Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins. AIM OF THE STUDY: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VT-A) in comparison to a structurally similar lectin and thionin. MATERIALS AND METHODS: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively. RESULTS: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin®Salicis and Tiliae and a strong with Iscucin®Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml - 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested. CONCLUSION: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

Activation of RAW264.7 mouse macrophage cells in vitro through treatment with recombinant ricin toxin-binding subunit B: involvement of protein tyrosine, NF-κB and JAK-STAT kinase signaling pathways.

Ricin toxin-binding subunit B (RTB) is a galactose-binding lectin protein. In the present study, we investigated the effects of RTB on inducible nitric oxide (NO) synthase (iNOS), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, as well as the signal transduction mechanisms involved in recombinant RTB-induced macrophage activation. RAW264.7 macrophages were treated with RTB. The results revealed that the mRNA and protein expression of iNOS was increased in the recombinant RTB-treated macrophages. TNF-α production was observed to peak at 20 h, whereas the production of IL-6 peaked at 24 h. In another set of cultures, the cells were co-incubated with RTB and the tyrosine kinase inhibitor, genistein, the phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, the p42/44 inhibitor, PD98059, the p38 inhibitor, SB203580, the JNK inhibitor, SP600125, the protein kinase C (PKC) inhibitor, staurosporine, the JAK2 inhibitor, tyrphostin (AG490), or the NOS inhibitor, L-NMMA. The recombinant RTB-induced production of NO, TNF-α and IL-6 was inhibited in the macrophages treated with the pharmacological inhibitors genistein, LY294002, staurosporine, AG490, SB203580 and BAY 11-7082, indicating the possible involvement of protein tyrosine kinases, PI3K, PKC, JAK2, p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in the above processes. A phosphoprotein analysis identified tyrosine phosphorylation targets that were uniquely induced by recombinant RTB and inhibited following treatment with genistein; some of these proteins are associated with the downstream cascades of activated JAK-STAT and NF-κB receptors. Our data may help to identify the most important target molecules for the development of novel drug therapies.

Nigrina b: una proteína inactivadora de ribosomas no tóxica del saúco. Utilidad farmacéutica en la construcción de inmunotoxinas y conjugados para la terapia del cáncer* / Nigrin b: a ribosome-inactivating protein from elder. Pharmaceutical utility in the construction of inmmunotoxins and conjugates for cancer therapy

El saúco posee una colección de proteínas inactivadoras de ribosomas, en particular las nigrinas, que parecen ser responsables de su toxicidad. La nigrina b (corteza) posee isoformas en frutos (nigrina f) y hojas (nigrina l) que se encuentran a mayor concentración en las fases iniciales del desarrollo. Nigrina b es 103-104 veces menos tóxica que la ricina, una proteína inactivadora de ribosomas relacionada estructuralmente con la nigrina b y extremadamente tóxica presente en Ricinus communis L., que se utiliza para la construcción de inmunotoxinas para la terapia del cáncer. Nigrina b se internaliza en las células superiores por una vía intracelular distinta a la de la ricina independiente de temperatura y de brefeldina A. La administración de dosis letales de nigrina b produce lesiones intestinales irreversibles específicas por destrucción de las criptas y desaparición del epitelio intestinal lo que ocasiona hemorragias intestinales letales. Los datos sobre estructura primaria de las nigrinas indican que la diferencia de toxicidad con la ricina se basa en cambios de aminoácidos clave en los dominios de fijación de galactosa en las cadenas B de ambas proteínas. Esta característica de la nigrina b es de enorme utilidad en la construcción de los denominados "proyectiles mágicos" o fármacos inteligentes capaces de interaccionar y destruir blancos específicos. Como ejemplos de dichos proyectiles mágicos se han construido conjugados transferrina- nigrina b/ebulina l que han mostrado ser efectivos contra células cancerosas que sobreexpresan el receptor de transferrina y una inmunotoxina antitumoral contra el CD105 de ratón que identifica a la células CD105+ y las destruye de manera selectiva tanto in vitro como in vivo. Ello convierte a la nigrina b en una herramienta útil en la inmunotoxiterapia del cáncer (AU)

A new antigenic epitope appears in the catalytic subunit of viscumin during intracellular transport.

The plant toxin viscumin (60 kD) consists of B- ("binding") and A- ("active") subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody-toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody-toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of (1)H-NMR spectroscopy.

Differences in endocytosis and intracellular sorting of ricin and viscumin in 3T3 cells.

