Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells

Aspirin consumption has been reported to be able to reduce colorectal cancer risk in humans and in animal models of colon carcinogenesis. Although the mechanism involved in such an effect is not yet clear, both prostaglandin-dependent and -independent effects have been proposed. Using HT-29 Glc(-/+)cells, which originate from a human colon adenocarcinoma, we demonstrated in this study a dose-dependent effect of millimolar concentration of aspirin on cell growth that was concomitant with a rapid accumulation of the cells in the G0/G1 phase, followed by an accumulation in the G2/M phase and by a minor increase in the proportion of cells undergoing nuclear condensation. Cell membrane integrity and cell release into the culture medium were not affected by this treatment. The aspirin effects were apparently unrelated to prostaglandin biosynthesis inhibition, since although these cells were found to express high levels of cyclooxygenase 1 (COX-1) and low levels of COX-2 proteins, they did not produce any measurable net amounts of prostaglandins, based on both utilization of radiolabelled arachidonic acid and the radioimmunoassay of prostaglandins E2 and F2 alpha. In contrast, we identified polyamine biosynthesis as a cellular target of aspirin, since the treatment of HT-29 Glc(-/+) cells with aspirin reduced the flux of L-ornithine through ornithine decarboxylase, an effect that could not be explained by an acute action of the drug on the ornithine decarboxylase catalytic activity. Since polyamine biosynthesis is strictly necessary for HT-29 cell growth, our data suggest that reduced flux through ornithine decarboxylase may participate in the antiproliferative activity of aspirin towards colonic tumoral cells. It is concluded that in HT-29 Glc(-/+) cells that are not functional for prostaglandin production, aspirin can affect cell growth, cell cycle, and polyamine biosynthesis without affecting cell membrane integrity.     

Adaptative increase of ornithine production and decrease of ammonia metabolism in rat colonocytes after hyperproteic diet ingestion

Chronic high-protein consumption leads to increased concentrations of NH(4)(+)/NH(3) in the colon lumen. We asked whether this increase has consequences on colonic epithelial cell metabolism. Rats were fed isocaloric diets containing 20 (P20) or 58% (P58) casein as the protein source for 7 days. NH(4)(+)/NH(3) concentration in the colonic lumen and in the colonic vein blood as well as ammonia metabolism by isolated surface colonic epithelial cells was determined. After 2 days of consumption of the P58 diet, marked increases of luminal and colonic vein blood NH(4)(+)/NH(3) concentrations were recorded when compared with the values obtained in the P20 group. Colonocytes recovered from the P58 group were characterized at that time and thereafter by an increased capacity for l-ornithine and urea production through arginase (P < 0.05). l-Ornithine was mostly used in the presence of NH(4)Cl for the synthesis of the metabolic end product l-citrulline. After 7 days of the P58 diet consumption, however, the ammonia metabolism into l-citrulline was found lower (P < 0.01) when compared with the values measured in the colonocytes recovered from the P20 group despite any decrease in the related enzymatic activities (i.e., carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase). This decrease was found to coincide with a return of blood NH(4)(+)/NH(3) concentration in colonic portal blood to values close to the one recorded in the P20 group. In response to increased NH(4)(+)/NH(3) concentration in the colon, the increased capacity of the colonocytes to synthesize l-ornithine is likely to correspond to an elevated l-ornithine requirement for the elimination of excessive blood ammonia in the liver urea cycle. Moreover, in the presence of NH(4)Cl, colonocytes diminished their synthesis capacity of l-citrulline from l-ornithine, allowing a lower cellular utilization of this latter amino acid. These results are discussed in relationship with an adaptative process that would be related to both interorgan metabolism and to the role of the colonic epithelium as a first line of defense toward luminal NH(4)(+)/NH(3) concentrations.     

Effects of agmatine accumulation in human colon carcinoma cells on polyamine metabolism, DNA synthesis and the cell cycle

Putrescine, spermidine and spermine are low molecular polycations that play important roles in cell growth and cell cycle progression of normal and malignant cells. Agmatine (1-amino-4-guanidobutane), another polyamine formed through arginine decarboxylation, has been reported to act as an antiproliferative agent in several non-intestinal mammalian cell models. Using the human colon adenocarcinoma HT-29 Glc(-/+) cell line, we demonstrate that agmatine, which markedly accumulated inside the cells without being metabolised, exerted a strong cytostatic effect with an IC50 close to 2 mM. Agmatine decreased the rate of L-ornithine decarboxylation and induced a 70% down-regulation of ornithine decarboxylase (ODC) expression. Agmatine caused a marked decrease in putrescine and spermidine cell contents, an increase in the N1-acetylspermidine level without altering the spermine pool. We show that agmatine induced the accumulation of cells in the S and G2/M phases, reduced the rate of DNA synthesis and decreased cyclin A and B1 expression. We conclude that the anti-metabolic action of agmatine on HT-29 cells is mediated by a reduction in polyamine biosynthesis and induction in polyamine degradation. The decrease in intracellular polyamine contents, the reduced rate of DNA synthesis and the cell accumulation in the S phase are discussed from a causal perspective.     

