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PubMed1995

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in Ht29 colonic carcinoma cells.

Cl- secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5'-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n=6) inhibited ATP (0.1 mmol.l-1) induced increases in whole-cell conductance (Gm). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol.l-1) still induced a significant increase in Gm from 1.2 +/- 0.3 nS to 4.7 +/- 1 nS (n = 24). This suggests that ATP induces a cation influx with a conductance of approximately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n = 9). When Ba2+ (5 mmol.l-1) and 4,4'-diisothiocyanato-stilbene-2-2'-disulphonic acid (DIDS, 0.1 mmol.l-1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl- currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol.l-1) reduced the membrane current (Im) significantly from 86 +/- 14 pA to 54 +/- 11 pA (n = 13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5-10 mmol.l-1 1,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The zero-current membrane voltage (Vm) and Im (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30-120 s after membrane rupture. Vm depolarised significantly from -33 +/- 2 mV to -12 +/- 1 mV, and Im fell significantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly ( Im) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Im of 5.0 +/- 1.2 pA (n = 12); removal of Na+ in the absence of Ca2+ caused a Im of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol.l-1 Na+ and 1 mmol.l-1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.

Adenosine Triphosphate↗
PubMed2002

Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis.

The HT29 adenocarcinoma is a common model of epithelial cell differentiation and colorectal cancer and its death is an oft-analyzed response to TNF family receptor signaling. The death event itself remains poorly characterized and here we have examined the involvement of caspases using pan-caspase inhibitors. zVAD-fmk did not block death of HT29 cells in response to activation of the Fas, TRAIL, TNF, TWEAK and LTbeta receptors. The secondary induction of TNF or the other known bona fide death inducing ligands did not account for death following LTbeta receptor activation indicating that TNF family receptors can trigger a caspase-independent death pathway regardless of the presence of canonical death domains in the receptor. To provide a frame of reference, the phenotype of HT29 death was compared to four other TNF family receptor triggered death events; Fas induced Jurkat cell apoptosis, TNF/zVAD induced L929 fibroblast necrosis, TNF induced death of WEHI 164 fibroblastoid cells and TNF/zVAD induced U937 death. The death of HT29 and U937 cells under these conditions is an intermediate form with both necrotic and apoptotic features. The efficient coupling of TNF receptors to a caspase-independent death event in an epithelial cell suggests an alternative approach to cancer therapy.

Active Transport, Cell Nucleus↗
PubMed1992

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.

Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.

Base Sequence↗
PubMed2006

Protein phosphatase-2A associates with and dephosphorylates keratin 8 after hyposmotic stress in a site- and cell-specific manner.

Keratins 8 and 18 (K8 and K18) are regulated by site-specific phosphorylation in response to multiple stresses. We examined the effect and regulation of hyposmotic stress on keratin phosphorylation. K8 phospho-Ser431 (Ser431-P) becomes dephosphorylated in HT29 cells, but hyperphosphorylated on other K8 but not K18 sites in HRT18 and Caco2 cells and in normal human colonic ex vivo cultures. Hyposmosis-induced dephosphorylation involves K8 but not K18, K19 or K20, occurs preferentially in mitotically active cells, and peaks by 6-8 hours then returns to baseline by 12-16 hours. By contrast, hyperosmosis causes K8 Ser431 hyperphosphorylation in all tested cell lines. Hyposmosis-induced dephosphorylation of K8 Ser431-P is inhibited by okadaic acid but not by tautomycin or cyclosporine. The PP2A catalytic subunit co-immunoprecipitated with K8 and K18 after hyposmotic stress in HT29 cells, but not in HRT18 or Caco2 cells where K8 Ser431 becomes hyperphosphorylated. K8 Ser431-P dephosphorylation after hyposmosis was independent of PP2A levels but correlated with increased PP2A activity towards K8 Ser431-P. Therefore, hyposmotic stress alters K8 phosphorylation in a cell-dependent manner, and renders K8 Ser431-P a physiologic substrate for PP2A in HT29 cells as a result of PP2A activation and the physical association with K8 and K18. The divergent hyposmosis versus hyperosmosis K8 Ser431 phosphorylation changes in HT29 cells suggest that there are unique signaling responses to osmotic stress.

