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[Immunomodulating mistletoe therapy by lectin standardization: a double-edged sword?].

Taking advantage of an unique, legally generous regulation, proprietary anthroposophic and phytotherapeutic mistletoe preparations are on the market in Germany. One constituent of the extract, the galactoside-specific lectin, is a potent biological response modifier in a very narrow low-dose range. Although the clinical implications of the lectin effects remain to be rigorously defined, this activity already prompted companies to eliminate the common batch-to-batch variations in favor of standardization, keeping the lectin content, which could otherwise vary drastically, purportedly constant. Based on literature data, immunomodulation by the lectin involves enhanced secretion of multifunctional proinflammatory cytokines such as IL-6. The apparently context-dependent ambivalence of their actions includes capacity to serve as autocrine and paracrine tumor growth and survival factors for a wide variety of tumor cell types in vitro and in vivo, as illustrated by the literature presented. The potential for clinical risks is indicated to be non-negligible, e.g. for lymphomas, advanced-stage melanomas and renal cell carcinomas. Moreover, negative effects of immunomodulatory lectin or extract treatment have already been reported. To reliably prove clinical efficacy and exclude lack of undesired side effects for each tumor class and stage, it is mandatory to evaluate the performance of this experimental therapy modality exclusively in relevant preclinical settings and strictly controlled clinical studies to obey the generally accepted rule: primum non nocere.

Adjuvants, Immunologic↗

Survival of glioma patients after complementary treatment with galactoside-specific lectin from mistletoe.

Malignant glioma patients were prospectively enrolled into a clinical trial. All the patients were provided with the internationally recommended oncologic standard treatment (neurosurgery, radiation, basic clinical care according to protocol and indication) and randomly divided into a treatment group (receiving complementary immunotherapy with a galactoside-specific lectin from mistletoe, ML-1) and a control group (without additional complementary treatment). Whereas the beneficial effects of ML-1 treatment on immunological rescue and quality of life have been recently shown, evaluation of relapse free/overall survival was performed after a 50 months follow up time. Non-stratified analysis of all the patients revealed non-relevant prolongation of relapse-free intervals/overall survival time for the treatment group. However, analysis of stratified stage III/IV glioma patients demonstrated: 1. a tendency for a prolongation of relapse-free survival for patients of the treatment group (17.43 +/- 8.2 months) vs. the control group (10.45 +/- 3.9 months) 2. a statistically significant (BRESLOW p = 0.035) prolongation of the overall survival for the treatment group (20.05 +/- 3.5 months) as compared to the control group (9.90 +/- 2.1 months). These promising data warrant confirmation in a GCP-based prospectively randomized (multicenter) study, which is currently under consideration.

Adjuvants, Immunologic↗

Antitumoral effect of recombinant mistletoe lectin on chemically induced urinary bladder carcinogenesis in a rat model.

BACKGROUND: The objective of this study was to determine the effect of intravesically applied, recombinant, galactoside specific mistletoe lectin (rML) on chemically induced tumor development in the urinary bladder of rats. METHODS: For tumor induction, rats were treated with four biweekly 1.5 mg doses of N-methyl-N-nitrosourea (NMU) intravesically (Weeks 0, 2, 4, and 6). The control group (n = 39 + 17 rats) received no other treatment. The four therapy groups also received rML twice weekly according to one of the following instillation regimens: 1) 30 ng rML per instillation from Week 8 to Week 13 (Group a: n = 14 rats), 2) 150 ng rML per instillation from Week 8 to Week 13 (Group b: n = 23 + 15 rats), 3) 30 ng rML per instillation from Week 14 to Week 19 (Group c: n = 22 rats), and 4) 150 ng rML per instillation from Week 14 to Week 19 (Group d: n = 19 rats). After the rats were asphyxiated at Week 21, the urinary bladders were excised in toto and examined histopathologically. To study the immunomodulatory effects of intravesically applied rML, 17 animals from the control group and 15 animals from Group b were asphyxiated at Week 13, and urinary bladder tissue was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction analysis for mRNA expression of interferon-gamma, interleukin-10, and Fas ligand. RESULTS: By Week 21, atypical hyperplasia and neoplastic transformation were found in 82% of the animals in the control group. In contrast, in all four cohorts that were treated with rML, significantly lower rates of atypical hyperplasia and neoplastic transformation were found (Group a, 50%; Group b, 52%; Group c, 45%; and Group d, 42%). By Week 13, in the bladder tissue of 15 rML-treated animals from Group b, lower expression of interleukin-10 mRNA was measured, whereas the expression levels of interferon-gamma mRNA and Fas ligand mRNA were comparable to those of 17 animals from the control group. CONCLUSIONS: The current data provide evidence for an inhibitory effect of rML on experimental urothelial carcinogenesis that does not seem to be due to interferon-gamma and/or interleukin-10 dependent mechanisms.

