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.     

Modulation of CYP3A4 expression by ceramide in human colon carcinoma HT-29 cells

Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C(6)-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C(6)-dihydroceramide, a biologically inactive analog of C(6)-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-alpha (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.     

Global phosphoproteome of HT-29 human colon adenocarcinoma cells

Phosphorylation events in cellular signaling cascades triggered by a variety of cellular stimuli modulate protein function, leading to diverse cellular outcomes including cell division, growth, death, and differentiation. Abnormal regulation of protein phosphorylation due to mutation or overexpression of signaling proteins often results in various disease states. We provide here a list of protein phosphorylation sites identified from HT-29 human colon adenocarcinoma cell line by immobilized metal affinity chromatography (IMAC) combined with liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis. In this study, proteins extracted from HT-29 whole cell lysates were digested with trypsin and carboxylate groups on the resulting peptides were converted to methyl esters. Derivatized phosphorylated peptides were enriched using Fe(3+)-chelated metal affinity resin. Phosphopeptides retained by IMAC were separated by high performance liquid chromatography (HPLC) and analyzed by electrospray ionization-quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. We identified 238 phosphorylation sites, 213 of which could be conclusively localized to a single residue, from 116 proteins by searching MS/MS spectra against the human protein database using MASCOT. Peptide identification and phosphorylation site assignment were confirmed by manual inspection of the MS/MS spectra. Many of the phosphorylation sites identified in our results have not been described previously in the scientific literature. We attempted to ascribe functionality to the sites identified in this work by searching for potential kinase motifs with Scansite (http://scansite.mit.edu) and obtaining information on kinase substrate selectivity from Pattern Explorer (http://scansite.mit.edu/pe). The list of protein phosphorylation sites identified in the present experiment provides broad information on phosphorylated proteins under normal (asynchronous) cell culture conditions. Sites identified in this study may be utilized as surrogate bio-markers to assess the activity of selected kinases and signaling pathways from different cell states and exogenous stimuli.     

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

AimsStress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved.Main methodsThe effect of adrenaline on HT-29 cell proliferation was determined by [³H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E₂ (PGE₂) release were determined by zymography and enzyme immunoassay, respectively.Key findingsAdrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE₂ release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β₁- and β₂-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE₂ release in HT-29 cells.SignificanceThese results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β₁- and β₂-adrenoceptors by a COX-2 dependent pathway.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

Sphingoid bases and ceramide induce apoptosis in HT-29 and HCT-116 human colon cancer cells

Complex dietary sphingolipids such as sphingomyelin and glycosphingolipids have been reported to inhibit development of colon cancer. This protective role may be the result of turnover to bioactive metabolites including sphingoid bases (sphingosine and sphinganine) and ceramide, which inhibit proliferation and stimulate apoptosis. The purpose of the present study was to investigate the effects of sphingoid bases and ceramides on the growth, death, and cell cycle of HT-29 and HCT-116 human colon cancer cells. The importance of the 4,5-trans double bond present in both sphingosine and C(2)-ceramide (a short chain analog of ceramide) was evaluated by comparing the effects of these lipids with those of sphinganine and C(2)-dihydroceramide (a short chain analog of dihydroceramide), which lack this structural feature. Sphingosine, sphinganine, and C(2)-ceramide inhibited growth and caused death of colon cancer cells in time- and concentration-dependent manners, whereas C(2)-dihydroceramide had no effect. These findings suggest that the 4,5-trans double bond is necessary for the inhibitory effects of C(2)-ceramide, but not for sphingoid bases. Evaluation of cellular morphology via fluorescence microscopy and quantitation of fragmented low-molecular weight DNA using the diphenylamine assay demonstrated that sphingoid bases and C(2)-ceramide cause chromatin and nuclear condensation as well as fragmentation of DNA, suggesting these lipids kill colon cancer cells by inducing apoptosis. Flow cytometric analyses confirmed that sphingoid bases and C(2)-ceramide increased the number of cells in the A(0) peak indicative of apoptosis and demonstrated that sphingoid bases arrest the cell cycle at G(2)/M phase and cause accumulation in the S phase. These findings establish that sphingoid bases and ceramide induce apoptosis in colon cancer cells and implicate them as potential mediators of the protective role of more complex dietary sphingolipids in colon carcinogenesis.     

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.     

DCC regulates cell adhesion in human colon cancer derived HT-29 cells and associates with ezrin

The deleted in colorectal cancer (DCC) gene encodes a 170- to 190-kDa protein of the Immunoglobulin superfamily. Firstly identified as a tumor suppressor gene in human colorectal carcinomas, the main function for DCC has been described in the nervous system as part of a receptor complex for netrin-1. Moreover, roles in mucosecretory cell differentiation and as inducer of apoptosis have also been reported. DCC knockout mice supported a crucial role for this gene in axonal migration, yet questioned its implication in tumor suppression and mucosecretory differentiation. The work presented here demonstrates that a DCC-transfected HT-29 colonic human cell line (HT-29/DCC) displays an increase in cell-cell adhesion to the detriment of cell-matrix interactions: HT-29/DCC cells exhibit more and better-structured desmosomes while focal adhesions and hemidesmosomes are disrupted. HT-29/DCC cells show no changes in adherent junctions but upon treatment with TPA, HT-29/DCC cells show resistance to scattering, and maintain E-cadherin in the membrane. In addition, the actin cytoskeleton is affected in HT-29/DCC cells: stress fibers are disrupted while cortical actin remains intact. We identified a putative ERM-M (ezrin/radixin/moesin and merlin) binding domain in the juxtamembrane region of the DCC protein. In vitro pull-down assays demonstrate the interaction of the DCC cytoplasmic domain with the N-terminal region of ezrin and merlin, and co-immunoprecipitation assays in transiently DCC-transfected COS-1 cells showed that the interaction between DCC and ezrin also takes place in vivo. Altogether, our results suggest that DCC could regulate cell adhesion and migration through its association with ERM-M proteins.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cell growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were almost completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.     

