hymecromone and Colonic-Neoplasms

A lysosome-accessing nanoprobe is designed for recognition of lysosomal neuraminidases (Lyso-Neus), which can cleave the 4-methylumbelliferone moieties of the substrate from the nanoprobe, and lead to the escape of the moieties from acidic lysosomes into the neutral cytosol assisted by cationic poly(ethyleneimine) to light up the pH-responsive fluorescence for visual detection and dynamic tracking of Lyso-Neu activity in living cells.

Topics: Cell Survival; Colonic Neoplasms; Cytosol; Fluorescence; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Hymecromone; Image Processing, Computer-Assisted; Light; Lysosomes; Microscopy, Confocal; Nanoparticles; Neuraminidase; Tumor Cells, Cultured

Focal adhesion kinase (FAK), hyaluronan (HA), and hyaluronan synthase-3 (HAS3) have been implicated in cancer growth and progression. FAK inhibition with the small molecule inhibitor Y15 decreases colon cancer cell growth in vitro and in vivo. HAS3 inhibition in colon cancer cells decreases FAK expression and activation, and exogenous HA increases FAK activation. We sought to determine the genes affected by HAS and FAK inhibition and hypothesized that dual inhibition would synergistically inhibit viability. Y15 (FAK inhibitor) and the HAS inhibitor 4-methylumbelliferone (4-MU) decreased viability in a dose dependent manner; viability was further inhibited by treatment with Y15 and 4-MU in colon cancer cells. HAS inhibited cells treated with 2 μM of Y15 showed significantly decreased viability compared to HAS scrambled cells treated with the same dose (p < 0.05) demonstrating synergistic inhibition of viability with dual FAK/HAS inhibition. Microarray analysis showed more than 2-fold up- or down-regulation of 121 genes by HAS inhibition, and 696 genes by FAK inhibition (p < 0.05) and revealed 29 common genes affected by both signaling. Among the genes affected by FAK or HAS3 inhibition were genes, playing role in apoptosis, cell cycle regulation, adhesion, transcription, heatshock and WNT pathways. Thus, FAK or HAS inhibition decreases SW620 viability and affects several similar genes, which are involved in the regulation of tumor survival. Dual inhibition of FAK and HAS3 decreases viability to a greater degree than with either agent alone, and suggests that synergistic inhibition of colon cancer cell growth can result from affecting similar genetic pathways.

Topics: Aniline Compounds; Antineoplastic Agents; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hymecromone; Structure-Activity Relationship; Tumor Cells, Cultured

Dansyl fluoride (Dan-F), an active site directed fluorescent inhibitor of guanidinobenzoatase (GB), has been used for the location of tumour cells in frozen sections of human squamous cell carcinoma and colonic carcinoma tissues. The tumour cell surfaces having active GB bind Dan-F and fluoresce blue. The surrounding normal epithelial lung cell surfaces fail to bind Dan-F and hence lack fluorescence, whilst the normal colon cell surfaces have another isoenzymic form of GB, bind Dan-F and fluoresce blue. Kinetic studies have shown that Dan-F is an irreversible inhibitor of GB, and Dan-GB complexes are not dissociated with SDS and high salt concentration. However hydroxylamine (1 M) can dissociate Dan-GB complexes in the presence of 0.1% SDS, both on membrane-bound and in free solution. These studies suggest that Dan-F is a potent inhibitor of GB, and in very low concentration (3 x 10(-8) M) can be used as a novel fluorescent probe for the location of tumour cells in histological sections of human tissues.

Topics: Aminacrine; Binding Sites; Carboxylic Ester Hydrolases; Colonic Neoplasms; Coloring Agents; Dansyl Compounds; Endopeptidases; Enzyme Activation; Histocytochemistry; Humans; Hydroxylamine; Hydroxylamines; Hymecromone; Isoenzymes; Lung Neoplasms; Microscopy, Fluorescence; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence

Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon.

Topics: Carboxylic Ester Hydrolases; Cell Membrane; Chromatography, Affinity; Colonic Neoplasms; Dansyl Compounds; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Fluorescent Dyes; Heparin; Humans; Hymecromone; Isoenzymes; Kinetics; Microscopy, Fluorescence; Pentosan Sulfuric Polyester; Protease Inhibitors; Protein Conformation; Suramin; Trypan Blue; Xanthenes

A factor in colon carcinogenesis might be the partial defeat in colon epithelial cells of the protective enzymic barrier against xenobiotics, via bile acid inhibition of enzymes that detoxify mutagens. The applicability of aspects of this concept to glucuronosyltransferase, a phenol detoxification enzyme, was tested in a colon cancer cell line. Inhibition of glucuronidation of the test substrate, 4-methylumbelliferone, occurred at bile acid concentrations found in faecal water, and depended on pH for some bile acids. Lithocholate was the most inhibitory: the concentration causing 50% inhibition of the initial rate of glucuronidation (IC50) was about 3 microM at pH 7.4 and at pH 6.2. The inhibitory potency of deoxycholate and chenodeoxycholate increased when pH decreased, but still remained less than that of lithocholate: the IC50 for deoxycholate was 88.5 microM at pH 7.4, and 14.8 microM at pH 6.2, and for chenodeoxycholate the IC50 was 67.4 microM at pH 7.4, and 21.7 microM at pH 6.2. Cholate did not cause appreciable inhibition. The inhibitory effects were additive when lithocholate was present together with either deoxycholate or chenodeoxycholate. The results provide a mechanism for the comutagenicity of bile acids, a feature of which is the inter-relation of bile acid comutagenicity specifically with mutagens that are inactivated by a bile acid-inhibitable enzyme. The results are also in accord with the view that high concentrations of bile acids in solution in faecal water, especially lithocholate, are a risk factor for colon cancer.

Topics: Bile Acids and Salts; Chenodeoxycholic Acid; Colon; Colonic Neoplasms; Deoxycholic Acid; Dose-Response Relationship, Drug; Fluoresceins; Fluorescence; Fluorescent Dyes; Glucuronates; Glucuronic Acid; Glucuronosyltransferase; Glutathione Transferase; Humans; Hymecromone; Lithocholic Acid; Permeability; Tumor Cells, Cultured; Xenobiotics

Topics: Adenocarcinoma; alpha-Glucosidases; Animals; Cell Line; Colonic Neoplasms; Disaccharides; Glucosidases; Glucosides; Glycogen; Humans; Hymecromone; Kinetics; Maltose; Mice; Mice, Nude