okadaic-acid and Colonic-Neoplasms

Diarrhetic shellfish poisoning (DSP) is a syndrome caused by the intake of shellfish contaminated with a group of lipophilic and thermostable toxins, which consists of okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2). These toxins are potent protein Ser/Thr phosphatase inhibitors, mainly type 1 protein phosphatase (PP1) and type 2A protein phosphatase (PP2A). Different effects have been reported at the cellular, molecular and genetic levels. In this study, changes in cell survival and cell mobility induced by OA, DTX-1 and DTX-2 were determined in epithelial cell lines of the colon and colon cancer. The cell viability results showed that tumoral cell lines were more resistant to toxins than the nontumoral cell line. The results of the functional assays for testing cell migration, evaluation of cell death and the expression of proteins associated with cell adhesion showed a dual effect of toxins since in the nontumoral cell line, a greater induction of cell death, presumably by anoikis, was detected. In the tumoral cell lines, there was an induction of a more aggressive phenotype characterized by increased resistance to toxins, increased migration and increased FAK activation. In tumoral cell lines of colon cancer, OA, DTX-1/DTX-2 induce a more aggressive phenotype.

Topics: Animals; Carcinogens; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Colonic Neoplasms; Focal Adhesion Kinase 1; Humans; Inhibitory Concentration 50; Okadaic Acid; Protein Phosphatase 2

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1.

Topics: Cell Line, Tumor; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Hydroxamic Acids; Okadaic Acid; Phosphorylation; Plicamycin; Protein Synthesis Inhibitors; Repressor Proteins; Retinoblastoma Protein; Sp1 Transcription Factor; Transcription, Genetic

Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites.

Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Brain; Brain Chemistry; Cell Line; Colonic Neoplasms; Cricetinae; Enzyme Inhibitors; Epitopes; HT29 Cells; Humans; Immunoblotting; Immunohistochemistry; Keratin-18; Keratin-8; Keratins; Kidney; Liver; Liver Diseases; Mice; Mutation; Okadaic Acid; Phosphorylation; Rabbits; Serine; Stomach Neoplasms; Transfection; Vaccination; Vanadates

Some lines of colon cancer cells are forced to undergo differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA). The increases in activities of both protein tyrosine phosphatase (PTP) and protein tyrosine kinase (PTK) have been reported to be associated with the TPA-induced differentiation of HL-60 leukemia cells. In the present study, a 2-fold increase in PTP activity was observed in SW620 human colon cancer cells after 30 min of TPA treatment; a maximal level (4- to 5-fold) was reached at 60 min and continued for more than 6 hr. In addition, two TPA-induced differentiated characteristics, morphological alteration and release of cellular surface proteoglycan, were effectively blocked by PTP inhibitors, such as sodium orthovanadate (50 microM), zinc chloride (100 microM), and iodoacetate (250 microM), but not by the protein serine/threonine phosphatase inhibitor okadaic acid (20 nM). On the other hand, although TPA induced a transient slight increase in PTK activity (1.4-fold) at 60 min, four PTK inhibitors (genistein, herbimycin A, tyrphostin-23 and quercetin) had different effects on the TPA-induced release of cell surface proteoglycan. Genistein (60 microM) potentiated this process, but in contrast, quercetin (45 microM) could partially inhibit the TPA effect. Taken together, these observations suggest that both PTP and PTK activities were increased in SW620 cells in response to TPA; however, the activation of PTP seems to be preferentially required for the TPA-induced differentiation of SW620 human colon cancer cells.

Topics: Cell Differentiation; Chlorides; Colonic Neoplasms; Enzyme Activation; Ethers, Cyclic; Genistein; Humans; Isoflavones; Okadaic Acid; Protein Tyrosine Phosphatases; Proteoglycans; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Zinc Compounds

Butyrate is a potent differentiating agent present in high concentrations in colonic lumen as a result of metabolic breakdown of dietary fibre and, as such, may directly influence colonic cancer progression. We have investigated the effects of butyrate on an enzyme system important in colonic tumour progression, the plasminogen-activating system, in a poorly differentiated colon cancer cell. Butyrate was found to induce a rapid and transient increase in plasminogen activator inhibitor type 1 (PAI-1) mRNA while concomitantly suppressing the constitutive production of both urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) mRNA transcripts. We have investigated the mechanisms involved in mediating these effects by run-on transcription and RNA stability analyses. Our data show that PAI-1 mRNA induction occurs through both regulation of the stability of the alternately spliced 3.3 kb PAI-1 mRNA transcript and induction of the 2.4 kb PAI-1 mRNA transcript. Studies using modulators of signal transduction pathways demonstrate that induction of PAI-1 mRNA synthesis is independent of protein kinase C but dependent on the activation of protein kinase A. Suppression of uPA mRNA by butyrate was found to occur by down-regulation of gene transcription through a process independent of de novo protein synthesis. The transcription rate of the uPAR gene was not modulated by butyrate, but rapid turnover of the uPAR gene by butyrate was dependent on ongoing protein synthesis. Our results demonstrate that butyrate can effect rapid changes in the expression of genes of the plasminogen-activating system through several different mechanisms in a gene-specific manner.

Topics: Adenocarcinoma; Butyrates; Colonic Neoplasms; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Down-Regulation; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; In Vitro Techniques; Okadaic Acid; Plasminogen Activator Inhibitor 1; Protein Kinase C; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Transcription, Genetic; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

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.

Topics: 1-Methyl-3-isobutylxanthine; Adenocarcinoma; Bucladesine; Calcimycin; Colforsin; Colonic Neoplasms; DNA Probes; Ethers, Cyclic; Gene Expression Regulation, Neoplastic; Humans; Intestinal Mucosa; Ionomycin; Ionophores; Kinetics; Mucins; Okadaic Acid; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

The aim of the present study was to elucidate the mechanism responsible for the high mannose glycoprotein instability in undifferentiated HT-29 cells (a human colon cancer cell line) reported previously. The results presented here are consistent with lysosomal degradation of these molecular species. In addition inhibitors of the autophagic-lysosomal degradative pathway (3-methyladenine, okadaic acid and asparagine) dramatically block the degradation of proteins and N-linked glycoproteins in undifferentiated HT-29 cells. The main conclusions of this work are: 1- the autophagic-lysosomal pathway is responsible for the high mannose glycoprotein degradation in undifferentiated HT-29 cells; 2- this degradative pathway exists in differentiated cells but is greatly reduced (3.5-4 fold); 3- the HT-29 cell line is a new model to investigate the molecular regulation of autophagy.

Topics: Adenine; Adenocarcinoma; Asparagine; Autophagy; Carbon Radioisotopes; Carcinogens; Cell Differentiation; Cell Line; Chloroquine; Colonic Neoplasms; Ethers, Cyclic; Glycoproteins; Humans; Kinetics; Leucine; Lysosomes; Mannose; Okadaic Acid; Polysaccharides; Tritium; Tumor Cells, Cultured

Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU.. Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro.. Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates.. We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity.. Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro.. Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.

Topics: Breast Neoplasms; Colonic Neoplasms; Drug Synergism; Ethers, Cyclic; Fluorouracil; Humans; Levamisole; Melanoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Skin Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vanadates