Mistletoe Lectin Research Articles

NCCAM/NCI phase I study of mistletoe extract and gemcitabine in patients with advanced solid tumors.

2559 Background: European mistletoe (viscum album L.) extracts (EMEX) are used for cancer treatment in Europe. Clinical efficacy remains controversial. Controlled safety and toxicity data are lacking. Objectives of this novel phase I dose-escalation study of an EMEX/gemcitabine (GEM) combination were 1) safety, toxicity, and MTD in patients with advanced solid cancers (ASC), 2) neutrophil count recovery, 3) formation of mistletoe lectin antibodies (ML ab), 4) plasma concentrations of IL-6, IL-12, IFNg, and TNF-a, 5) clinical response. Methods: ASC patients (n=44, breast 12, pancreas 10, colorectal 17, NSCLC 5, f:m=21:23, average age 55 years) received escalating doses of EMEX (HELIXOR A, Helixor GmbH, Rosenfeld, Germany) during stage 1 (20-250 mg s.c. daily) and increasing doses of GEM during stage 2 (750 - 1680 mg/m2 over 30 minutes i.v. on day 1 and 8, 3-week cycle) for 3 cycles. GEM plasma concentrations (Cp, baseline and week 8 (stable EMEX dose for 7 days) were determined during stage 1 by HPLC with UV detection. Plasma cytokine and MLab concentrations were measured by ELISA. Results: Five dose-limiting toxicities were observed: grade 4 neutropenia (2), grade 4 thrombocytopenia, grade 3 cellulitis, and grade 4 acute renal failure. GEM 1380 mg/m2 and EMEX 250 mg were the MTD. Of 44 patients 24 developed Non-neutropenic fever and flu-like syndrome. GEM pharmacokinetics were unaffected by EMEX. All patients developed ML3 IgG antibodies. Plasma cytokine concentrations were minimally affected. ANC values showed a trend to increase between baseline and C2 in stage I. Partial response and stable disease were found in 6% and 42% of patients, respectively. All partial responses occurred in patients with pancreatic cancer. Median survival was 190 days. Compliance with EMEX injections varied from 78.6-100%. Conclusions: The EMEX/GEM combination demonstrated limited toxicity, no alterations of GEM Cp during infusion of EMEX, clinical benefit in 48% of patients, good tolerability and excellent EMEX compliance. Addition of EMEX may allow for use of higher doses of GEM and increase the ANC nadir. No effects on the measured cytokines were observed. The long-term utility, safety, and tolerability of EMEX plus GEM warrant further study. No significant financial relationships to disclose.

A phase II multicenter study on CY-503 in the treatment of patients with unresectable stage IV metastatic melanoma after failure of previous chemotherapy.

e19015 Background: CY-503 is a recombinant mistletoe lectin ( INN: aviscumine) leading to catalytical inactivation of ribosomes, thereby inhibiting protein synthesis. Dependent on dosage immunomodulatory and cytotoxic effects are induced. In patients (pts.) with stage IV metastatic melanoma CY-503 could potentially lead to an antitumoral response. Methods: Pts. with stage IV metastatic melanoma (ECOG performance score 0-1), who had failed from one or more previous systemic chemotherapies, were eligible in this trial. Pts. with severe organ dysfunction or brain metastasis were excluded. Pts. received CY-503 350ng SC twice weekly for a minimum of 8 weeks (2 cycles). Pts. without disease progression were continued. The primary study endpoints were progression free survival (PFS) and overall survival (OS). Tumor assessment included CT and MRI scans every 8 weeks, which were centrally reviewed. Results: Between April 2008 and June 2009, 32 pts. were enrolled, one being ineligible. 31 pts. (16m; 15f) with a median age of 67 years (range: 20-85y) were evaluable. Pts. were belonging to the following stage IV categories: M1a/b/c = 4/5/22, and median number of prior treatments was 2 (range: 1- 6), 7 pts. (23%) were pretreated with ≥ 3 lines. (84%) received at least 8 weeks of treatment; 4 pts. withdrew prior to 8 weeks due to clinically overt disease progression, and 1 pt. due to a severe adverse event (SAE). At 8 weeks 11 pts. with stable disease (SD) were observed (disease control rate: 36% ITT) with a median PFS of 3.75 months (95% CI: 3,6-3,9 months). The median PFS in the ITT population was 2.0 months (95% CI: 1.9–2.1 months). Median OS had not been reached at the time of evaluation. Local reactions at the injection site were the main toxicities. Two severe adverse events with fatal outcome (1 pt. with cardiac arrhythmia, 1 pt. with stroke) could not clearly be related to the study medication. Conclusions: CY-503 showed a modest clinical activity in pts. with advanced melanoma. Side effects were mostly limited to local reactions at injection site. Given this heavily pretreated population, further investigations on the use of CY-503 in the treatment of malignant melanoma might be warranted. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cytavis Biopharm GmbH Cytavis Biopharm GmbH Cytavis Biopharm GmbH Cytavis Biopharm GmbH

