alpha-chymotrypsin and Colonic-Neoplasms

The present concept of the intestinal microflora has been reviewed with stressing the fact that it represents an entity with a weight comparable to one of the larger organs of the body. It is composed of about 500 different strains, many of which as yet have not been isolated because 95% of them are anaerobic and fastidious in their growth requirements. The intimate relationship of the flora to the surface epithelium of the intestines and the Lieberkühn's crypts are brought forward. The production of germfree animals is reviewed as an accounting of the most obvious differences between the germfree animal and its conventional counterparts, as far as gastroenterology is concerned. This includes the protective function of the normal intestinal flora, the caecum enlargement in germfree rodents, the influence of the flora on the metabolism of bilirubin, intestinal mucin, and pancreatic enzymes. Differences found in germfree animals due to the absence of the microbial flora related to the metabolism of fatty acids, bile acids, cholesterol and steroid hormones are also reviewed. The germfree animal has even proven to be an important research tool in such fields as carcinogenesis and studies of Crohn's disease and ulcerative colitis. There is need of a wider use of the germfree animal as a baseline in studies on the interference of the intestinal microbial flora with factors and conditions of great physiological and clinical importance.

Topics: Anaerobiosis; Animals; Bile Acids and Salts; Bilirubin; Carcinogens; Cholesterol; Chymotrypsin; Colitis, Ulcerative; Colon; Colonic Neoplasms; Crohn Disease; Fatty Acids; Germ-Free Life; Humans; Mice; Mucins; Neoplasms, Experimental; Pancreatic Elastase; Rats; Trypsin

Spirulina platensis is an excellent source of proteins (>60%) that can be hydrolyzed into bioactive peptides.. In this study, whole proteins of Spirulina platensis were extracted and hydrolyzed using three gastrointestinal endopeptidases (pepsin, trypsin and chymotrypsin). Subsequently, gel filtration chromatography was employed to separate hydrolysates, and four fractions (Tr1-Tr4) were obtained. Among them, Tr2 showed the strongest anti-proliferation activities on three cancer cells (MCF-7, HepG-2 and SGC-7901), with IC. Taken together, these peptides possessed anti-proliferation activities on cancer cells and low cytotoxicity on normal cells, suggesting that they might serve as a natural anticancer agent for nutraceutical and pharmaceutical industries. © 2016 Society of Chemical Industry.

Topics: Algal Proteins; Amino Acid Sequence; Anticarcinogenic Agents; Bacterial Proteins; Cell Line; Cell Line, Tumor; Cell Proliferation; China; Chymotrypsin; Colonic Neoplasms; Dietary Supplements; Drug Discovery; Hepatocytes; Humans; Molecular Weight; Neoplasms; Oligopeptides; Pepsin A; Peptide Fragments; Protein Hydrolysates; Spirulina; Trypsin

We have identified alkylresorcinols (ARs) as the major active components in wheat bran against human colon cancer cell growth (HCT-116 and HT-29) using a bioassay-guided approach. To further study the structure-activity relationships, 15 ARs and their intermediates (1-15) were synthesized expediently by the modified Wittig reaction in aqueous media, and six 5-alkylpyrogallols and their analogues (16-21) were prepared by the general Grignard reaction. The synthetic AR analogues were evaluated for activities against the growth of human colon cancer cells HCT-116 and HT-29 and the chymotrypsin-like activity of the human 20S proteasome. Our results found that (1) AR C13:0 and C15:0 (13 and 14) had the greatest inhibitory effects in human colon cancer cells HCT-116 and HT-29, while decreasing or increasing the side chain lengths diminished the activities; (2) two free meta-hydroxyl groups at C-1 and C-3 on the aromatic ring of the AR analogues greatly contributed to their antitumor activity; (3) the introduction of a third hydroxyl group at C-2 (20 and 21) into the aromatic ring of the AR analogues yielded no significant enhancement in activity against HCT-116 cells and decimated the effects against HT-29 cells, but dramatically increased the activity against the chymotrypsin-like activity of the human 20S proteasome; and (4) AR C11:0 (12) was found to have the greatest effect in a series of AR C9:0-C17:0 against the chymotrypsin-like activity of the human 20S proteasome.