Caco-2 Cells↗
PubMed2000

Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors.

LPS elicits several immediate proinflammatoy responses in peripheral blood leukocytes via a recently described pathway including CD14, Toll-like receptors (TLR), serine-threonine kinases, and NF-kappaB transcription factor. However, the functional responses of intestinal epithelial cells (IEC) to stimulation with LPS are unknown. Expression of mRNA and protein for CD14 and TLRs were assessed by RT-PCR, immunoblotting, and immunohistochemistry in mouse and human IEC lines. LPS-induced activation of signaling pathways (p42/p44 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), p38, p65, NF-kappaB) were assessed by immunoblotting and gel shifts. CD14 mRNA and protein expression were not detectable in IEC. However, human TLR2, TLR3, and TLR4 mRNA were present in IEC. TLR4 protein was expressed in all cell lines; however, TLR2 protein was absent in HT29 cells. Immunofluorescent staining of T84 cells demonstrated the cell-surface presence of the TLRs. LPS-stimulation of IEC resulted in activation (>1.5-fold) of the three members of the MAPK family. In contrast, LPS did not significantly induce activation of JNK and p38 in CMT93 cells, p38 in T84 cells and MAPK and JNK in HT29 cells. Downstream, LPS activated NF-kappaB in IEC in a time-, dose-, and serum-dependent manner. IEC express TLRs that appear to mediate LPS stimulation of specific intracellular signal transduction pathways in IEC. Thus, IEC may play a frontline role in monitoring lumenal bacteria.

Animals↗
PubMed2002

The c-kit tyrosine kinase inhibitor STI571 for colorectal cancer therapy.

The c-kit tyrosine kinase inhibitor STI571 exhibits a substantial therapeutic activity in patients with chronic myeloid leukemia and gastrointestinal stromal tumors respectively associated with constitutive activation of the BCR-ABL and c-kit tyrosine kinases. Human colorectal tumors also express the c-kit proto-oncogene. The present study focuses on the anticancer activity of STI571 in human colorectal tumor cells in vitro and in vivo. The c-kit receptor was identified as a M(r) 145,000 immunoreactive band in human colon cancer cells HT29, HCT8/S11, and HCT116. Cellular invasion induced by 10 ng/ml stem cell factor (EC(50) = 3 ng/ml) in HT29 cells was blocked by 1 micro M STI571 (IC(50) = 56 nM) and pharmacological inhibitors of several oncogenic signaling pathways, namely, phosphatidylinositol 3-kinase (LY294002), Rho GTPases (Clostridium botulinum exoenzyme C3 transferase), and Rho-kinase (Y27632). STI571 inhibited HT29 cell proliferation (IC(50) = 6 micro M) and induced apoptosis in vitro. These cellular effects were associated with a decrease in tumor growth. We also demonstrated that stem cell factor is a proangiogenic factor in vivo and in vitro. These encouraging results warrant further preclinical investigations and clinical trials on the use of the c-kit inhibitor STI571 as a chemotherapeutic agent in colon cancer prevention and in treatment of advanced colorectal cancers associated with liver metastases.

Animals↗
PubMed2003

Repression of MUC2 gene expression by butyrate, a physiological regulator of intestinal cell maturation.

Sodium butyrate (NaB) inhibits proliferation, stimulates apoptosis, and promotes differentiation of human colon cancer cells along the absorptive phenotype. In vitro, butyrate induces a switch from cells with a secretory to an absorptive phenotype. Here, we report that NaB specifically represses the expression of the MUC2 gene, a differentiation marker of the secretory goblet cell lineage, in forskolin- and 12-O-tetradecanoylphorbol 13-acetate-induced HT29 cells, and Cl.16E cells, a clonal derivative of HT29 cells that spontaneously differentiates into goblet cells. Thus, NaB repression is independent of the nature of the stimulus that triggers MUC2 expression. Further, repression was independent of new protein synthesis. Our results suggest that inhibition of MUC2 is linked to the ability of butyrate to repress histone deacetylase activity, since trichostatin A, another inhibitor of histone deacetylases, also inhibited MUC2 expression in induced HT29 cells. Finally, we demonstrate that the NaB effect is specific for this marker of the secretory cell lineage, since carcinoembryonic antigen, which is expressed in both the secretory and absorptive cells, is induced by NaB. Thus, the NaB repression of a definitive function of the secretory cell lineage is a further mechanism, in addition to the effects on proliferation and apoptotic pathways, through which butyrate can regulate intestinal homeostasis.