Pharmacokinetics of natural mistletoe lectins after subcutaneous injection

Knowledge of natural mistletoe lectins (nML) pharmacokinetics can be regarded as essential for further rational studies with mistletoe preparations. Studies with intravenous application of a recombinant type II ribosome inactivating protein (rML) analogous to nML revealed a short half-life of about 13 min in cancer patients. This open-label, phase I, monocenter clinical trial was performed in order to describe the pharmacokinetics of nML. In 15 healthy male volunteers aged 18-42 years, nML were detected with a modified sandwich immuno-polymerase chain reaction (PCR) technique (Imperacer, Chimera Biotec) after single subcutaneous injection of a mistletoe extract (abnobaVISCUM(R) Fraxini 20 mg) with marketing authorization containing about 20 microg nML/ml. Secondary objectives were safety and the number of activated natural killer cells (CD54(+)/CD94(+)). In none of the volunteers were nML detectable before the injection, and in all volunteers, nML were detected in serum samples after injection. Individual variability, however, was large. Mean and median peak concentrations were reached 1 and 2 h after injection, respectively. In some volunteers, nML were still detectable at the final investigation 2 weeks after injection. The injection resulted in fever and flu-like symptoms in all volunteers, but no serious adverse events occurred. All symptoms and local reactions at the injection site completely disappeared within a range of 4-95 days. The number of activated natural killer (NK) cells did not change. Natural ML from abnobaVISCUM Fraxini 20 mg are detectable in serum after a single subcutaneous injection. Detectability is considerably longer compared with intravenously administered rML. The subcutaneous injection of this preparation without usual pretreatment with lower doses results in short-lasting fever and other flu-like symptoms.

TLR4-mediated activation of mouse macrophages by Korean mistletoe lectin-C (KML-C)

Korean mistletoe lectin (KML-C) is an adjuvant that activates systemic and mucosal immune cells to release cytokines including TNF-α, which induces immunity against viruses and cancer cells. Although the immunomodulatory activity of KML-C has been well established, the underlying mechanism of action of KML-C has yet to be explored. When mouse peritoneal macrophages were treated with KML-C, both transcription and translation of TLR4 were upregulated. KML-C-induced TLR4 downstream events were similar to those activated by LPS: the upregulation of interleukin-1 receptor-associated kinase-1 (IRAK1); resulting in macrophage activation and TNF-α production. When TLR4 was blocked using a TLR4-specific neutralizing antibody, TNF-α production from the macrophages was significantly inhibited. Moreover, TLR4-deficient mouse macrophages treated with KML-C also secreted greatly reduced level of TNF-α secretion. Finally, TLR4 molecules were co-precipitated with KML-C, to which agarose beads were conjugated, indicating that those molecules are associated. These data indicate that KML-C activates mouse macrophages to secrete TNF-α by interacting with the TLR4 molecule and activating its signaling pathways.

