A new development of triterpene acid-containing extracts from Viscum album L. displays synergistic induction of apoptosis in acute lymphoblastic leukaemia

Objectives: Aqueous Viscum album L. extracts are widely used for anti‐cancer therapies. Due to their low solubility, triterpenes (which are known to act on cancers), do not occur in aqueous extracts in significant amounts. Using cyclodextrins, we have found it possible to solubilize mistletoe triterpene acids and to determine their effects on acute lymphoblastic leukaemia (ALL) in vitro and in vivo. Materials and methods: A C.B‐17/SCID model of pre‐B ALL (NALM‐6) was used to test efficacy and mechanisms of treatment with lectin‐ and triterpene acid containing preparations in vivo. Cytotoxicity of increasing concentrations of V. album L. preparations was assessed in vitro. Apoptosis was determined using mitochondrial membrane potential measurements, annexin V/PI, western blot analyses and caspase inhibitor assays. Results: Solubilized triterpene acid‐ or lectin‐containing V. album L. extracts inhibited cell proliferation and demonstrated cytotoxic properties in vitro. Annexin V/PI and mitochondrial membrane potential assays indicated that dose‐dependent induction of apoptosis was the main mechanism. Combination (viscumTT) of lectin‐ (viscum) and triterpene‐containing (TT) extracts resulted in greatest induction of apoptosis. Furthermore, caspase activity demonstrated that these extracts were able to induce apoptosis through both caspase‐8 and ‐9 dependent pathways. In vivo experimentation showed that treatment of mice with viscumTT combination prolonged mean survival to 50.5 days compared to 39.3 days in the phosphate‐buffered saline group. Conclusion: Here for the first time, we have demonstrated that either solubilized triterpene acids or lectins and combinations thereof, induce dose‐dependent apoptosis in the ALL cell line NALM‐6 via caspase‐8 and ‐9 dependent pathways.     

Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins (“TT” extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous “viscum” extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20% in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin.     

Novel aminoalkaloids from European mistletoe (Viscum album L.)

The European white-berry mistletoe (Viscum album L.) has remained an important medicinal plant for millennia. Preparations of the plant have found application in the treatment of cancer and the anticancer activity of mistletoe extracts has been ascribed to the presence of lectins, viscotoxins and alkaloids. However, the alkaloids of this species have hitherto remained unidentified because of their claimed extreme lability. Here we report on the isolation and characterisation of the novel aminoalkaloids 4,5,4′-trihydroxy-3,3′-iminodibenzoic acid (1) and 4,5,4′,5′-tetrahydroxy-3,3′-iminodibenzoic acid (2) from V. album L. The compounds define a novel group of aminoalkaloids and are the first novel alkaloids ever identified in any mistletoe species. The structures were established using a combination of several 2D NMR spectroscopic techniques and high resolution mass spectrometry.     

Distribution, frequency and host patterns of European mistletoe (Viscum album subsp. album) in the major city of Lodz, Poland

The frequency of parasitism of the European mistletoe, Viscum album L. subsp. album, in the city of Lodz, a typical major city in Poland, was investigated. The infection prevalence and intensity of the mistletoe Viscum album subsp. album on its main host, Acer saccharinum as a function of host size was also investigated. The parasite showed a strong preference for alien, planted tree species (i.e. A. saccharinum, Populus×canadensis and Robinia pseudoacacia). In 2009–2011, V. album subsp. album was observed on 28 host taxa of trees and shrubs, which represents the highest diversity of host trees in a single locality in the Poland. Within the studied area 2147 trees were infested by mistletoe. The distribution of mistletoes (V. album subsp. album) among A. saccharinum hosts is significantly aggregated. The intensity of mistletoe infection in the silver maple trees was affected by the individual tree characteristics, such as the height of the tree. The overall level of aggregation as indicated by the variance to mean ratio of mistletoe numbers per host fell within the midrange of values found in other published studies of host-mistletoe interactions. The higher mistletoe infection prevalence in taller trees results from differential dispersal of mistletoe seeds to tall trees as well as differential survival of established mistletoes on tall trees. The incidence of mistletoe was higher in city centre (zone of high density development) than it was on the outskirts of a city (outer marginal zone). It was found that the abundant occurrence of mistletoe was recorded in the stands of increased nitrogen input, while other stands have little or no mistletoe infection present. Thus, this mistletoe species uses both passive and active uptake, which may be a selective advantage in a nutrient-poor environment or on a nutrient-deficient host species.     

