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.     

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.     

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.     

HIP/RPL29 antagonizes VEGF and FGF2 stimulated angiogenesis by interfering with HS-dependent responses

HIP/RPL29 is a heparan sulfate (HS) binding protein with diverse activities including modulation of heparanase (HPSE) activity. We examined HIP/RPL29's ability to modulate actions of HS-binding growth factors (HBGFs) in angiogenesis. Between 1 and 2.5 microg/ml (ca. 60-150 nM), HIP/RPL29 inhibited HBGF-stimulated endothelial cell tube formation. Aortic explant outgrowth also was inhibited, but at higher concentrations (40 microg/ml). At this concentration, HIP/RPL29 had no effect on HBGF-stimulated MAPK phosphorylation or VEGF-stimulated receptor-2 phosphorylation at site Y-996. Partial inhibition occurred at VEGF receptor-2 site Y951, associated with cell migration. HBGF displacement from HS-bearing perlecan domain I showed that HIP/RPL29 released 50% of bound HBGF at 20 microg/ml, a dose where endothelial tube formation is inhibited. Similar FGF2 release occurred at pH 5.0 and 7.0, conditions where HPSE is highly and residually active, respectively. We considered that HIP/RPL29 inhibits HPSE-dependent release of HS-bound HBGFs. At pH 5.0, release of soluble HS was inhibited by 64% at concentrations of 5 microg/ml and by 77% at 40 microg/ml, indicating that HIP/RPL29 antagonizes HPSE activity. At concentrations up to 40 microg/ml (ca. 2.5 microM) where angiogenic processes are inhibited, release of FGF2 occurred in the presence of HPSE and HIP/RPL29. The majority of this FGF2 is not bound to soluble HS. Studies of HIP/RPL29 binding to HS indicated that many structural features of HS are important in modulation of HBGF activities. Our findings suggest that inhibition of angiogenic processes by HIP/RPL29 involves attenuation of the formation of soluble, biologically active HBGF:HS complexes that activate HBGF receptors.     

Polyethylene glycol induces apoptosis in HT-29 cells: potential mechanism for chemoprevention of colon cancer

Recent experimental evidence suggests that polyethylene glycol (PEG) is a highly effective chemopreventive agent against colon cancer; however, the mechanism(s) remain largely unexplored. To further elucidate this issue, we evaluated the effect of PEG on two human colon cancer cell lines. PEG treatment resulted in a dose- and time-dependent reduction in cell number without alteration in markers of cell proliferation. However, there was a dramatic and specific, concentration-dependent induction of apoptosis, with 50 mM PEG rendering approximately half the cells apoptotic. This corresponded with a 17-fold induction in the expression of the pro-apoptotic protein, prostate apoptosis response-4. Our data suggest that induction of apoptosis may be responsible, at least in part, for the ability of PEG to prevent experimental colon cancer.     

Hyperthermia in combination with oxidative stress induces autophagic cell death in HT-29 colon cancer cells

The purpose of this study was to evaluate the mechanism of ROS-induced hyperthermic cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 degrees C) in the presence of tert-butyl hydroperoxide (t-BOOH). t-BOOH combined with hyperthermia significantly decreased cell viability as compared with t-BOOH or hyperthermia alone. This decrease in cell numbers was associated with retardation in the S phase transit and not through apoptosis. Cell death was noted to be accompanied by specific features characteristic of autophagy: the presence of cytoplasmic autophagic vacuoles; autophagosome membrane association of microtubule-associated protein light chain 3; accumulation of acidic vesicular organelles; and increased incorporation of MDC in the autophagosome. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.     

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.     

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.     

Endoplasmic reticulum calcium transport ATPase expression during differentiation of colon cancer and leukaemia cells

The calcium homeostasis of the endoplasmic reticulum (ER) is connected to a multitude of cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Calcium is accumulated in the ER by various biochemically distinct sarco/endoplasmic reticulum calcium transport ATPase isoenzymes (SERCA isoforms). Experimental data indicate that the SERCA composition of some carcinoma and leukaemia cell types undergoes significant changes during differentiation, and that this is accompanied by modifications of SERCA-dependent calcium accumulation in the ER. Because ER calcium homeostasis can also influence cell differentiation, we propose that the modulation of the expression of various SERCA isoforms, and in particular, the induction of the expression of SERCA3-type proteins, is an integral part of the differentiation program of some cancer and leukaemia cell types. The SERCA content of the ER may constitute a new parameter by which the calcium homeostatic characteristics of the organelle are adjusted. The cross-talk between ER calcium homeostasis and cell differentiation may have some implications for the better understanding of the signalling defects involved in the acquisition and maintenance of the malignant phenotype.     