Adjuvants, Immunologic↗

Binding of mistletoe lectins to cutaneous malignant melanoma: implications for prognosis and therapy.

BACKGROUND: Glycoconjugates, as detected by lectin histochemistry, have been implicated in metastasis formation in many neoplasias. However, no data concerning the three mistletoe lectins (MLs) and the spread of malignant melanoma have been published. MATERIALS AND METHODS: The binding status of ML-I, -II and -III was histochemically assessed in 100 malignant melanomas and correlated with metastasis in a 10 year follow-up period. Furthermore, the staining intensity of the three MLs, scored from negative (-) to very intense (+ + +), was evaluated. RESULTS: Kaplan-Meier analsis revealed that very intense binding (+ + +) of ML-I was positively-correlated with metastasis (p=0.044). CONCLUSION: Since ML-I is specific for galactose, high density galactose expression in malignant melanoma is a predictor of poor prognosis.

Adjuvants, Immunologic↗

Changes in immunological characteristics of white blood cells after administration of standardized mistletoe extract.

After administering standardized mistletoe extract, Viscum album L, (Iscador injections of 0.1 mg twice and 1.0 mg in defined intervals) the functional characteristics of microcirculation and immunological behavior of the white blood cells in different target tissues (derma, intestine) were investigated in healthy volunteers by vital microscopic investigation over 13 days of observation. The investigations showed a temporarily improved function of the microcirculation and an increased adhesion and transmigration of white blood cells in the target tissue areas. This observation was evaluated as a biologically relevant immunomodulation. Further investigations under pathophysiological conditions with regard to complementary administration of the test substance (e.g. to cancer patients) appear promising.

Antineoplastic Agents, Phytogenic↗

Anticancer activity of rViscumin (recombinant mistletoe lectin) in tumor colonization models with immunocompetent mice.

The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe lectin) were investigated in two mouse tumor models. After intravenous inoculation with RAW-117-P or L-1 sarcoma cells in Balb/c mice, rViscumin was given s.c. at non-toxic doses ranging from 0.3 to 150 ng rViscumin/kg. One set of experiments was performed to investigate the survival of rViscumin-treated animals. Another set was carried out to analyze the effect of rViscumin treatment on the number of tumor colonies in infiltrated lungs (RAW-117P) or liver (L-1) and the activation of immune cell subsets, respectively. An overall prolonged survival time after treatment with rViscumin and a reduction in the number of tumor colonies after administration of certain rViscumin doses was observed. Immunophenotyping of the peripheral leukocytes of treated mice revealed increased numbers of T-lymphocytes, pan-NK cells and activated monocytes. The results indicate that rViscumin has antineoplastic properties and might therefore be a promising candidate in cancer therapy.

Adjuvants, Immunologic↗

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.

Animals↗

Impact of mistletoe lectin binding in breast cancer.

BACKGROUND: One of the most often used complementary or alternative anticancer therapeutics for breast cancer is mistletoe extract with Viscum album lectin (VAA-1) as the leading component. The aim of this study was to determine the correlation between VAA-1 binding in breast cancer and disease outcome. MATERIALS AND METHODS: VAA-1 binding to either tumour cells or tumour infiltrating inflammatory cells was examined by immunohistochemical staining of paraffin tissue sections from breast cancer patients (n=226). RESULTS: About 34% of breast cancer tumour cells displayed a strong VAA-1 binding. Kaplan-Meier statistics revealed an inverse correlation between disease outcome and presence of strong VAA-1 binding to tumour cells. CONCLUSION: As binding of VAA-1 indicates the presence of galactose molecules in saccharide structures of the cytoplasm or cell membrane, we conclude that the amount of galactose-containing glycoproteins, mucoproteins and glycolipids in breast cancer tumour cells is related to disease outcome.

Breast Neoplasms↗

[Determination of primary structure of a novel peptide from mistletoe and its antitumor activity].

AIM: To study the antitumor peptide components in the stems and leaves of mistletoe (Viscum coloratum (Kom.) Nakai), the primary structure of the novel peptide was elucidated. METHODS: Cation exchange, gel filtration and HPLC were employed for isolation and purification. Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry was used to determine the mass. The complete amino acid sequence of the novel peptide was obtained by Edman degradation combined with enzyme digestion. The antitumor activity of the peptide in vitro was studied with MTT method. RESULTS: The primary stucture of the peptide named as viscotoxin B2 is KSCCKNTTGRNIYNTCRFAGGSRERCAKLSGCKIISASTCPSDYPK. The IC50 value of viscotoxin B2 on the Rat Osteoblast-like Sarcoma 17/2.8 cells in vitro is 1.6 mg x L(-1). CONCLUSION: Viscotoxin B2 in V. coloratum, which has high similarity with viscotoxins from V. album, showed antitumor activity.

Amino Acid Sequence↗

Binding of recombinant mistletoe lectin (aviscumine) to resected human adenocarcinoma of the lung.

BACKGROUND: Lectins, carbohydrate proteins, bind to glycoconjugates of all mammalian cells, including cancer cells. Aberrant glycosylation, detected by lectin histochemistry, can predict outcome in some tumour entities. One such lectin is aviscumine (recombinant mistletoe lectin). Aviscumine has cytotoxic effects and can therefore be used as anti-tumour therapy. MATERIALS AND METHODS: Lectin histochemistry with aviscumine was performed on primary tumour sections from resected adenocarcinoma of the lung. Staining results were then correlated with the clinical course of the patients. RESULTS: Most of the adenocarcinomas (92.5%) bound aviscumine. Kaplan-Meier analysis revealed no correlation between aviscumine binding and progression-free survival or overall survival. CONCLUSION: These results suggest that for the selected group of patients with adenocarcinoma of the lung aviscumine binding activity can not serve as a prognostic factor. More strikingly, however, aviscumine binds to malignant cells in 92.5% of the patients. This is an indicator for the use of aviscumine as a possible target for tumour therapy.

Adenocarcinoma↗

Antiproliferative effects of mistletoe (Viscum album L.) extract in urinary bladder carcinoma cell lines.

BACKGROUND: The aim of the study was to evaluate the antiproliferative potency of Viscum album extract (VA-E) in human bladder carcinoma cell lines with regard to its possible use for intravesical therapy of superficial bladder cancer. MATERIALS AND METHODS: Proliferation (MTT-test or 3H-thymidine incorporation), necrotic disintegration (3H-thymidine release of prelabelled cells) and portions of apoptotic and/or necrotic cells (Annexin-V binding, propidium iodide (PI) labelling and DNA-fluorescence profiles by flow cytometry) were measured in four different human bladder carcinoma cell lines (T24, TCCSUP, J82 and UM-UC3) cultured in vitro. RESULTS: Antiproliferative effects of VA-E were observed in the four bladder carcinoma cell lines tested. Metabolic activity could also be completely abrogated by short-time contact of the cells with VA-E. Apoptosis and necrosis, as underlying mechanisms of action, were differentially expressed by the different cell lines. CONCLUSION: VA-E and cytotoxic proteins, i.e., mistletoe lectins (ML) and viscotoxins (VT), were able to block the growth of bladder carcinoma cells. Together with the immunomodulating properties of VA-E, the observed antiproliferative potency might give a rationale for the topical intravesical application of VA-E for the treatment of superficial bladder cancer.