N-glycosylation modification of proteins is an early marker of the enterocytic differentiation process of HT-29 cells

The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.     

Kallikrein-related peptidase signaling in colon carcinoma cells: targeting proteinase-activated receptors

We hypothesized that kallikrein-related peptidase 14 (KLK14) is produced by colonic tumors and can promote tumorigenesis by activating proteinase-activated receptors (PARs). We found that KLK14 is expressed in human colon adenocarcinoma cells but not in adjacent cancer-free tissue; KLK14 mRNA, present in colon cancer, leads to KLK14 protein expression and secretion; and KLK14 signals viaPAR-2 in HT-29 cells to cause (1) receptor activation/internalization, (2) increases in intracellular calcium, (3) stimulation of ERK1/2/MAP kinase phosphorylation, and (4) cell proliferation. We suggest that KLK14, acting via PAR-2, represents an autocrine/paracrine regulator of colon tumorigenesis.     

Adaptative metabolic response of human colonic epithelial cells to the adverse effects of the luminal compound sulfide

Hydrogen sulfide (H₂S), a bacterial metabolite present in the lumen of the large intestine, is able to exert deleterious effects on the colonic epithelium. The mechanisms involved are still poorly understood, the reported effect of sulfide being its capacity to reduce n-butyrate β-oxidation in colonocytes. In this work, we studied both the acute effect of the sodium salt of H₂S on human colonic epithelial cell metabolism and the adaptative response of these cells to the pre-treatment with this agent. Using the human colon carcinoma epithelial HT-29 Glc^−/+ cell model, we found that the acute effect of millimolar concentrations of NaHS was to inhibit l-glutamine, n-butyrate and acetate oxidation in a dose-dependent manner. Using micromolar concentrations of NaHS, a comparable effect but largely reversible was observed for O₂ consumption and cytochrome c oxidase activity. Pre-treatment with 1 mM NaHS induced several adaptative responses. Firstly, increased lactate release and decreased cellular oxygen consumption evidenced a Pasteur-like effect which only partly compensated for the altered mitochondrial ATP production. Thus, a decrease in the proliferation rate with a constant adenylate charge was observed. Secondly, in these pre-treated cells, NaHS induced a hypoxia-like effect on cytochrome c oxidase subunits I and II which were decreased. Thirdly, a mild uncoupling of mitochondrial respiration possibly resulting from an increase of UCP2 protein was observed. The NaHS antimitotic activity was not due to cellular apoptosis and/or necrosis but to a proportional slowdown in all cell cycle phases. These results are compatible with a metabolic adaptative response of the HT-29 colonic epithelial cells to sulfide-induced O₂ consumption reduction which, through the maintenance of a constant energetic load and an increased mitochondrial proton leak, would participate in the preservation of cellular viability.     

Butyrate metabolism in human colon carcinoma cells: implications concerning its growth-inhibitory effect

Butyrate and acetate are bacterial metabolites present in the large intestine lumen. Although butyrate is well known to inhibit the in vitro proliferation of human colon carcinoma cells in a process involving the hyperacetylation of specific nuclear histones, little is known about the possible link between butyrate metabolism and its growth-inhibitory effect. In a previous study (Leschelle et al., 2000, Eur J Biochem 267: 6435-6442), we showed that butyrate accumulates and is metabolized in HT-29 Glc(-/+) cells without increasing oxygen consumption. In the present study, using the same cell line incubated with (14)C-labeled butyrate, we determined that a minor part of (14)C from butyrate was recovered in nuclear histones. Unlike butyrate, acetate exerted no effect on cell growth but was a precursor for overall net histone acetylation. Although butyrate was able to increase the cellular AMP/ADP ratio, it did not affect the ATP cell content or the adenylate charge or the oxidation of endogenous L-glutamine. Butyrate oxidation was found to be markedly sensitive to the presence of other substrates with D-glucose decreasing this oxidation and L-malate stimulating it. Furthermore, in the presence of L-malate, the growth-inhibitory effect of butyrate was significantly weaker than in its absence. From these data, we conclude that the metabolism of butyrate downstream acetyl-CoA synthesis is not involved in the butyrate antiproliferative effect. The suggestion that butyrate metabolism in mitochondria is not used in these cells as a fuel but acts as a regulator of butyrate free concentrations (thus limiting its action upon cellular targets), is discussed.     

