Overexpression of protein kinase C in HT29 colon cancer cells causes growth inhibition and tumor suppression.

By using a retrovirus-derived vector system, we generated derivatives of the human colon cancer cell line HT29 that stably overexpress a full-length cDNA encoding the beta 1 isoform of rat protein kinase C (PKC). Two of these cell lines, PKC6 and PKC7, displayed an 11- to 15-fold increase in PKC activity when compared with the C1 control cell line that carries the vector lacking the PKC cDNA insert. Both of the overexpresser cell lines exhibited striking alterations in morphology when exposed to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Following exposure to TPA, PKC6 and PKC7 cells displayed increased doubling time, decreased saturation density, and loss of anchorage-independent growth in soft agar; but these effects were not seen with the C1 cells. Also, in contrast to the control cells, the PKC-overproducing cells failed to display evidence of differentiation, as measured by alkaline phosphatase activity, when exposed to sodium butyrate. In addition, the PKC-overexpresser cells displayed decreased tumorigenicity in nude mice, even in the absence of treatment with TPA. These results provide the first direct evidence that PKC can inhibit tumor cell growth. Thus, in some tumors, PKC might act as a growth-suppressor gene.     

Retinoic acid receptor alpha mediates growth inhibition by retinoids in human colon carcinoma HT29 cells.

Although retinoids have been suggested to inhibit chemically induced colon carcinogenesis, the molecular mechanisms underlying retinoid-mediated growth regulation in colon carcinoma cells are unknown. Therefore, we investigated the biological effects of retinoids on growth in HT29 colon carcinoma cells. All-trans retinoic acid (ATRA) treatment of HT29 cells resulted in a profound inhibition of anchorage-independent growth without biochemical or morphological evidence for induction of differentiation. Treatment with the selective RARalpha agonist Ro 40-6055 completely mimicked the effects of ATRA on growth and transactivation of a betaRAREx2-luciferase reporter construct, while RARbeta- and gamma-specific analogues were ineffective. Furthermore, ATRA-regulated growth and transactivation could be completely blocked by a RARalpha-selective receptor antagonist. Thus, ATRA potently inhibits anchorage-independent growth in HT29 cells and this effect is mainly if not exclusively mediated by the retinoic acid receptor alpha.     

Insulin controls key steps of carbohydrate metabolism in cultured HT29 colon cancer cells.

Effects of insulin on key steps of carbohydrate metabolism were investigated in cultured HT29 colon cancer cells by two different approaches, i.e. incubation of the cells either in the absence or in the presence of glucose in the medium. In glucose-deprived cells, insulin decreased glycogen breakdown, but did not affect polysaccharide levels when glucose was present. Glycogen synthase became activated after insulin treatment in both conditions, even though the activation was more evident when glucose was omitted. No effect on glycogen phosphorylase activity was evident under our experimental conditions. In cells incubated with glucose, the hormone stimulated in a dose-dependent manner the rates of glucose uptake and lactate release. Concomitantly with the increase in glycolytic rate, insulin caused a strong increase in fructose 2,6-bisphosphate. This effect was not observed in the absence of glucose. It is concluded that the carbohydrate metabolism of cultured HT29 cells responds to insulin, making this biological model suitable for investigations in vitro on the mechanism of insulin action.     

Growth and intestinal differentiation are independently regulated in HT29 colon cancer cells

