cultivars were pre-screened for anti-proliferation of HCT116 and HT29 cells along with consideration of the impact of heat processing on cancer cell viability. Mandelup and Jurien showed significant inhibition of HCT116 cells, whereas the highest inhibition of HT29 cell proliferation was attained by Jurien and Mandelup. Processing decreased the anti-proliferation activity drastically. Lupin cultivars Mandelup, Barlock, and Jurien (dose: 300 μg/mL) induced early and late apoptosis of colon cancer cells in Annexin V-FITC assay. The mechanism of apoptosis was explored, which involves boosting of caspases-3/7 activation and intracellular reactive oxygen species (ROS) generation in HCT116 cells (Mandelup and Barlock) and HT29 cells (Jurien and Mandelup). Thus, the findings showed that lupin cultivars arrest cell cycles by inducing apoptosis of colorectal carcinoma cells triggered by elevated ROS generation and caspases-3/7 activation.

Synthesis, characterization, and biological properties of novel Schiff bases containing pentafluorophenyl hydrazine.

In the present study, new Schiff bases derived from pentafluorophenyl-hydrazine (L1, L2, L3, L4) were synthesized and their structures were characterized by 1 H nuclear magnetic resonance spectroscopy (NMR), 13 C NMR, 19 F NMR, fourier transform infrared spectroscopy, and elemental analysis methods. Then, the anticancer activities of the obtained compounds were investigated using three human cancer cell lines (A2780, over; Caco-2, colon; and HT-29 colon carcinoma cell lines). According to the obtained cytotoxicity results, compound number L4 was found to have the highest anticancer activity in A2780 (over) and Caco-2 (colon) cell lines. Furthermore, in silico, ADMET properties, where studies play an important role in the development and prediction of drug compounds, were calculated using web-based platforms. In addition, molecular docking studies were performed to evaluate the binding interactions between the synthesized pentafluorophenyl-hydrazone compounds and the MDM2 protein (4JSC). Both in vitro and in silico results showed that the synthesized compounds could act as potent anticancer agents.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells

BackgroundSurface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA.ResultsC. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-β as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05).ConclusionThe results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Evaluation of Radiosensitive Effect of Tolmetin in Radiotherapy on Human Colonic Carcinoma Cell Line HT-29

Background: Radiotherapy is one of the cancer treatment methods. Although radioresistance and normal tissue toxicity limit the radiation therapy in certain anatomical locations, using some substances can be useful to increase radiosensitivity on cancer cells without cytotoxicity effect on normal cells. Objective: This study aimed to evaluate the radiosensitizing effect of tolmetin in radiotherapy treatment on human colon cancer cell line HT-29. Methods In this study, human colon cancer cells HT-29 in different groups were irradiated with 4 Gy x-ray, and tolmetin was administered in different concentrations (75, 100, and 150 μM). Then, the groups were compared with each other and with the control group by Micronucleus Assay (MN) and Nuclear Division Index (NDI). NDI investigated the cytotoxicity, and MN indicated the genotoxicity. Results: In the group receiving radiation, micronuclei increased significantly compared to the control group. In the group receiving tolmetin with a concentration of 75 and 100 μM, the number of micronuclei also increased compared to the control group. In all groups treated with tolmetin that also received radiation, a significant increase in micronuclei was observed, which was more noticeable at concentrations of 100 and 150 μM. At the same time, tolmetin at the studied concentrations did not change the NDI index. Conclusion: The present study demonstrates that tolmetin has a radiosensitizing effect on HT-29 colon cancer cells, which depends on the tolmetin concentration. In addition, tolmetin has no cytotoxic effect on this cell line.

Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells

Photodynamic therapy (PDT), a highly targeted therapy with acceptable side effects, has emerged as a promising therapeutic option in oncologic pathology. One of the issues that needs to be addressed is related to the complex network of cellular responses developed by tumor cells in response to PDT. In this context, this study aims to characterize in vitro the stressors and the corresponding cellular responses triggered by PDT in the human colon carcinoma HT29 cell line, using a new asymmetric porphyrin derivative (P2.2) as a photosensitizer. Besides investigating the ability of P2.2-PDT to reduce the number of viable tumor cells at various P2.2 concentrations and fluences of the activating light, we assessed, using qRT-PCR, the expression levels of 84 genes critically involved in the stress response of PDT-treated cells. Results showed a fluence-dependent decrease of viable tumor cells at 24 h post-PDT, with few cells that seem to escape from PDT. We highlighted following P2.2-PDT the concomitant activation of particular cellular responses to oxidative stress, hypoxia, DNA damage and unfolded protein responses and inflammation. A web of inter-connected stressors was induced by P2.2-PDT, which underlies cell death but also elicits protective mechanisms that may delay tumor cell death or even defend these cells against the deleterious effects of PDT.