Effects of polysaccharides derived from Orostachys japonicus on induction of cell cycle arrest and apoptotic cell death in human colon cancer cells

Crude Orostachys japonicus polysaccharide extract (OJP) was prepared by hot steam extraction. Polysaccharides (OJPI) were separated from OJP by gel filtration chromatography and phenol-sulfuric acid assay. The average molecular weight of the OJPI was 30-50 kDa. The anti-proliferative effect of OJPI on HT-29 human colon cancer cells was investigated via morphology study, cell viability assay, apoptosis assay, cell cycle analysis, and cDNA microarray. OJPI inhibited proliferation and growth of HT29 cells and also stimulated apoptosis in a dose- and time-dependent manner. In cell cycle analysis, treatment with OJPI resulted in a marked increase of cells in the G0 (sub G1) and G2/M phases. To screen for genes involved in the induction of cell cycle arrest and apoptosis, the gene expression profiles of HT-29 cells treated with OJPI were examined by cDNA microarray, revealing that a number of genes were up- or down-regulated by OJPI. Whereas several genes involved in anti-apoptosis, cell proliferation and growth, and cell cycle regulation were down-regulated, expression levels of several genes involved in apoptosis, tumor suppression, and other signal transduction events were up-regulated. These results suggest that OJPI inhibits the growth of HT-29 human colon cancer cells by various apoptosis-aiding activities as well as apoptosis itself. Therefore, OJPI deserve further development as an effective agent exhibiting anticancer activity.     

Purification of a new intestinal anti-proliferative factor from normal human small intestine.

Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti-proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti-proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT-29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT-29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase i.v. probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation.     

Fucosyltransferase III and sialyl-Lex expression correlate in cultured colon carcinoma cells but not in colon carcinoma tissue

The potential contribution of fucosyltransferases to the overexpression of sialyl-Le^x antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le^x. This modulation of sialyl-Le^x was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le^x, the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le^x moiety in HT-29 cells but not in human colon carcinoma tissue.     

Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.

Butyrate is a short chain fatty acid (SCFA) produced by bacterial fermentation of dietary fibers in the colon lumen which severely affects the proliferation of colon cancer cells in in vitro experiments. Although butyrate is able to interfere with numerous cellular targets including cell cycle regulator expression, little is known about butyrate metabolism and its possible involvement in its effect upon colon carcinoma cell growth. In this study, we found that HT-29 Glc-/+ cells strongly accumulated and oxidized sodium butyrate without producing ketone bodies, nor modifying oxygen consumption nor mitochondrial ATP synthesis. HT-29 cells accumulated and oxidized sodium acetate at a higher level than butyrate. However, sodium butyrate, but not sodium acetate, reduced cell growth and increased the expression of the cell cycle effector cyclin D3 and the inhibitor of the G1/S cdk-cyclin complexes p21/WAF1/Cip1, demonstrating that butyrate metabolism downstream of acetyl-CoA synthesis is not required for the growth-restraining effect of this SCFA. Furthermore, HT-29 cells modestly incorporated the 14C-labelled carbon from sodium butyrate into cellular triacylglycerols and phospholipids. This incorporation was greatly increased when D-glucose was present in the incubation medium, corresponding to the capacity of hexose to circulate in the pentose phosphate pathway allowing NADPH synthesis required for lipogenesis. Interestingly, when HT-29 cells were cultured in the presence of sodium butyrate, their capacity to incorporate 14C-labelled sodium butyrate into triacylglycerols and phospholipids was increased more than twofold. In such experimental conditions, HT-29 cells when observed under an electronic microscope, were found to be characterized by an accumulation of lipid droplets in the cytosol. Our data strongly suggest that butyrate acts upon colon carcinoma cells upstream of acetyl-CoA synthesis. In contrast, the metabolism downstream of acetyl-CoA [i.e. oxidation in the tricarboxylic acid (TCA) cycle and lipid synthesis] likely acts as a regulator of butyrate intracellular concentration.     

Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and μ-calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and μ-calpain may be involved in colon epithelial cell death induced by E. histolytica.     

