Stimulated HT-29 Cells Research Articles

Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches.

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.

Molecular Docking Identifies 1,8-Cineole (Eucalyptol) as A Novel PPARγ Agonist That Alleviates Colon Inflammation.

Inflammatory bowel disease, comprising Crohn's disease (CD) and ulcerative colitis (UC), is often debilitating. The disease etiology is multifactorial, involving genetic susceptibility, microbial dysregulation, abnormal immune activation, and environmental factors. Currently, available drug therapies are associated with adverse effects when used long-term. Therefore, the search for new drug candidates to treat IBD is imperative. The peroxisome proliferator-activated receptor-γ (PPARγ) is highly expressed in the colon. PPARγ plays a vital role in regulating colonic inflammation. 1,8-cineole, also known as eucalyptol, is a monoterpene oxide present in various aromatic plants which possess potent anti-inflammatory activity. Molecular docking and dynamics studies revealed that 1,8-cineole binds to PPARγ and if it were an agonist, that would explain the anti-inflammatory effects of 1,8-cineole. Therefore, we investigated the role of 1,8-cineole in colonic inflammation, using both in vivo and in vitro experimental approaches. Dextran sodium sulfate (DSS)-induced colitis was used as the in vivo model, and tumor necrosis factor-α (TNFα)-stimulated HT-29 cells as the in vitro model. 1,8-cineole treatment significantly decreased the inflammatory response in DSS-induced colitis mice. 1,8-cineole treatment also increased nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus to induce potent antioxidant effects. 1,8-cineole also increased colonic PPARγ protein expression. Similarly, 1,8-cineole decreased proinflammatory chemokine production and increased PPARγ protein expression in TNFα-stimulated HT-29 cells. 1,8-cineole also increased PPARγ promoter activity time-dependently. Because of its potent anti-inflammatory effects, 1,8-cineole may be valuable in treating IBD.

Postbiotic Lipoteichoic acid of probiotic Lactobacillus origin ameliorates inflammation in HT-29 cells and colitis mice

Lipoteichoic acid (LTA) is a key surface component of probiotic lactobacilli that is involved in important cellular functions including cross talk with the host immune cells. In this study, the anti-inflammatory and ameliorative properties of LTA from probiotic lactobacilli strains were assessed in in vitro HT-29 cells and in vivo colitis mice. The LTA was extracted with n-butanol and its safety was confirmed based on its endotoxin content and cytotoxicity in HT-29 cells. In the Lipopolysaccharide stimulated HT-29 cells, the LTA from the test probiotics evoked a visible but non-significant increase in IL-10 and decrease in TNF-α levels. During the colitis mice study, probiotic LTA treated mice showed substantial improvement in external colitis symptoms, disease activity score and weight gain. The treated mice also showed improvements in key inflammatory markers such as the gut permeability, myeloperoxidase activity and histopathological damages in colon, although non-significant improvements were recorded for the inflammatory cytokines. Furthermore, structural studies by NMR and FTIR revealed increased level of D-alanine substitution in the LTA of LGG strain over MTCC5690. The present study demonstrates the ameliorative effect of LTA as a postbiotic component from probiotics which can be helpful in building effective strategies for combating gut inflammatory disorders.

Design of a chimeric protein composed of FimH, FyuA and CNF-1 virulence factors from uropathogenic Escherichia coli and evaluation its biological activity and immunogenicity in vitro and in vivo.

Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most prevalent bacterial infections in humans. Antibiotic resistance among UPEC isolates is increasing, and designing an effective vaccine can prevent or reduce these infections. FimH adhesin, iron scavenger receptor FyuA, and cytotoxic necrotizing factor -1 (CNF-1) are among the most important virulence factors of UPEC strains. Thus, a novel multi-epitope protein composed of FimH, FyuA, and CNF-1 was designed to evaluate its biological activity and immunogenicity in vitro and in vivo, respectively. The final vaccine design had seven domains, including the N-terminal domain of FimH, four domains of FyuA, and two domains of CNF-1, as determined by immunoinformatics analysis. The results of tertiary structure prediction showed that the chimeric protein had a C-score of -0.25 and Z-score of -1.94. Molecular docking indicated that thirty six ligand residues of the chimeric protein interacted with 53 receptor residues of TLR-4 by hydrogen bonds and hydrophobic interactions. Analysis of protein expression by SDS-PAGE showed an approximately 44kDa band with different concentrations of IPTG which were confirmed by Western blot. According to ELISA results, the level of IL-8 produced by stimulated Ht29cells with the chimeric protein was significantly higher than the stimulated Ht29cells with CNF-1 alone and un-stimulated Ht29cells. Rabbits subcutaneously immunized with the chimeric protein admixed with Freund adjuvant induced higher level of serum IgG on day 14 after the first vaccination than control rabbits. Furthermore, the booster dose of the chimeric protein significantly enhanced the IgG levels as compared to day 14 and also controls. As, the chimeric protein has suitable B-cell epitopes and MHC-I and MHC-II binding epitopes to stimulate humoral and cellular immunity, it could be a promising vaccine candidate against UTIs caused by UPEC. Evaluating the multi-epitope protein in inducing humoral and cellular immune responses, as well as protection, is ongoing in the mice models.

Curcumin and capsaicin regulate apoptosis and alleviate intestinal inflammation induced by Clostridioides difficile in vitro

BackgroundThe dramatic upsurge of Clostridioides difficile infection (CDI) by hypervirulent isolates along with the paucity of effective conventional treatment call for the development of new alternative medicines against CDI. The inhibitory effects of curcumin (CCM) and capsaicin (CAP) were investigated on the activity of toxigenic cell-free supernatants (Tox-S) of C. difficile RT 001, RT 126 and RT 084, and culture-filtrate of C. difficile ATCC 700057.MethodsCell viability of HT-29 cells exposed to varying concentrations of CCM, CAP, C. difficile Tox-S and culture-filtrate was assessed by MTT assay. Anti-inflammatory and anti-apoptotic effects of CCM and CAP were examined by treatment of HT-29 cells with C. difficile Tox-S and culture-filtrate. Expression of BCL-2, SMAD3, NF-κB, TGF-β and TNF-α genes in stimulated HT-29 cells was measured using RT-qPCR.ResultsC. difficile Tox-S significantly (P < 0.05) reduced the cell viability of HT-29 cells in comparison with untreated cells. Both CAP and CCM significantly (P < 0.05) downregulated the gene expression level of BCL-2, SMAD3, NF-κB and TNF-α in Tox-S treated HT-29 cells. Moreover, the gene expression of TGF-β decreased in Tox-S stimulated HT-29 cells by both CAP and CCM, although these reductions were not significantly different (P > 0.05).ConclusionThe results of the present study highlighted that CCM and CAP can modulate the inflammatory response and apoptotic effects induced by Tox-S from different clinical C. difficile strains in vitro. Further studies are required to accurately explore the anti-toxin activity of natural components, and their probable adverse risks in clinical practice.

A recombinant Bifidobacterium bifidum BGN4 strain expressing the streptococcal superoxide dismutase gene ameliorates inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) is a gastrointestinal disease characterized by diarrhea, rectal bleeding, abdominal pain, and weight loss. Recombinant probiotics producing specific proteins with IBD therapeutic potential are currently considered novel drug substitutes. In this study, a Bifidobacterium bifidum BGN4-SK strain was designed to produce the antioxidant enzymes streptococcal superoxide dismutase (SOD) and lactobacillus catalase (CAT), and a B. bifidum BGN4-pBESIL10 strain was proposed to generate an anti-inflammatory cytokine, human interleukin (IL)-10. In vitro and in vivo efficacy of these genetically modified Bifidobacterium strains were evaluated for colitis amelioration.ResultsIn a lipopolysaccharide (LPS)-stimulated HT-29 cell model, tumor necrosis factor (TNF)-α and IL-8 production was significantly suppressed in the B. bifidum BGN4-SK treatment, followed by B. bifidum BGN4-pBESIL10 treatment, when compared to the LPS-treated control. Synergistic effects on TNF-α suppression were also observed. In a dextran sodium sulphate (DSS)-induced colitis mouse model, B. bifidum BGN4-SK treatment significantly enhanced levels of antioxidant enzymes SOD, glutathione peroxidase (GSH-Px) and CAT, compared to the DSS-only group. B. bifidum BGN4-SK significantly ameliorated the symptoms of DSS-induced colitis, increased the expression of tight junction genes (claudin and ZO-1), and decreased pro-inflammatory cytokines IL-6, IL-1β and TNF-α.ConclusionsThese findings suggest that B. bifidum BGN4-SK ameliorated DSS-induced colitis by generating antioxidant enzymes, maintaining the epithelial barrier, and decreasing the production of pro-inflammatory cytokines. Although B. bifidum BGN4-pBESIL10 exerted anti-inflammatory effects in vitro, the enhancement of IL-10 production and alleviation of colitis were very limited.

