Biological activity of mistletoe: in vitro and in vivo studies and mechanisms of action.

Mistletoe has been used as treatment of many diseases in traditional and folk medicine. To date, anticancer, immunomodulatory, cardiac, antidiabetic, hepatoprotective, neuropharmacological, antibacterial and antifungal properties of mistletoe extracts have been studied the most. In this review, we summarized in vitro and in vivo studies on the pharmacological activity of Viscum species. Furthermore, we proposed the possible mechanisms of action of this herb, which might include many signalling pathways. Mistletoe could regulate either similar or different targets in various pathways that act on membrane receptors, enzymes, ion channels, transporter proteins and transcriptional targets. Still, pharmacological activities of mistletoe have been investigated mainly for crude extracts. It is a new field for scientists to determined which chemical compounds are responsible for the individual biological activities of mistletoe and how these activities are achieved. As a result, mistletoe might become a source of new complementary therapies supporting the treatment of many diseases.

[Mistletoe in the treatment of cancer patients]. / Die Misteltherapie in der Behandlung von Patienten mit einer Krebserkrankung.

Mistletoe (Viscum album L.) continues to be the medical herb prescribed most frequently for cancer patients in German-speaking countries. Demand for this therapy often comes from patients themselves and requires careful consideration by the attending physician during consultation.In German-speaking countries, mistletoe extracts are available as approved drugs (based on monographs of the commissions C and E of the German Federal Institute for Drugs and Medical Devices). In Switzerland, treatment costs are generally covered by statutory health insurance. In Germany, coverage is limited to palliative care. In adjuvant cases, treating physicians can request coverage by the health insurance if patients suffer from side effects due to the antitumoral treatment.The spectrum of Viscum album extract includes mistletoe lectin I; II, and III, viscotoxins, flavonoids, amino acids, polysaccharides, and membrane lipids. Preclinical studies have demonstrated cytotoxic, apoptosis-inducing, and immunomodulatory effects.Many clinical studies indicate a supportive efficacy of mistletoe extracts in tumor patients, even though methodological quality is discussed controversially in many cases. Clinical data regarding effects on survival of patients is inconsistent; effects concerning quality of life as well as the tolerability of antitumoral treatments are evaluated more positively.In view of the high demand on the patient side and increasing scientific evidence, the general conditions for prescriptions should continue as well as the ongoing scientific evaluation.

Anti-cancer effects of enteric-coated polymers containing mistletoe lectin in murine melanoma cells in vitro and in vivo.

In this study, we evaluated the effects of Korean mistletoe (Viscum album L. var. coloratum) coated with a biodegradable polymer (Eudragit(®)) wall on the growth of mouse melanoma in vivo. Oral administration of 4% (430 mg/kg/day) enteric-coated mistletoe resulted in a significant reduction in tumor volume on day 14 compared to the negative control group in B16F10 melanoma-inoculated BDF1 mice. When we measured the survival rate, enteric-coated mistletoe-received mice had a higher survival rate after day 12. Also, we investigated the mechanism involving the cancer cell growth inhibition when melanoma cells were treated with Korean mistletoe lectin (Viscum album L. var. coloratum agglutinin, VCA) and its extract in vitro. As a result, a significant G0/G1 arrest was observed in both B16BL6 and B16F10 melanoma cells with VCA or mistletoe extract. In addition, VCA or mistletoe extract induced an increase in both early and late apoptosis in cells. When we studied the molecular mechanism, our results showed that VCA and mistletoe extract can increase activated multiple caspases (caspase-1, 3, 4, 5, 6, 7, 8, and 9), dose-dependently. We also found out that VCA and mistletoe treatment causes a significant decrease in the expression of procaspase-3 and 8.

Mistletoe lectin has a shiga toxin-like structure and should be combined with other Toll-like receptor ligands in cancer therapy.

Mistletoe extract (ME) is applied as an adjuvant treatment in cancer therapy in thousands of patients each year in Europe. The main immunostimulating component of mistletoe extract, mistletoe lectin, recently has been shown to be a pattern recognition receptor ligand and hence is binding to an important class of pathogen-sensing receptors. Pattern recognition receptor ligands are potent activators of dendritic cells. This activation is a prerequisite for a full-blown T-cell response against cancer cells. Pattern recognition receptor ligands are increasingly recognized as important players in cancer immunotherapy. We collect evidence from case studies on spontaneous regression, from epidemiology, from experiments in a mouse cancer model, and from protein structure comparisons to argue that a combination of mistletoe therapy with other pattern recognition receptor ligand substances leads to an increased immune stimulatory effect. We show that mistletoe lectin is a plant protein of bacterial origin with a 3D structure very similar to shiga toxin from Shigella dysenteriae, which explains the remarkable immunogenicity of mistletoe lectin. Secondly, we show that a combination of pattern recognition receptor ligands applied metronomically in a cancer mouse model leads to complete remission, while single pattern recognition receptor ligands slowed tumor growth. Taken together, we propose to combine mistletoe drugs with other pattern recognition receptor ligand drugs to increase its efficacy in adjuvant or even primary cancer therapy.