The polar-planar compound hexamethylene bisacetamide (HMBA) can inhibit HT29 colon carcinoma cell growth and induce a more benign phenotype, as defined by decreased anchorage-independent clonogenicity, loss of a cell surface malignancy marker, and decreased in vivo tumorigenicity. The principle aim of this study was to determine whether HMBA's effects on HT29 cell growth and biologic behavior correlate with effects on intestinal differentiation. Parallel studies were performed with sodium butyrate (NaBT), a potent inducer of intestinal differentiation HT29 cell growth, proliferation, and markers of intestinal differentiation were assayed after short- and long-term treatment with HMBA, NaBT, or the combination. Both 5 mM HMBA and 5 mM NaBT were potent inhibitors of monolayer growth; in combination their effects were nearly additive. Inhibition of DNA synthesis was detectable within 6 h of treatment and was preceded by down-regulation of c-myc expression. Soft agar clonogenicity was also decreased by 90%, > 99%, and > 99% by HMBA, NaBT, and the combination, respectively. Despite these parallel effects on growth and in vitro markers of a benign phenotype, effects on intestinal differentiation were discordant. NaBT induced significant increases in membrane-associated alkaline phosphatase activity, cytosolic mucin content, PAS⁺/diastase-resistant cells, and ultrastructural evidence of intestinal cell differentiation. HMBA not only failed to induce markers of intestinal differentiation, but attenuated NaBT's effects when used in combination. These data suggest that growth and intestinal differentiation may be independently regulated in HT29 cells. They also suggest that expression of intestinal markers of differentiation is not a prerequisite for the acquisition of a more benign phenotype. © 1994 Wiley-Liss, Inc.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells

Growth factor-mediated stimulation of epithelial cells induces the disassembly of E-cadherin-mediated cell-cell adhesion. We found that overexpression of a disintegrin and metalloprotease 9 (ADAM9) enhanced growth factor-mediated induction of endocytosis and dynamic recycling of E-cadherin in HT29 human colon cancer cells. In addition, ubiquitination and degradation of E-cadherin were reduced in these cells. ADAM9 constitutively interacted with E-cadherin, and the two proteins co-localized at the plasma membrane of HT29 cells. Administration of a metalloprotease inhibitor or overexpression of an ADAM9 mutant lacking metalloprotease activity attenuated growth factor-dependent endocytosis and recycling of E-cadherin as well as scattering of HT29 cells. These results suggest that the metalloprotease activity of ADAM9 mediates growth factor-induced endocytosis and dynamic recycling of E-cadherin and prevents E-cadherin degradation.     

Caffeic acid phenethyl ester mediates apoptosis in serum-starved HT29 colon cancer cells through modulation of heat shock proteins and MAPK pathways

Colorectal cancer (CRC) is among the most prevalent gastrointestinal cancers of epithelial origin worldwide, with over 2 million cases detected every year. Emerging evidence suggests a significant increase in the levels of inflammatory and stress-related markers in patients with CRC, indicating that oxidative stress and lipid peroxidation may influence signalling cascades involved in the progression of the disease. However, the precise molecular and cellular basis underlying CRC and their modulations during bioactive compound exposure have not yet been deciphered. This study examines the effect of caffeic acid phenethyl ester (CAPE), a natural bioactive compound, in HT29 CRC cells grown under serum-supplemented and serum-deprived conditions. We found that CAPE inhibited cell cycle progression in the G2/M phase and induced apoptosis. Migration assay confirmed that CAPE repressed cancer invasiveness. Protein localisation by immunofluorescence microscopy and protein expression by western blot analysis reveal increased expressions of key inflammatory signalling mediators such as p38α, Jun N-terminal kinase and extracellular signal-regulated kinase (ERK) proteins. Molecular docking data demonstrates that CAPE shows a higher docking score of −5.35 versus −4.59 to known p38 inhibitor SB203580 as well as a docking score of −4.17 versus −3.86 to known ERK1/2 inhibitor AZD0364. Co-immunoprecipitation data reveals that CAPE treatment effectively downregulates heat shock protein (HSP) expression in both sera-supplemented and limited conditions through its interaction with mitogen-activated protein kinase 14 (MAPK14). These results suggest that stress induction via serum starvation in HT29 CRC cells leads to the induction of apoptosis and co-ordinated activation of MAPK-HSP pathways. Molecular docking studies support that CAPE could serve as an effective inhibitor to target p38 and MAPK compared to their currently known inhibitors.     

Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells.