Glycine-extended gastrin stimulates proliferation and inhibits apoptosis in colon cancer cells via cyclo-oxygenase-independent pathways

Glycine-extended gastrin (G-Gly) is an end product of processing of the progastrin precursor peptide that has a different spectrum of activity to amidated gastrin. G-Gly promotes cell proliferation in normal and malignant colonic epithelium but the mechanisms responsible are poorly understood. Prostaglandins produced by the cyclo-oxygenase (COX) enzymes have been implicated as downstream mediators of several growth factors, and COX inhibitors such as non-steroidal anti-inflammatory drugs inhibit the proliferation and invasiveness of colonic cancer and reduce the incidence of colon cancer. We have examined the mechanisms of the actions of G-Gly in HT-29 colon cancer cells. G-Gly induced a dose-dependent increase in cell proliferation that was insensitive to inhibition of either COX-1 or COX-2, but was abolished by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. G-Gly did not increase prostaglandin E2 production. Celecoxib induced apoptosis and reduced viable cell numbers in a COX-independent manner. G-Gly significantly reduced serum-starvation and celecoxib-induced apoptosis and this effect was also blocked by inhibition of the p38 MAP kinase, ERK and NF-kappaB pathways. Stimulation of HT-29 cells with G-Gly led to a rapid increase in ERK and p38 MAP kinase phosphorylation and increased nuclear translocation of active NF-kappaB. Activation of NF-kappaB was independent of ERK and p38 MAP kinase. G-Gly stimulates proliferation and inhibits apoptosis in colon cancer cells via COX-independent and ERK-, p38 MAP kinase-, and NF-kappaB-dependant pathways. Locally and systemically produced G-Gly may be important in reducing the beneficial effects of chemopreventative agents in colon cancer.     

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.     

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.     

MicroRNA-451 regulates AMPK/mTORC1 signaling and fascin1 expression in HT-29 colorectal cancer

The earlier studies have shown that Fascin1 (FSCN1), the actin bundling protein, is over-expressed in colorectal cancers, and is associated with cancer cell progression. Here, we aimed to understand the molecular mechanisms regulating FSCN1 expression by focusing on mammalian target of rapamycin (mTOR) signaling and its regulator microRNA-451. We found that microRNA-451 was over-expressed in multiple colorectal cancer tissues, and its expression was correlated with mTOR complex 1 (mTORC1) activity and FSCN1 expression. In cultured colorectal cancer HT-29 cells, knockdown of FSCN1 by RNAi inhibited cell migration and proliferation. Activation of mTORC1 was required for FSCN1 expression, HT-29 cell migration and proliferation, as RAD001 and rapamycin, two mTORC1 inhibitors, suppressed FSCN1 expression, HT-29 cell migration and proliferation. Meanwhile, forced activation of AMP-activated protein kinase (AMPK), the negative regulator of mTORC1, by its activators or by the genetic mutation, inhibited mTORC1 activation, FSCN1 expression, cell migration and proliferation. In HT-29 cells, we found that over-expression of microRNA-451 inhibited AMPK activation, causing mTORC1 over-activation and FSCN1 up-regulation, cells were with high migration ability and proliferation rate. Significantly, these effects by microRNA-451 were largely inhibited by mTORC1 inhibitors or the AMPK activator AICAR. On the other hand, knockdown of miRNA-451 by the treatment of HT-29 cells with miRNA-451 antagomir inhibited mTORC1 activation and FSCN1 expression. The proliferation and migration of HT-29 cells after miRNA-45 knockdown were also inhibited. Our results suggested that the over-expressed microRNA-451 in colon cancer cells might inhibit AMPK to activate mTORC1, which mediates FSCN1 expression and cancer cell progression.     

Neuromedin B and its receptor are mitogens in both normal and malignant epithelial cells lining the colon

