sodium-dodecyl-sulfate and Colonic-Neoplasms

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Colonic Neoplasms; Drugs, Chinese Herbal; HCT116 Cells; Humans; Male; Mice; Mice, Inbred C57BL; RAW 264.7 Cells; Receptors, Interleukin-6; Signal Transduction; Sodium Dodecyl Sulfate; STAT3 Transcription Factor

Fungi represent a significant proportion of the gut microbiota. Aberrant immune responses to fungi are frequently observed in inflammatory bowel diseases (IBD) and colorectal cancer (CRC), and mutations in the fungal-sensing pathways are associated with the pathogenesis of IBD. Fungal recognition receptors trigger downstream signaling via the common adaptor protein CARD9 and the kinase SYK. Here we found that commensal gut fungi promoted inflammasome activation during AOM-DSS-induced colitis. Myeloid cell-specific deletion of Card9 or Syk reduced inflammasome activation and interleukin (IL)-18 maturation and increased susceptibility to colitis and CRC. IL-18 promoted epithelial barrier restitution and interferon-γ production by intestinal CD8

Topics: Animals; CARD Signaling Adaptor Proteins; Cells, Cultured; Colitis; Colonic Neoplasms; Disease Models, Animal; Fungi; Gastrointestinal Microbiome; Humans; Inflammasomes; Inflammatory Bowel Diseases; Interleukin-18; Intestinal Mucosa; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Signal Transduction; Sodium Dodecyl Sulfate; Syk Kinase

High-fat-diet (HFD) consumption is associated with colon cancer risk. However, little is known about how the lipid composition of a HFD can influence prooncogenic processes. We examined the effects of three HFDs differing in the percentage of total calories from saturated fat (SF) (6, 12, and 24% of total caloric intake), but identical in total fat (40%), and a commercially available Western diet (26 and 41% saturated and total fat, respectively) on colon cancer development using the azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model. A second dose-response experiment was performed using diets supplemented with the saturated-fatty-acid (SFA)-rich coconut oil. In experiment 1, we found an inverse association between SF content and tumor burden. Furthermore, increased SF content was associated with reduced inflammation, increased apoptosis, and decreased proliferation. The second dose-response experiment was performed to test whether this effect may be attributed to the SF content of the diets. Consistent with the initial experiment, we found that high SF content was protective, at least in male mice; there was a decrease in mortality in mice consuming the highest concentration of SFAs. To explore a potential mechanism for these findings, we examined colonic mucin 2 (Muc2) protein content and found that the HFDs with the highest SF content had the greatest concentration of Muc2. Our data suggest that high dietary SF is protective in the AOM/DSS model of colon cancer, which may be due, at least in part, to the ability of SF to maintain intestinal barrier integrity through increased colonic Muc2.

Topics: Animals; Apoptosis; Azoxymethane; Cell Proliferation; Colonic Neoplasms; Diet, High-Fat; Dietary Fats; Fatty Acids; Female; Male; Mice; Mice, Inbred C57BL; Sodium Dodecyl Sulfate

Uncontrolled canonical Wnt signalling supports colon epithelial tumour expansion and malignant transformation. Understanding the regulatory mechanisms involved is crucial for elucidating the pathogenesis of and will provide new therapeutic targets for colon cancer. Epsins are ubiquitin-binding adaptor proteins upregulated in several human cancers; however, the involvement of epsins in colon cancer is unknown. Here we show that loss of intestinal epithelial epsins protects against colon cancer by significantly reducing the stability of the crucial Wnt signalling effector, dishevelled (Dvl2), and impairing Wnt signalling. Consistently, epsins and Dvl2 are correspondingly upregulated in colon cancer. Mechanistically, epsin binds Dvl2 via its epsin N-terminal homology domain and ubiquitin-interacting motifs and prohibits Dvl2 polyubiquitination and degradation. Our findings reveal an unconventional role for epsins in stabilizing Dvl2 and potentiating Wnt signalling in colon cancer cells to ensure robust colon cancer progression. The pro-carcinogenic role of Epsins suggests that they are potential therapeutic targets to combat colon cancer.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Animals; Azoxymethane; Binding Sites; Colitis; Colon; Colonic Neoplasms; Dishevelled Proteins; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Mice; Mice, Knockout; Phosphoproteins; Primary Cell Culture; Protein Binding; Protein Interaction Domains and Motifs; Protein Stability; RNA, Small Interfering; Sodium Dodecyl Sulfate; Wnt Signaling Pathway; Xenograft Model Antitumor Assays