Effects of Taro (Colocasia esculenta) Water-Soluble Non-Starch Polysaccharide, Lactobacillus acidophilus, Bifidobacterium breve, Bifidobacterium infantis, and Their Synbiotic Mixtures on Pro-Inflammatory Cytokine Interleukin-8 Production

In the past decades, the regulation of pro-inflammatory cytokine production, including interleukin-8 (IL-8), has been the goal of many targeted therapeutic interventions for Necrotising enterocolitis (NEC), a gastrointestinal disease commonly associated with a very low birth weight in preterm infants. In this study, the ability to regulate the production of IL-8 of the water-soluble non-starch polysaccharide (WS-NSP) from taro corm (Tc-WS-NSP) extracted using a conventional (CE) or improved conventional (ICE) extraction method, of the probiotics Lactobacillus acidophilus, Bifidobacterium breve, and Bifidobacterium infantis, and their synbiotic mixtures were evaluated. The TNF-α stimulated HT-29 cells were incubated with undigested or digested Tc-WS-NSPs (CE or ICE), probiotics, and their synbiotic mixtures with Klebsiella oxytoca, an NEC-positive-associated pathogen. Overall, the synbiotic mixtures of digested Tc-WS-NSP-ICE and high bacterial concentrations of L. acidophilus (5.57 × 109), B. breve (2.7 × 108 CFU/mL), and B. infantis (1.53 × 108) demonstrated higher (42.0%, 45.0%, 43.1%, respectively) ability to downregulate IL-8 compared to the sole use of Tc-WS-NSPs (24.5%), or the probiotics L. acidophilus (32.3%), B. breve (37.8%), or B. infantis (33.1%). The ability demonstrated by the Tc-WS-NSPs, the probiotics, and their synbiotics mixtures to downregulate IL-8 production in the presence of an NEC-positive-associated pathogen may be useful in the development of novel prophylactic agents against NEC.

Cell-Free Lactobacillus casei 21L10 Modulates Nitric Oxide Release and Cell Proliferation/Cell Death in Lipopolysaccharide-Challenged HT-29 Cells.

Lactobacillus casei (L. casei) is one of the probiotic strains that may influence intestinal injury and inflammation in nonspecific intestinal diseases. We aimed to evaluate the effect of cell-free Lactobacillus casei 21L10 supernatant (LC) on the cell line HT-29 challenged with lipopolysaccharide (LPS) in order to modulate production of NO, cell proliferation, and apoptosis. Cell line HT-29 was stimulated with LPS in the presence or absence of LC. Our results showed that LC from L. casei 21L10 did not affect the viability of unstimulated HT-29 cells line. HT-29 cell line treatment with LC caused significant decrease of LPS induced NO production after 3h, and 24h, but not after 48h. Proliferation activity of LPS stimulated HT-29 cell line analysed with MTT assay significantly decreased after 24h and 48h, but not after 3h. The majority of LPS stimulated HT-29 cell line treated with LC showed annexin V/PI positivity at 48h survival, which corresponded to late apoptotic/necrotic cell features. The observed differences suggest that cell-free L. casei 21L10 supernatant could participate in attenuation of LPS-induced inflammation, and may exhibit anti-proliferative and pro-apoptotic/necrotic effects. This study provides pilot data for the further development of L. casei exoproducts as an anti-inflammatory or anti-proliferative agent for the treatment of inflammatory and cancer diseases in gut. However, more data is needed before final conclusions of L. casei cell-free supernatant's efficacy can be drawn.