Ricin and viscumin are heterodimeric protein toxins. Their A-chain is enzymatically active and removes an adenine residue from the 28S rRNA, the B-chain has lectin activity and binds to terminal galactose residues of cell surface receptors. The toxins reveal a high degree of identity in their amino acid sequences. Nevertheless, uptake into 3T3 cells occurs via different receptors and endocytotic pathways. This has been revealed by enzyme linked based analysis of ricin competition with viscumin, and by fluorochrome-labeled toxins (viscumin-FITC, ricin-Alexa 568), which were added simultaneously or separately to living cells. Then the uptake was followed by confocal laser scanning microscopy. Ricin immediately is delivered to the tubular and vesicular structures of endosomes in the perinuclear area while viscumin becomes endocytosed into small vesicles preferentially in the cell periphery. After about 60 min both these toxins may be found in tubo-vesicular structures of endosomes where the sorting process can directly be observed. The fact that this sorting takes place is a strong argument for the assumption that the toxins are bound to membrane proteins, either to their original receptors or to other proteins inside the endosomal compartment exhibiting terminal galactose residues. The toxins are biologically fully active as has been proven by binding and by toxicity experiments, thus the differences in targeting do not arise from labeling.

Allergen cross-reactivity between proteins of the latex from Hevea brasiliensis, seeds and pollen of Ricinus communis, and pollen of Mercurialis annua, members of the Euphorbiaceae family.

Allergen cross-reactions among three strongly sensitizing Euphorbiaceae species, i.e., the rubber tree (Hevea brasiliensis), castor bean (Ricinus communis), and the Mediterranean weed Mercurialis annua were studied in Finnish patients (n = 25) allergic to natural rubber latex (NRL), but with no known exposure to castor bean or M. annua, and French patients allergic to castor bean (n = 26) or to M. annua (n = 9), but not to NRL. In immunoglobulin E (IgE)-immunoblotting, 28% of NRL-allergic patient sera recognized castor bean seed and 48% reacted to castor bean pollen proteins. Likewise, 35% of the NRL-allergic patient sera bound to M. annua pollen allergens. Nineteen percent of castor bean-allergic patients showed IgE to NRL and 8% to M. annua proteins. Sera from patients allergic to M. annua reacted in 44% to NRL, in 56% to castor bean seed, and in 78% to castor bean pollen proteins. In immunoblotting, castor bean seed extract inhibited the binding of NRL-reactive IgE to 20 kDa, 30 kDa of NRL, and 55 kDa of proteins; NRL extract, in turn, inhibited the binding of castor bean-reactive IgE to 14, 21-22, 29, and 32-34 kDa of castor bean proteins. In ELISA inhibition, NRL extract inhibited 33% of the binding of M. annua--reactive IgE of pooled sera to M. annua pollen. In conclusion, allergen cross-reactivity in vitro was observed among three botanically related Euphorbiaceae members, H. brasiliensis, R. communis, and M. annua, but the molecular specificity of the observed cross-reactions as well as their clinical significance remains to be elucidated. Allergen cross-reactivity should be taken into account in diagnostic work.

Relationship of the CD22 immunotoxin dose and the tumour establishment in a SCID mice model.

Immunotoxins (ITs) may be very potent to erradicate tumour growth in vivo. We investigated the influence of the IT-dose, in relation to the establishment of the tumour, on the anti-tumour activity of CD22-recombinant (rec) ricin A for a disseminated tumour (Ramos) in SCID mice. Furthermore, the enhancement of the IT cytotoxicity in vivo by chloroquine was assessed. CD22-rec ricin A appeared to be highly effective. Paralysis of the hind legs was significantly delayed by a very low IT-dose of 2 microg administered intravenously (i.v.) 7 days after i.v. inoculation of the tumour cells. Even a dose of 30 microg administered 21 days after inoculation of the target cells significantly delayed the onset of paralysis up to 8 days compared with the median paralysis time (MPT) of the control group. The efficacy of treatment was obviously influenced by the establishment of the tumour, the tumour load and localisation. The anti-tumour activity of 10 and 30 microg IT diminished when the IT was administered after increasing the time lag following inoculation of tumour cells. Delaying IT administration resulted in growth of solid tumours. This implies that cells migrate to sanctuaries protected from the IT indicating that the anti-tumour activity was influenced by the accessibility of the IT to the target cells. The in vivo anti-tumour activity of CD22-rec ricin A could not be enhanced by simultaneously administered chloroquine, despite the continuous infusion with an intraperitoneally (i.p.) implanted mini-osmotic pump. Ex vivo experiments revealed that the maximally tolerated serum concentration (3.9 microM) was too low to be effective. In conclusion, CD22-rec ricin A is highly effective for in vivo treatment of B-cell malignancies, in particular if treatment is started when the tumour load is low and before migration takes place to poorly accessible sanctuaries.

Mistletoe lectin A-chain unfolds during the intracellular transport.

