ucn-1028-c and Colonic-Neoplasms

Mucin alterations are a common feature of colonic neoplasia, and alterations in MUC2 mucin have been associated with tumor progression in the colon. Bile acids have been linked to colorectal carcinogenesis and mucin secretion, but their effects on mucin gene expression in human colon carcinoma cells is unknown. Human colon carcinoma cells were treated chenodeoxycholate > ursodeoxycholate). Treatment with the putative chemopreventive agent curcumin, which decreased AP-1 activity, also decreased MUC2 transcription. Cotransfection with a dominant negative AP-1 vector decreased MUC2 transcription, confirming the significance of AP-1 in MUC2 induction by deoxycholate. Calphostin C, a specific inhibitor of protein kinase C (PKC), greatly decreased bile acid-induced MUC2 transcription and AP-1 activity, whereas inhibitors of MAP kinase had no effect.. Bile acids induced mucin expression in human colon carcinoma cells by increasing MUC2 transcription through a process involving MAP kinase-independent, PKC-dependent activation of AP-1.

Topics: Antineoplastic Agents; Chenodeoxycholic Acid; Cholagogues and Choleretics; Cholic Acids; Colonic Neoplasms; Curcumin; Deoxycholic Acid; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinases; Mucin-2; Mucins; Naphthalenes; Promoter Regions, Genetic; Protein Kinase C; RNA, Messenger; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation; Ursodeoxycholic Acid

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.

Topics: Acetophenones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Benzopyrans; Carbazoles; Caspases; Colonic Neoplasms; Drug Synergism; Enzyme Inhibitors; Humans; Indoles; Membrane Glycoproteins; Mitochondria; Naphthalenes; Protein Kinase C; Protein Kinase C-delta; Protein Kinase C-epsilon; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhesion molecule of the immunoglobulin family behaving as a tumor inhibitory protein in colon, prostate, liver, endometrial and breast cancers. Inhibition of tumor development is dependent upon the presence of the long 71-73 amino acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have recently defined a number of cis-acting motifs within the long cytoplasmic domain participating in tumor cell growth inhibition. These are Tyr488, corresponding to an Immunoreceptor Tyrosine-based Inhibition Motif, as well as the three terminal lysine residues of the protein. In this study, we provide evidence that treatment with phorbol esters leads to increased phosphorylation of in vivo (32)P-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cells transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the protein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin kinase had only minimal effects on the levels of basal or PMA-induced Ser phosphorylation. Furthermore, PMA treatment of the CT51 cells induced cell spreading and cellular relocalization of the CEACAM1-L protein. Since Ser503 has been described as a PMA-induced phosphorylation site in other cell systems, we investigated whether Ser503 was involved in these responses in mouse intestinal cells. No differences were noticed in the basal or the PMA-induced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala mutant CEACAM1-L. However, we provide evidence that Ser503 participates in CEACAM1-L-mediated tumor inhibition as its mutation to an Ala led to in vivo tumor development, contrary to the tumor inhibitory phenotype observed with the wild-type CEACAM1-L protein.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Animals; Antigens, CD; Antigens, Differentiation; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoembryonic Antigen; Carcinogenicity Tests; Carcinoma; Cell Adhesion Molecules; Colonic Neoplasms; Cyclic AMP; Enzyme Inhibitors; Epithelial Cells; Female; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Naphthalenes; Point Mutation; Protein Kinase C; Serine; Staurosporine; Tetradecanoylphorbol Acetate; Thionucleotides; Tumor Cells, Cultured