Protective Effects of Korean Mistletoe Lectin on Radical-Induced Oxidative Stress

The radical scavenging effects and protective activities against oxidative stress of Korean mistletoe (Viscum album coloratum) lectin were investigated in vitro and with a cellular system using LLC-PK(1) renal epithelial cells. The Korean mistletoe lectin (KML) showed 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with an IC(50) value of 42.6 microg/ml. It also exerted nitric oxide (NO), superoxide anion (O(2)(-)), and hydroxyl radical scavenging activities in concentration-dependent manners. These results suggest that KML is a promising antioxidant by scavenging free radicals. Furthermore, under the LLC-PK(1) cellular model, the cells showed declines in viability and increases in lipid peroxidation through oxidative stress induced by sodium nitroprusside (SNP) and pyrogallol, generators of NO and O(2)(-), respectively. However, KML significantly and dose-dependently inhibited cell cytotoxicity and lipid peroxidation. In addition, 3-morpholinosydnonimnie (SIN-1), a generator of peroxynitrite (ONOO(-)) formed by simultaneously releases of NO and O(2)(-), caused cytotoxicity, lipid peroxidation, and NO overproduction in the LLC-PK(1) cells while KML ameliorated ONOO(-)-induced oxidative damage. Furthermore, overexpressions of cyclooxygenase-2 and inducible NO synthase induced by SIN-1 were observed, but KML down-regulated the expression levels of both genes. KML also reduced SIN-1-induced nuclear factor kappa B expression and the phosphorylation of inhibitor kappa B alpha in LLC-PK(1) cells. These results indicate that KML has protective activities against oxidative damage induced by free radicals.

Safety and immunological effects of Iscucin® Populi and Viscum Mali—A placebo-controlled study

Background Mistletoe preparations Iscucin ® Populi (IP) and Viscum Mali (VM, both WALA GmbH) are licensed in Germany as supportive cancer medication within the concept of Anthroposophical Medicine. Safety and immunological effects in humans of these preparations were for the first time investigated in this study. Methods A 3-armed randomized study in healthy volunteers was performed. The probands injected in increasing doses either IP (strength F, G and H, each for 4 weeks) or VM (strength D3, D2 and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice weekly for a total of 12 weeks. Clinical and safety controls were performed weekly. Immunological outcome parameters (differential blood count, lymphocyte differentiation, interleukin (IL)-6) were analysed every 4 weeks. Results A total of 71 probands were included (IP=30, VM=21, placebo=20), 69 finished the study regularly and were analysed. Application of IP strength G and H caused strong local reactions at the site of injection. In parallel they resulted in distinct eosinophilia ( p p Discussion and conclusion Eosinophilia as an effect of mistletoe-lectin (ML) containing mistletoe preparations is well known and was pronounced due to the high content of ML in IP strength G and H. Most of the probands did not tolerate the full dose of IP strength G and H due to local reactions. Repeated injections induced, however, a certain tolerance (smaller local reactions, less eosinophilia despite inreasing dosages), which was accompanied by induction of mistletoe lectin antibodies in all subjects receiving IP (data not shown). Therefore, IP strength G and H should in general be used only after pretreatment with lower dosages. For the first time there was an increase of T-helper cells during application of mistletoe preparations in a placebo-controlled study. This could be beneficial for the treatment of immunosuppressed cancer patients. Eosinophils play a role in host response against cancer and eosinophilia correlates with a better prognosis in a variety of cancer types. Whether eosinophilia during treatment with IP has a clinical benefit for cancer patients has to be further investigated. Both mistletoe preparations have been shown to be safe. VM induces only mild local reactions and is used as mild counter stimulant in patients with painful osteoarthritis.

Immunomodulating effects of Korean mistletoe lectin in vitro and in vivo

The immunomodulatory effects of Korean mistletoe lectin (KML), one of the major active components in Viscum album L. var. coloratum, were investigated in vitro in immune cell proliferation and natural killer (NK) cell- and macrophage-mediated cytotoxicity, and in vivo in the forced swim test and cold stress. In mitogen-induced lymphocyte proliferation of murine splenocytes, concanavalin A and lipopolysaccharide significantly increased the proliferation of T cell and B cell lymphocytes, respectively. KML exposure increased lymphocyte proliferation in response to mitogen. KML also increased the splenic NK cell and macrophage activities in vitro. Exposure to KML increased production of cytokines such as interleukin-1 and interleukin-6 by macrophages. Two-week treatment with KML (30, 100, 300 and 600 μg/kg) increased the recruitment of lymphocytes, monocytes and macrophages. In the forced swim test, the immobility time was significantly attenuated by treatment with KML (300 and 600 μg/kg). In a cold stress experiment, spleen and thymus weight increased in KML-treated mice, while the weight of adrenal gland was lower than that in vehicle-treated mice. The levels of serum aminotransferases, lactate dehydrogenase and alkaline phosphatase were decreased by KML treatment. KML treatment also induced increases in the percentages of CD4 + and CD8 + cells in thymus. Our results suggest that KML enhances the immune system through modulation of lymphocytes, NK cells, and macrophages.