Protein conformation during intracellular routing and translocation of the ribosome-inactivating proteins was investigated on hybridomas producing monoclonal antibodies (monAbs) against mistletoe lectin (ML). Decrease in the toxin activity towards these hybridomas is accounted for by the intracellular interaction of monAbs and the toxin resulting in the interruption of enzymatic subunit translocation into the cytosol. Obtained monAbs interacted with denatured ML A-chain (MLA) and a panel of MLA synthetic octapeptides linked to the surface of polyethylene pins. Enzyme-linked immunosorbent assay (ELISA) shows that monAbs recognize five epitopes in denatured MLA. Treatment of MLA by 3 M of guanidine hydrochloride leads to appearance of the epitopes. Hybridoma TA7 has been shown to be insensitive to cytotoxic action of ML. TA7 monAb as we have shown recognizes epitope 101-105, FTGTT, and inhibits the liposome aggregation induced by MLA. A study of the cytotoxicity of ML and ricin for the hybridomas revealed that the unfolding of A-chain is probably required for intracellular transport and cytotoxic activity of ML.

Effects of castor bean extract and ricin A-chain on ovulation and implantation in rabbits.

The anti-implantation and antiovulation effects of castor bean extract (CBE) and ricin A-chain (RAC) were evaluated in rabbits. Both CBE and RAC, administered intraperitoneally on days 5-9 of pregnancy, exhibited a pronounced decrease in maternal body weight gain and in death of all fetuses. A significant (p < 0.01) decrease of implantation sites resulted after rabbits were treated with RAC on the first 6 consecutive days of pregnancy. When female rabbits were treated with RAC for 10 consecutive days followed by human chorionic gonadotropin (hCG) (50 IU/kg intravenously), there was a 30% reduction in the number of corpora lutae. These data clearly indicate that CBE and RAC possess potent effects on implantation and ovulation in rabbits. The protein contents of castor bean extract, separated by polyacrylamide gel electrophoresis, revealed the presence of several protein bands, ricin toxin being a major constituent of the extract.

Membrane fusion mediated by ricin and viscumin.

The ribosome inactivating plant proteins (RIPs) ricin and viscumin but not Ricinus communis agglutinin are able induce vesicle-vesicle fusion. A model is suggested in which the toxicity of the RIPs is partially determined by their fusogenicity. Herein, fusion is hypothesized to allow the RIPs to leak across endocytic vesicles to approve their access to cytoplasmic ribosomes.

Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity.

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

Opposing neuroendocrine actions of the natriuretic peptides: C-type and A-type natriuretic peptides do not interact with the same hypothalamic cells controlling prolactin secretion.

The two major members of the family of natriuretic peptides (NPs) in brain, A-type natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) exert opposing actions on the neuroendocrine regulation of prolactin (PRL) secretion. We have targeted for compromise and destruction cells within the diencephalon which bear receptors for ANP (NRP-A receptors), CNP (NRP-B receptors), or both peptides (NPR-C receptors) using novel cytotoxin cell targeting methodology in order to determine if the neuroendocrine effects of these two peptides are exerted on similar cell systems. In animals pretreated with ANP conjugated to the cytotoxic A chain of ricin, central administration of a dose of ANP which is known to inhibit PRL secretion did not alter PRL levels in plasma; however, subsequent administration of CNP elicited the stimulation of PRL secretion. In rats pretreated with CNP-ricin A chain conjugate, a treatment we hypothesize targets for destruction CNP responsive cells, ANP injection did inhibit PRL secretion, while the stimulatory effect of CNP was absent. These results suggest that the neuroendocrine effects of these two natriuretic peptides on PRL secretion are expressed on different cellular elements of the hypothalamo-pituitary axis. Furthermore, they reveal that neither peptide acts directly on the tuberoinfundibular dopamine system since pretreatment with either cytotoxin conjugate failed to alter basal PRL levels. Thus ANP and CNP do not appear to express opposing actions on the same cell systems, suggesting the recruitment of each peptide individually by differing, unique stimuli for PRL release.

C-type natriuretic peptide mediates the hypothalamic actions of the natriuretic peptides to inhibit luteinizing hormone secretion.

Both A- and C-type natriuretic peptides (ANP and CNP, respectively) significantly reduce LH secretion when injected into the third cerebral ventricle of conscious rats. To establish which natriuretic peptide receptor subtype transduces these inhibitory messages, we have employed novel cytotoxin cell targeting techniques to selectively destroy cells in the hypothalamus that respond to ANP or CNP. Rats pretreated with ANP conjugated to the toxic A-chain of the plant cytotoxin ricin failed 1 week later to respond to central injection of ANP with the normal inhibition of LH secretion. These rats did, however, respond with significant inhibition of LH secretion to central injection of CNP. In fact, the LH inhibition observed after CNP injection was significantly greater than that expressed after similar injection of CNP in rats pretreated with unconjugated ricin A-chain (toxin control). Those control rats displayed significant reduction of LH levels in response to ANP injection as well. Plasma LH levels were not significantly affected by central administration of either ANP or CNP in rats pretreated with ricin A-chain conjugated to CNP. These results further demonstrate the power of this novel technology and provide positive evidence supporting our hypothesis that ANP exerts its LH-inhibiting effect by displacing endogenous CNP from clearance receptors within the brain. This endogenous CNP, then, like exogenously applied CNP, activates the guanyl cyclase-B receptors on cells, which are part of the network controlling the release of LHRH.