Bombesin-like peptides are uniformly thought to act as mitogens in cancer. Yet by studying human tissues, we have recently shown that bombesin and its mammalian homologue gastrin-releasing peptide act as morphogens, promoting tumor differentiation when aberrantly upregulated in colon cancer. In contrast, little is known about the bombesin-like peptide neuromedin B (NMB) and its receptor (NMB-R) in the human gastrointestinal tract. We therefore studied their presence and function in normal and malignant human colonic epithelia. Anti-NMB monoclonal antibodies were made against keyhole limpet hemocyanin (KLH)-conjugated human NMB, whereas anti-NMB-R antibodies were raised in rabbits against KLH-conjugated peptides corresponding to the third intracellular loop and COOH-terminal tail of the receptor protein. NMB antibody recognized two bands at approximately 1.2 kDa and approximately 1.5 kDa. NMB-R antibodies recognized a band at 80 kDa (predicted 43 kDa); whereas treatment with the deglycosylating agent peptide-N-glycosidase generated bands at 65, 47, and 43 kDa. By immunohistochemistry, both NMB and NMB-R were expressed in normal and cancerous colonic epithelial tissues. In cancer, the amount of NMB was similar to that expressed by proliferating epithelial cells located within the crypt. In contrast, NMB-R expression was increased in cancer, with higher levels detected in better differentiated tumor cells. To assess NMB function, proliferation was determined in the nonmalignant human colonic epithelial cell line NCM-460 and in the colon cancer cell lines Caco-2 and HT-29. Exogenously added NMB was 50-100% more efficacious than gastrin-releasing peptide in causing tumor cell proliferation, whereas only NMB increased NCM-460 cell proliferation. These findings indicate that NMB and its receptor are coexpressed by proliferating cells in which they act in an autocrine fashion with similar and modest potency in both normal and malignant colonic epithelial cells.     

Synergistic effect of indomethacin and NGX6 on proliferation and invasion by human colorectal cancer cells through modulation of the Wnt/β-catenin signaling pathway

This study was designed to investigate whether indomethacin and NGX6 synergistically inhibit the growth and invasiveness of human colon cancer cells (HT-29 and SW620) and to elucidate the molecular mechanism of their action. Cell proliferation was assessed by MTT assay. Cell apoptosis was assessed by acridine orange/ethidium bromide staining (AO–EB) and annexin-V-FITC/PI assay. Invasive behaviors of colorectal cancer cells were examined by cell adhesion, migration, and invasion assays. Gap junctional intercellular communication (GJIC) was assessed by the scrape-loading/dye transfer technique. The subcellular localization and expression of β-catenin protein was examined by immunofluorescence staining and western blot analysis, respectively. Indomethacin and NGX6 had a synergistic effect on inhibiting proliferation and invasiveness of colon cancer HT-29 and SW620 cells, restoring GJIC of HT-29 and SW620, and suppressing translocation of β-catenin from the nucleus and cytoplasm to the plasma membrane. However, they did not have synergistic effects on enhancing apoptosis and suppressing extracellular matrix adhesion of HT-29 and SW620 cells. Indomethacin and NGX6 inhibit the proliferation and invasiveness of HT-29 and SW620 colon cancer cells by attenuating the WNT/ß-catenin signaling pathway.     

EGCG 对人结肠癌HT-29 细胞凋亡的影响以及对MMP-2，RECK 的 调节作用

目的:观察表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)对人结肠癌HT-29细胞增殖和凋亡的影响,并探讨其对MMP-2,RECK的调节作用。方法:体外培养人结肠癌HT-29细胞,MTT比色法检测EGCG对HT-29细胞的生长抑制作用;Histone/DNA ELISA检测细胞凋亡;FITC标记Annexin-V/PI双染流式细胞术分析凋亡细胞百分率;Western Blot和RT-PCR方法检测EGCG对MMP-2,RECK蛋白和mRNA表达的影响。结果:EGCG呈浓度和时间依赖性抑制HT-29细胞的增殖,并且增加HT-29细胞Histone/DNA碎片的渗漏;EGCG诱导HT-29细胞凋亡百分率增高;EGCG抑制MMP-2蛋白和mRNA的表达,促进RECK蛋白和mRNA的表达。结论:EGCG抑制人结肠癌HT-29细胞的增殖,促进其凋亡,并且呈浓度和时间依赖性;其作用机制可能与其下调MMP-2蛋白和mRNA的表达、上调RECK蛋白和mRNA的表达有关。     

Peroxisome proliferator-activated receptor gamma overexpression suppresses proliferation of human colon cancer cells

Peroxisome proliferator-activated receptor gamma (PPARγ) plays an important role in the differentiation of intestinal cells and tissues. Our previous reports indicate that PPARγ is expressed at considerable levels in human colon cancer cells. This suggests that PPARγ expression may be an important factor for cell growth regulation in colon cancer. In this study, we investigated PPARγ expression in 4 human colon cancer cell lines, HT-29, LOVO, DLD-1, and Caco-2. Real-time polymerase chain reaction (PCR) and Western blot analysis revealed that the relative levels of PPARγ mRNA and protein in these cells were in the order HT-29>LOVO>Caco-2>DLD-1. We also found that PPARγ overexpression promoted cell growth inhibition in PPARγ lower-expressing cell lines (Caco-2 and DLD-1), but not in higher-expressing cells (HT-29 and LOVO). We observed a correlation between the level of PPARγ expression and the cells' sensitivity for proliferation.