Fluorescent spherical mesoporous silica nanoparticles loaded with emodin: Synthesis, cellular uptake and anticancer activity

The natural product emodin (EO) exhibits anti-inflammatory, antiangiogenesis and antineoplastic properties in vitro and in vivo. Due to its biological properties as well as its fluorescence, EO can be useful in pharmacology and pharmacokinetics. To enhance its selectivity to cancer cells, EO was loaded into non-fluorescent and novel fluorescent spherical mesoporous nanoparticles bearing N-methyl isatoic anhydride (SNM~M) or lissamine rhodamine B sulfonyl moieties (SNM~L). The propylamine functionalized mesoporous silica nanomaterial (SNM) were characterized by powder X-ray diffraction (XRD), nitrogen gas sorption, scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence spectroscopy, thermogravimetric analysis (TGA) and UV spectroscopy. The cytotoxicity of EO-loaded nanoparticles was tested against the human colon carcinoma cell line HT-29. Non-loaded SNM did not affect cell proliferation, whereas those loaded with EO were at least as efficient as EO alone. It could be shown by fluorescence microscopy that the uptake of silica nanomaterial by the tumor cells occurred within 2 h and the release of EO occurred within 48 h of treatment. Flow cytometry and Western blot analysis showed that SNM containing EO induced apoptosis in HT-29 cells.

Combined Targeted Proteomics and Oxylipin Metabolomics for Monitoring of the COX-2 Pathway.

The important role of inducible cyclooxygenase-2 (COX-2) in several diseases necessitates analytical tools enabling thorough understanding of its modulation. Analysis of a comprehensive oxylipin pattern provides detailed information about changes in enzyme activities. In order to simultaneously monitor gene expression levels, a targeted proteomics method for human COX-2 is developed. With limits of detection and quantification down to 0.25 and 0.5 fmol (on column) the method enables sensitive quantitative analysis via LC-MS/MS within a linear range up to 2.5pmol. Three housekeeping proteins are included in the method for data normalization. A tiered approach for method development comprised of in silico and experimental steps is described for choosing unique peptides and selective and sensitive SRM transitions while avoiding isobaric interferences. This method combined with a well-established targeted oxylipin metabolomics method allows to investigate the role of COX-2 in the human colon carcinoma cell lines HCT-116, HT-29, and HCA-7. Moreover, the developed methodology is used to demonstrate the time-dependent prostanoid formation and COX-2 enzyme synthesis in lipopolysaccharide-stimulated human primary macrophages. The described approach is a helpful tool which will be further used as standard operation procedure, ultimately aiming at comprehensive targeted proteomics/oxylipin metabolomics strategies to examine the entire arachidonic acid cascade.

Trichothecenes from an Endophytic Fungus Alternaria sp. sb23.

Three new (alterchothecenes A - C, 1: -3: ) and 3 known (4: -6: ) trichothecenes, along with 9 known compounds (7: -15: ), were isolated from the culture of Alternaria sp. sb23, an endophytic fungus separated from the root of Schisandra sphenanthera Rehd. et Wils. Their structures were elucidated by spectroscopic analyses, and the absolute configurations of 1: -3: were determined through comparison of the experimental electronic circular dichroism (ECD) spectra and optical rotations with similar analogues. In vitro cytotoxicity tests of compounds 1: -6: against human HT-29 colon carcinoma and human MCF-7 breast cancer cell lines indicated that 4: -6: exhibited significant cytotoxic effects, with IC50 values ranging from 0.89 to 9.38 µM. And the potential of compounds 1: -6: as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) sensitizers in HT-29 cells was evaluated. The results revealed that combination treatment of TRAIL with compounds 1: -6: synergistically decreased cell viability compared with the sole treatment with those compounds.