The activity of two immunotoxins composed of monoclonal antibody MoAb-16 and A-chain of ricin (MoAb-16-RTA) or A-chain of mistletoe lectin I (MoAb-16-MLIA).

The A-chains of ricin obtained from Ricinus communis or mistletoe lectin I from Viscum album were coupled to the monoclonal, anti-L1210V antibody MoAb-16, using SPDP as a cross linking agent. The cytotoxic activity in vitro of these immunotoxins was compared. Each of two immunotoxins tested, applied in vitro for 1 h in appropriate doses, caused irreversible inhibition of leukemic L1210 cells proliferation. Unexpectedly, MoAb-16-MLIA immunotoxin appeared to be cytotoxic to normal bone marrow progenitor cells, as observed in NCFUS tests. Moreover, this immunotoxin revealed cytotoxic effect to the P388 leukemia cells which do not share the antigen, common within L1210 leukemia cells, detected by MoAb-16 antibody.

Effect of immunotoxins against Trypanosoma cruzi.

Immunotoxins were constructed with IgG antibodies against Trypanosoma cruzi surface antigens by hybridization with abrin (ITA) and ricin (ITR) A chains. The biological activity of the hybrid macromolecules was tested on the parasite forms. Motility of parasite forms was lost in vitro after incubation with ITR. In general, killing of the parasite with ITR was more efficient than with ITA. Inhibition of protein synthesis after incubation with either ITR or ITA, measured by 3H-leucine incorporation, confirmed the parasite immobilization experiments. The lethal effect was potentiated when the immunotoxins were used in the presence of 2.5 mM ammonium chloride. T. cruzi antibodies specific to cell surface antigens are excellent drug carriers that can be delivered to the target cell. However, ITR and ITA did not reduce parasitemia or increase survival of mice infected with T. cruzi.

Reduced turnover of the elongation factor EF-1 X ribosome complex after treatment with the protein synthesis inhibitor II from barley seeds.

The effect of the protein synthesis inhibitor II from barley seeds (Hordeum sp.) on protein synthesis was studied in rabbit reticulocyte lysates. Inhibitor treatment of the lysates resulted in a rapid decrease in amino acid incorporation and an accumulation of heavy polysomes, indicating an effect of the inhibitor on polypeptide chain elongation. The protein synthesis inhibition was due to a catalytic inactivation of the large ribosomal subunit with no effect on the small subparticle. The inhibitor-treated ribosomes were fully active in participating in the EF-1-dependent binding of [14C]phenylalanyl-tRNA to poly(U)-programmed ribosomes in the presence of GTP and the binding of radioactively labelled EF-2 in the presence of GuoPP[CH2]P. Furthermore, the ribosomes were still able to catalyse peptide-bond formation. However, the EF-1- and ribosome-dependent hydrolysis of GTP was reduced by more than 40% in the presence of inhibitor-treated ribosomes, while the EF-2- and ribosome-dependent GTPase remained unaffected. This suggests that the active domains involved in the two different GTPases are non-identical. Treatment of reticulocyte lysates with the barley inhibitor resulted in a marked shift of the steady-state distribution of the ribosomal phases during the elongation cycle as determined by the ribosomal content of elongation factors. Thus, the content of EF-1 increased from 0.38 mol/mol ribosome to 0.71 mol/mol ribosome, whereas the EF-2 content dropped from 0.20 mol/mol ribosome at steady state to 0.09 mol/mol ribosome after inhibitor treatment. The data suggest that the inhibitor reduces the turnover of ribosome-bound ternary EF-1 X GTP X aminoacyl-tRNA complexes during proof-reading and binding of the cognate aminoacyl-tRNA by inhibiting the EF-1-dependent GTPase.

Ribosomal core-particles as the target of ricin.

Core-particles and split-proteins were prepared by treatment with ethanol and NH4Cl of control and ricin-treated Artemia salina ribosomes. No modifications of the ricin-treated split-proteins was detected by polyacrylamide-gel electrophoresis. Moreover, the split-proteins from ricin-treated ribosomes complemented control core-particles in poly(U)-directed phenylalanine polymerization. Conversely, ricin-treated core-particles remained totally inactive when supplemented with control split-proteins.