Oligoclonal in vitro response of CD4 T cells to vesicles of mistletoe extracts in mistletoe-treated cancer patients

 Mistletoe (Viscum album) extracts are widely used in adjuvant cancer therapy. We have investigated the in vitro responsiveness of T cells from mistletoe-treated cancer patients and untreated healthy donors to various preparations of mistletoe extracts. Proliferation of peripheral blood mononuclear cells from treated but not from untreated patients was observed in response to therapeutically used mistletoe extracts prepared from apple (mali) or pine (pini) host trees. The strongest proliferation was induced by a vesicle preparation of mali extract. Activation was strongly inhibited by interleukin-10. Using a newly developed flow-cytometry assay, we determined that cell growth was restricted to CD4 T cells. Analysis with a panel of monoclonal antibodies against the variable region of the T cell receptor β chain (Vβ) revealed an oligoclonal pattern of CD4 T cell activation. These results indicate that therapeutic administration of mistletoe extracts sensitizes a restricted set of CD4 T lymphocytes in mistletoe-treated patients. Received: 25 May 1996 / Accepted: 9 January 1997     

Cloning and expression of mistletoe lectin III B-subunit

Aqueous extracts of mistletoe (Viscum album L.) contain toxic proteins (lectins) MLI (viscumin), MLII, and MLIII. We previously cloned the gene encoding MLIII precursor. In the present study, a gene fragment encoding the carbohydrate-binding subunit of mistletoe toxic lectin MLIII was cloned and expressed in Escherichia coli cells. The structure and immunochemical properties of recombinant MLIII B-subunit were investigated using a panel of monoclonal antibodies against ML-toxins. Sugar-binding activity of recombinant MLIII B-subunit was determined by ELISA. Amino acid sequence analysis of the cloned MLIII compared with known mistletoe toxins and other ribosome inactivating type II proteins (ricin, abrin a, and nigrin b B-subunits) revealed essential features of the recombinant MLIIIB primary structure that could determine sugar specificity of the lectin as well as immunomodulating and anti-tumor properties of mistletoe extracts.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 378–389.Original Russian Text Copyright © 2005 by Pevzner, Agapov, Pfueller, Pfueller, Maluchenko, Moisenovich, Tonevitsky, Kirpichnikov.     

Naturapotheke Mistel

The mistletoe in the un‐conventional and the molecular medicine The mistletoe is an evergreen semi‐parasite which is growing on trees. It is impressive by its unique appearance, its world‐wide dissemination and the great variety of its chemical components and active substances. Only in newer times the mistletoe with its ingredients have found a way into the un‐conventional, and finally into the molecular medicine. The spectrum of the active ingredients is rather well‐known now based on the extensively investigated European mistletoe (Viscum album).     

Nuclear DNA Metabolism in the Tip of the Cortical Strands of Viscum album L

The tips of the cortical strands of Viscum album were investigatedby autoradiographic and cytophotometric methods. It is shown that DNA synthesis and mitotic activity of the sub-apicalmeristematic cells are constant during the whole year. Theirnuclear DNA content varies from 2C to 4C values. The elongated cells which cover the meristematic zone are characterizedby no DNA synthesis, no mitotic activity and a 2C nuclear DNAcontent. They are ‘blocked’ in the presyntheticphasc G₁ of their cellular cycle, without polyploidy. The relationship between polyploidy and secretion is discussed. Viscum album, mistletoe, nucleus, DNA, polyploidy     

Cloning and Expression of Catalytic Subunit of MLIII, the Ribosome-Inactivating Protein from Viscum album

We have cloned the gene encoding a precursor of mistletoe (Viscum album) toxin MLIII. Analyses of nucleotide and deduced amino acid sequences of this gene revealed significant differences between MLI and MLIII preprotoxin genes. Immunochemical properties of recombinant A-subunit expressed in Escherichia coli and renatured were investigated using a panel of monoclonal antibodies raised against three mistletoe toxins (MLI, MLII, and MLIII). Ribosome-inactivating activity of recombinant MLIII A-subunit was detected in cell-free lysate of rabbit reticulocytes.     

Synergistic anticancer effects of lectin and doxorubicin in breast cancer cells

We studied the effects, either combined or alone, of lectin from Korean mistletoe (Viscum album var. coloratum agglutinin, VCA) and doxorubicin (DOX) in MCF-7 (estrogen receptor-positive) and MDA-MB231 (estrogen receptor-negative) human breast cancer cells. When VCA and DOX were combined, a strong synergistic effect was shown in cell growth inhibition, compared to VCA or DOX treatment alone. In quantitative apoptosis studies analyzed by flow cytometry, a combination of two agents showed an increase in apoptosis in both cells, compared to agents alone. Also, pro-apoptotic proteins including Bax, Bik, and Puma were increased in both cells, and the survival factor Bcl-2 was inhibited in MCF-7 cells when drugs were combined. Furthermore, VCA combined with DOX mediated S phase arrest, accompanied with a decrease of cell number at G0/G1 phase. This suggests that VCA and DOX combination may possibly lead to a novel strategy for the treatment of breast cancer.     