Transition metal complexes of α-aminophosphonates part II: Synthesis, spectroscopic characterization, and in vitro anticancer activity of copper(II) complexes of α-aminophosphonates

A one pot procedure was used to synthesize two new derivatives of α-aminophosphonates. Novel copper(II) complexes of α-aminophosphonates were synthesized by coordinating different copper salts with the newly synthesized α-aminophosphonates. Their structures were characterized by different spectral and analytical techniques. Evaluation of the metal-free ligands HL1, HL2, and their Cu(II) complexes against human colon carcinoma HT-29 cell lines was performed, using cisplatin as a reference drug. The results indicated that the complexes of the ligand HL1 exhibited enhanced anticancer activity, while ligand HL2 complexes showed decreased anticancer activity.

Effect of Pectin on the Expression of Proteins Associated with Mitochondrial Biogenesis and Cell Senescence in HT29-Human Colorectal Adenocarcinoma Cells.

Mitochondria dynamic is regulated by different proteins, maintaining a balance between fission and fusion. An imbalance towards mitochondrial fission has been associated with tumor cell proliferation. The aim of this study was to analyze whether pectin modifies the viability of human colon cancer cells and the expression of proteins involved in mitochondrial fusion and fission. The human colon carcinoma cell line HT29 cells was growth in 10% fetal bovine serum in the absence and presence of pectin. Pectin reduced HT29 cell viability in a concentration-dependent manner, reaching a plateau at 150~300 μmol/L pectin. The presence of 200 μmol/L pectin reduced the expression of dynamin-related protein-1 and increased expression of the mitochondrial fusion-associated proteins mitofusin-1 and 2. Expression of cyclin B1, a protein involved in G2/M transition, was found decreased in pectin-incubated HT29 cells. Moreover, expression of p53 protein, the amount of p53 in the nucleous and β-galactosidase activity, which are all biomarkers for cellular senescence, were significantly higher in pectin-incubated HT29 cells than in HT29 cells incubated without pectin. Expression of the protein B-cell lymphoma 2 (Bcl-2) homologous antagonist/killer was increased in response to incubation with pectin. However, incubation with pectin did not affect expression of Bcl-2-associated X protein or Bcl-2, or the caspase-3 activity. Overall, we concluded that pectin reduces the viability of human HT29 colon cancer cells, which is accompanied with a shift in the expression of proteins associated with mitochondrial dynamics towards mitochondrial fusion. Moreover, incubation with pectin favors cellular senescence over apoptosis in HT29 cells.

Potent antitumor activity of Laccaic acid and Phenethyl isothiocyanate combination in colorectal cancer via dual inhibition of DNA methyltransferase-1 and Histone deacetylase-1

Despite an incessant advancement in the treatment of Colorectal Cancer (CRC), 40% of individuals suffer from disease relapse which marks the need for new treatment options. In this study we report synergistic antitumor activity of Laccaic acid (LA) and Phenethyl isothiocyanate (PEITC) combination in colorectal cancer via dual inhibition of DNA methyltransferase-1 and Histone deacetylase-1. Efficacy of combination was evaluated in both in-vitro and in-vivo experiments using human colon carcinoma cell line HT29 and 1,2-dimethyl hydrazine induced colon cancer rat model, respectively. In the cell line studies treatment with combination showed reduced cell viability with apoptotic cell death compared to individual treatment groups which showed necrotic cell death. IC50 value for combination (0.478 μM) was much lower than LA (6.08 μM), PEITC (11.88 μM) and 5-Fluorouracil (9.88 μM). In the in-vivo study combination group significantly attenuated number of aberrant crypt foci, fecal consistency score, IL-6, TNF-α, DNMT1 and HDAC1 levels, histological findings and mortality as compared to negative control. Further, toxic effects produced by combination were significantly less. Results suggest potent synergistic efficacy of Laccaic acid and Phenethyl isothiocyanate combination in colorectal cancer.

A study of the impact of inhibitors of DNA binding-1 on proliferation and migration in human colon carcinoma cells.