Butyrates↗
PubMedDate not supplied

Cytokine regulation of ezrin expression in the human colon cancer cell line HT29.

Ezrin is a membrane-cytoskeleton linker protein and belongs to the TERM family. It has been implicated in the membrane ruffling, motility, and metastatic process of tumour cells. This study examined the effects of a range of cytokines on the expression of ezrin in the human colon cancer cell line, HT29. Levels of ezrin were determined by Northern and Western blotting and indirect immunofluorescence. We report that IL-2, IL-8, IL-10 and IGF-I had an inhibitory effect on the expression, whereas EGF and IL-11 enhanced cellular ezrin levels. Immunofluorescence confirmed that these changes were seen both in cytosol and generalised membrane. It is concluded that ezrin expression in tumour cells can be regulated by cytokines and this bears importance in the understanding of its role in tumour biology.

Cytokines↗
PubMed1995

Selective toxicity of compounds naturally present in food toward the transformed phenotype of human colorectal cell line HT29.

It has previously been observed that allyl isothiocyanate, a compound naturally present in the diet, is more cytotoxic toward the human colorectal adenocarcinoma cell line HT29 in its control transformed state than after exposure to sodium butyrate or to dimethylformamide, which slow growth and induce differentiation (detransformation). In the present study, a range of other dietary compounds were assayed for such selective toxicity. These compounds were chosen as constituents of foodstuffs that have been identified from epidemiologic studies as being potentially antitumorigenic and also as having anticarcinogenic activity in experimental models. Benzyl and phenethyl isothiocyanate, benzyl thiocyanate, and quercetin showed decreased toxicity towards HT29 after detransformation of the cells by one or both treatments, whereas no change was observed in the sensitivity to diallyl sulfide or diallyl disulfide. It is proposed that the presence of such selectively toxic compounds in the diet may inhibit the development of tumors by interfering with the growth of preneoplastic lesions while having little effect on normal cells. The cumulative effects of these inhibitions may contribute to the chemopreventive properties of the parent foodstuffs observed in epidemiologic studies.

Adenocarcinoma↗
PubMed1993

Regulated expression of an intestinal mucin gene in HT29 colonic carcinoma cells.

We have investigated the regulation of the intestinal mucin gene MUC2 in HT29 cells. Surprisingly, sodium butyrate, an effective inducer of aspects of colonic cell differentiation in HT29 cells, fails to induce MUC2 during short-term exposure, despite the fact that it has been used to select stably differentiated clones of HT29 that resemble goblet cells and produce mucin. However, 12-O-tetradecanoylphorbol-13-acetate and forskolin, which trigger the protein kinase C- and A-dependent signal transduction pathways, respectively, are potent inducers of MUC2 gene expression. 12-O-Tetradecanoylphorbol-13-acetate and forskolin operate through distinct mechanisms, with the former requiring de novo protein synthesis and the latter not. Experiments using specific protein kinase inhibitors suggest that both inducers operate by triggering their respective signal transduction pathways. Nuclear runoff analyses suggest that post-transcriptional (rather than transcriptional) mechanisms are important in the accumulation of MUC2 mRNA. Finally, we show that in several cell lines from human mucinous tumors, characterized by elevated levels of mucin production, MUC2 expression is very high and constitutive compared to forskolin-treated HT29 cells. Thus, the different regulation of MUC2 in HT29 cells and in mucinous tumor cell lines may reflect molecular pathways that characterize colon carcinomas of different histology and pathology.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Short-term suramin treatment followed by the removal of the drug induces terminal differentiation of HT29-D4 cells.