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Topics: Adenocarcinoma; Antigens, Neoplasm; Carcinogens; Cell Adhesion; Cell Movement; Collagen; Colonic Neoplasms; Drug Combinations; Enzyme Induction; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Laminin; Metalloendopeptidases; Mucin 5AC; Mucin-2; Mucin-3; Mucins; Naphthalenes; Neoplasm Invasiveness; Neoplasm Proteins; Protein Kinase C; Proteoglycans; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The adhesion of anti-CD3-activated mouse T cells (AK-T cells) to syngeneic colon adenocarcinoma (MCA-38) cells is mediated principally through the integrin VLA-4 (alpha4beta1). We investigated the signalling pathways through which this adhesive interaction might be regulated. The protein tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) markedly inhibited the adhesion of AK-T cells to MCA-38 cells. Furthermore, pretreatment of the AK-T cells alone (but not the MCA-38 targets) with MDHC inhibited adhesion to a comparable extent as when MDHC was present during the assay. Calphostin C, an inhibitor of protein kinase C, also inhibited the adhesion of AK-T cells to MCA-38 monolayers. However, the phosphatidylinositol 3-kinase inhibitor wortmannin failed to alter AK-T cell adhesion to MCA-38 tumour cells. Inhibition of protein kinase A with the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate had no effect on adhesion, but the adenylyl cyclase activator forskolin and the cell-permeable cAMP analogues 8-Br-cAMP and dibutyryl-cAMP significantly suppressed adhesion. Pretreatment of AK-T cells alone with forskolin also inhibited adhesion. The adhesion of AK-T cells to MCA-38 tumour targets is therefore promoted by protein tyrosine kinases and protein kinase C, but inhibited by cAMP-dependent pathways, and the predominant location of the regulatory pathways is within the effector cell.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Cell Adhesion; Cells, Cultured; Colonic Neoplasms; Cyclic AMP; Enzyme Inhibitors; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Naphthalenes; Phosphatidylinositol 3-Kinases; Protein Kinase C; Protein-Tyrosine Kinases; Receptor-CD3 Complex, Antigen, T-Cell; Signal Transduction; Spleen; T-Lymphocytes; Tumor Cells, Cultured

The human colon cancer cell lines HCT 116 (poorly differentiated) and GEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-alpha). The secretion of TGF-alpha was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-alpha. Cells were metabolically labeled with 35S-cysteine and the formation of the mature 6 kDa TGF-alpha polypeptide from the 17 kDa pro-TGF-alpha precursor was determined. The conversion of pro-TGF-alpha was complete in 2-4 hr with the HCT 116 cells showing faster kinetics of TGF-alpha formation than GEO cells. HCT 116 cells secreted more TGF-alpha than GEO cells and the rate and extent of formation of TGF-alpha was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-alpha by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), stimulated the release of TGF-alpha into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-beta 1 specifically, our results suggest a potential role for PKC-beta in the processing of pro-TGF-alpha by these 2 human colon carcinoma cell lines.

Topics: Colonic Neoplasms; Humans; Isoenzymes; Naphthalenes; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

Transforming growth factor-beta 1 (TGF-beta 1) regulates the expression of the carcinoembryonic antigen (CEA) gene family in the human colon carcinoma cell line Moser. The mechanisms through which it acts, however, are unknown. In this communication, several lines of evidence are presented to show that the induction of CEA expression and secretion (collectively called CEA responses) by TGF-beta 1 is associated with protein kinase C (PKC) pathway of signal transduction. Treatment of intact cells with the PKC-specific inhibitor calphostin C down-modulated cellular PKC phosphotransferase activity and blocked the induction of the CEA responses by TGF-beta 1. Depletion of PKC by treatment of intact cells with phorbol ester also blocked the action of TGF-beta 1. The induction of the CEA responses by TGF-beta 1 was also blocked by the protein kinase inhibitor 1-(isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), which also inhibited cellular PKC activity. However, TGF-beta 1 did induce the CEA responses in intact cells treated with the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), the calmodulin-dependent phosphodiesterase inhibitor calmidazolium, the diacylglycerol kinase inhibitor R59 022, and the G-protein inhibitors cholera toxin and pertussis toxin. Treatment of intact cells with TGF-beta 1 induced a rapid and transient increase in PKC phosphotransferase activity. TGF-beta 1, however, was unable to induce PKC enzymatic activity in cells pretreated with calphostin C. Therefore, it is concluded that TGF-beta 1 regulates the CEA responses through a signal transducing pathway associated with PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Carcinoembryonic Antigen; Colonic Neoplasms; Humans; Isoquinolines; Naphthalenes; Piperazines; Polycyclic Compounds; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured