Abstract. In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositolanchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; K D = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-flee ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo-and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.T RANSFERRIN (TF) 1 is the major serum glycoprotein that transports iron to most tissues in mammals. Diferric-TF (holo-TF) binds to a specific cell-surface receptor, the ligand-receptor complex is endocytosed and delivered to endosomes (for reviews see Crichton and Charloteaux-Wauters, 1987;Huebers and Finch, 1987). The low pH of endosomes triggers the release of iron from holo-TF but the resulting iron-free TF (apo-TF) remains bound to the receptor and is recycled back to the cell surface where, at neutral pH, apo-TF dissociates from the receptor. The human TF receptor is a transmembrane glycoprotein composed of two identical disulphide-linked subunits of 90 kD; each monomer can bind one molecule of TF. The intracellular domain of the receptor subunits contains specific signals for uptake in clathrin-coated vesicles (Trowbridge et al., 1993).African trypanosomes, the causative agents of sleeping sickness in humans and Nagana in cattle, are unicellular, flagellated protozoa that live extracellularly in the blood Address all correspondence to P. Overath,
The term ribosome-inactivating protein (RIP) is used to denominate proteins mostly of plant origin, which have N-glycosidase enzymatic activity leading to a complete destruction of the ribosomal function. The discovery of the RIPs was almost a century ago, but their usage has seen transition only in the last four decades. With the advent of antibody therapy, the RIPs have been a subject of extensive research especially in targeted tumor therapies, which is the primary focus of this review. In the present work we enumerate 250 RIPs, which have been identified so far. An attempt has been made to identify all the RIPs that have been used for the construction of immunotoxins, which are conjugates or fusion proteins of an antibody or ligand with a toxin. The data from 1960 onwards is reviewed in this paper and an extensive list of more than 450 immunotoxins is reported. The clinical reach of tumor-targeted toxins has been identified and detailed in the work as well. While there is a lot of potential that RIPs embrace for targeted tumor therapies, the success in preclinical and clinical evaluations has been limited mainly because of their inability to escape the endo/lysosomal degradation. Various strategies that can increase the efficacy and lower the required dose for targeted toxins have been compiled in this article. It is plausible that with the advancements in platform technologies or improved endosomal escape the usage of tumor targeted RIPs would see the daylight of clinical success.
Saponins are plant glycosides with favorable anti-tumorigenic properties. Several saponins inhibit tumor cell growth by cell cycle arrest and apoptosis with IC50 values of up to 0.2 microM. We discuss diverse groups of saponins (dioscins, saikosaponins, julibrosides, soy saponins, ginseng saponins and avicins) investigated in relation to tumor therapy and focus on cellular and systemic mechanisms of tumor cell growth inhibition both in vitro and in vivo. The review also describes saponins in combination with conventional tumor treatment strategies, which result in improved therapeutic success. Some combinations of saponins and anti-tumorigenic drugs induce synergistic effects with potentiated growth inhibition.
Saponin Targeted toxin A B S T R A C TTumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects.This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR.We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells.We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity.Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641.We further demonstrate that SA1641 did not influence the plasma membrane permeability,
The most potent immunotoxins (ITs) developed to date contain bacterial or plant cytotoxic components. As these are potentially immunogenic in man, human proteins are preferred for the long-term treatment of cancer. We have developed the first humanized IT for the treatment of CD64 malignancies such as acute myeloid leukemia. The bacterially expressed IT is composed of a humanized anti-CD64 single chain fragment [h22(scFv)] genetically fused to the human RNase angiogenin. As angiogenin lacks a dedicated translocation domain responsible for the higher potency of bacterial and plant-derived toxins, we have incorporated a recombinant adapter that contains a synthetic translocation domain flanked by proteolytically cleavable endosomal and cytosolic consensus sites. Although insertion of the adapter increased the cytotoxicity by up to 20-fold, serum stability was markedly reduced. Therefore, we designed a modified adapter variant with the endosomal-cleavable peptide deleted. The IT containing the truncated adapter showed significantly higher cytotoxicity than the adapter-free IT and superior serum stability to facilitate the potential applications in patients.
Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.
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