Our previous studies have shown that the Galbeta1-3GalNAcalpha- (Thomsen-Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem. 274, 4890-4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 degrees C followed by culture of the cells at 37 degrees C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 +/- 2% of ABL in the medium and 14 +/- 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells.     

Carbohydrate metabolism in HT29 colon cancer cells cultured in a glucose free medium supplemented with inosine

1. Carbohydrate metabolism was studied in HT29 human colon cancer cells cultured in a glucose free medium supplemented with 2.8 mM inosine (HT29ino cells) in comparison with standard HT29 cells grown in the permanent presence of glucose (HT29Glc + cells) and with HT29Glc- cells which are adapted to grow permanently without glucose. 2. Inosine allows the standard cells to grow when glucose is lacking but surprisingly stops the growth of HT29Glc- cells. 3-mercaptopicolinate, an inhibitor of PEP-carboxykinase, does not hinder HT29ino cells to grow, which shows that gluconeogenesis from aspartate or pyruvate is not essential. It suggests that enough carbohydrate is supplied by the ribose moiety of inosine. 3. While standard HT29Glc + cells are highly glycolytic, it is not the case of HT29ino or HT29Glc- cells when glucose is given for few hours. When glucose is present for 24 hr or more, glycolytic rate increases in HT29ino cells and glycogen accumulates. 4. It is found that the pattern of enzymes activities related to carbohydrate metabolism in HT29ino cells is closer to that of HT29Glc + cells rather than to that of HT29Glc- cells. However, phosphofructokinase-1 activity, measured with saturating concentration of Fru-2,6-diP, is significantly lower in HT29ino cells. 5. Binding rate of hexokinase to mitochondria is similar in the three cell-lines. However, in HT29Glc- cells, bound hexokinase easily utilizes ATP generated by the mitochondria. By contrast, in HT29Glc+ and HT29ino cells, bound hexokinase is much more active with exogenous ATP, suggesting a functional defect in the mitochondria from these two latter cells.     

Insulin induces PFKFB3 gene expression in HT29 human colon adenocarcinoma cells

Fructose 2,6-bisphosphate is present at high concentrations in many established lines of transformed cells. It plays a key role in the maintenance of a high glycolytic rate by coupling hormonal and growth factor signals with metabolic demand. The concentration of fructose 2,6-bisphosphate is controlled by the activity of the homodimeric bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2). We report here the PFKFB-3 gene expression control by insulin in the human colon adenocarcinoma HT29 cell line. The incubation of these cells with 1 microM insulin resulted in an increase in the PFK-2 mRNA level after 6 h of treatment, this effect being blocked by actinomycin D. Furthermore, insulin induced ubiquitous PFK-2 protein levels, that were evident after a lag of 3 h and could be inhibited by incubation with cycloheximide.     

Role of caspase activation in butyrate-induced terminal differentiation of HT29 colon carcinoma cells

Colon epithelial cells have a defined life span and undergo terminal differentiation as they mature and migrate to the luminal surface. The differentiation process can be induced in cultured colon cancer cells by sodium butyrate, which induces expression of various differentiation markers followed subsequently by cell death. In the present study, HT29 colorectal carcinoma cells were shown to undergo butyrate-induced caspase activation that was mainly produced through a mitochondrial pathway. Inhibition of caspase activation, either by peptide pan caspase inhibitor Z-VAD-FMK, by caspase 9 inhibitor Z-LEHD-FMK, or by overexpression of Bcl-XL, also inhibited the expression of differentiation markers. These findings suggest (a) that terminal differentiation of HT29 colon carcinoma cells is tightly linked to caspase activation and (b) that increased expression of anti-apoptotic members of the Bcl-2 family of proteins, as well as other inhibitors of caspase activation, has the potential to inhibit terminal differentiation and thereby may contribute to the progression of colon cancer.     

Genetically tagged putative differentiation intermediates derived from undifferentiated HT29 human colon carcinoma cells.

