Lactobacillus plantarum AS1 binds to cultured human intestinal cell line HT-29 and inhibits cell attachment by enterovirulent bacterium Vibrio parahaemolyticus

Aim: Lactobacillus plantarum AS1 was incubated with HT‐29 adenocarcinoma cell line to assess its adhesion potency and examined for its inhibitory effect on the cell attachment by an enterovirulent bacterium Vibrio parahaemolyticus. Methods and Results: Lactobacillus plantarum AS1 attached efficiently to HT‐29 cells as revealed by scanning electron microscopy and bacterial adhesion assay. Lactobacillus plantarum AS1 significantly reduced V. parahaemolyticus attached to HT‐29 cells by competition, exclusion and displacement mode. Lactobacillus plantarum AS1 seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and protein factors on the bacteria and eukaryotic cell surface. Conclusion: Strain Lact. plantarum AS1 inhibits the cell attachment of a pathogen V. parahaemolyticus by steric hindrance mechanism. Also, antibacterial factors such as bacteriocins, lactic acid and exopolysaccharides could be involved. Significance and Impact of the Study: The ability to inhibit the adhesion of V. parahaemolyticus to intestinal cell line warrants further investigation to explore the use of probiotic strain Lact. plantarum AS1 in the management of gastroenteritis caused by V. parahaemolyticus.     

Characterization of a spontaneously polarizing HT-29 cell line,HT-29/cl.f8

Summary A cloned cell line that spontaneously polarizes in standard glucose-containing media was derived from a single cell of the adenocarcinoma cell line HT-29. The cloned line, designated HT-29/cl.f8, has remained stable over 2 yr in culture, maintained high transepithelial resistance (300 ohm cm² or higher), and correctly sorted influenza virus and vesicular stomatitis virus to apical or basolateral domains, respectively. The newly cloned cells also displayed apical microvilli, tight junctions, and desmosomes, the morphological characteristics of mature epithelia. The cloned HT-29/cl.f8 cells function as epithelial enterocytes as shown by the apical expression of intestinal alkaline phosphatase, the expression of vimentin and cytokeratin, and lack of expression of mucin. We propose that the newly cloned HT-29/cl.f8 cells offer a viable alternative for studies of enterocyte function that will readily yield interpretable data not complicated by cell alterations due to the presence of drugs or chemicals that induce differentiation.     

The influence of type I collagen on the growth and differentiation of the human colonic adenocarcinoma cell line HT-29 in vitro

HT-29 Human colonic adenocarcinoma cells when grown on a plastic substratum were anaplastic in appearance and failed to express any morphological or biochemical features that were characteristic of intestinal differentiation. Growth of HT-29 cells subcutaneously in the flank of immune deprived mice gave rise to morphologically heterogeneous tumors which were poorly differentiated but contained approximately 11% of cells with an intestinal phenotype: these showed features typical of cell polarization with well-developed microvilli, tight junctional complexes and desmosomes between adjacent cells. The transfer of cells from plastic onto either a fixed (designated 'non-released') or floating (designated 'released') type I collagen gel induced some morphological features typical of intestinal differentiation; for example goblet-like cells were observed after 9 days, but biochemical markers of differentiation were expressed only modestly. The continued subculture of HT-29 cells on collagen type I gels, which were either attached to the plastic or floating in the medium, induced some morphological features of intestinal differentiation and changes in the activity of brush border-associated enzymes. Alkaline phosphatase activity was enhanced from 1.3 x 10(-3) mumoles/mg/min for cells cultured on plastic substrata to 2.1 x 10(-3) mumoles/mg/min when gels were non-released, and 2.9 x 10(-3) mumoles/mg/min when gels were released after 12 days of culture. This was confirmed by electron microscopical visualization of alkaline phosphatase activity. Elevated levels of aminopeptidase activity were also observed on day 12 (plastic = 26 milliunits/mg; non-released gel = 41 milliunits/mg; released gel = 36 milliunits/mg). Similarly, changes occurred in the secretion of carcinoembryonic antigen from 0.96 x 10(-2) micrograms/mg/48 hours by cells cultured on plastic to 2.3 x 10(-2) micrograms/mg/48 hours by cells cultured on floating collagen gels. The effects of permitting HT-29 cells to undergo polarization were tested by culture on inert filter inserts: morphological features of intestinal differentiation were observed although this did not occur until after 21 days. These studies show that optimization of the growth conditions of anaplastic cells in vitro may provide cultures more representative of the tumor in vivo. This model system may be useful for cell biological and pharmacological studies of colon carcinoma.     

