The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression

Mucin expression was studied during proliferation and differentiation of the enterocyte-like Caco-2 and goblet cell-like LS174T cell lines. Caco-2 cells express mRNAs of MUC1, MUC3, MUC4 and MUC5A/C whereas MUC2 and MUC6 mRNAs are virtually absent. Furthermore, MUC3 mRNA is expressed in a differentiation dependent manner, as is the case for enterocytes. Concomitantly MUC3 protein precursor (550 kDa) was detected in Caco-2 cells. In LS174T cells mucin mRNAs of MUC1, MUC2 and MUC6 are constitutively expressed at high levels, whereas MUC3, MUC4 and MUC5A/C mRNAs are present at low levels. At the protein level LS174T cells express the goblet cell specific mucin protein precursors MUC2, MUC5A/C and MUC6 with apparent molecular masses of about 600 kDa, 470/500 kDa and 400 kDa respectively. MUC3 protein is not detectable. Furthermore, human gallbladder mucin protein (470 kDa precursor), of which the gene has not yet been identified, is expressed in LS174T cells. In addition, synthesis and secretion of the goblet cell specific mature MUC2, MUC5A/C and human gallbladder mucin was demonstrated in LS174T cells. It is concluded that Caco-2 and LS174T cell lines provide excellentin vitro models to elucidate the cell-type specific mechanisms responsible for mucin expression.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DMEM Dulbecco's modified Eagle's medium - EMEM Eagle's minimum essential medium - Endo-H endo--N-acetylglucosaminidase H - HGBM human gallbladder mucin - dpc days past confluence - PBS phosphate buffered saline     

Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer

To compare the differences in MUC2 and MUC5AC mRNA among four colon cancer cell lines and to identify the best in vitro models for studying mucin expression, quantitative real-time polymerase chain reaction was used to measure the expression of MUC2 and MUC5AC mRNA in Caco-2, HT29, LoVo, and LS174T cell lines. The levels of MUC2 mRNA expression in the four colon cancer cell lines ranked in order of mRNA abundance were: LS174T > LoVo > HT-29 > Caco-2. In contrast to MUC2, the abundances of MUC5AC mRNA were in the order: Caco-2 > HT-29 > LS174T > LoVo. Caco-2 (highest level of MUC5AC mRNA) and LS174T (highest level of MUC2 mRNA) were used to investigate the phenotypes. Morphologically, Caco-2 cells were larger with low electron density mucus-storing vacuoles, many cell surface microvilli, and no obvious intercellular spaces between cells, compared to LS174T cells. The proliferative and invasive capacities of LS174T cells were significantly higher than those of Caco-2 cells. Caco-2 and LS174T cells provide excellent in vitro models for studying mucin expression in colon cancer.     

Tributyrin enhances the cytotoxic activity of interleukin-2/interleukin-12 stimulated human natural killer cells against LS 174T colon cancer cells in vitro

Tributyrin has been shown to be cytostatic to tumor cells by inducing differentiation and apoptosis. On the other hand, immunological NK cells can kill tumor cells, particularly when stimulated with interleukin-2 (IL-2) and/or interleukin-12(IL-12). However, little is known about whether and how both antitumor mechanisms act together, although in vivo such an interaction must exist. Here we demonstrate in vitro, that pretreatment of human LS 174T colon cancer cells with nontoxic concentrations of tributyrin augments the sensitivity to spontaneous NK cell activity two-fold. However, when NK cells have been activated with an optimized combination of IL-2 and IL-12, the immunocytotoxicity increases up to five-fold (from 14% to 70%), versus a 3.8-fold increase against untreated cancer cells. These effects are accompanied by increased IFN-γ secretion and decreased TGF-β₁ secretion. Tributyrin is found to be a potent inducer of ICAM-1, LFA-3 and Fas on target cells corresponding to an increase of the FasL expression by IL-2/IL-12 on the effector cells. Our data suggest a synergistic link between induction of tumor cell differentiation and immunological defense mechanisms that may provide a rational basis for the improvement of clinical protocols, especially for colon cancer. Received: 6 November 2000 / Accepted: 15 December 2000     

Suppression of mucin 2 enhances the proliferation and invasion of LS174T human colorectal cancer cells

Altered expression of MUC2 (mucin 2) is related to tumour development in colorectal cancer. Colorectal mucinous carcinomas are positive for MUC2 expression, whereas MUC2 is down-regulated in non-mucinous adenocarcinomas. In the present study, we down-regulated MUC2 expression by RNAi (RNA interference) and investigated the in vitro and in vivo effects on the proliferation and invasion/migration potential of the LS174T human colorectal cancer cells. The LS174T cell line is a goblet-cell-like colorectal cancer cell line that continuously produces high levels of MUC2. Inhibition of MUC2 expression in vitro by transfection of LS174T cells with the recombinant plasmid pcDNA6.2-GW/EmGFP-miR-MUC2 led to the production of a stably transfected MUC2-RNAi LS174T cell line. The proliferation and invasion/migration of MUC2-RNAi cells in vitro were significantly higher than those in control cells, as assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide], colony formation and transwell assays. Subcutaneous injection of MUC2-RNAi LS174T cells into nude mice resulted in the development of subcutaneous tumours visible to the naked eye after 1 week. The growth rate of tumours derived from MUC2-RNAi LS174T cells was greater than that of tumours derived from control cells. Ki67 and matrix metalloproteinase-9 proteins were detected by immunohistochemistry in the xenografts. The expression levels of these proteins were higher in the MUC2-RNAi-derived xenografts than in xenografts derived from control cells. Although the role of MUC2 in colorectal tumorigenesis is not fully understood, these results strongly suggest a relationship between the proliferation and invasion of LS174T cells and the expression of MUC2.     