The HT29 colonic carcinoma cell line has proven to be a very practical tool for modelling aspects of colonic cell differentiation and toxification by chemotherapeutic agents. As an approach to subclone and clarify molecular events involved in sublineage maturation, non-differentiated HT29 cells were electroporated with a dominant marker gene (NeoR) to convey aminoglycoside resistance (G418R). Transfectants surviving passage in glucose-G418 medium were >200 times the abundance of transient G418R cells of controls. Genomic analysis showed that each clonal type was unique in NeoR integration pattern while mitochondrial DNA copy was relatively unchanged. All of the randomly generated NeoR clones resembled the parental phenotype, but some over-produced the mucin, secretory cell type or the cell death phenotype after culturing in 2 mM sodium butyrate medium. Re-exposure to glucose medium restored the parental-like phenotype.     

An endogenous colon mitosis inhibitor reduces proliferation of colon carcinoma cells (HT 29) in serum-restricted medium

Extracts of mouse intestine contain a colonic epithelial mitosis inhibitor that has recently been purified and identified as a tripeptide (pGlu-His-GlyOH). In order to elucidate further the biological characteristics of this peptide, the effect of the tripeptide on cell proliferation in a human colon carcinoma cell line (HT 29) was examined. The incorporation of tritiated thymidine was significantly reduced at 20-30 h after addition of the tripeptide. The dose-response relationship was bell-shaped with loss of inhibitory effect at high or low doses. The number of cells were significantly reduced at a peptide concentration of 10(-8) M at 24 h, but not at 48 or 72 h after addition of the peptide. The inhibition was reversible, and was only observed when the cells were grown in a serum-restricted medium (1%). The inhibitory effect was abolished by increasing the serum content to 10% or adding insulin to the medium.     

15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is up-regulated by flurbiprofen and other non-steroidal anti-inflammatory drugs in human colon cancer HT29 cells

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit prostaglandin synthetic enzyme, cyclooxygenases (COXs), as well as to exhibit anti-tumor activity although at much higher concentrations. 15-Hydroxyprostaglandin dehyrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore examined. Flurbiprofen and several other NSAIDs were found to induce 15-PGDH expression in human colon cancer HT29 cells. Flurbiprofen, the most active one, was also shown to induce 15-PGDH expression in other types of cancer cells. Induction of 15-PGDH expression appeared to occur at the stage of mRNA as levels of 15-PGDH mRNA were increased by flurbiprofen in HT29 cells. Levels of 15-PGDH were also found to be regulated at the stage of protein turnover. MEK inhibitors, PD98059 and U-0126, which inhibited ERK phosphorylation were shown to elevate 15-PGDH levels very significantly. These inhibitors did not appear to alter 15-PGDH mRNA levels but down-regulate matrix metalloproteinase-9 (MMP-9). This protease was shown to degrade and inactivate 15-PGDH suggesting that elevation of 15-PGDH levels could be due to inhibition of MMP-9 expression by these inhibitors. Similarly, flurbiprofen was also demonstrated to inhibit ERK activation and to down-regulate MMP-9 expression. Furthermore, flurbiprofen was shown to induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9. The turnover of 15-PGDH was found to prolong in the presence of flurbiprofen as compared to that in the absence of this drug. Taken together, these results indicate that flurbiprofen up-regulates 15-PGDH by increasing the expression and decreasing the degradation of 15-PGDH in HT29 cells.     

Identification of transglutaminase substrates in HT29 colon cancer cells: use of 5-(biotinamido)pentylamine as a transglutaminase-specific probe.

A biotinamine probe, 5-(biotinamido)pentylamine, was used for biotin-labeling of proteins in HT29 colon cancer cell extracts by endogenous transglutaminase activity. The biotin-labeled protein substrates were isolated and recovered by avidin-affinity chromatography. The proteins were separated using SDS-polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced. Amino acid sequence data identified human fructose-1,6-bisphosphate aldolase A, an intracellular protein, as a substrate for cellular transglutaminase.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.