This study aims to explore the effect of an inhibitor of DNA binding-1 (Id-1) on the proliferation and migration of human colon carcinoma cell line SW480 and HT-29. SW480 and HT-29 cells transfected with Id-1-interference sequence were assigned to the experimental groups (inhibition groups 1 and 2), and SW480 and HT-29 cells with blank interference sequence (blank groups) and blank load transfection (blank load groups) were assigned as the control groups. The expression of Id-1 in six groups was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation in vitro was assessed by MTT assay. RT-PCR and Western blot results demonstrated that the mRNA and protein expressions of Id-1 in the inhibition group 1 were lower than those in the blank group 1 and blank load group 1. RT-PCR and Western blot results revealed that the mRNA and protein expressions of Id-1 were lower in the inhibition group 2 than in the blank group 2 and blank load group 2. The results of the growth curve revealed that proliferation ability was significantly weaker from the third day in the inhibition groups 1 and 2 than in the blank group and blank load group. Transwell chamber experiment and Matrigel invasion assay revealed that the number of Transwell cells significantly decreased in the inhibition groups 1 and 2 than in the blank groups and blank load groups (P < 0.01). Id-1 significantly promotes the proliferation and migration of human colon carcinoma cell lines SW480 and HT-29.

Antiproliferative and Cytotoxic Activity of Xanthohumol and Its Non-Estrogenic Derivatives in Colon and Hepatocellular Carcinoma Cell Lines.

Xanthohumol (XN), a prenylated flavonoid found in hops, inhibits growth in a variety of cancer cell lines; however, its use raises concerns as gut microbiota and the host’s hepatic cytochrome P450 enzymes metabolize it into the most potent phytoestrogen known, 8-prenylnaringenin (8-PN). The XN derivatives dihydroxanthohumol (DXN) and tetrahydroxanthohumol (TXN) are not metabolized into 8-PN and they show higher tissue concentrations in vivo compared with XN when orally administered to mice at the same dose. Here we show that DXN and TXN possess improved anti-proliferative activity compared with XN in two colon (HCT116, HT29) and two hepatocellular (HepG2, Huh7) carcinoma cell lines, as indicated by their respective IC50 values. Furthermore, XN, DXN, and TXN induce extensive apoptosis in all these carcinoma cell lines. Finally, TXN induces G0/G1 cell cycle arrest in the colon carcinoma cell line HT29. Our findings suggest that DXN and TXN could show promise as therapeutic agents against colorectal and liver cancer in preclinical studies without the drawback of metabolism into a phytoestrogen.

Indonesian Pasuruan propolis extract does not exert antiproliferation and pro-apoptotic effect on human colon carcinoma cell line HT-29

Purpose : To evaluate the anti-cancer activity of Indonesian Pasuruan propolis extract (PPE) against HT-29 human colon carcinoma cell line. Methods : HT-29 cells were cultured and treated in different concentrations of PPE (50, 100, 200, 400 μg/mL) for 24 h. The cells were evaluated by several indicators such as cell proliferation and apoptosis as well as the expression of protein Ki67, p53, cyclin D1, and Bcl-xL. Results : Administration of PPE inhibited cell proliferation of HT-29 in concentration-dependent manner but the effect was not significant (p = 0.842); the results were similar with regard to the expression of Ki67 in HT-29 cells (p = 0.953). Administration of 400 μg/mL PPE insignificantly decreased cyclin D1 expression (p = 0.149). The concentration of PPE at 50, 100, and 200 μg/mL induced cell apoptosis of HT-29 cells initially, but the level of apoptosis subsequently decreased (p = 0.416). Furthermore, the expressions of p53 and Bcl-xL decreased following treatment with PPE at 50, 100 and 200 μg/mL but increased for the PPE 400 μg/mL group (p = 0.000). Conclusion : PPE reduced the expressions of p53, Ki67, cyclin D1, and Bcl-xL insignificantly as it it generally failed inhibit cell proliferation and promote cell apoptosis of HT-29 cells. Keywords : Bcl-xL, Colon cancer, Cyclin D1, Human colon adenocarcinoma cell line HT-29, Proliferation marker Ki67, Tumor protein p53