The microbe-derived short chain fatty acid butyrate targets miRNA-dependent p21 gene expression in human colon cancer

Colonic microbiota ferment non-absorbed dietary fiber to produce prodigious amounts of short chain fatty acids (SCFAs) that benefit the host through a myriad of metabolic, trophic, and chemopreventative effects. The chemopreventative effects of the SCFA butyrate are, in part, mediated through induction of p21 gene expression. In this study, we assessed the role of microRNA(miRNA) in butyrate's induction of p21 expression. The expression profiles of miRNAs in HCT-116 cells and in human sporadic colon cancers were assessed by microarray and quantitative PCR. Regulation of p21 gene expression by miR-106b was assessed by 3' UTR luciferase reporter assays and transfection of specific miRNA mimics. Butyrate changed the expression of 44 miRNAs in HCT-116 cells, many of which were aberrantly expressed in colon cancer tissues. Members of the miR-106b family were decreased in the former and increased in the latter. Butyrate-induced p21 protein expression was dampened by treatment with a miR-106b mimic. Mutated p21 3'UTR-reporter constructs expressed in HCT-116 cells confirmed direct miR-106b targeting. Butyrate decreased HCT-116 proliferation, an effect reversed with the addition of the miR-106b mimic. We conclude that microbe-derived SCFAs regulate host gene expression involved in intestinal homeostasis as well as carcinogenesis through modulation of miRNAs.     

<Emphasis Type="Italic">Listeria </Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> internalins bind to the human intestinal mucin MUC2

Listeria monocytogenes cross the intestinal barrier causing systemic infections with high mortality rates. Intestinal infection triggers release of intestinal mucus. We show that three L. monocytogenes internalins, InlB, InlC and InlJ all bound to MUC2 (the major component of intestinal mucus), but not to the cell surface mucin MUC1. Binding was strongest to InlB>InlC>InlJ (P < 0.001). Listerial internalins are characterized by their internalin domain, composed by leucine rich repeats (LRR) followed by an immunogloblin-like region. We report here that the internalin domain of the InlJ protein also bound MUC2, suggesting that an internalin domain is sufficient to bind to MUC2.     

The expression of human FUT1 in HT-29/M3 colon cancer cells instructs the glycosylation of MUC1 and MUC5AC apomucins

Recently, we have reported that in normal gastric epithelium, the expression of gastric apomucins MUC5AC and MUC6 is associated with the specific expression of type 1 and type 2 Lewis antigens, and FUT2 and FUT1 fucosyltransferases, respectively. Until now, there are no data demonstrating the direct implication of specific glycosyltransferases in the specific patterns of apomucin glycosylation.HT29/M3 colon cancer cell line express MUC1, MUC5AC, type 1 Lewis antigens and FUT2 but not type 2 structures and FUT1, as it occurs in the epithelial cells of the gastric superficial epithelium. These cells were transfected with the cDNA of human FUT1, the -1,2-fucosyltransferase responsible for the synthesis of type 2 Lewis antigens, to assess the implication of FUT1 in the glycosylation of MUC1 and MUC5AC.The M3-FUT1 clones obtained express high levels of type 2 Lewis antigens: H type 2 and Ley antigens. Immunoprecipitation of MUC1 and MUC5AC apomucins gives the direct evidence that FUT1 catalyses the addition of -1,2-fucose to these apomucins, supporting the hypothesis that the pattern of apomucin glycosylation is not only instructed by the mucin primary sequence but also by the set of glycosyltransferases expressed in each specific cell type.     

Decrease in uptake of arachidonic acid by indomethacin in LS174T human colon cancer cells; a novel cyclooxygenase-2-inhibition-independent effect

Nonsteroidal anti-inflammatory drugs (NSAIDs) have chemopreventive activity and may be suitable for treatment of colorectal cancer. A popular and potent NSAID, indomethacin, is known to cause serious side-effects, for this reason its therapeutic usefulness is limited. However, these side-effects are likely to be attributed to the additional effects of indomethacin besides its cyclooxygenase inhibition. In this study, we examined the effect of indomethacin on arachidonic acid uptake using LS174T human colon cancer cells. We here show that treatment of LS174T cells with indomethacin reduced arachidonic acid uptake as well as reduced expressions of fatty acid translocase/CD36 and peroxisome proliferators-activated receptor γ. Since arachidonic acid is a major substrate of inflammatory mediators such as prostaglandins and leukotrienes, we believe this novel effect of indomethacin may apply to new treatment strategies that aim to suppress these mediators by decreasing the uptake of their substrates, which would eventually inhibit colorectal cancer malignancy.     