Uses of plant lectins in bioscience and biomedicine.

New research directions in the last decade have led to major developments in the uses of plant lectins in bioscience and biomedicine. Major advances have been made in our understanding how lectins in the diet can act on the gastrointestinal tract and the physiological consequences of their actions, and how they can modulate body- and organ metabolism, the immune system and the gut microflora. Particularly striking progress has been made in unravelling the effects, often beneficial, of both orally- and parenterally administered lectins, including lectins of Viscum album-, Phaseolus vulgaris-, Robinia pseudoacacia, Agaricus bisporus, etc on tumours and in cancer therapy. Results have also made it possible to devise and try out other beneficial applications of plant lectins as gut-, metabolic- and hormonal regulators, immune reagents, probiotic/prebiotic oral supplements and to develop methods based on the oral application of lectins to protect the intestines against the often lethally harmful effects of chemo- and radiotherapy. With the development of genetically modified (GM) plants by transferring the genes of some of the natural insecticidal lectins such as the various Bacillus thuringiensis lectin-Cry toxins or some insecticidal plant lectins to major crop plants, a possible new avenue in plant protection may have opened up.

AB-type lectin (toxin/agglutinin) from mistletoe: differences in affinity of the two galactoside-binding Trp/Tyr-sites and regulation of their functionality by monomer/dimer equilibrium.

Viscumin of mistletoe (Viscum album L.) has a concentration-dependent activity profile unique to plant AB-toxins. It starts with lectin-dependent mitogenicity and then covers toxicity and cell agglutination, associated with shifts in the monomer/dimer equilibrium. Each lectin subunit harbors two sections for ligand contact. In the dimer, the B-chain sites in subdomain 2 gamma (designated as the Tyr-sites) appear fully accessible, whereas Trp-sites in subdomain 1 alpha are close to the dimer interface. It is unclear whether both types of sites operate similarly in binding glycoligands in solution. By systematically covering a broad range of lactose/lectin ratio in isothermal titration calorimetry, we obtained evidence for two sites showing dissimilar binding affinity. Intriguingly, the site with higher affinity was only partially occupied. To assign the observed properties to the Trp/Tyr-sites, we next performed chemically induced dynamic nuclear polarization measurements of Trp and Tyr accessibility. A Tyr signal, but not distinct Trp peaks, was recorded when testing the dimer. Lactose-quenchable Trp peaks became visible on the destabilization of the dimer by citraconylation, intimating Trp involvement in ligand contact in the monomer. Fittingly, Tyr acetylation but not mild Trp oxidation reduced the dimer hemagglutination activity and the extent of binding to asialofetuin-Sepharose 4B. Altogether, the results attribute lectin activity in the dimer primarily to Tyr-sites. Full access to Trp-sites is gained on dimer dissociation. Thus, the monomer/dimer equilibrium of viscumin regulates the operativity of these sites. Their structural divergence affords the possibility for differences in ligand selection when comparing monomers (Tyr- and Trp-sites) with dimers (primarily Tyr-sites).

Antifungal effects and mechanism of action of viscotoxin A3.

Viscotoxins are cationic proteins, isolated from different mistletoe species, that belong to the group of thionins, a group of basic cysteine-rich peptides of approximately 5 kDa. They have been shown to be cytotoxic to different types of cell, including animal, bacterial and fungal. The aim of this study was to obtain information on the cell targets and the mechanism of action of viscotoxin isoform A3 (VtA3). We describe a detailed study of viscotoxin interaction with fungal-derived model membranes, its location inside spores of Fusarium solani, as well as their induced spore death. We show that VtA3 induces the appearance of ion-channel-like activity, the generation of H2O2, and an increase in cytoplasmic free Ca2+. Moreover, we show that Ca2+ is involved in VtA3-induced spore death and increased H2O2 concentration. The data presented here strongly support the notion that the antifungal activity of VtA3 is due to membrane binding and channel formation, leading to destabilization and disruption of the plasma membrane, thereby supporting a direct role for viscotoxins in the plant defence mechanism.

Some medicinal plants as immunostimulant for fish.

Immunostimulant effects of the dietary intake of various medicinal plant extracts on fish, rainbow trout (Oncorhynchus mykiss), were investigated. For this purpose fish were fed with diets containing aqueous extracts of mistletoe (Viscum album), nettle (Urtica dioica), and ginger (Zingiber officinale). Food containing lyophilized extracts of these plants as 0.1 and 1% was used at a rate of 2% of body weight per day for three weeks. At the end of the experimental period, various parameters of non-specific defence mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leukocytes and total plasma protein level were examined. Specific growth rates (SGRs) and condition factors (CFs) of the fish were also measured. Plant materials tested for immunostimulatory food additives caused an enhanced extracellular respiratory burst activity (P<0.001) compared to the control group. Especially the rainbow trout fed with a diet containing 1% aqueous extract of powdered ginger roots for three weeks exhibited a significant non-specific immune response. Phagocytosis and extracellular burst activity of blood leukocytes were significantly higher in this group than those in the control group. All plant extracts added to fish diet increased the total protein level in plasma except 0.1% ginger. The highest level of plasma proteins was observed in the group fed with 1% ginger extract containing feed.