The antimicrobial and anticancer activities of an antimicrobial peptide (AMP) KL15 obtained through in silico modification on the sequences of 2 previously identified bacteriocins m2163 and m2386 from Lactobacillus casei ATCC 334 by us have been studied. While significant bactericidal effect on the pathogenic bacteria Listeria, Escherichia, Bacillus, Staphylococcus, Enterococcus is exerted by KL15, the AMP can also kill 2 human adenocarcinoma cells SW480 and Caco-2 with measured IC50 as 50 μg/ml or 26.3 μM. However, the IC50 determined for KL15 on killing the normal human mammary epithelial cell H184B5F5/M10 is 150 μg/ml. The conformation of KL15 dissolved in 50% 2,2,2-trifluroroethanol or in 2 large unilamellar vesicle systems determined by circular dichroism spectroscopy appears to be helical. Further, the cell membrane permeability of treated SW480 cells by KL15 appears to be significantly enhanced as studied by both flow cytometry and confocal microscopy. As observed under a scanning electron microscope, the morphology of treated SW480 cells is also significantly changed as treating time by 80 μg/ml KL15 is increased. KL15 appears to be able to pierce the cell membrane of treated SW480 cells so that numerous porous structures are generated and observable. Therefore, KL15 is likely to kill the treated SW480 cells through the necrotic pathway similar to some recently identified AMPs by others.

Topics: Adenocarcinoma; Amino Acid Sequence; Antimicrobial Cationic Peptides; Bacterial Proteins; Caco-2 Cells; Cell Line, Tumor; Circular Dichroism; Colonic Neoplasms; Computer Simulation; Humans; Inhibitory Concentration 50; Microscopy, Electron, Scanning; Molecular Sequence Data; Phosphorylcholine; Protein Conformation; Sodium Dodecyl Sulfate

Control of colorectal cancer needs to be tailored to its etiology. Tumor promotion mechanisms in colitis-associated colon cancer differ somewhat from the mechanisms involved in hereditary and sporadic colorectal cancer. Unlike sporadic or inherited tumors, some experimental models show that colitis-associated colon tumors do not require cyclooxygenase (COX) expression for progression, and non-steroidal anti-inflammatory drugs (NSAIDs) which prevent sporadic or inherited colon cancer do not prevent colitis-associated colon cancer. We report that myeloperoxidase (MPO), an ancestor of the COX isoenzymes, is a determinant of colitis-associated colon tumors in Apc(Min/+) mice. During experimentally induced colitis, inhibition of MPO by resorcinol dampened colon tumor development. Conversely, in the bowels of Apc(Min/+) mice without colitis, resorcinol administration or 'knockout' of MPO gene coincided with a slight, but discernible increase in colon tumor incidence. Acrolein, a by-product of MPO catalysis, formed a covalent adduct with the phosphatase tensin homolog (PTEN) tumor suppressor and enhanced the activity of the Akt kinase proto-oncogene in vitro and in vivo. Thus, MPO may be an important determinant of diet and inflammation on colon cancer risk via its effect on endogenous exposure to oxidants and acrolein. We propose a hypothetical model to explain an apparent dichotomy between colon tumor occurrence and MPO inhibition in inflamed versus non-inflamed colons.

Topics: Acrolein; Animals; Colitis; Colonic Neoplasms; Female; Gene Expression; Inflammation; Male; Mice; Mice, Transgenic; Oxidation-Reduction; Peroxidase; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Resorcinols; RNA, Small Interfering; Sodium Dodecyl Sulfate

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.

Topics: Antigens; Biopsy; Blotting, Western; Colonic Neoplasms; Fixatives; Formaldehyde; Humans; Immunohistochemistry; Neoplasm Proteins; Paraffin; Paraffin Embedding; Proteomics; Sodium Dodecyl Sulfate; Temperature; Tissue Fixation

Capillary sodium dodecyl sulfate (SDS)-DALT electrophoresis (SDS-DALT-CE) refers to CE separation of proteins based on their size; DALT is the abbreviation for Dalton, the unit used to describe molecular weight. In this work, seven proteins from 18 to 116 kDa were denatured by SDS, labeled by 3-(2-furoyl) quinoline-2-carboxaldehyde, separated by SDS-DALT-CE in polyethylene oxide sieving matrix, and detected by laser-induced fluorescence (LIF) in a sheath flow cuvette. This method was combined with detergent differential fractionation, which is a protein fractionation method using a series of detergent-containing buffers to sequentially extract protein fractions from cells, to analyze the proteins in HT29 human colon adenocarcinoma cells. In addition, on-column labeling was demonstrated for protein analysis by SDS-DALT-CE with LIF, and applied to analysis of proteins in a single HT29 cancer cell. Most proteins had molecular masses from 10 to 120 kDa. Similar protein profiles were obtained for single cells and protein extract of a large cell population.