Targeting miR-21 enhances the sensitivity of human colon cancer HT-29 cells to chemoradiotherapy in vitro

5-Fluorouracil (5-FU) is a classic chemotherapeutic drug that has been widely used for colorectal cancer treatment, but colorectal cancer cells are often resistant to primary or acquired 5-FU therapy. Several studies have shown that miR-21 is significantly elevated in colorectal cancer. This suggests that this miRNA might play a role in this resistance. In this study, we investigated this possibility and the possible mechanism underlying this role. We showed that forced expression of miR-21 significantly inhibited apoptosis, enhanced cell proliferation, invasion, and colony formation ability, promoted G1/S cell cycle transition and increased the resistance of tumor cells to 5-FU and X radiation in HT-29 colon cancer cells. Furthermore, knockdown of miR-21 reversed these effects on HT-29 cells and increased the sensitivity of HT-29/5-FU to 5-FU chemotherapy. Finally, we showed that miR-21 targeted the human mutS homolog2 (hMSH2), and indirectly regulated the expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD). These results demonstrate that miR-21 may play an important role in the 5-FU resistance of colon cancer cells.     

Mithramycin inhibits etoposide resistance in glucose-deprived HT-29 human colon carcinoma cells

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha, rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.     

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.     

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.     

Effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro

Colon cancer is the third most frequent cancer in humans. Carica papaya leaves are vegetable foods consumed by most people around the world; it has potential as an anticancer. Therefore it is of interest to investigate the effect of Carica papaya on beta catenin and Wnt mRNA expression in human colon cancer (HT-29) cells in vitro. Human Colon cancer cell line (HT-29) was purchased from the National Centre for Cell Sciences, Pune, India. Cell viability test was done by MTT assay. Gene expression analysis was done by Real Time-PCR. The obtained data were analyzed statistically by one-way analysis of variance and Duncan''s multiple range test with Graph Pad Prism version 5 to analyze the significance of individual variations among the control and experimental groups. The significance was considered at p<0.05 level in Duncan''s test. Carica papaya caused a marked increase in cell death in a dose dependent manner. At the end of 48 hours, maximum inhibition was at 300 and 400 µg/ml. Carica papaya has significantly reduced the mRNA expression of Wnt and beta catenin (p<0.05). Data showed that Carica papaya leaf extract has anticancer activity on Colon cancer cell lines (HT-29).     

Tumor suppressor gene NGX6 induces changes in protein expression profiles in colon cancer HT-29 cells

Nasopharyngeal carcinoma-associated gene 6 (NGX6; syn. transmembrane protein 8B, TMEM8B) is a recently identified tumor suppressor gene. The underlying mechanisms by which the gene inhibits tumor development are not completely known. To further understand the function of the gene's protein product NGX6, in the present study, we employed two-dimensional difference gel electrophoresis to analyze the protein expression profiles of colon cancer HT-29 cells stably transfected with the gene NGX6. The differentially expressed proteins were selected and identified by matrix-assisted laser desorption/ionization coupled with time-of-flight tandem mass spectrometry. The results showed that 12 proteins were down-regulated and 4 were up-regulated in NGX6-transfected HT-29 cells, compared with vector-transfected HT-29 cells. The MS results were verified by western blot. Bioinformatic analysis showed that these proteins are involved in cell proliferation, metastasis, apoptosis, cytoskeletal structure, metabolism, and signal transduction, suggesting that NGX6 may inhibit colon cancer through the regulation of these biological processes.     

Cyclooxygenase-independent effects of aspirin on HT-29 human colon cancer cells, revealed by oligonucleotide microarrays

Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation of human colon cancer cells in vitro. Transmission electron microscope detected morphological features of apoptosis in the aspirin-treated (5 mM, 72 h) HT-29 cells in which cyclooxygenoase-2 is catalytically inactive. We investigated aspirin-induced genome-wide expression changes in HT-29 cells and further studied the time- and concentration-dependent expression changes in 374 apoptosis-related genes, which is the first to show stimulation of genome-wide expression of HT-29 cells by aspirin. The most marked effects of aspirin are on ribosome assembly and rRNA metabolism, which could explain why the quasi-apoptotic morphological changes are not accompanied by a classical DNA ladder. These findings demonstrate that aspirin induces apoptosis in HT-29 cells, bolstering the hypothesis that apoptosis may be a mechanism by which NSAIDs inhibit colon carcinogenesis.