Topics: Alkylation; Antineoplastic Agents, Phytogenic; Cell Division; Chymotrypsin; Colonic Neoplasms; Dietary Fiber; HCT116 Cells; HT29 Cells; Humans; Proteasome Endopeptidase Complex; Pyrogallol; Resorcinols; Serine Proteinase Inhibitors; Structure-Activity Relationship

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C(2)C(12) myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 20S proteasome alpha-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 microM). At a concentration of 4 nM, PIF induced a transient decrease in IkappaBalpha levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IkappaBalpha, an NF-kappaB inhibitory protein, returned to normal after 60 min. Depletion of IkappaBalpha from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-kappaB/IkappaB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-kappaB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-kappaB inhibitor peptide SN50, suggesting that NF-kappaB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-kappaB accumulation in the nucleus.

Topics: Animals; Blood Proteins; Blotting, Western; Cachexia; Cells, Cultured; Chymotrypsin; Colonic Neoplasms; Cysteine Endopeptidases; DNA Primers; Eicosapentaenoic Acid; Electrophoretic Mobility Shift Assay; Female; Fungal Proteins; Gene Expression Regulation; I-kappa B Proteins; Mice; Mice, Knockout; Multienzyme Complexes; Muscle Fibers, Skeletal; NF-kappa B; NF-KappaB Inhibitor alpha; Proteasome Endopeptidase Complex; Proteins; Proteoglycans; Transcription Factors; Ubiquitins

In our course of screening for novel proteasome inhibitors, TMC-95A and its diastereomers, TMC-95B to D, were isolated from the fermentation broth of Apiospora montagnei Sacc. TC 1093. TMC-95A inhibited the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of 20S proteasome with IC50 values of 5.4nM, 200nM, and 60nM, respectively. TMC-95B inhibited these activities to the same extent as TMC-95A, while the inhibitory activities of TMC-95C and D were 20 to 150 times weaker than that of TMC-95A and B. TMC-95A did not inhibit m-calpain, cathepsin L, and trypsin at 30 microM, suggesting its high selectivity for proteasome. Taxonomy of the producing strain is also described.

Topics: Chymotrypsin; Colonic Neoplasms; Cysteine Endopeptidases; Fermentation; HL-60 Cells; Humans; Hydrolysis; Inhibitory Concentration 50; Mitosporic Fungi; Multienzyme Complexes; Peptides; Peptides, Cyclic; Protease Inhibitors; Proteasome Endopeptidase Complex; Trypsin; Tumor Cells, Cultured

Bcl-2 expression was studied in a human colon cell line (HT-29) and in human colonic biopsies by Western and Northern blotting. Polyclonal antibodies raised against the Bcl-2 protein detected the expected 26 KDa protein in human colon. However, although Bcl-2 mRNA was present, the 26 KDa Bcl-2 protein was absent in HT-29 cells. Instead, a 30 KDa protein band strongly reacting with anti-Bcl-2 antibodies was found in HT-29 cells, and also in human colon, tonsil, and some other tissues. Alkaline phosphatase shifted the 30 KDa protein to the 26 KDa position in a time-dependent manner. 32P-labeling of HT-29 cells showed that the 30 KDa protein was phosphorylated. A 27 KDa phosphorylated protein was also immunoprecipitated by anti-Bcl-2 antibody. Phosphopeptide mapping showed that the 27 KDa protein contained a minimum of 3 and the 30 KDa protein at least an additional four phosphorylation sites. Phosphoamino acid analysis revealed that both the 30 KDa and 27 KDa proteins were phosphorylated on serine residues. These findings strongly suggest that the 30 KDa protein is a phosphorylated form of Bcl-2, which is widely distributed in human tissues.

Topics: Binding Sites; Blotting, Northern; Blotting, Western; Chymotrypsin; Colonic Neoplasms; Humans; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Biosynthesis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Tumor Cells, Cultured

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.

Topics: Adenocarcinoma; Animals; Cell Differentiation; Chromatography, High Pressure Liquid; Chymotrypsin; Colonic Neoplasms; Cytosol; Epithelial Cells; Epithelium; Humans; Insulin; Insulin-Like Growth Factor I; Male; Rats; Rats, Sprague-Dawley; Trypsin

In human intestinal malignancy, alterations occur in the expression of mucins defined by the MUC-2 gene. These changes include the unmasking of epitopes in the mucin protein core. In order to probe these modifications associated with mucins of the malignant phenotype, a monoclonal antibody (MAb) was developed against synthetic peptide with a sequence based upon that of the protein core of the MUC-2 mucin. The antibody (designated 996) was shown to recognize a high-molecular-weight glycoprotein from colonic carcinoma tissue. The material reacted uniformly with Concanavalin A but variably with other lectins, indicating heterogeneity in the associated oligosaccharide side chains. The protein core was accessible both to 996 antibody binding and to degradation with proteases. Immunization with the affinity-purified mucin-like material elicited antibodies reactive with both the immunogen and the synthetic peptides, confirming the immunogenic character of protein-core determinants. Epitope mapping studies, using synthetic peptides in solution and synthetic peptides tethered to the heads of plastic pins, indicated that the minimum epitope for the 996 antibody is a tetramer of T G T Q. Antibody interaction with the glutamine (Q) residue was determined to be of major importance in the antigen-antibody reaction. The findings illustrate the characterization of an anti-peptide antibody which may be used to probe alterations in MUC-2 mucin expression associated with human intestinal malignant disease.