Interaction of STAT3 and RelB modulates MMP-1 in colon cancer

BackgroundMMP-1 (Matrix metalloproteinase-1) promotes carcinogenesis and distant metastasis in different cancers. Regulation of MMP-1 could occur at multiple levels: epigenetically, post-transcriptionally, or post-translationally. An increasing body of evidence supports that the cytoplasmic transcription factor STAT3 (signal transducer and activator of transcription 3) is activated constitutively in a variety of cancers wherein it significantly affects the growth of tumors and also facilitates metastasis. In addition, STAT3 has been found to regulate nuclear activity pro-inflammatory transcriptional factor, NF-κB signaling, especially, the alternative one (RelB/p100) by directly interacting with them Method and ResultsIn this proof of concept study, we tested the hypothesis that STAT3 interacts with RelB to promote tumor invasion by positively regulating MMP-1 in colon cancer. We found that RelB and STAT3 were constitutively localized in the nucleus of colon cancer in surgically-resected specimens with use of Western blot analysis, which was further confirmed by immunofluorescence (IF) staining in colon carcinoma cell line HT29. We further observed that STAT3/RelB knockdown resulted in reduced MMP-1. Our results from chromatin immunoprecipitation studies further established that association between RelB and MMP-1 promoter decreased when STAT3 was depleted, and conversely, STAT3 association with MMP-1 decreased with the knockdown of RelB. ConclusionThese results suggest that STAT3 and ReB constitute a minimal activator complex for positive regulation of MMP-1 in colon cancer

Revisiting the thiosemicarbazonecopper(II) reaction with glutathione. Activity against colorectal carcinoma cell lines

Thiosemicarbazones (TSCs), and their copper derivatives, have been extensively studied mainly due to the potential applications as antitumor compounds. A part of the biological activity of the TSC-CuII complexes rests on their reactivity against cell reductants, as glutathione (GSH). The present paper describes the structure of the [Cu(PTSC)(ONO2)]n compound (1) (HPTSC=pyridine-2-carbaldehyde thiosemicarbazone) and its spectroscopic and magnetic properties. ESI studies performed on the reaction of GSH with 1 and the analogous [{Cu(PTSC*)(ONO2)}2] derivative (2, HPTSC*=pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) show the absence of peaks related with TSC-Cu-GSH species. However GSH-Cu ones are detected, in good agreement with the release of CuI ions after reduction in the experimental conditions. The reactivity of 1 and 2 with cytochrome c and myoglobin and their activities against HT-29 and SW-480 colon carcinoma cell lines are compared with those shown by the free HPTSC and HPTSC* ligands.

Cytotoxicity of portoamides in human cancer cells and analysis of the molecular mechanisms of action.

Portoamides are cyclic peptides produced and released by the cyanobacterial strain Phormidium sp. presumably to interfere with other organisms in their ecosystems ("allelopathy"). Portoamides were previously demonstrated to have an antiproliferative effect on human lung carcinoma cells, but the underlying mechanism of this activity has not been described. In the present work, the effects of portoamides on proliferation were examined in eight human cancer cell lines and two non-carcinogenic cell lines, and major differences in sensitivities were observed. To generate hypotheses with regard to molecular mechanisms of action, quantitative proteomics using 2D gel electrophoresis and MALDI-TOF/TOF were performed on the colon carcinoma cell line HT-29. The expression of proteins involved in energy metabolism (mitochondrial respiratory chain and pentose phosphate pathway) was found to be affected. The hypothesis of altered energy metabolism was tested in further experiments. Exposure to portoamides resulted in reduced cellular ATP content, likely due to decreased mitochondrial energy production. Mitochondrial hyperpolarization and reduced mitochondrial reductive capacity was observed in treated cells. Furthermore, alterations in the expression of peroxiredoxins (PRDX4, PRDX6) and components of proteasome subunits (PSB4, PSA6) were observed in portoamide-treated cells, but these alterations were not associated with detectable increases in oxidative stress. We conclude that the cytotoxic activity of portoamides is associated with disturbance of energy metabolism, and alterations in mitochondrial structure and function.