Effect of sulindac sulfide on metallohydrolases in the human colon cancer cell line HT-29

Matrix metalloproteinase 7 (MMP7), a metallohydrolase involved in the development of several cancers, is downregulated in the Apc(Min/+) colon cancer mouse model following sulindac treatment. To determine whether this effect is relevant to the human condition, HT-29 human colon cancer cells were treated with sulindac and its metabolites, and compared to results obtained from in vivo mouse studies. The expression of MMP7 was monitored. The results demonstrated that sulindac sulfide effectively downregulated both MMP7 expression and activity. Furthermore, activity-based proteomics demonstrated that sulindac sulfide dramatically decreased the activity of leukotriene A4 hydrolase in HT-29 cells as reflected by a decrease in the level of its product, leukotriene B4. This study demonstrates that the effect of sulindac treatment in a mouse model of colon cancer may be relevant to the human counterpart and highlights the effect of sulindac treatment on metallohydrolases.     

Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29

Autophagy is a major catabolic process allowing the renewal of intracellular organelles by which cells maintain their homeostasis. We have previously shown that autophagy is controlled by two transduction pathways mediated by a heterotrimeric Gi3 protein and phosphatidylinositol 3-kinase activities in the human colon cancer cell line HT-29. Here, we show that 3-methyladenine, an inhibitor of autophagy, increases the sensitivity of HT-29 cells to apoptosis induced by sulindac sulfide, a nonsteroidal anti-inflammatory drug which inhibits the cyclooxygenases. Similarly, HT-29 cells overexpressing a GTPase-deficient mutant of the G(alpha i3) protein (Q204L), which have a low rate of autophagy, were more sensitive to sulindac sulfide-induced apoptosis than parental HT-29 cells. In both cell populations we did not observe differences in the expression patterns of COX-2, Bcl-2, Bcl(XL), Bax, and Akt/PKB activity. However, the rate of cytochrome c release was higher in Q204L-overexpressing cells than in HT-29 cells. These results suggest that autophagy could retard apoptosis in colon cancer cells by sequestering mitochondrial death-promoting factors such as cytochrome c.     

Carbachol induces oscillations in membrane potential and intracellular calcium in a colonic tumor cell line, HT-29

The patch-clamp technique was used to study the effects ofcarbachol (CCh) on HT-29 cells. During CCh exposure, the cells (n = 23) depolarized close to theequilibrium potential forCl(;48 mV) and the membrane potential then started to oscillate(16/23 cells). In voltage-clamp experiments, similar oscillations inwhole cell currents could be demonstrated. The whole cell conductanceincreased from 225 ± 25 pS in control solution to 6,728 ± 1,165 pS (means ± SE, n = 17). Insubstitution experiments (22 mMCl in bath solution, = 0 mV), the reversal potential changed from 41.6 ± 2.2 mV(means ± SE, n = 9) to 3.2 ± 2.0 mV (means ± SE, n = 7).When the cells were loaded with the calcium-sensitive fluorescent dye,fluo 3, and simultaneously patch clamped, CCh caused a synchronousoscillating pattern of fluorescence and membrane potential. Incell-attached patches, the CCh-activated currents reversed at arelative membrane potential of 1.9 ± 3.7 mV (means ± SE,n = 11) with control solution in thepipette and at 46.2 ± 5.3 mV (means ± SE,n = 10) with a 15 mMCl solution in the pipette.High K⁺ (144 mM) did not changethe reversal potential significantly (P  0.05, n = 8). In inside-out patches,calcium-dependent Clchannels could be demonstrated with a conductance of 19 pS(n = 7). It is concluded that CChcauses oscillations in membrane potential that involvecalcium-dependent Clchannels and a K⁺ permeability.