Topics: Adsorption; Amino Acid Sequence; Antibodies, Monoclonal; Antibody Specificity; Chymotrypsin; Colonic Neoplasms; Concanavalin A; Epitopes; Glutamine; Humans; Immunoblotting; Molecular Sequence Data; Mucin-2; Mucins; Neoplasm Proteins; Trypsin

Incubation of monolayers of HT29-D4 cells (a clone of the human colonic adenocarcinoma cell line HT29) in the presence of 17.5 microM cycloheximide resulted in an increase in the number of vasoactive intestinal peptide (VIP) binding sites at the cell surface without any change in the affinity of receptor for its ligand. The increase in 125I-VIP-binding capacity was dose-dependent between 0.35 microM and 17.5 microM cycloheximide and was correlated with the inhibition of protein biosynthesis. At higher concentrations of drug (17.5-100 microM) a plateau corresponding to a twofold increase in VIP-binding capacity was reached independently of the extent of protein synthesis inhibition. We found that VIP receptors of HT29-D4 cells with such an enhanced binding capacity behaved like those of control cells with respect to receptor internalization and recycling (i.e. the cycle of occupied receptors was insensitive to cycloheximide). After inactivation of 90% of cell-surface VIP receptors by alpha-chymotrypsin, we observed a biphasic kinetic of reappearance of VIP-binding sites. 40% of VIP-binding sites reappeared very quickly (less than 5 min) and 100% within 17 h. The fast recovery of VIP receptors was probably due to the deployment of new binding sites from an intracellular pool. The rate and extent of recovery of these receptors were similar in control cells and in cycloheximide-treated cells. However, the slow recovery was inhibited in cycloheximide-treated cells probably because a pool of immature receptors was depleted by the drug before the alpha-chymotrypsin treatment. Our data are consistent with the existence of two different intracellular pathways of occupied and unoccupied VIP receptors.

Topics: Adenocarcinoma; Binding Sites; Cell Line; Chymotrypsin; Colonic Neoplasms; Cycloheximide; Dose-Response Relationship, Drug; Humans; Kinetics; Methionine; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoembryonic Antigen; Chymotrypsin; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Male; Neoplasms; Rectal Neoplasms; Trypsin Inhibitors

Using common purification procedures CEA was eluted as a symmetrical peak after gel-chromatography with a molecular weight (mw) of 180,000. The purity was assessed by the Ouchterlony test and immunoelectrophoresis, and by SDS-PAGE, where only one precipitation line and one band were obtained. However, two weak bands with a mw of 45,000 and 58,000 appeared, when iodinated CEA preparations were analyzed on SDS-PAGE. It was impossible to separate these proteins from CEA by a great variety of purification procedures. Out of several different immune sera, only two, anti-alpha-antitrypsin and anti-alpha-1-antichymotrypsin, reacted with the proteins. Furthermore, antisera against these protease inhibitors also immunoprecipitated the typical 180,000 mw band of CEA. This was also true for CEA prepared by other laboratories. We have purified and partially characterized both proteins. Although reacting with antisera against alpha-1-antitrypsin and alpha-1-antichymotrypsin they were not identical to the protease inhibitors, because they possess a different N-terminal amino acid sequence than published for them. However, the comparison of their sequences to 1900 total protein sequences made by computer search revealed a strong homology of the N-terminal sequence of the 45,000 mw protein with an internal sequence of alpha-1-antitrypsin. For the 58,000 mw protein no significant homology was found.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Amino Acid Sequence; Amino Acids; Autoradiography; Carcinoembryonic Antigen; Chemical Phenomena; Chemistry; Chromatography; Chymotrypsin; Colonic Neoplasms; Cross Reactions; Electrophoresis, Polyacrylamide Gel; Humans; Immunodiffusion; Immunoelectrophoresis; Molecular Weight; Neoplasm Proteins

The light microscopic, electron microscopic and histochemical features of a highly malignant colonic tumor resected from a 39 year old man are presented. The tumor was composed predominantly of undifferentiated cells with focally admixed neuroendocrine, exocrine and squamous cells, occasionally arranged in an organoid manner. Histochemically the tumor contained argyrophilic cells as well as cells that reacted positively with the antibodies to alpha-1-antitrypsin, alpha-1-antichymotrypsin, carcinoembryonic antigen and lysozyme. The term "stem cell carcinoma of the intestine" is proposed for this highly malignant tumor composed of undifferentiated cells exhibiting only focally their multidirectional developmental capacity.