Suramin is an anti-cancer drug which induces the differentiation of the human colon cancer clone HT29-D4. Yet chronic suramin treatment of these cells eventually leads to a marked disturbance of the lysosomal system, which consists in an accumulation of hypertrophied autophagic vacuoles and the occurrence of lamellated inclusion bodies. We report here the effect of a prime treatment of HT29-D4 cells with suramin during various periods of time, followed by the removal of the drug and a subsequent culture in suramin-free medium. A prime treatment of cells in the presence of the drug for 2 days or 4 days was found ineffective to induce the organization of cells into polarized monolayers. On the contrary, a prime treatment of cells for 5 days is sufficient to allow the cellular organization to proceed normally toward a fully polarized monolayer, without any lysosomal damage. The cells did not require the continuous presence of suramin to develop an electrical resistance and a transepithelial potential difference. Moreover the basolateral localization of HLA class I molecules was achieved 9 days after the removal of the drug from the culture medium. Finally prime treatment of cells in the presence of suramin for times longer than 5 days induced the morphological, biochemical, and electrophysiological differentiation of HT29-D4 cells. However, in this case, severe lysosomal disturbances were constantly observed. These data demonstrate that the impaired lysosomal system is a post-differentiation event due to prolonged exposure of the cells to suramin. A metabolic analysis of HT29-D4 cells primed for various times with the drug showed that differentiated cells have a reduced glycolytic activity and this suggests an action of suramin at the level of autocrine growth factors which are known to regulate glucose uptake and degradation.

Cell Count↗
PubMed1995

Tumour necrosis factor-alpha induces morphological and functional alterations of intestinal HT29 cl.19A cell monolayers.

TNF-alpha is a widely distributed proinflammatory cytokine, involved in many disease states. Although it has widely distributed effects, a precise mechanism of action has never been described, in particular at the epithelial level. Morpho-functional changes of the intestinal epithelial monolayer HT29 cl.19A exposed to TNF-alpha were therefore assessed, using electron microscopy (including freeze-fracture replica analysis), as well as measurement of mannitol, Na+ and horseradish peroxidase fluxes across intestinal HT29 cl.19A cell monolayers using Ussing chambers. TNF-alpha receptors were induced on HT29 cl.19A cells by a small non-toxic dose of IFN-gamma (5 U/ml). After 4 h of the combined presence of TNF-alpha (10 ng/ml) and IFN-gamma (5 U/ml), the tight junction structure was altered as shown by a significant decrease in the average strand number measured in the apico-basal direction (5.50 +/- 2.70 vs 3.73 +/- 1.39 in control and treated cells respectively, P < 0.0001) and by a significant decrease in junctional depth (0.27 +/- 0.14 and 0.17 +/- 0.10 microns in control and treated cells respectively, P < 0.0001). These results are in agreement with a decrease in number of 'kiss' sites between contiguous membranes of TNF-alpha treated cells observed in ultrathin sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane Permeability↗
PubMed1999

Effect of eicosapentaenoic acid on the proliferation and incidence of apoptosis in the colorectal cell line HT29.

Fish oil has been shown to reduce the induction of colorectal cancer in animal models by a mechanism which may involve suppression of mitosis, increased apoptosis, or both. We used the human colonic adenocarcinoma cell line HT29 to explore the effects of the long-chain n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) on cell proliferation and death in vitro. Cells were cultured in media containing EPA at 5, 10, and 15 microg/mL. Cell number and thymidine incorporation were used to quantify proliferation, and cell cycle effects were studied using flow cytometry. Gel electrophoresis, annexin-V binding, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells were treated as two distinct populations. In the presence of EPA at 10 and 15 microg/mL there was a marked reduction in the growth rate of adherent HT29 colonies, owing to an increased detachment of adherent cells. After treatment with 10 or 15 microg/mL EPA the proportion of adherent cells in S-phase increased, indicating either a block in late S-phase or early G2. Floating cells showed evidence of extensive DNA cleavage, but the proportion of floating cells with sub GO DNA content declined on treatment with 10 or 15 microg/mL EPA even though the number of floating cells increased. We conclude that EPA does not inhibit mitosis of adherent cells, but increases the rate at which they become detached from the substrate, probably at an early stage in the initiation of apoptosis. This mechanism may be analogous to "anoikis," or induction of apoptosis in response to loss of cell contact, and may contribute to the anticarcinogenic effects of fish oil in vivo.

Acridine Orange↗
PubMedDate not supplied

Alterations in mitochondrial and apoptosis-regulating gene expression in photodynamic therapy-resistant variants of HT29 colon carcinoma cells.

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragment-associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3' region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.