Dense crowns of the hemiparasitic mistletoe Viscum album L. exhibit shrub-like growth and high dry matter turnover

Growth characteristics and crown parameters of the evergreen, hemiparasitic mistletoe Viscum album were studied using a growth model based on the regular dichotomous growth and conservancy of branching patterns. Data were collected from one final harvest of 13 individuals of three different subspecies growing on the hosts Betula pendula, Abies alba and Pinus sylvestris. Compared to Viscum album ssp. album growing on deciduous hosts, the two subspecies abietis and austriacum (syn. laxum) on evergreen hosts showed longer average leaf life spans, but leaves continuously increased in area over time up to an age of at least 4 years irrespective of host. Relative growth rates in plant dry matter (RGR; 0.53–0.61 g g⁻¹ a⁻¹) were in the same range as of deciduous shrubs in central Europe but clearly higher than in mature forest trees. However, this growth is hidden within the spherical crowns of mistletoes, as annual increase in crown volume is extraordinarily low, resulting in very high leaf area densities (LAD; 7.7–17.6 m² m⁻³), not found in any other woody species. Besides differences in leaf size and phenology, the three subspecies exhibit similar patterns of partitioning. As in other mistletoe species, significant parts of dry matter are regularly allocated into fruit production (10–15%). Regarding the generally lower net photosynthesis rates in mistletoes as compared to their hosts, it is questioned, whether photoautotrophic carbon gain of the leaves is sufficient to maintain the observed high relative growth rates.     

Direct cryopreservation of winter-acclimated buds of Dracocephalum austriacum (Lamiaceae) from field material

Mistletoes are semiparasite plants containing pharmaceutical proteins with applications in cancer treatment. Previous research has demonstrated that somaclonal variation can lead to the biosynthesis of novel proteins from mistletoe callus cultures. The protein content of Viscum album subsp. abietis tissues and biotechnologically propagated calluses, was analyzed to identify proteins with putative anticancer properties. In addition, evolutionary relations among linked species to Viscum were studied. Calluses were propagated from stem explants. The protein extracts mass spectra were processed with Proteome Discoverer and a search was performed using as reference the Uniprot V. album reviewed database. A phylogenetic tree was reconstructed using the LG amino acid substitution model by homologous sequences for Beta galactoside-specific lectin 2. The homology modeling of the Beta-galactoside-specific lectin 2 was carried out using Modeller software. Considerable differences were observed by comparing the protein content of the calluses and the maternal tissues. Four mistletoe lectins, six viscotoxins and the chitin binding lectin-cbML were identified within the species tissues. An in silico phylogenetic and structural study provides insights to the role of these lectins and the mechanism of semiparasite survival and evolution, towards a novel anticancer and immune system modulation pipeline. Callogenesis exhibited protein biosynthesis alterations and novel protein isoforms expression. Phyllogenetic analysis revealed evolutionary relations primarily within the Viscum genus and other species containing 2-ribosome inactivating proteins. The homology modeling of the mistletoe lectin 2 revealed possible structure related anticancer properties. In conclusion, mistletoe calluses were shown to possess a unique protein biosynthetic profile compared to donor plant tissues.

     

Assessment of Shape Changes of Mistletoe Berries: A New Software Approach to Automatize the Parameterization of Path Curve Shaped Contours

Photographs of mistletoe (Viscum album L.) berries taken by a permanently fixed camera during their development in autumn were subjected to an outline shape analysis by fitting path curves using a mathematical algorithm from projective geometry. During growth and maturation processes the shape of mistletoe berries can be described by a set of such path curves, making it possible to extract changes of shape using one parameter called Lambda. Lambda describes the outline shape of a path curve. Here we present methods and software to capture and measure these changes of form over time. The present paper describes the software used to automatize a number of tasks including contour recognition, optimization of fitting the contour via hill-climbing, derivation of the path curves, computation of Lambda and blinding the pictures for the operator. The validity of the program is demonstrated by results from three independent measurements showing circadian rhythm in mistletoe berries. The program is available as open source and will be applied in a project to analyze the chronobiology of shape in mistletoe berries and the buds of their host trees.     