Indomethacin decreases EP2 prostanoid receptor expression in colon cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) can decrease the risk of colorectal cancer; however, it has not been established if this effect is solely through their ability to inhibit cyclooxygenase (COX). In this study the effects of indomethacin, a potent NSAID and nonselective COX inhibitor, was examined in LS174T human colon cancer cells. These cells were found to express EP2 prostanoid receptors, but not the EP1, EP3 or EP4 subtypes. Pretreatment of LS174T cells with indomethacin produced a complete inhibition of prostaglandin E(2) (PGE(2)) stimulated cyclic AMP (cAMP) formation in a dose dependent manner with an IC(50) of 21 microM. Interestingly, the inhibition of PGE(2)-stimulated cAMP formation by indomethacin was accompanied by a decrease in EP2 mRNA expression and by a decrease in the whole cell specific binding of [(3)H]PGE(2). Thus, treatment of LS174T cells with indomethacin causes a down regulation of EP2 prostanoid receptors expression that may be independent of COX inhibition.     

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line

We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A_2A receptor (A_2AR) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3′,5′-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A_2AR expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A_2AR agonists may have therapeutic relevance.     

2-Carba cyclic phosphatidic acid inhibits lipopolysaccharide-induced prostaglandin E2 production in a human macrophage cell line

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that contains a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Using mouse models for multiple sclerosis (cuprizone-induced demyelination and experimental autoimmune encephalomyelitis) and traumatic brain injury, we revealed that cPA and its metabolically stabilized cPA derivative, 2-carba-cPA (2ccPA), have potential to protect against neuroinflammation. In this study, we investigated whether 2ccPA has anti-inflammatory effect on peripheral immune function or not using inflammation-induced macrophages-like cell line, THP-1 monocytes differentiated by phorbol 12-myristate 13-acetate (PMA). Lipopolysaccharide (LPS)-stimulated THP-1 cells were found to have higher expression of the mRNAs of several inflammation-related cytokines and of the enzyme cyclooxygenase-2 (Cox-2); however, when THP-1 cells were stimulated by LPS in the presence of 2ccPA, the increase in the expression of pro-inflammatory cytokine and Cox-2 mRNA was attenuated. 2ccPA treatment also decreased the amount of prostaglandin E2 (PGE2) produced by LPS-stimulated THP-1 cells and decreased expression of the mRNA of prostaglandin E receptor 2 (EP2, PTGER2), a PGE2 receptor that mediates inflammation. These results indicate that 2ccPA has anti-inflammatory properties.     

Glycosylation of the two <Emphasis Type="Italic">O</Emphasis>-glycosylated domains of human MUC2 mucin in patients transposed with artificial urinary bladders constructed from proximal colonic tissue

Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2–6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.     

Lipotropes regulate bcl-2 gene expression in the human breast cancer cell line,MCF-7

Lipotropes, a methyl group containing nutrients, including choline, methionine, folic acid, and vitamin B(12), are essential nutrients for humans. They are important methyl donors that interact in the metabolism of one-carbon units and are essential for the synthesis and methylation of deoxyribonucleic acid. The purpose of this study was to examine the effects of excess lipotropes on the growth of a human breast cancer cell line, MCF-7, and normal mammary cells, MCF-10A, in culture. Both cell lines were grown in basal culture medium for 24 h and then switched to medium supplemented with 50 times the amount of each lipotrope as basal culture medium (control). Although there were no significant differences in growth between treatments in either cell line, gene array and Northern analysis revealed that expression of bcl-2 was decreased in lipotrope-treated MCF-7 cells. The ability to induce tumor cell death could have many uses in the prevention and treatment of cancer. Bcl-2 regulates apoptosis and has been shown to directly affect the sensitivity of cancer cells to chemotherapy agents, and it is suggested that strategies designed to block Bcl-2 might prove useful in sensitizing tumor cells to chemotherapy-induced apoptosis. This study shows that although excess lipotropes do not inhibit the growth of breast cancer cells, they can down-regulate the bcl-2 gene, suggesting that lipotropes may increase the susceptibility of breast cancer cells to anticancer drugs.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.     

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo.In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.     

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage.     

双歧杆菌脂磷壁酸对结肠癌细胞CD44v6和MMP-2表达的影响

目的研究双歧杆菌脂磷壁酸(LTA)对结肠癌细胞中CD44v6与基质金属蛋白酶2(MMP-2)表达的影响,探讨其在抑制结肠癌转移中的作用。方法结肠癌LoVo细胞及HT-29细胞用含50 mg/L双歧杆菌LTA的培养液培养24 h后,RT-PCR和免疫细胞化学染色检测CD44v6和MMP-2在结肠癌细胞中的表达变化。结果结肠癌LoVo细胞及HT-29细胞中CD44v6和MMP-2的mRNA和蛋白质均呈高表达,经双歧杆菌LTA处理后,其表达均明显下降,与对照组比较,差异有非常显著性(P0.01)。结论双歧杆菌LTA可能通过下调CD44v6和MMP-2的表达来抑制结肠癌的转移。