ABSTRACT
The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe lectin) were investigated in two mouse tumor models. After intravenous inoculation with RAW-117-P or L-1 sarcoma cells in Balb/c mice, rViscumin was given s.c. at non-toxic doses ranging from 0.3 to 150 ng rViscumin/kg. One set of experiments was performed to investigate the survival of rViscumin-treated animals. Another set was carried out to analyze the effect of rViscumin treatment on the number of tumor colonies in infiltrated lungs (RAW-117P) or liver (L-1) and the activation of immune cell subsets, respectively. An overall prolonged survival time after treatment with rViscumin and a reduction in the number of tumor colonies after administration of certain rViscumin doses was observed. Immunophenotyping of the peripheral leukocytes of treated mice revealed increased numbers of T-lymphocytes, pan-NK cells and activated monocytes. The results indicate that rViscumin has antineoplastic properties and might therefore be a promising candidate in cancer therapy.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antineoplastic Agents/pharmacology , Plant Preparations , Plant Proteins , Sarcoma, Experimental/drug therapy , Toxins, Biological/pharmacology , Animals , Drug Screening Assays, Antitumor , Immunocompetence , Liver Neoplasms, Experimental/drug therapy , Liver Neoplasms, Experimental/immunology , Liver Neoplasms, Experimental/secondary , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Lung Neoplasms/secondary , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/immunology , Male , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Recombinant Proteins/pharmacology , Ribosome Inactivating Proteins, Type 2 , Sarcoma, Experimental/immunology , Sarcoma, Experimental/secondary , Tumor Cells, CulturedABSTRACT
Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase.