Chrysophanol Attenuates Manifestations of Immune Bowel Diseases by Regulation of Colorectal Cells and T Cells Activation In Vivo.

Inflammatory bowel disease (IBD) is an immune disorder that develops due to chronic inflammation in several cells. It is known that colorectal and T cells are mainly involved in the pathogenesis of IBD. Chrysophanol is an anthraquinone family member that possesses several bioactivities, including anti-diabetic, anti-tumor, and inhibitory effects on T cell activation. However, it is unknown whether chrysophanol suppresses the activity of colorectal cells. In this study, we found that chrysophanol did not induce cytotoxicity in HT-29 colorectal cells. Pre-treatment with chrysophanol inhibited the mRNA levels of pro-inflammatory cytokines in tumor necrosis factor-α (TNF-α)-stimulated HT-29 cells. Western blot analysis revealed that pre-treatment with chrysophanol mitigates p65 translocation and the mitogen-activated protein kinase (MAPK) pathway in activated HT-29 cells. Results from the in vivo experiment confirmed that oral administration of chrysophanol protects mice from dextran sulfate sodium (DSS)-induced IBD. Chrysophanol administration attenuates the expression of pro-inflammatory cytokines in colon tissues of the DSS-induced IBD model. In addition, we found that oral administration of chrysophanol systemically decreased the expression of effector cytokines from mesenteric lymph nodes. Therefore, these data suggest that chrysophanol has a potent modulatory effect on colorectal cells as well as exhibiting a beneficial potential for curing IBD in vivo.

SER-287, AN INVESTIGATIONAL MICROBIOME THERAPEUTIC FOR PATIENTS WITH ACTIVE MILD-TO-MODERATE ULCERATIVE COLITIS, PRODUCES REMISSION ASSOCIATED METABOLITES, REDUCES INTESTINAL EPITHELIAL IL-8 SECRETION AND PROTECTS EPITHELIAL BARRIER FUNCTION IN VITRO

Abstract Background In a Phase 1b study of mild-to-moderate ulcerative colitis (UC) (Henn et al. Gastro 2020), oral daily dosing of SER-287, a purified consortium of Firmicutes spores administered after vancomycin pre-conditioning, led to significantly higher rates of clinical remission compared to placebo (40% versus 0%, respectively; p=0.024). Engraftment, defined as SER-287 spore germination and growth of metabolically-active bacteria, was associated with remission and shifts in disease relevant microbe-associated metabolites. To better understand the mechanism underlying these favorable clinical observations, we evaluated metabolite production by SER-287 and their impact on specific inflammatory pathways and barrier epithelial integrity in vitro. Methods Three SER-287 lots and a negative control pro-inflammatory consortium including three strains isolated from UC subjects (Escherichia coli, Bifidobacterium dentium, and Streptococcus parasanguinis) were grown in vitro in a complex medium designed to mimic the nutrient composition found in the human colon. Culture supernatants were assessed for the presence of anti-inflammatory and remission associated metabolites by GC-MS or LC-MS. Anti- and pro-inflammatory activities were determined by measuring IL-8 secretion, an inflammatory cytokine whose expression is elevated in UC patients, by HT29 epithelial cells after incubation with bacterial supernatants in the presence or absence of TNF-α. Epithelial integrity was assayed by measuring FITC-dextran leakage in a trans-well culture of differentiated primary human colonic epithelial cells challenged with IFN-γ in the presence or absence of culture supernatants. Results When cultured in vitro, all SER-287 lots tested produced the anti-inflammatory short-chain fatty acids butyrate and propionate and other metabolites associated with remission in the Phase 1b trial, including tryptophan metabolites, bile acids and medium-chain fatty acids. SER-287 culture supernatants significantly reduced IL-8 secretion in TNF-α stimulated HT29 cells while the pro-inflammatory consortium increased IL-8 secretion compared to TNF-α alone (Figure 1A). SER-287 lots induced minimal IL-8 secretion in the absence of TNF-α while the pro-inflammatory consortium strongly induced IL-8 (Figure 1B). SER-287 supernatants significantly decreased IFN-γ-mediated barrier disruption, while the pro-inflammatory consortium was not protective (Figure 2). Conclusions Production of remission-associated metabolites, reduction of epithelial inflammation, and protection from epithelial barrier damage are shown to be pharmacological properties of SER-287 which may underlie the mechanism of action of this microbiome therapeutic for mild-to-moderate UC.