Topics: Adult; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoembryonic Antigen; Carcinoma; Chymotrypsin; Colonic Neoplasms; Histocytochemistry; Humans; Immunologic Techniques; Keratins; Male; Microscopy, Electron; Muramidase; Staining and Labeling

Lysozyme, alpha 1-Antichymotrypsin and alpha 1-Antitrypsin were demonstrated by an immunoperoxidase technique (PAP) in malignant cells of adenocarcinomas of the stomach but not of the large intestine. Lymph-node metastases showed identical immunoreactivity to that of the primary tumour. Neoplasms arising from the cardia, the body and the pyloric antrum of the stomach showed different immunostaining reactions. It seems that these differences partly reflect the distribution of lysozyme, alpha 1-Antichymotrypsin and alpha 1-Antitrypsin in the normal gastric mucosa. The usefulness of our findings in the identification of the primary tumour in cases of lymph node metastases of unknown origin, is also discussed.

Topics: Adenocarcinoma; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Cardia; Chymotrypsin; Colonic Neoplasms; Gastric Mucosa; Histocytochemistry; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Muramidase; Pyloric Antrum; Stomach Neoplasms

The alpha-protein which we previously detected in PCA extracts of malignant tumours, normal tissues, sera and other body fluids has been further tested. The alpha-protein was identified by immunological methods as alpha1-antichymotrypsin. Experiments by crossed immunoelectrophoresis showed that this protein cross-reacts with the betaE-protein found in the PCA extracts. The betaE-protein shows heterogeneity in molecular size, electrophoretic mobility and it has been demonstrated that it shares antigenic determinants with the CEA or betaI-molecule. Cross-reaction between betaE and other glycoproteins found in the PCA extract of normal or malignant tissue or serum was not detected.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Chromatography, Gel; Chymotrypsin; Colonic Neoplasms; Cross Reactions; Epitopes; Humans; Immune Sera; Immunoelectrophoresis; Neoplasm Proteins; Perchlorates

The molecular weight fractions of 10,000-50,000 daltons prepared from "used" medium obtained during cultivation of human colon carcinoma cells (SW-48) in vitro inhibited the proliferation and DNA synthesis of these cells. Fractions exceeding 50,000 daltons were not inhibitory; those less than 10,000 daltons were cytotoxic. The inhibitory fraction did not affect either proliferation of human fibroblasts or transformation of human lymphocytes in vitro. Similar fractions from the colon mucosa of other species inhibited the proliferation of SW-48 cells, whereas extracts of dog jejunum or lung did not. This mitotic inhibition was completely reversible and could be destroyed by preincubation with trypsin. Therefore, colon cells appear to contain a cell- (but not species) specific, endogenous mitotic inhibitor or chalone.

Topics: Animals; Cattle; Cell Division; Cells, Cultured; Chymotrypsin; Colonic Neoplasms; Culture Media; DNA, Neoplasm; Dogs; Fibroblasts; Growth Inhibitors; Humans; Lymphocytes; Molecular Weight; Ribonucleases; Swine; Trypsin

Topics: Adenocarcinoma; Adenosine Triphosphatases; Alkaline Phosphatase; Carboxypeptidases; Carcinoembryonic Antigen; Chymotrypsin; Colonic Neoplasms; Cystinyl Aminopeptidase; Esterases; Glucuronidase; Humans; Liver Neoplasms; Neoplasm Metastasis; Sulfatases

Topics: Adolescent; Adult; Chymotrypsin; Colectomy; Colitis, Ulcerative; Colon; Colonic Neoplasms; Female; Gastrointestinal Hemorrhage; Humans; Ileostomy; Intestinal Mucosa; Intestinal Polyps; Male; Middle Aged; Necrosis; Protease Inhibitors; Secretory Rate; Trypsin; Trypsin Inhibitors

Topics: Chymotrypsin; Colitis, Ulcerative; Colonic Neoplasms; Endoscopes; Fiber Optic Technology; Humans; Intestinal Polyps; Magnesium Sulfate; Methods; Premedication; Rectal Neoplasms; Rectum; Sigmoidoscopes