     

In vivo delayed rejection of tumors and inhibition of delayed - type hypersensitivity by HT-29 human colonic adenocarcinoma cell line

Summary Cis-diamminedichloroplatinum (II) (cis-DDP) and its structural analogue cis-diamminediaquoplatinum(II) nitrate (cis-aq) were complexed via an intermediate dextran carrier to antibodies specifically reactive with B lymphoma cells (38C-13). The potential use of these drugs in site-directed immunotargeting was evaluated. The two platinum(II) compounds were previously shown to form pharmacologically active complexes with carboxymethyl dextran (CM-dex). For the purpose of preparing drug-antibody complexes, CM-dex was first conjugated to idiotypic antibodies that recognize a specific membrane IgM on the B lymphoma cells. The conjugates were prepared by a modified water-soluble carbodiimide method in which N-hydroxysuccinimide was used to enhance the coupling reaction. The conjugation was followed by separation of the CM-dex-IgG conjugates from unconjugated CM-dex or IgG. The platinum(II) compounds were then complexed to the CM-dex-IgG resulting in complexes carrying up to 50 mole drug/mole IgG. Both cis-DDP and cis-aq complexes of CM-dex-antibody conjugates maintained most of the original cell-binding activity of the antibodies. An in vitro assay was used to demonstrate selective binding to tumor cells in which the target cells were treated with specific immune complexes and washed before culture. In this assay the specific complexes showed preferential cytotoxicity for the B lymphoma cells in comparison to the free drugs, drug CM-dex, or nonspecific immune complexes.     

Conjugated linoleic acid inhibits cell proliferation and ErbB3 signaling in HT-29 human colon cell line

Conjugated linoleic acid (CLA) has chemoprotective properties in experimental cancer models, and in vitro studies have shown that CLA inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 have been implicated in the development of colon cancer, and both proteins are expressed at high levels in the HT-29 cell line. Activation of ErbB2/ErbB3 heterodimers is regulated by the ErbB3 ligand heregulin. To examine CLA regulation of HT-29 cell proliferation and apoptosis and the influence of CLA on the ErbB3 signaling pathway, HT-29 cells were cultured in the presence of CLA and/or heregulin. CLA inhibited DNA synthesis and induced apoptosis of HT-29 cells. Although the addition of heregulin-alpha led to an increase in cell number, it was not able to counteract the negative growth regulatory effect of CLA. Immunoprecipitation/Western blot studies revealed that CLA inhibited heregulin-alpha-stimulated phosphorylation of ErbB2 and ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3-kinase) to the ErbB3 receptor, ErbB3-associated PI3-kinase activities, and phosphorylation of Akt. CLA decreased ErbB2 and ErbB3 mRNA and protein levels in a dose-dependent manner. In conclusion, we demonstrate that CLA inhibits cell proliferation and stimulates apoptosis in HT-29 cells and that this may be mediated by its ability to downregulate ErbB3 signaling and the PI3-kinase/Akt pathway.     

Apoptosis and cell cycle arrest of human colorectal cancer cell line HT-29 induced by vanillin

Background: Vanillin is responsible for the flavor and smell of vanilla, a widely used flavoring agent. Previous studies showed that vanillin could enhance the repair of mutations and thus function as an anti-mutagen. However, its role in cancer, a disease that is closely related to mutation has not yet been fully elucidated. Methods: Hence, this study investigated the cytolytic and cytostatic properties of vanillin against HT-29, a human colorectal cancer cell line. Methods used including cell viability assay, acridine orange (AO)–ethidium bromide (EB) double staining cell morphological analysis, Cell cycle analysis, annexin V–propidium iodide apoptosis test and 5-bromo-2-deoxyuridine (BrdU)-labeling cell proliferation assay. Results: Results showed that apoptosis was induced by vanillin and the IC₅₀ for HT-29 and NIH/3T3 normal cell lines were 400 μg/ml and 1000 μg/ml, respectively. Different concentrations of vanillin arrest cell cycle at different checkpoints. 5-Bromo-2-deoxyuridine-labeling cell proliferation assay showed that G0/G1 arrest was achieved at lower concentration of vanillin (200 μg/ml) while cell cycle analysis by flow cytometer showed that G2/M arrest occurs at higher concentration of vanillin (1000 μg/ml). Conclusion: Cytolytic and cytostatic effects shown by vanillin showed that it could be a useful colorectal cancer preventive agent. Further in vivo study should be carried out to confirm that similar effects could happen in animals.     