Dysregulation of GABAergic Signalling Contributes in the Pathogenesis of Diarrhea-predominant Irritable Bowel Syndrome.

Background/AimsDiarrhea-predominant irritable bowel syndrome (IBS-D) is a prevalent functional bowel disorder. Abdominal pain, discomfort and altered intestinal habits are the salient features of IBS-D. Low grade inflammation and altered neurotransmitters are the 2 recently identified factors contributing to the pathogenesis of IBS-D, but their role and interactions has not been elucidated in detail. Here we investigate the potential role of γ-aminobutyric acid (GABA) in regulating gut inflammation during IBS-D.MethodsBlood samples and colonic mucosal biopsies from clinically diagnosed IBS-D patients and controls were collected. Levels of GABA were measured in serum samples through enzyme-linked immunosorbent assay (ELISA). Expression of GABAergic system and proinflammatory cytokines were analyzed in biopsy samples by reverse transcriptase polymerase chain reaction (RT-PCR). Effect of GABA and its antagonist on the expression of proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated HT-29 cells was examined through RT-PCR.ResultsELISA data revealed diminished level of GABA in IBS-D patients as compared to controls. RT-PCR analysis showed altered GABAergic signal system in IBS-D patients as compared to controls. GABA reduced the expression of proinflammatory cytokines in LPS stimulated HT-29 cells, whereas bicuculline methiodide (GABA antagonist) upregulated the expression of same cytokines in LPS stimulated HT-29 cells.ConclusionsOur sets of data indicate that diminished level of GABA and altered GABAergic signal system contributes to pathogenesis of IBS-D by regulating inflammatory processes. These results provide novel evidence for anti-inflammatory role of GABA in IBS-D patients by altering the expression of pro-inflammatory cytokines.

P-196 - Anti-angiogenic action of leukotriene-C4 induced 15-hydroxyprostaglandin dehydrogenase in colon cancer cells is a TNF-α dependent phenomenon

Introduction: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2 (COX-2), which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2 and is often down-regulated in cancer. Interesting enough, CRC patients expressing high levels of Cysteinyl leukotriene receptor 2 (CysLTR2) have a good prognosis and therefore, we investigated a potential link between CysLTR2-signaling and the tumor suppressor 15-PGDH in colon cancer cells. In the other hand, TNF-α is considered as the main regulator of COX-2 and mPGES-1 that contribute to the increased synthesis of PGE2, which is inhibited by overexpressed 15-PGDH. Level of the pro-tumorigenic PGE2 is increased in CRC, previously attributed to increased production via TNF-α mediated COX-2 up-regulation but more recently attributed to decreased catabolism due to down-regulation of 15-PGDH. Methods: HT-29 and Caco-2 cells were employed for this study. Unstimulated and LTC4 stimulated cells were compared for various markers through qRT-PCR as well as immunoblotting analysis. Vibrant-DiI labeled HT-29 cells were used for the zebrafish metastasis model. Visualization of the metastatic spread was observed using the light sheet microscopy (SPIM) and tissue sections were analyzed by immunohistochemistry for specific markers. Conditioned media from the unstimulated and LTC4 stimulated HT-29 cells were used to analyze the endothelial tube formation in HUVEC. Results: Elevation of 15-PGDH expression by leukotriene C4 (LTC4), a CysLTR2 ligand, exhibited anti-tumor activity in colon cancer cells with significant phosphorylation of β-catenin and down-regulation of anti-apoptotic marker Bcl-2 with concurrent activation of CASPASE-3 expression. We also observed a dramatic down-regulation of TNF-α on mRNA level and NF-κβ on both mRNA as well as protein level with LTC4 induced 15-PGDH in a TNF-α dependent manner. Moreover, TNF-α regulated anti-angiogenic action of 15-PGDH in HT-29 and Caco-2 colon cancer cells by depleting the mRNA level of MMP-2 and MMP-9 and protein level of VEGFR-1. Furthermore, we also observed disrupted tube formation in HUVEC with LTC4 induced 15-PGDH which is also governed by TNF-α. The angiogenesis study in transgenic zebrafish, Tg(fli1a: EGFP) embryo model suggested significant decrease in early and late metastasis with distinct disruption in the intersegmental vessel in the LTC4 induced 15-PGDH treated group. IHC analysis of Ki-67 exhibited decrease with LTC4 and very interestingly CASPASE-3 showed increase in the stimulated group which supported our in vitro observation. Conclusion: Hence, restoration of 15-PGDH expression through CysLTR2-signaling promotes the anti-angiogenic action against colon cancer cells, indicating an anti-tumor as well as the anti-metastatic efficacy of CysLTR2-signaling.