Oleanolic acid rich solubilized triterpene extracts from mistletoe induce apoptotic and necrotic cell death of murine B16.F10 melanoma cells

Oleanolic acid (OA) is an almost water insoluble (<0.02µg/ml), naturally occurring pentacyclic triterpenoid, which has a variety of biological effects (e.g. anti-cancer and anti-inflammatory). OA is the main component (˜80%) of triterpene extracts from European mistletoe, which does not contain mistletoe lectins and viscotoxins. The toxic solvent DMSO is normally used for in vitro administration of OA. This restricts the use of OA due to limited solubility of OA in DMSO and toxicity of DMSO itself.

Extracellular Signal-Regulated Kinase Is a Major Enzyme in Korean Mistletoe Lectin-Mediated Regulation of Macrophage Functions

Abstract － Korean mistletoe lectin (KML) is the major component found in Viscum album var. (coloratum) , displaying anti-cancer and immunostimulating activities. Even though it has been shown to boost host immune defense mechanisms, the regulatory roles of KML on the functional activation of macrophages have not been fully elucidated. In this study, regulatory mechanism of KML on macrophage-mediated immune responses was examined in terms of KML-mediated signaling event. KML clearly induced mRNA expression of tumor necrosis factor (TNF)-α, the generation of reactive oxygen species (ROS) and phagocytic uptake in RAW264.7 cells. All of these events were strongly suppressed by U0126, whereas TNF-α mRNA was not diminished by SB203580 and SP600125, indicating ERK as a central enzyme managing KML-induced up-regulation of macrophage functions. Indeed, KML strongly induced the phosphorylation of ERK in a time-dependent manner without altering its total level. Therefore, these data suggest that ERK may be a major signaling enzyme with regulatory property toward various KML-mediated macrophage responses.Keywords: Korean mistletoe lectin,

A new detection method of mistletoe lectin I by surface plasmon resonance

Mistletoe lectin I (ML-I) is considered as the main effective substance in the extract of mistletoe and is currently widely applied in cancer therapy. The Plasmonic® surface plasmon resonance (SPR) device was used to establish an innovative, simple and rapid assay for detection of ML-I in buffer, serum and injection drugs. In the reported sandwich immunoassay, specific anti-ML-I A chain antibodies were used as capture and detection antibodies. Trials were undertaken to compare the influence of detection signals by using four different types of functionalised chips. It was found that alkylsilane-coated chip got the highest detection signal and showed the lowest unspecific binding of the detection antibody. Hence the assays were established on alkylsilane-coated chips in both buffer system and serum system. For the buffer system, it was measured as a detection range of 7.5–100μgmL−1 and lower limit of detection of 1.0μgmL−1. In the serum system, the linear standard calibration curve indicated that the detection limit of ML-I by SPR was 1.5μgmL−1. The method also was verified to quantify the contents of mistletoe extract in commercially available injection drugs. In this novel approach, no laborious sample preparation is required. And it is less time-consuming compared to other biotechniques normally used at present. The system can be utilised not only for exploring properties of ML-I in biological research but also for the quality control of medicinal products of mistletoe in the pharmaceutical industry.

Immunomodulatory effect of a galactoside-specific mistletoe lectin

Immunomodulatory effect of a galactoside-specific mistletoe lectin

Glycan Tagging to Produce Bioactive Ligands for a Surface Plasmon Resonance (SPR) Study via Immobilization on Different Surfaces

Suitable glycan derivatives will find immediate application in the study of their interactions. Here, we present an efficient synthetic strategy to introduce a fluorescent tag functionalized with an amino group into a model disaccharide structure (lactose). This strategy led to the maintenance of bioactivity, checked by the study of the interaction of this bioconjugate with a plant lectin (mistletoe lectin 1) by NMR spectroscopy, computational docking, and surface plasmon resonance (SPR). To demonstrate the versatility of this approach, we immobilized the new glycan derivatives on different surfaces, and a comparative analysis is presented and can be successfully used for biomimetic carbohydrate-protein interaction studies on the SPR biochip.