Activation of caspase cascades in Korean mistletoe (Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells.

Mistletoe lectins are of high biological activity and exert cytotoxic effects. We have previously shown that Korean mistletoe, Viscum album var. coloratum, lectin-II specifically induces apoptotic cell death in cancer cells, not normal lymphocytes. The destructive mechanism by mistletoe lectins on tumor cells was mediated by activation of c-JUN N-terminal kinase (JNK)/stress-activated protein kinase. Herein, we investigated the involvement of caspase cascade and its proteolytic cleavage effects on biosubstrates of human myeloleukemic U937 cells by D-galactoside and N-acetyl-galactosamine-specific Korean mistletoe lectin-II. Mistletoe lectin-II induced ladder pattern DNA fragmentation and activation of caspase-3, -8, and -9 of U937 cells, but not caspase-1 protease, in a time- and dose-dependent manner. Consistent with catalytic activation of protease, both poly(ADP-ribose) polymerase (PARP) and protein kinase C-delta (PKC-delta) are also cleaved in mistletoe lectin-II-treated U937 cells. An inhibitor of caspase-3-like protease, DEVD-CHO peptide, significantly inhibited mistletoe lectin-II-induced apoptosis, PARP cleavage, and fragmentation of DNA. These results provide the evidence that Korean mistletoe lectin-II induces apoptotic death of U937 cells via activation of caspase cascades.

Induction of mitochondrial Apo2.7 molecules and generation of reactive oxygen-intermediates in cultured lymphocytes by the toxic proteins from Viscum album L.

We analysed mitochondrial alterations in human lymphocytes incubated with toxins exerting RNA and/or protein synthesis/transport inhibitory activity. We found that all toxins known to affect macromolecule synthesis, such as ricin from Ricinus communis, mistletoe lectin I (ML I) from Viscum album, cycloheximide, actinomycin D, and brefeldin A but also the thionins from Viscum album (viscotoxins; VT) generated reactive oxygen intermediates (ROI) and induced expression of newly described mitochondrial membrane proteins Apo2.7, however, with different kinetics. Apart from a rapid permeabilisation of cell membranes by the VT with swelling of mitochondria, loss of their cristae and ROI generation within 2-4 h, the majority of the cells may have received a distinct 'death signal' resulting in an induction of Apo2.7 molecules within 24 h. In contrast, protein synthesis/transport inhibition may signal for apoptosis within 24 h by decreasing distinct 'survival promotors' which remain to be characterised.

Influence of polysaccharides from Viscum album L. on human lymphocytes, monocytes and granulocytes in vitro.

BACKGROUND: An acidic arabinogalactan from European mistletoe (Viscum album L, VAL; 1.34 x 10(6) Dalton) was studied in detail because its immunological properties are poorly characterised. MATERIALS AND METHODS: Flow cytometric studies focussed on PS-activated proliferation of human lymphocytes measured via incorporation of bromo-deoxyuridine (BrdU), granulocyte phagocytosis via ingestion of FITC-labelled E.coli, and respiratory burst via oxidation of dihydrorhodamine 123 to rhodamine 123. Cytokines were detected in the cell culture supernatants by ELISA. RESULTS: PS, in contrast to mistletoe lectins (ML), significantly stimulated proliferation of CD4+ T-cells but not CD8+ and CD19+ cells. However, ML influenced PS-mediated stimulation, with a synergistic effect in one and an inhibitory effect in another individual. Furthermore, IFN-gamma release was significantly enhanced by PS, favouring a T-helper cell type-1 cytokine pattern, further IL-6 was significantly stimulated, while granulocyte activity was not affected. CONCLUSIONS: VAL-PS exert yet unknown stimulatory activities, especially on specific CD4+ T-cells which may be influenced by other extract components like the ML. These components may contribute to the anti-tumour effect of VAL.

Effect of immunomodulation with galactoside-specific mistletoe lectin on experimental listeriosis.

BALB/c-mice (n = 10 per experimental group) were intravenously infected with 5 x 10(4) and 5 x 10(5) viable cells of Listeria monocytogenes SLCC 4013. About 50-80 h after this challenge, all mice of the untreated control groups succumbed to their infection. Pretreatment of experimental animals on the optimal immunoactive schedule with the galactoside-specific mistletoe lectin (ML-1; 1 ng/kg body weight; days 1, 4, 5, 6 before challenge), however, evidently reduced the lethality of listerial infection (survival rate 60%).

[Study of plant lectins from Viscum album using monoclonal antibodies]. / Issledovanie rastitel'nykh lektinov iz omely beloi s pomoshch'iu monoklonal'nykh antitel.

Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

Fine sugar specificity of the mistletoe (Viscum album) lectin I.

The behaviour of N-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmasked N-acetyllactosamine sequences. However, substitution of at least one of the N-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylated N-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal alpha 1-3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15 M galactose in the starting buffer.