Eclipta prostrata Improves DSS-Induced Colitis through Regulation of Inflammatory Response in Intestinal Epithelial Cells.

Eclipta prostrata (EP) and its compounds are known to have several pharmacological effects including anti-inflammatory effects. In the present study, we demonstrated that EP improves the dextran sulfate sodium (DSS)-induced colitis symptoms such as body weight loss, colon length shortening and disease activity index. In DSS-induced colitis tissue, EP controls the protein expressions of cyclooxygenase-2 (COX-2) and hypoxia inducible factor-1[Formula: see text] (HIF-1[Formula: see text]). In addition, the release of prostaglandin E2 and vascular endothelial growth factor-A were significantly reduced by EP administration. EP also inhibited COX-2 and HIF-1[Formula: see text] expressions in the tumor necrosis factor-[Formula: see text] stimulated HT-29 cells. These inhibitory effects of EP occurred by reducing the phosphorylation of I[Formula: see text]B and the translocation of the nuclear factor-[Formula: see text]B (NF-[Formula: see text]B). Additionally, we found through HPLC analysis that wedelolactone, which is an inhibitor of NF-[Formula: see text]B transcription, was contained in water extract of EP. These results indicate that EP can improve colitis symptoms through the modulation of immune function in intestinal epithelial cells and suggests that EP has the potential therapeutic effect to intestinal inflammation.

Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells.

Novel dietary agents for colon cancer prevention and therapy are desired. Kaempferol, a flavonol, has been reported to possess anticancer activity. However, little is known about the molecular mechanisms of the anticancer effects of kaempferol. The aim of this study was to determine the inhibitory effect of kaempferol on growth factor-induced proliferation and to elucidate its underlying mechanisms in the HT-29 human colon cancer cell line. To assess the effects of kaempferol and/or growth factors [insulin-like growth factor (IGF)-I and heregulin (HRG)-β], cells were cultured with or without 60 μmol/L kaempferol and/or 10 nmol/L IGF-I or 20 μg/L HRG-β. Cell proliferation, DNA synthesis, and apoptosis were determined by a cell viability assay, a [(3)H]thymidine incorporation assay, and Annexin-V staining, respectively. Western blotting, immunoprecipitation, and an in vitro kinase assay were conducted to evaluate expression and activation of various signaling molecules involved in the IGF-I receptor (IGF-IR) and ErbB3 signaling pathways. IGF-I and HRG-β stimulated HT-29 cell growth but did not abrogate kaempferol-induced growth inhibition and apoptosis. Kaempferol reduced IGF-II secretion, HRG expression and phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. Kaempferol reduced IGF-I- and HRG-β-induced phosphorylation of the IGF-IR and ErbB3, their association with p85, and phosphatidylinositol 3-kinase (PI3K) activity. Additionally, kaempferol inhibited IGF-I- and HRG-β-induced phosphorylation of Akt and ERK-1/2. The results demonstrate that kaempferol downregulates activation of PI3K/Akt and ERK-1/2 pathways by inhibiting IGF-IR and ErbB3 signaling in HT-29 cells. We suggest that kaempferol could be a useful chemopreventive agent against colon cancer.