Administration, Intravesical↗

Comparative studies on internalization of gold-labelled mistletoe lectin I, its subunits, as well as of an immunotoxin in murine L 1210 leukemia cells.

The binding and internalization of gold-labelled mistletoe lectin I (ML I), its A (ML I-A) and B (ML I-B) subunits, as well as of an immunotoxin consisting of an monoclonal anti L 1210 antibody and the cytotoxic A chains of ML I, were studied on murine L 1210 leukemia cells by a preembedding electron microscopic technique. We found that receptor-mediated endocytosis differs remarkably between the whole lectin, its subunits, and the immunotoxin. Whereas ML I, its A chain, as well as the immunotoxin are internalized by coated pits/coated vesicles or in combination with uncoated membranes, the B chain is exclusively endocytosed via uncoated deep invaginations of the cell membrane. The endocytosis via clathrin-coated or uncoated vesicles is discussed taking into account the binding and internalization kinetics of ligand-receptor complexes related with the movement by the cytoskeleton.

Animals↗

Enhancement of the cytotoxicity of mistletoe lectin-1 (ML-1) by high pH or perturbation in Golgi functions.

We have studied the cytotoxicity of Mistletoe lectin-1 (ML-1), a cytotoxic protein produced by Viscum album, in CHO and V79 cells and in mutant cell lines altered in Golgi functions or in endosomal acidification. In wild-type CHO cells, cytotoxicity of ML-1 was greatly enhanced by ammonium chloride or nigericin. A CHO mutant defective in endosomal acidification (DMPR-2), which is resistant to diphtheria toxin, modeccin and Pseudomonas aeruginosa exotoxin A and hypersensitive to ricin, showed increased sensitivity to ML-1. MonR-31 and MF-1 are monensin- and compactin-resistant mutants derived from CHO and V79 cell lines, respectively, and are presumably altered in Golgi functions. The cytotoxicity of ML-1 was found to be increased in both MonR-31 and MF-1 cells as compared with their parental cells. These results indicate that the effects of chemicals or mutations altering endosomal acidification and Golgi functions on the cytotoxicity of ML-1 are similar to those on ricin cytotoxicity. Our results suggest that the cytotoxicity of ML-1 is enhanced by an increase in endosomal pH, as well as by chemicals or mutations altering the structure/functions of the Golgi regions. Like ricin, the intoxication process of ML-1 may involve the Golgi regions.

Ammonium Chloride↗

Relationship between internalization kinetics and cytotoxicity of mistletoe lectin I to L1210 leukaemia cells.

We have taken two different approaches to the study of the entry of mistletoe lectin I (MLI) into murine L1210 leukaemia cells. As detected by cellular protein synthesis and DNA synthesis inhibition, the lectin was cytotoxic to L1210 leukaemia cells. Inhibition of [3H]leucine and [3H]thymidine incorporation into L1210 cells by MLI was found dose dependent in a concentration range from 10(-16) to 10(-12) mol/ml. The kinetics of cellular protein synthesis inhibition by MLI was concentration dependent, too. Using preembedding electron microscopy, the binding and intracellular routing of the gold-labelled lectin (MLI.Au15) were studied. MLI was internalized into L1210 leukaemia cells by two different pathways: via coated pits to coated vesicles and via long enclosed invaginations of the plasma membrane.

Animals↗

Increased secretion of tumor necrosis factors alpha, interleukin 1, and interleukin 6 by human mononuclear cells exposed to beta-galactoside-specific lectin from clinically applied mistletoe extract.