Repression of protein translation and mTOR signaling by proteasome inhibitor in colon cancer cells

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of ³⁵S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.     

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.     

Modulation of glutathione level during butyrate-induced differentiation in human colon derived HT-29 cells

Glutathione plays an important role in various cellular functions including cell growth and differentiation. In the present study, cell differentiation was induced by butyrate in human colon cell line HT-29 and cellular thiol status was assessed. It was observed that butyrate-induced differentiation was associated with decrease in cellular GSH level and this was prominent at early stages of differentiation. Buthionine sulfoximine (BSO), a specific cellular GSH depleting agent, did not induce differentiation in cells but potentiated the differentiation induced by butyrate. Both BSO and butyrate individually and together inhibited cell growth. These studies suggest that cellular GSH level is modulated in butyrate-induced differentiation and decrease of GSH at the initial stage might facilitate cellular differentiation.     

Dictyopteris undulata extract induces apoptosis in human colon cancer cells

The present study investigated the cytotoxic and apoptotic effects of an ethanol extract derived from the marine brown alga Dictyopteris undulata against human colon adenocarcinoma cells. The Dictyopteris undulata extract (DUE) showed cytotoxic activity against SW480 cells in a dose-dependent manner, with 50% inhibition of cell viability at a concentration of 40 μg/mL. DUE also induced programmed cell death in SW480 cells, as evidenced by apoptotic body formation, DNA fragmentation, an increase in the population of apoptotic sub-G1 phase cells, and mitochondrial membrane depolarization. Moreover, DUE significantly modulated the expression of apoptosisassociated proteins, resulting in a decrease in B cell lymphoma-2 expression and an increase in Bcl-2-associated X protein expression, as well as the activation of caspase-9 and caspase-3. Furthermore, DUE showed apoptotic cell death in two other colon cancer cell lines, SNU407 and HT29. These observations suggest that DUE may prove useful as a therapeutic agent for the attenuation of colon cancer.     

A new EGFR inhibitor induces apoptosis in colon cancer cells

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, mAbs and EGFR tyrosine kinase inhibitors seemed to be the most promising. However they have demonstrated scarce utility in therapy, the former being effective only at toxic doses, the latter resulting inefficient in colon cancer. This paper presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphthoquinone core as shikonin, an agent with great anti-tumor potential. In HT29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 microM, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer.     

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.     

Activity and properties of alpha-L-fucosidase are dependent on the state of enterocytic differentiation of HT-29 colon cancer cells

Previously we have demonstrated an impairment in the activity of alpha-L-fucosidase in colon tumours. In order to establish an in vitro model to study this enzyme in colon cancer, we have determined the activity and properties of the enzyme during the differentiation of HT-29 colon cancer cells. Cultures were committed to differentiate into enterocyte-like cells by placing them in a culture medium without glucose for 18-21 days. The state of differentiation was evaluated by assaying the activity of enterocytic marker enzymes, and the acquisition of enterocyte morphology was assessed by electron microscopy. The alpha-L-fucosidase activity was determined using a fluorometric method. Intracellular levels of alpha-L-fucosidase activity are lower in non-differentiated cells (3.0 +/- 1.01 U/mg) than in differentiated ones (9.2 +/- 4.09 U/mg) (P < 0.001). This variation is not due to a greater secretion of the enzyme to the culture medium, and properties such as pH optimum or the affinity towards substrate are not dependent on differentiation. The enzyme however, is more stable at acidic pH and at high temperatures, and V(max) is higher in differentiated cells. Moreover, in undifferentiated cells the enzyme is mainly in a monomeric form whereas multimeric forms of the enzyme appear only upon differentiation. Most of these changes are very similar to those previously observed between normal colon tissue and colon tumours. Thus, we suggest that differentiation of HT-29 colon cancer cells could be used as a model to study the alterations of the enzyme alpha-L-fucosidase during the progression of the tumoural process.     

Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.