Kaempferol Downregulates Insulin-like Growth Factor-I Receptor and ErbB3 Signaling in HT-29 Human Colon Cancer Cells

Background:Novel dietary agents for colon cancer prevention and therapy are desired. Kaempferol, a flavonol, has been reported to possess anticancer activity. However, little is known about the molecular mechanisms of the anticancer effects of kaempferol. The aim of this study was to determine the inhibitory effect of kaempferol on growth factor-induced proliferation and to elucidate its underlying mechanisms in the HT-29 human colon cancer cell line.Methods:To assess the effects of kaempferol and/or growth factors [insulin-like growth factor (IGF)-I and heregulin (HRG)-β], cells were cultured with or without 60 μmol/L kaempferol and/or 10 nmol/L IGF-I or 20 μg/L HRG-β. Cell proliferation, DNA synthesis, and apoptosis were determined by a cell viability assay, a [3H]thymidine incorporation assay, and Annexin-V staining, respectively. Western blotting, immunoprecipitation, and an in vitro kinase assay were conducted to evaluate expression and activation of various signaling molecules involved in the IGF-I receptor (IGF-IR) and ErbB3 signaling pathways.Results:IGF-I and HRG-β stimulated HT-29 cell growth but did not abrogate kaempferol-induced growth inhibition and apoptosis. Kaempferol reduced IGF-II secretion, HRG expression and phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. Kaempferol reduced IGF-I- and HRG-β-induced phosphorylation of the IGF-IR and ErbB3, their association with p85, and phosphatidylinositol 3-kinase (PI3K) activity. Additionally, kaempferol inhibited IGF-I- and HRG-β-induced phosphorylation of Akt and ERK-1/2.Conclusions:The results demonstrate that kaempferol downregulates activation of PI3K/Akt and ERK-1/2 pathways by inhibiting IGF-IR and ErbB3 signaling in HT-29 cells. We suggest that kaempferol could be a useful chemopreventive agent against colon cancer.

Antioxidant activity induced by main polyphenols present in edible artichoke heads: influence of in vitro gastro-intestinal digestion

Artichoke (Cynara cardunculus (L.) subsp. scolymus Hayek) is an important component of the Mediterranean diet, and a good source of health-promoting compounds, including polyphenols. The main phenolic compounds of artichoke are 5-O-caffeoylquinic acid (chlorogenic acid), and 3,5-O-, 1,5-O-dicaffeoylquinic acids and its beneficial effects are linked to their antioxidant activity that can be affected by their stability under gastro-intestinal conditions, during digestion. This study aimed to assess the antioxidant activity induced by artichoke head extract, and its main individual polyphenols, on a stimulated HT-29 cell line, using a DCFH-DA probe. In addition, influences of in vitro digestion on artichoke head and its individual polyphenols towards antioxidant activity, were tested. Artichoke extract showed higher antioxidant activity than the individual polyphenols, even after in vitro gastro-intestinal digestion. In addition, in vitro digestion did not modify the antioxidant activity of artichoke polyphenols, except for 1,5-O-dicaffeoylquinic acid, which proved to be the least active.

Mucosal toll-like receptor 3-dependent synthesis of complement factor B and systemic complement activation in inflammatory bowel disease.

Recent studies link Toll-like receptor 3 (TLR3) to the pathogenesis of inflammatory bowel disease (IBD). Screening TLR3-agonist response in an intestinal epithelial cell line, we found complement factor B mRNA (CFB) potently upregulated and went on to further study localization of complement factor B synthesis and systemic activation of complement in ulcerative colitis and Crohn's disease. In a transcriptome analysis of poly (I:C) stimulated HT-29 cells, we found CFB highly upregulated downstream of TLR3. We sought to confirm CFB upregulation in a microarray gene expression analysis on colonic biopsies from an IBD population (n = 133). Immunohistochemical staining and in situ hybridization were done to identify cellular sources of factor B and CFB. Systemic complement activation was assessed in plasma (n = 18) using neoepitope-based enzyme linked immunosorbent assay. CFB mRNA and protein were abundantly expressed in the colonic epithelial cell line, and synthesis enhanced by the poly (I:C) TLR3 ligand. In inflamed versus normal colonic mucosa of ulcerative colitis and Crohn's disease, CFB mRNA was the most significantly overexpressed gene and the mRNA abundance ratio was among the 50 highest. Epithelial cells were the dominating site of factor B expression. Systemic complement activation was significantly higher in active than in nonactive IBD. This study is the first to link TLR3 to activation of the alternative complement pathway. Complement factor B is potently upregulated locally in IBD in addition to having a possible central role in systemic complement activation. This suggests a prominent role for complement in IBD pathogenesis.