A beta-galactoside-specific lectin from proprietary mistletoe extract, recently reported to exhibit immunomodulatory potency in vivo (T. Hajto, K. Hostanska, and H J. Gabius, Cancer Res. 49: 4803-4808, 1989), induced increased secretion of tumor necrosis factor alpha, interleukin 1, and interleukin 6 in cultures of human peripheral blood mononuclear cells. The enhancement of secretion, determined independently by bioassays and enzyme-linked immunosorbent assay-based quantitation, was caused by selective protein-carbohydrate interaction, as revealed by the strict dependence on the presence of the carbohydrate-binding subunit of the specific lectin-binding sugar as well as anti-lectin antibodies. Increased cytokine levels in serum of patients after injection of optimal lectin doses corroborated the in vitro results. Thus, these data provide an explanation for the increases in cellular parameters of the host defense system in vivo, which presents a further step toward their potentially beneficial clinical exploitation in standardized regimens.

Animals↗

Effects of lectin I from mistletoe (ML I) and its isolated A and B chains on human mononuclear cells: mitogenic activity and lymphokine release.

The interaction of lectin I from mistletoe (Viscum album) (ML I) and its isolated A and B chains with mononuclear cells from healthy human donors was investigated with respect to proliferation capacity (mitogenicity) and monokine/lymphokine factor (MSF) production. The factor produced was studied by means of the electrophoretic mobility inhibition assay using guinea pig macrophages as indicator cells. ML I and its B chain exhibited comparable inhibitory effects on the proliferation of mononuclear cells (MNC, lymphocytes) that differed in quantity only. After 72 h the tritiated thymidine incorporation had diminished in comparison to the control over a concentration range from 10(-7) to 10(-11) mol/l (ML I) or from 3 X 10(-7) to 3 X 10(-9) mol/l (B chain), respectively. The ML I was about 30 times more active than the B chain. Moreover, MSF production could be substantiated for the B chain at a concentration of 3 X 10(-8) mol/l. In contrast to the B chain, the A chain stimulated the MNC to blastogenic transformation at concentrations of 3 X 10(-8) and 3 X 10(-9) mol/l. The stimulation index was much lower than after PHA stimulation. The same concentrations induced the production of lymphokine (MSF). The lymphokine activity on the indicator cells could be inhibited by L-fucose. Perhaps both cytotoxicity and lymphocyte stimulation are important for anti-tumor activity after application in vivo.

Humans↗

[Histamine receptor-bearing lymphocytes. III. Suppression of immune reactions by killing of histamine receptor-bearing lymphocytes with a conjugate of histamine and the A chain of mistletoe lectin I].

Conjugates of the A-chain of the mistletoe lectin I and histamine but not A-chain per se inhibit the capacity of spleen cells of the mouse to induce antibody response or graft-versus-host reaction by 90%. The conjugates killed 27% of the spleen cells, 60% of the T-, and 15% of the B-lymphocytes. The receptors for histamine could be shown to be of the H2-type, because only H2-antagonists inhibited the toxic influence of the conjugates. The results suggest the hypothesis that not only suppressor cells but at least the majority of the immunocompetent cells bear receptors for histamine. The A-chain-histamine conjugate represents an "affinotoxin" which is cytotoxic only after affinity binding on receptors of the target cell.

Animals↗

[Effect of mistletoe lectin therapy on serum level of defined serum proteins (acute phase proteins) in tumor patients].

The influence of galactoside-specific mistletoe lectin (ML-1) administration on defined acute phase reactants in the serum of cancer patients (mammary carcinoma, n = 4; larynx carcinoma, n = 11; TNM-stages II to IV; after appropriate surgery, chemotherapy, radiation) was studied. Regular subcutaneous injections of the optimal doses of ML-1 (1 mg/kg body weight, twice a week) yielded statistically significant increases of certain acute phase reactants (C-reactive protein, haptoglobin, coeruloplasmin, C3-complement, albumin, immunoglobulin IgM) after four weeks of treatment. However, serum concentrations of transferrin, C4-complement and the immunoglobulins IgG and IgA were found within the biological range (means +/- 2 s). The increase of acute phase reactants after administration of ML-1 correlates positively with the activity of lymphatic cells (e.g. expression of IL-2 and HLA-DR receptors) in FACS (fluorescence-activated cell sorter) staining experiments and indicates the immunoreactive potency of this substance which may be speculated to be cytokine-induced.

Acute-Phase Proteins↗