1,4-Disubstituted-5-hydroxy-3-methylpyrazoles and some derived ring systems as cytotoxic and DNA binding agents. Synthesis, in vitro biological evaluation and in silico ADME study

Some novel polysubstituted pyrazoles, bipyrazoles and pyranopyrazoles, supported with various chemotherapeutically-active pharmacophores, were synthesized and biologically evaluated for their cytotoxic potential. Fifteen compounds (7–9, 12, 16, 17, 19, 21, 22, 26, 28, 30, 32, 33, and 34) exhibited variable degrees of cytotoxic activity against a panel of three cancer cell lines, among which the analogs 16, 17, 21, 26, and 34 showed a considerable broad spectrum cytotoxic potential, with special effectiveness against the colon HT29 and breast MCF7 cancer cell lines. In particular, compounds 16, 17, and 26 displayed double the activity of doxorubicin against colon carcinoma HT29 cell line, while the pyranopyrazole analog 34 was nearly equiactive with the reference cytotoxic agent. Meanwhile, the analogs 16 and 17 were nearly equipotent to doxorubicin against breast MCF7 cell line. DNA-binding activities of the most active compounds were in agreement with the obtained anticancer activity, where compounds 16, 17, 26, and 34 displayed the highest affinity. In silico calculations of molecular properties revealed that most of the active compounds comply with Lipinski’s RO5 and Veber’s criteria for good bioavailability suggesting good drug-likeness properties upon oral administration.

Probiotic Mixture Golden Bifido Prevents Neonatal Escherichia coli K1 Translocation via Enhancing Intestinal Defense.

Escherichia coli (E. coli) K1 sepsis and meningitis is a severe infection characterized by high mortality in neonates. Successful colonization and translocation across the intestinal mucosa have been regarded as the critical steps for E. coli K1 sepsis and meningitis. We recently reported that the probiotic mixture, Golden Bifido (containing live Lactobacillus bulgaricus, Bifidobacterium, and Streptococcus thermophilus, LBS) has a preventive role against neonatal E. coli K1 bacteremia and meningitis. However, the interaction between the neonatal gut barrier, probiotics and E. coli K1 is still not elucidated. The present study aims to investigate how LBS exerts its protective effects on neonatal gut barrier during E. coli K1 infection. The beneficial effects of LBS were explored in vitro and in vivo using human colon carcinoma cell lines HT-29 and rat model of neonatal E. coli K1 infection, respectively. Our results showed that stimulation with E. coli K1 was able to cause intestinal barrier dysfunction, which were reflected by E. coli K1-induced intestinal damage and apoptosis of intestinal epithelial cells, reduction of mucin, immunoglobulin A (IgA) and tight junction proteins expression, as well as increase in intestinal permeability, all these changes facilitate E. coli K1 intestinal translocation. However, these changes were alleviated when HT-29 cells were treated with LBS before E. coli K1 infection. Furthermore, we found that LBS-treated neonatal rats (without E. coli K1 infection) have showed higher production of mucin, ZO-1, IgA, Ki67 in intestinal mucosa as well as lower intestinal permeability than that of non-treated rats, indicating that LBS could accelerate the development of neonatal intestinal defense. Taken together, our results suggest that enhancement of the neonatal intestinal defense to fight against E. coli K1 translocation could be the potential mechanism to elucidate how LBS confers a protective effect against neonatal E. coli K1 bacteremia and meningitis. This indirect mechanism makes LBS exert preventive effect on most of gut-derived pathogenic infections rather than only E. coli.

Methyleugenol and selected oxidative metabolites affect DNA-Damage signalling pathways and induce apoptosis in human colon tumour HT29 cells

Previously the food carcinogen methyleugenol was found to be cytotoxic and genotoxic in multiple cell lines and in primary hepatocytes. In this study, the question addressed was whether methyleugenol and the selected oxidative metabolites, 1′-hydroxymethyleugenol, methyleugenol-2′,3′-epoxide and 3′-oxomethylisoeugenol trigger a DNA damage response in the human colon carcinoma HT29 cell line. Most notably investigations by flow cytometry revealed that the metabolites induce an accumulation of HT29 cells in the G2 phase of the cell cycle. DNA damage response is characterised by a time-delayed phosphorylation of ATM (ataxia-telangiectasia, mutated)/ATR (ATM- and Rad3-related) kinases and checkpoint kinase 1 after 2 h of incubation, and the tumour suppressor protein p53 only after 24 h of incubation. The test compounds induced apoptotic cell death indicated by cleavage of caspase 3 and poly-(ADP-ribose)-polymerase after a prolonged incubation time up to 72 h. In addition, activation of ATM/ATR-signalling cascade might contribute to apoptosis induction to a certain extent. However, clarification of this relationship awaits experimental confirmation.