Mistletoe lectin modulates intestinal epithelial cell-derived cytokines and B cell IgA secretion

A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anti-cancer activity, was isolated from Korean mistletoe. In this study, IEC-6 rat intestinal epithelial cells and IM-9 human B-cells were cultured to determine the effect of VCA on cytokine and immunoglobulin (Ig) secretion. In lipopolysaccharide (LPS)-stimulated IEC-6 cells, VCA significantly shifted the interleukin (IL)-2, IL-5, IL-6, and tumor necrosis factor-alpha (TNF-alpha) secretion toward a more immunostimulatory response. Since intestinal epithelial cell-derived secretions may be capable of affecting local B cell Ig production in a variety of ways, we mimicked this condition by deriving a 2-day culture supernatant from IEC-6 cell line which was treated VCA in the presence or absence of LPS, and adding these supernatants to cultures of IM-9 human B cells. As a result, IgA secretion was significantly enhanced at in the presence of VCA at 10(-8)-10(-4) microg/mL. This study suggests that cytokines derived from IEC by VCA may create an environment which may contribute to the enhancement of IgA secretion seen in mucosal tissues. Overall, the induction of cytokines in intestinal epithelial cells, and IgA in B cells by Korean mistletoe lectin could indicate an enhanced immunosurveillance to prevent intestinal infections or other intestinal pathologies.

Effects of Japanese mistletoe lectin on cytokine gene expression in human colonic carcinoma cells and in the mouse intestine

Mistletoe lectins have various biological activities including anti-cancer and immunomodulatory effects. We previously isolated a lectin (ML-J) from Japanese mistletoe. In the present study, we examined the effects of ML-J on cytokine gene expression in human colon adenocarcinoma Caco-2 cells and in the mouse intestine. The results of reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction indicated that ML-J caused an upregulation of the gene expression of the proinflammatory cytokines interleukin (IL)-8, tumor necrosis factor-alpha (TNF-alpha) and IL-6 in Caco-2 cells and TNF-alpha and IL-6 in the duodenum. This study provides the first example to show that a perorally administered plant lectin affects gene expression in the duodenum.

Transport of mistletoe lectin by M cells in human intestinal follicle-associated epithelium (FAE) In vitro

Purified mistletoe lectins are known to have cytotoxic and stimulating activities in the immune system. Mistletoe extract has been given subcutaneously because of its unstablity and poor absorption in the Gastrointestinal (GI) tract. A hallmark of M cells is their capacity to internalize material from the lumen and to transfer it efficiently to the underlying lymphoid cells. Although lectins are the prime candidates for oral vaccine delivery, the mechanisms whereby lectins are taken up, transported by M cells, and affect underlying immune cells remain poorly understood. In this study, uptake mechanism of Korean mistletoe lectin (Viscum album L. var. coloratum aggulutinin, VCA) across the human FAE (follicle associated epithelium) was investigated. An inverted FAE model of co-culture was obtained by a co-culture system of Caco-2 cells and human Raji B lymphocytes, and VCA transport across the in vitro model of human FAE was investigated. There was a greater transport of VCA across FAE monolayer cells than that of Caco-2 monolayer cells. These observations will be useful to assess the transport of other orally administered material in the GI tract.

European and Far East mistletoes: Potential in adjuvant cancer therapy

Semiparasitic plants, mistletoes, distributed in Europe and East-Asia mainly, China, Korea and Japan have long been recognised as therapeutic herbs. Since the 1920s, extracts from European mistletoe (Viscum album L) have been popular in Europe as an unconventional cancer treatment. These extracts have been used in adjuvant cancer therapy because their immunostimulatory, cytostatic/cytotoxic and DNA stabilising activities. The main biological activities are addressed to sugar binding proteins, the Mistletoe Lectins, and to micro proteins named viscotoxins. The mistletoe lectins are members of the family of toxic lectins and recognize sugars and glycoconjugates containing galactose and/or N-acetyl-galactosamine groups. In vitro and in vivo assays confirm that the mistletoe lectins, isolated from the plant and compared with recombinant forms, play an important role as biologically active principles in mistletoe extracts. Limited experimental evidence indicates that other components like glycosides, polysaccharides, amines etc, may also have anticancer activity. Under discussion in Europe is the principal question of using holo plant extracts or single component lectin preparations: wild type lectins or recombinant ones in cancer therapy. Finally problems of preparation, characterization and standardization of commercial mistletoe preparations and evaluation of biological activities are discussed.