Topics: Adenocarcinoma; Colonic Neoplasms; Electrophoresis, Capillary; Fluorescence; Humans; Lasers; Molecular Weight; Neoplasm Proteins; Reproducibility of Results; Sodium Dodecyl Sulfate; 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

The expression of the mucin-bound sialyl-Lewisx epitope is increased in the tissue of most colorectal carcinomas and in the sera of about 30% of tumor patients. In colon cancer, a portion of the sialyl-Lex groups detectable with the monoclonal antibody AM-3 is located on MUC1 (C. Hanski et al., Cancer Res., 53: 4082-4088, 1993). In order to characterize the major colon carcinoma-associated sialyl-Lex-positive glycoprotein components, the tissue- and serum-derived antigens were investigated. The buoyant densities of the sialyl-Lewisx-positive antigens from tumor and normal colonic tissues and from sera of patients with colon carcinoma and healthy donors correspond to that of mucins (1.40 g/ml). The sialyl-Lex-positive mucins purified from both tissues elute under nonreducing conditions in the void volume of a Sepharose CL-2B column, indicating a molecular mass more than 2 x 10(7) daltons. They yield in immunoblot after SDS gel electrophoresis under reducing conditions a main band at an apparent M(r) 880,000. Radioactive labeling revealed that the band at M(r) 880,000 is the major protein component in sialyl-Lewisx-positive mucins both from tumor and normal colonic tissue. In sera of colon carcinoma patients, the sialyl-Lex moiety is also detectable mainly on a M(r) 880,000 glycoprotein band and, additionally, on a M(r) 140,000 molecule as well as on alpha 1-acid glycoprotein. Sera from healthy donors exhibited only a sialyl-Lex-positive glycoprotein with the apparent M(r) 140,000. Sandwich ELISA as well as immunoblots of mucins purified from the colon carcinoma cell line LS174T indicated that the sialyl-Lex moiety migrating in the M(r) 880,000 band is located on MUC2 protein core. Together, these data suggest that sialyl-Lex antigen in colon, colon carcinoma, and the sera of patients with this tumor is located on the MUC2 molecule, consisting of several subunits with an apparent M(r) 880,000, linked via disulfide bridges. The increase of sialyl-Lex expression in colon carcinomas appears to be mainly due to a more frequent transfer of sialyl-Lex moieties onto the mucin core in tumor tissue.

Topics: Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Chromatography; Colon; Colonic Neoplasms; Colorectal Neoplasms; Electrophoresis; Humans; Immunoblotting; Molecular Weight; Mucins; Oligosaccharides; Sialyl Lewis X Antigen; Sodium Dodecyl Sulfate

The ascitic fluid of a patient with colon cancer was found to contain an inactive cathepsin B-like enzyme. The inactive enzyme with a molecular weight of 40 kDa was converted by pepsin treatment into an active form with a molecular weight of 28 kDa as revealed by Sephadex G-75 gel chromatography. The inactive cathepsin B-like enzyme was considered to represent a precursor form and not an enzyme-inhibitor complex. The activated cathepsin B-like enzyme resembled human liver cathepsin B in its enzymatic characteristics. Cysteine proteinase inhibitor activity was also detected in the same ascitic fluid, and it was separated into two main forms by Sephadex G-75 gel chromatography. The high molecular weight inhibitor fractions reacted with antiserum against alpha-CPI and the low molecular weight fractions reacted with antiserum against cystatin B.