Apoptosis↗
PubMed1995

Promoter regulation of a differentially expressed gene in the human colonic epithelial cell lines HT29-18 and HT29-18-C1.

Gene A4 is transcriptionally activated upon enterocyte differentiation of the human colonic epithelial cell line HT29-18 and its highly differentiated subclone HT29-18-C1 [Oliva et al., Arch. Biochem. Biophys. 302 (1993) 183-192]. To characterize the mechanisms regulating the differential transcription of A4, we analyzed its immediate 5'-flanking region for regulatory elements. Promoter-linked transfection experiments of progressively deleted A4 5'-flanking sequences fused to the bacterial cat reporter gene suggest the presence of one negative and two positive DNA elements within the first 371 bp of the A4 promoter (pA4). DNase I footprint and electrophoretic mobility shift assays demonstrate that one positive element which contains the core binding sequence for the transcription factor, Sp1, mediates an equal level of transcription in the two cell types. The second positive element, localized between nucleotide positions--169 and -152, contains a sequence previously unrecognized as a transcription factor-binding site. This element mediates a twofold increase in the activity of pA4 in HT29-18-C1, as compared to HT29-18. Furthermore, nuclear extracts obtained from HT29-18-C1 contain a higher binding activity for this element than those from HT29-18. Southwestern blot analysis suggests that the protein interacting with this element has an estimated molecular mass of 50 kDa. We conclude that this protein may be involved in the differential regulation of A4 in these intestinal cell lines.

Base Sequence↗
PubMed1997

Pretreatment by tubulin agents decreases C-MYC induction in human colon carcinoma cell line HT29-D4.

For HT29-D4 cell line, we confirmed the interaction between c-Myc protein and microtubules by immunoprecipitation. We then studied the effect of antimitotic agents, nocodazole and the taxoids [paclitaxel (taxol) and docetaxel (taxotere)] on c-myc oncogene expression. The expression was analyzed by RT-PCR and Western blot. Taxol (1 microM), taxotere (1 microM) and nocodazole (3 microM) inhibited by 30-50% the c-myc induction produced by growth factors in culture medium. According to the flow cytometry analysis, the inhibition is not linked to the mitotic block. These results observed for both stabilizing and depolymerizing agents suggest that microtubular system is involved in c-myc expression more through its dynamic properties which influence signal transduction and intracellular transports than through its direct interaction with c-Myc protein.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Effect of 5-fluorouracil on methotrexate transport and cytotoxicity in HT29 colon adenocarcinoma cells.

Exposure of the human colon adenocarcinoma HT29 cells to different concentrations of 5-fluorouracil (5FU) for 24 h resulted in a dose-dependent increase in the cellular glutathione (GSH) level which remained elevated up to 72 h following 5FU treatment. Pretreatment of HT29 cells with 5FU was found to enhance the uptake of methotrexate (MTX) as compared to control cells. Administration of MTX 24 h after 5FU was found to be synergistic, whereas administration of MTX 24 h before 5FU or together with 5FU was not found to be synergistic. The results of this study suggest that the increase in cellular GSH level as a result of 5FU pretreatment may play a role in the enhancement of MTX uptake and the cytotoxicity of both drugs in HT29 cells.

Adenocarcinoma↗
PubMed1994

Further evidence of different lactoferrin and transferrin binding sites on human HT29-D4 cells. Effects of lysozyme, fucose and cathepsin G. Comparison with transferrin.

We have defined by using competition experiments the nature of specific lactoferrin binding sites, probably responsible for the previously observed stimulatory growth effect of the iron binding protein on HT29-D4 cells. Lysozyme, albumin and fucose do not affect lactoferrin binding showing that the binding of the protein is mediated neither by electrostatic forces nor by fucose. Iron-free and iron-loaded protein produce similar effects, demonstrating that the metal is not involved in the protein recognition. Similar results are observed for transferrin. A specific binding inhibition of lactoferrin by cathepsin G, a leukocyte proteinase, is observed, suggesting the existence of a common receptor for lactoferrin and cathepsin G on HT29-D4 cells. These results and the fact that tumor tissues are more often infiltrated by inflammatory cells such as polymorphonuclear leukocytes could evoke an unexpected role for leukocytes, possibly mediated in part by these two proteins, on the proliferative cancer effect.

Binding Sites↗