epidermal-growth-factor and Hemolysis

The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models.. Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using (18)F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography.. In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05).. This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.

Topics: Animals; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Dianthus; Disease Models, Animal; Drug Therapy, Combination; Epidermal Growth Factor; Hemolysis; Humans; Immunotoxins; Male; Mice; Positron-Emission Tomography; Ribosome Inactivating Proteins, Type 1; Saponins; Tumor Burden; Xenograft Model Antitumor Assays

Human tissue-type plasminogen activator (t-PA) is cleared rapidly from the circulation by hepatic receptors, one of which recognizes a site in the epidermal growth factor-like domain of the molecule. To define this site more precisely, we have used oligonucleotide-mediated mutagenesis to introduce amino acid substitutions at specific positions located in turns that connect antiparallel beta-sheets in the epidermal growth factor-like domain. Mutated t-PA proteins with amino acid substitutions of the tyrosine residue at position 67 showed markedly lower rates of endocytosis and degradation by cultured cells of the rat hepatoma (H4) line that express a specific receptor for t-PA, and their half-life in the circulation of rats was extended significantly because of a reduction in the rate of the rapid alpha-phase of clearance. The enzymatic properties and fibrinolytic activity of these mutants in vitro were not significantly different from those of wild-type t-PA. We conclude that tyrosine 67 comprises a key determinant in the clearance of t-PA by a specific hepatic receptor.

Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Cell Line; Electrophoresis, Polyacrylamide Gel; Endocytosis; Epidermal Growth Factor; Hemolysis; Humans; Kinetics; Liver Neoplasms, Experimental; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Protein Conformation; Rats; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Recombinant Proteins; Substrate Specificity; Tissue Plasminogen Activator; Transfection; Tyrosine

The biological effects of EGF and PDGF growth factors on A172 and hEGFr-3T3 cell lines were studied using RBC induced cytolysis and polyacrylamide-gelatin gel electrophoresis assays. The authors report that growth factor-induced cytotoxicity in these cells is mediated by proteolytic enzymes. Treatment of A172 cells with either EGF or PDGF resulted in marked increase of their cytotoxicity (Release Index = 150%). Similarly, RBC induced release index by hEGFr-3T3 cells was elevated to 420% in the presence of 3.4 pM of EGF. However, in A172 cells, PDGF did not have a significant effect on DNA and protein synthesis indicating that stimulation of proteolytic activity is independent of the growth factor signaling pathway. Growth factor induced cytotoxicity was significantly reduced by protease inhibitors in both cell lines. Using EDTA and leupeptin several proteolytic species were identified and localized to cellular membranes as evidenced by polyacrylamide-gelatin electrophoresis assay. These data suggest that growth factors regulate the activation or secretion of proteolytic enzymes in cancer cells and may mediate the invasive and metastatic behavior of these cells.

Topics: Amiloride; Animals; Cell Line; Cell Survival; Cells, Cultured; Cycloheximide; Enzyme Activation; Epidermal Growth Factor; Hemolysis; Humans; Leupeptins; Mice; Peptide Hydrolases; Platelet-Derived Growth Factor; Tosyllysine Chloromethyl Ketone; Tumor Cells, Cultured