Parasitic Bacteria and Fungi on Common Mistletoe (Viscum album L.) and Their Potential Application in Biocontrol

This study was carried out to identify pathogenic bacteria and fungi on mistletoe (Viscum album L.) and investigate their potential use in biological control of this parasitic plant. For this purpose, a total of 48 fungal isolate and 193 bacterial strains were isolated from contaminated V. album during the summers 2005–2006. The isolated bacterial strains and fungal isolates were identified by using the Sherlock Microbial Identification System (MIS; Microbial ID, Newark) and microscopic methods, respectively. The bacterial strains that induced hypersensitive reaction (HR) on tobacco (Nicotiana tabacum L.) and fungal isolates were tested for pathogenicity on young shoots of mistletoe by using injection methods. The pathogenic bacterial strains and fungal isolates were also tested for their activity against mistletoe using spray methods. Five bacterial strains (two Burkholderia cepacia, one each of Bacillus megaterium, Bacillus pumilus and Pandoraea pulminicola) were HR and pathogenicity positive when injected but none of them when sprayed on mistletoe. When fungi were injected, 32 isolates were pathogenic but only thirteen when sprayed on mistletoe. Alternaria alternata VA?‐202, VA?‐205, VA?‐217 and Acremonium kiliense VA‐11 fungal isolates were the most effective ones and caused strong disease symptoms on mistletoe. The present study is the first report on the efficiency of potential biocontrol agents against mistletoe in Turkey.     

Leaf-spot disease on European mistletoe (<Emphasis Type="Italic">Viscum album</Emphasis>) caused by <Emphasis Type="Italic">Phaeobotryosphaeria visci</Emphasis>: a potential candidate for biological control

Viscum album (European mistletoe), a perennial, evergreen, hemiparasitic shrub, infects a wide range of woody species. It adversely affects the height and diameter of growth and it is associated with increased mortality of its hosts. There is no effective control methods against it. We have found a specific hyperparasitic fungus, which can completely destroy European mistletoe by infecting its branches, leaves and berries. Both morphological and molecular identification, based on ribosomal internal transcribed spacer sequences (rDNA-ITS), established its identity as Phaeobotryosphaeria visci. Our analysis also revealed unexpected ITS variability, as compared to the previous studies, that needs to be considered in identifying of this pathogen. Because of its efficient pathogenicity this fungus might be a good candidate for biological control of mistletoe.     

Generalist birds govern the seed dispersal of a parasitic plant with strong recruitment constraints

Mistletoes constitute instructive study cases with which to address the role of generalist consumers in the study of plant–animal interactions. Their ranges of safe sites for recruitment are among the most restricted of any plant; therefore, frugivores specializing in mistletoe have been considered almost indispensable for the seed dispersal of these parasitic plants. However, the absence of such specialists in numerous regions inhabited by many mistletoe species raises the question of whether unspecialized vectors may successfully disperse mistletoe seeds to narrowly defined safe sites. Using the European mistletoe Viscum album subsp. austriacum as a study case, we recorded a broad range of 11 bird species that disperse mistletoe seeds. For these species, we studied the mistletoe-visitation rate and feeding behavior to estimate the quantity component of dispersal effectiveness, and the post-foraging microhabitat use, seed handling, and recruitment probabilities of different microhabitats as a measure of the quality component of effectiveness. Both endozoochory and ectozoochory are valid dispersal mechanisms, as the seeds do not need to be ingested to germinate, increasing seed-dispersal versatility. Thrushes were the most effective dispersers, although they were rather inefficient, whereas small birds (both frugivores and non-frugivores) offered low-quantity but high-quality services for depositing seeds directly upon safe sites. As birds behave similarly on parasitized and non-parasitized hosts, and vectors have broad home ranges, reinfection within patches and the colonization of new patches are ensured by an ample assemblage of generalist birds. Thus, a parasitic plant requiring precision in seed dispersal can rely on unspecialized dispersers.     

Induction of apoptosis by the mistletoe lectins: A review on the mechanisms of cytotoxicity mediated by Viscum album L.

This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.     

Binding of the plant hormone kinetin in the active site of Mistletoe Lectin I from Viscum album

The crystal structure of the ribosome inhibiting protein Mistletoe Lectin I (ML-I) derived from the European mistletoe, Viscum album, in complex with kinetin has been refined at 2.7 Å resolution. Suitably large crystals of ML-I were obtained applying the counter diffusion method using the Gel Tube R Crystallization Kit (GT-R) on board the Russian Service Module on the international space station ISS within the GCF mission No. 6, arranged by the Japanese aerospace exploration agency (JAXA). Hexagonal bi-pyramidal crystals were grown during three months under microgravity. Before data collection the crystals were soaked in a saturated solution of kinetin and diffraction data to 2.7 Å were collected using synchrotron radiation and cryogenic techniques. The atomic model was refined and revealed a single kinetin molecule in the ribosome inactivation site of ML-I. The complex demonstrates the feasibility of mistletoe to bind plant hormones out of the host regulation system as part of a self protection mechanism.     

The incidence of mistletoe (<Emphasis Type="Italic">Viscum album</Emphasis> ssp. <Emphasis Type="Italic">abietis</Emphasis>) on silver fir (<Emphasis Type="Italic">Abies alba</Emphasis>) in Croatia

The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers. The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence. The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.