Rapid lateral diffusion of lectin-labelled glycoconjugates in the human colonic adenocarcinoma cell line HT29

The lateral diffusion of lectin-labelled glycoconjugates was studied in the human colon carcinoma cell line HT29 using fluorescence photobleaching techniques. HT29 cells were grown in either Dulbecco's modified Eagle's medium with glucose (25 mM; DMEM-Glu) or with galactose (25 mM; DMEM-Gal). Cell cultivation in the DMEM-Gal medium was assumed to promote a transformation of the cells to become small-intestinal-like with characteristic microvilli and associated enzymes. The diffusion of glycoconjugates labelled with fluoresceinated Triticum vulgaris agglutinin (Wheat germ agglutinin; WGA), Ricinus communis agglutinin-I (RCA-I), Concanavalia ensiformis agglutinin (ConA), Ulex europaeus agglutinin-I (UEA-I) and Arachis hypogaea agglutinin (PNA) was in all cases rapid, with a diffusion constant (D) ranging between 0.4 and 0.8×10^-8 cm² s^-1. As a comparison the diffusion of the fluorescent synthetic lipid analog diI-C₁₄ was characterized by D=0.8 – 1.0 × 10^–8 cm² s^-1. The diffusion of lectin-labelled surface components could not be related to the presence of microvilli on HT29 cells grown in DMEM-Gal, which ought to yield an apparently lower diffusion rate. The results indicate either that surface glycoconjugates in HT29 cells are dominated by glycolipid, or that the labelled glycoproteins are more or less free to diffuse in the plane of the membrane.     

乳杆菌DM9811发酵液提取物中RNA组分对人结肠癌细胞系HT-29增殖影响及分子机制研究

目的观察乳杆菌DM9811发酵液提取物中RNA组分对人结肠癌细胞系HT-29增殖的影响,探讨其对肠道肿瘤细胞的作用及其分子机制,为阐明乳杆菌与宿主相互作用规律的分子机制奠定基础。方法应用MTT方法研究不同时间不同浓度RNA组分对HT-29增殖的影响,应用流式细胞术、RT-PCR研究RNA组分对HT-29细胞周期的影响。结果乳杆菌DM9811发酵液中RNA组分能抑制HT-29细胞增殖,并呈现出时间-剂量依赖性;RNA组分作用于HT-29细胞24 h、48 h时,细胞周期G0/G1期所占比例明显上升,S期所占比例明显降低(P0.01),细胞周期调控因子CDK6、p27Kip1、p53的表达升高,CDK2、CDK4、PCNA的表达降低。结论乳杆菌DM9811代谢产物RNA在体外具有抑制癌细胞周期活性。     

Origin of microcells in the human sarcoma cell line HT-1080.

The aim of this study was to investigate the development of microcells in the human sarcoma cell line HT-1080 after interference with thiophosphamidum. We found that damaged interphase macrocells located at the projection of the nucleolus may form one or several microcells. The micronuclei of the microcells intensively incorporate the thymidine analogue 5-bromo-2'-deoxyuridine and strongly express argyrophilic nucleolar organiser region proteins. At an early phase of the development, the micronuclei contain fragmented DNA, but in subsequent phases, the micronuclei accumulate polymeric DNA, simultaneously with an increase in their size. After desintegration of the damaged macrocell, the microcells appear in the intercellular space. The microcells can enter mitosis and they strongly express the lung resistance protein. Electron microscopic observations suggest that coiled bodies are involved in the development of the microcells. Since the observed path of microcell formation differs from apoptotic cell fragmentation into apoptotic bodies, we propose a new term for this microcell development: sporosis. We suggest that self-renewal of the tumour stem cells is likely based on sporosis.     

A mucus-secreting human colonic epithelial cell line responsive to cholinergic stimulation

The human colonic epithelial cell line Cl.16E grows in culture as a polarized monolayer which differentiates at confluency into typical goblet cells secreting their mucin content into the culture medium. Polyclonal antibodies raised against these mucins were used in an ELISA to measure the amount of mucins secreted by the Cl.16E cells. Carbachol caused a transient and significant increase in mucus secretion with a maximal stimulation occurring at 30 min. A dose-dependent effect was found with a maximal stimulation with 10-3M carbachol. This effect was inhibited by atropine. These results indicate that the effects of carbachol are mediated by muscarinic receptors present on mucus-secreting epithelial cells.     

Activation of phosphatidylinositol turnover by neurotensin receptors in the human colonic adenocarcinoma cell line HT29

Association of neurotensin to its receptor in HT29 cells increases the intracellular concentration of inositol phosphates. A rapid (20–30 s), transient stimulation of inositol trisphosphate (275% of the basal level) and inositol bisphosphate (420%) is first observed, followed by a slower, stable increase in inositol monophosphate (170%). Half-maximal stimulation of the three inositol phosphates was obtained with 50–100 nM neurotensin. These results indicate that neurotensin is able to regulate intracellular Ca²⁺ levels in HT29 cells by using inositol trisphosphate as a second messenger.     