Structure of mistletoe lectin I from Viscum album in complex with the phytohormone zeatin

The crystal structure of mistletoe lectin I (ML-I) isolated from the European mistletoe Viscum album in complex with the most active phytohormone zeatin has been analyzed and refined to 2.54 Å resolution. X-ray suitable crystals of ML-I were obtained by the counter-diffusion method using the Gel-Tube R crystallization kit (GT-R) onboard the Russian Service Module on the international space station ISS. High quality hexagonal bipyramidal crystals were grown during 3 months under microgravity conditions. Selected crystals were soaked in a saturated solution of zeatin and subsequently diffraction data were collected applying synchrotron radiation. A distinct F o- F c electron density has been found inside a binding pocket located in subunit B of ML-I and has been interpreted as a single zeatin molecule. The structure was refined to investigate the zeatin–ML-I interactions in detail. The results demonstrate the ability of mistletoe to protect itself from the host transpiration regulation by absorbing the most active host plant hormones as part of a defense mechanism.

Mistletoe Lectins: Carbohydrate-Specific Apoptosis Inducers and Immunomodulators

Mistletoe lectin I (ML-I) is a heterodimeric ribosome-inactivating protein composed of a sialic acid-specific Bchain that binds to cell surfaces, and an A-chain with the capacity to depurinate a critical adenosine in the 28S ribosomal RNA. ML-1, in purified or recombinant form, exerts an immunomodulatory effect on neutrophils and macrophages/ monocytes in the low-dose range, while at high doses, it induces apoptosis in both normal and tumoral cells. While mistletoe extracts are widely used as cancer adjuvant therapy, recombinant ML-I (rAviscumin) is a candidate antineoplastic agent that has successfully passed Phase I clinical trials. In immunodeficient mouse models, the efficacy of recombinant ML-I was demonstrated for ovarian carcinoma, melanoma and various hematological malignant cell lines. The clinical potential of recombinant ML-I as a non-mutagenic and non-genotoxic molecule is high and could be used to potentiate classical anti-neoplastic drugs. Its capacity to induce apoptosis in cancer cell lines lacking p53 allows considering its use against genetically unstable and highly metastatic cancers. The mechanisms of apoptosis induced by ML-I probably involves intracellular pathways akin to those described as the ribotoxic stress response that directly target the mitochondrion.

Treatment results with fermented mistletoe (Viscum album L.) extract as part of long-term supportive care in patients with primary non- metastatic colorectal carcinoma

Mistletoe lectin is a potent biohazard. Lectin activity in the toxic dimer primarily originates from the 2γ-subdomain (Tyr-site) of the B-subunit. Crystallographic information on lectin–sugar complexes is available only at acidic pH, where lectin activity is low. Thus, we mapped ligand-binding properties including comparison to ricin’s Tyr-site at neutral pH. Using these results and molecular dynamics simulations, a local conformational change was rendered likely. The obtained structural information is valuable for the design of potent inhibitors.

Domain versatility in plant AB-toxins: Evidence for a local, pH-dependent rearrangement in the 2γ lectin site of the mistletoe lectin by applying ligand derivatives and modelling

Mistletoe lectin is a potent biohazard. Lectin activity in the toxic dimer primarily originates from the 2γ-subdomain (Tyr-site) of the B-subunit. Crystallographic information on lectin–sugar complexes is available only at acidic pH, where lectin activity is low. Thus, we mapped ligand-binding properties including comparison to ricin’s Tyr-site at neutral pH. Using these results and molecular dynamics simulations, a local conformational change was rendered likely. The obtained structural information is valuable for the design of potent inhibitors.