Allyl isothiocyanate inhibits cell metastasis through suppression of the MAPK pathways in epidermal growth factor-stimulated HT29 human colorectal adenocarcinoma cells

Allyl isothiocyanate (AITC) has been found to present sources from consumed cruciferous vegetables. AITC is known to possess pharmacological and anticancer activities. The present study was designed to test the hypothesis that AITC suppressed the invasion and migration of epidermal growth factor (EGF)-stimulated HT29 cells and to elucidate the mechanisms for the antimetastatic abilities in vitro. The invasion and migration of EGF-stimulated HT29 cells were determined individually by Transwell cell invasion and wound-healing assays. Our results showed that AITC effectively inhibited both the invasive and migratory ability of HT29 cells. Furthermore, AITC downregulated the protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and mitogen-activated protein kinases (MAPKs) (p-JNK, p-ERK and p-p38) by western blot analysis in HT29 cells following EGF induction. Thus, the metastatic responses in AITC-treated HT29 cells after EGF stimulation were mediated by the MMP-2/-9 and MAPK signaling pathways. We also used gene expression microarrays to investigate the gene levels related to cell growth, G-protein coupled receptor, angiogenesis, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, transcription and translation, EGFR and PKB/mTOR signals. In summary, it is possible that AITC suppresses the invasion and migration of EGF-induced HT29 cells, resulting from MMP-2/-9 and MAPKs. Hence, AITC may be beneficial in the treatment of human colorectal adenocarcinoma in the future.

Cytotoxic and anti-inflammatory effects of onion peel extract on lipopolysaccharide stimulated human colon carcinoma cells

The present study investigated the cytotoxic activity of ethanol extract of onion peel (OPE) in HT-29 human colon carcinoma cells. High-performance liquid chromatography (HPLC) analysis was performed to determine the amounts of phenolic acids and flavonoids in OPE. In addition, the influence of OPE on antioxidant- and inflammation-associated gene expression was also determined in a model of lipopolysaccharide (LPS)-stimulated HT-29 cells. HPLC analysis showed that OPE contained well-known antioxidant compounds, including p-coumaric acid, vanillic acid, epicatechin, and morin. After incubation with OPE, HT-29 cells showed either a loss of normal nuclear architecture or detachability from each other. The cytotoxic effects of OPE on HT-29 cells were confirmed by MTT and LDH release assays. LPS-induced oxidative conditions effectively downregulated TNF-α mRNA expression in OPE pretreated HT-29 cells compared with cells only stimulated with LPS. In addition, the expression of heme oxygenase-1 (HO-1) and glutathione S-transferase (GSTs) detoxification genes (i.e., GSTM1, GSTT1, and GSTP1) was upregulated after treatment with LPS at sublethal concentrations. However, the LPS-induced mRNA expression of HO-1 and GSTs was significantly attenuated by treatment with OPE. Therefore, onion peel extract is a promising component of future nutraceuticals and value-added products.

Anti-inflammatory activity of patchouli alcohol in RAW264.7 and HT-29 cells

Patchouli alcohol (PA) is a chemical compound extracted from patchouli which belongs to the genus Pogostemon, herb of mint family. Recently, it has been reported that PA inhibits the production of inflammatory mediators. However, the biological mechanisms of PA for anti-inflammatory activities have not been studied. In this study, we investigated whether PA decreases the production of inflammatory mediators through downregulation of the NF-κB and ERK pathway. Our data indicated that PA inhibits the over-expression of iNOS and IL-6 in protein and mRNA levels in LPS-stimulated RAW264.7 and TNF-α stimulated HT-29 cells. PA inhibited IκB-α degradation and p65 nuclear translocation, and subsequently suppressed transcriptional activity of NF-κB in LPS-stimulated RAW264.7 and TNF-α-stimulated HT-29 cells. In addition, PA inhibited LPS- or TNF-α-stimulated ERK1/2 activation by decreasing phosphorylation of ERK1/2. These findings suggest that PA shows anti-inflammatory activities through suppressing ERK-mediated NF-κB pathway in mouse macrophage and human colorectal cancer cells.