Aldosterone binding in the human colon carcinoma cell line HT29: correlation with cell differentiation

The purpose of the present study is to investigate aldosterone binding to human colon carcinoma HT29 cells. These cells grow as undifferentiated epithelial cells when cultured under standard conditions (Dulbecco's MEM; 10% FCS). Modification of the culture medium induced two types of differentiation: (1) enterocytic differentiation when glucose (25 mM) is replaced by galactose (5 mM) (2) mucus secreting cells in FCS free medium. Aldosterone specific binding was detected in the cytosolic fraction of enterocyte-differentiated cells. This binding corresponded to a single class of sites with affinity, specificity and anion-exchange chromatographic behaviour similar to those observed in the epithelial crypts of human colon. These putative aldosterone receptors were also detected in mucus secreting cells that are derived from the enterocyte-differentiated cells. Enterocytic differentiation appears thus to be a necessary step for the "induction" of aldosterone receptors in HT29 cells.     

Parathyroid hormone-related protein overexpression in the human colon cancer cell line HT-29 enhances adhesion of the cells to collagen type I

The present experiments examined the potential ability of parathyroid hormone-related protein (PTHrP) to influence growth of the human colon cancer cell HT-29 and the ability of the cell to adhere to several extracellular matrix (ECM) proteins found in normal tissues. Addition of PTHrP analogs, PTHrP (1-34), PTHrP (67-86), or PTHrP (107-139), to HT-29 cells in culture did not influence cell growth or the adhesion of the cells to wells coated with fibronectin, laminin, or collagen type I. Likewise, in HT-29 cells induced to overexpress PTHrP by stable transfection with PTHrP cDNA, compared to vector-transfected control HT-29 cells, no effect on cell growth occurred. However, in the transfected cells, the increased production of PTHrP significantly enhanced cell adhesion to type I collagen but not to fibronectin or laminin. The results raise the possibility that PTHrP might play a role in colon tumor invasion and metastasis by influencing cell adhesion to specific extracellular matrix proteins.     

Synthesis of Sansalvamide A derivatives and their cytotoxicity in the MSS colon cancer cell line HT-29

We report the synthesis of thirty-six Sansalvamide A derivatives, and their biological activity against colon cancer HT-29 cell line, a microsatellite stable (MSS) colon cancer cell-line. The thirty-six compounds can be divided into three subsets, where the first subset of compounds contains L-amino acids, the second subset contains D-amino acids, and the third subset contains both D- and L-amino acids. Five compounds exhibited excellent inhibitory activity (>75% inhibition). The structure-activity relationship (SAR) of the compounds established that a single D-amino acid in position 2 or 3 gave up to a 10-fold improved cytotoxicity over Sansalvamide A peptide. This work highlights the importance of residues 2 and 3 and the role of D-amino acids in the extraordinary SAR for this compound class.     

N-methylformamide effects on cell proliferation and antigenic pattern in HT-29 colon carcinoma cell line

The effects of the differentiating agent N-methylformamide (NMF) on cell proliferation and antigenic pattern of HT-29 colon carcinoma cells have been investigated. The cell line was cultured in the presence, or absence, of 1% NMF and tested for the above mentioned characteristics, both in vitro and after injection into nude mice. The percentage of cells in the various cell cycle compartments was estimated by flow cytometry. The presentation on the cell surface of molecules such as tumour associated antigens (TAAs), HLA class I molecules and epidermal growth factor receptor (EGF-R) was analysed by ELISA, flow cytometry and immunohistochemistry. Results demonstrate that NMF impairs HT-29 cell proliferation with a remarkable accumulation in the G0/G1 phases, as well as inducing a modification of the membrane antigenic pattern. The presence of NMF in the culture medium decreases the TAAs and EGF-R whereas HLA antigen maintains the same level of positivity in the two cell lines. These alterations are consistent with a different behaviour in vivo of the tumours originated from NMF treated and untreated cells. Tumours derived from NMF treated cells show a delay in the appearance and low levels of immunodetectable carcinoembryonic antigen (CEA) molecules.     

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2

Abstract Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.