Induction of the PERK-eIF2α-ATF4 Pathway in M1 Macrophages under Endoplasmic Reticulum Stress.

Translation inhibition can activate two cell death pathways. The first pathway is activated by translational aberrations, the second by endoplasmic reticulum (ER) stress. In this work, the effect of ribosome-inactivating protein type II (RIP-II) viscumin on M1 macrophages derived from the THP-1 cell line was investigated. The number of modified ribosomes was evaluated by real-time PCR. Transcriptome analysis revealed that viscumin induces the ER stress activated by the PERK sensor.

Specificity of viscumin revised. As probed with a printed glycan array.

Viscumin, a lectin used in anti-cancer therapy, was originally considered as ßGal recognizing protein; later, an ability to bind 6'-sialyl N-acetyllactosamine (6'SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galß1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral ß-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a ß-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Intracellular Transport of Ribosome-Inactivating Proteins Depends on Annexin 13.

In the present study, we assessed the role of annexin 13 membrane-binding protein (ANXA13) in the intracellular transport of vesicles containing type II ribosome-inactivating proteins (RIP-IIs). A modified human intestinal epithelial cell line HT29 was used, in which the expression of ANXA13 was significantly reduced. The cytotoxic effect of ricin and viscumin was evaluated by modification of 28S ribosome RNA. The observed differences in the activity of toxins on the parental and modified HT29 lines indicate that ANXA13 plays a different role in the intracellular transport of vesicles containing the RIP-IIs.

Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin.

The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.

Construction of eco-biosensor and its potential application for highly selective, sensitive and fast detection of viscumin.

Viscum album lectin 1 (Viscumin) is one of the most important plant-based protein of potential adjuvant in cancer treatment. Therefore, the use of nano-biosensor technology as a novel emerges of biosensors is crucial to detect this modal agent in pharmacological study. Molecular imprinted polymer using 9-mer peptides sequence (epitope) was applied as a template. Using ultraviolet light, hydrogen bonding attained between the functional monomer and epitope, leading to the formation of a molecularly imprinted polymer. In the following, the epitope was derived from the surface of the polymer by sodium dodecyl sulfate (SDS) 2.5% and acetic acid 0.6% w/w. Finally, the designed nano-biosensor was exposed to different concentrations of the epitope. The selectivity of the nano-biosensor was tested in complex matrices such as blood plasma and urine. The scatchard analysis was covered for a consequence of the dissociation constants and the numbers of binding sites. Based on the results, the designed nano-biosensor has a limit of detection of 0.117 ng/µl and limit of quantification of 0.517 ng/µl in PBS buffer, respectively. These amounts stood 0.5 ng/µl and 0.8 ng/µl for urine environment and 1.25 ng/µl and 5 ng/µl for human blood fresh frozen plasma in the presence of ricin as the most homologue of viscumin (ML1) in fixed concentration (12:1), respectively. The time of detection and optimum pH was 8.0 min and 7.4, respectively. Designed and synthesized nano-biosensor is adequately qualified to be used in diverse complex areas, due to good efficiency.

Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin.

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria.

[Ribosome Inactivation and the Integrity of the Intestinal Epithelial Barrier].

The mistletoe lectin viscumin (MLI) is a ribosome-inactivating protein from Viscum album widely used in cancer therapy. Its antitumor properties are due to its immunomodulating action, previously demonstrated in experiments involving intravenous, subcutaneous, and oral administration of viscumin. To investigate whether viscumin has a cytotoxic effect on the intestinal epithelium, its safety was assessed using (i) impedance spectroscopy to measure the integrity of the colorectal adenocarcinoma Caco-2 cell monolayer after exposure to viscumin and (ii) a novel technique of determining the portion of viscumin-inactivated ribosomes. It was shown that inactivation of at least 20% of the ribosomes within 6 h did not lead to disruption of the Caco-2 cell monolayer or alter the physicochemical parameters of enterocyte membranes.

Elimination of a Viscumin-Ferromagnetic Nanoparticles Conjugate from the Tumor Nodule in Mice.

External magnetic field is characterized by low toxicity and existence of magnetic properties, which contributes to an interest in the development of products from ferromagnetic nanoparticles (FNP) for antitumor therapy. Previously we synthesized a conjugate of ferromagnetic magnetite nanoparticles and viscumin (mistletoe lectin I, MLI), which exhibits the antitumor activity. Studying the pharmacological properties of this conjugate (FNP-MLI) was directed to the evaluation of FNP-MLI elimination after intratumor injection in mice. The elimination rate of FNP-MLI was much lower than that of native plant MLI. The presence of FNP-MLI was not accompanied by undesired changes in the tumor tissue. The use of a FNP-MLI conjugate allowed us to prolong the time of MLI presence in tissues without increasing the dose of exogenous lectin. These features contribute to the prolongation of an immunomodulatory effect of MLI.

Target Cell Glycosylation Determines the Biodistribution of Plant Lectin Viscumin.

We studied the possibility of using viscumin lectin (MLI) for targeted delivery of antitumor drugs. Affinity of MLI for more than 600 oligosaccharide structures was determined and the glycosylation profiles of cell surface in various mouse tissues were analyzed. It was found that biodistribution of MLI was determined by not only expression of oligosaccharides specifically recognized by the lectin in tissue cells, but also by the structure of glycan in general.

Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10-11-10-7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Preparation of Viscumin-Ferromagnetic Particles Conjugate and Study of Its Internalization by Human Glioblastoma A172 Cells.

Magnetite particles modified by polyethylene glycol with a molecular weight of 3 kDa and hydrodynamic diameter of ~60 nm were used. Plant lectin viscumin covalently immobilized on these nanoparticles retained its binding activity. Immunochemical characteristics of conjugated viscumin were evaluated using monoclonal antibodies. The resultant conjugate with a hydrodynamic diameter of 70 nm was used for studies of binding and internalization by target cells. Binding of viscumin and its conjugate was determined by receptors containing terminal galactose, while intracellular distribution varied. The model system presented in this study can be used for creation of drugs for target therapy.