Topics: Ascites; Ascitic Fluid; Cathepsin B; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Female; Humans; Hydrogen-Ion Concentration; Immunoblotting; Immunodiffusion; Immunoelectrophoresis; Middle Aged; Molecular Weight; Pepsin A; Sodium Dodecyl Sulfate; Substrate Specificity

The subendothelial extracellular matrix (ECM) mediates the attachment of both human Ewing's sarcoma and colon carcinoma cells. Attachment and flattening of the sarcoma cells was sensitive to heat treatment but not to periodate oxidation of the ECM, whereas the colon carcinoma cells attached and flattened over heated but not periodate-treated ECM. Such differential sensitivity to heat treatment and periodate oxidation was also observed using purified fibronectin and laminin, respectively, but the inhibition of cell attachment was greater than with a similarly treated ECM. It is therefore conceivable that fibronectin and laminin specifically mediate the attachment and flattening of Ewing's sarcoma and colon carcinoma cells to the ECM, but that other constituents may support this attachment either directly or via interaction and stabilization of adhesive glycoproteins in the ECM. The ECM-mediated morphological differentiation of adult rat oligodendrocytes was sensitive to periodate oxidation to a much higher extent than to heat treatment of the ECM. In contrast, both treatments had only a small effect on the ECM-induced proliferation of vascular endothelial cells. These results indicate that different constituents of the ECM may be held responsible for its effects on different parameters of cell behavior, and that various cell types respond differently to a given modification of the ECM.

Topics: Animals; Aorta, Thoracic; Cattle; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Chemical Phenomena; Chemistry; Clone Cells; Colonic Neoplasms; Cornea; Endothelium; Extracellular Matrix; Glutaral; Guanidine; Guanidines; Hot Temperature; Humans; Nitrous Acid; Oligodendroglia; Oxidation-Reduction; Periodic Acid; Protein Denaturation; Rats; Sarcoma, Ewing; Sodium Dodecyl Sulfate

Water-soluble and sodium dodecyl sulfate (SDS)-extractable fractions were prepared from feces of four patients with inflammatory and six with non-inflammatory bowel disease. Both types of fractions were run on polyacrylamide gel electrophoresis, followed by electroblotting on nitrocellulose sheets of the separated components. The antibody reactivities in serum and in the intestinal wall against the extracted and separated fecal components were investigated. A striking lack of reactivity was observed when serum was used as antibody source against the fecal extracts both in patients with inflamed and in those with non-inflamed intestinal mucosa. The locally produced gut-associated IgG reacted more intensely with SDS-extractable fecal components than with water-soluble components. A strong reaction between intestinal-wall IgG extracts and a water-soluble antigen of 46-48 kD in the feces of two colitis patients was, however, observed. No clear correlation between the single bands and the occurrence of inflammatory bowel disease could be established because of the small number of patients.

Topics: Adult; Aged; Antibody Formation; Antigen-Antibody Reactions; Antigens, Bacterial; Antigens, Viral; Colitis, Ulcerative; Colonic Neoplasms; Crohn Disease; Electrophoresis, Polyacrylamide Gel; Feces; Female; Humans; Immunoenzyme Techniques; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Intestinal Mucosa; Intestines; Male; Middle Aged; Sodium Dodecyl Sulfate

The effect of emulsifier (nonionic surfactant) on the production of adenocarcinoma by methylazoxymethanol acetate in the large intestine of rats was studied. Following emulsifier, sodium lauryl sulfate administration, many cases of undifferentiated adenocarcinoma consisting of anaplastic glandular cells were induced in the experimental groups. Lymphatic invasion by cancer cells was found in 3 cases and metastasized to other organs in 6 cases. On the contrary, the control group (administered methylazoxymethanol acetate only) revealed well-differentiated adenocarcinoma in many cases. This fact may be due to an emulsifier used as a vehicle for the chemical, and the emulsifier might activate the character of promotion to carcinogenisity as a secondary agent. By virtue of the strong penetrating property of the emulsifier, colonial carcinogenesis seems to be enhanced.

Topics: Adenocarcinoma; Animals; Azo Compounds; Body Weight; Colonic Neoplasms; Emulsions; Female; Male; Methylazoxymethanol Acetate; Neoplasms, Experimental; Rats; Rats, Inbred Strains; Sodium Dodecyl Sulfate

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.

Topics: Animals; Carrier Proteins; Centrifugation, Density Gradient; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Mice; Molecular Weight; Receptors, Retinoic Acid; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Sodium Dodecyl Sulfate; Sulfhydryl Compounds; Tretinoin

Topics: Amino Acid Sequence; Antigens; Antigens, Neoplasm; Autopsy; Colonic Neoplasms; Electrophoresis, Disc; Glycoproteins; Humans; Sodium Dodecyl Sulfate