Modulation of mistletoe (Viscum album L.) lectins cytotoxicity by carbohydrates and serum glycoproteins.

Three D-galactose and/or N-acetyl-D-galactosamine specific mistletoe lectins, ML I, ML II and ML III, were purified by affinity chromatography followed by cation exchange chromatography. These lectins were toxic for Molt 4 cells in culture at concentrations in the pg/ml range, ML III being the most cytotoxic. Carbohydrates able to bind to the B-chain of these lectins inhibited their toxic activity. The digalactosides Gal beta 1,2Gal beta-allyl and Gal beta 1,3Gal beta-allyl were 60 and 30 times, respectively, more potent than D-galactose in their ability to protect the cells from the ML I cytotoxicity. N-acetyl-D-galactosamine and rho-nitrophenyl N-acetylgalactosamine protected mainly from the toxic effects of ML II and III. Protection from cytotoxicity varied in the same order as the affinity of the tested carbohydrates for lectins. Serum glycoproteins particularly haptoglobin, but also alpha 1-acid glycoprotein and transferrin, notably inhibited the cytotoxicity of the lectins. This effect was due to the binding of lectin to the sugar moiety of the glycoprotein because deglycosylated haptoglobin did not have a protective activity on Molt 4 cells. Inhibition of the cytotoxicity of lectins by serum glycoproteins explains why mistletoe extracts containing lectins can be administered to cancer patients without harmful effects.

Modulation of cytotoxicity and enhancement of cytokine release induced by Viscum album L. extracts or mistletoe lectins.

Cytotoxic effects of Viscum album L. (mistletoe) extracts and mistletoe lectins were studied by light and electron microscopy. The first events observed were membrane perforation and protrusions typical for apoptosis. Inhibition of Molt 4 cell growth was obtained with lectin concentrations in the pg/ml range as long as cells were cultured in serum-free medium. Under this condition, mistletoe lectin-III was about 10 times more cytotoxic than mistletoe lectin-I; mistletoe lectin-II was in between. Lectin cytotoxicity was modulated by human serum from donors who had never been treated with mistletoe preparations and lectin-specific carbohydrates, added at the mmol/l range, particularly D-galactose (or beta-lactose) for mistletoe lectin-I and N-acetyl-galactosamine for mistletoe lectin-II and -III. In addition, at subtoxic concentrations, mistletoe lectin-I, -II and -III enhanced the production of cytokines (tumour necrosis factor-alpha, interleukin-1 alpha) by isolated human monocytes. The experimental results are discussed in relation to the treatment of cancer patients administered with mistletoe extracts.

Mistletoe lectins I, II and III induce the production of cytokines by cultured human monocytes.

The three mistletoe (Viscum album L.) lectins. ML I, ML II and ML III, were tested on their ability to enhance the secretion of the cytokines tumor necrosis factor (TNF)alpha, interleukin (IL)-1 alpha, IL-1 beta and IL-6 by human monocytes obtained from healthy donors. At lectin concentrations from 0.02 to 20/pg ml (100-10,000-fold lower than those showing toxic effects), stimulations of cytokine production several-fold over control values were observed. The immunoactivating concentrations by the three lectins were found different for each donor. At toxic concentrations, the amounts of IL-1 alpha, IL-1 beta and to a less extent of TNF alpha in monocytes supernatants were particularly high. The data are discussed in relationship with the cytotoxic and immunoactivating effects of mistletoe lectins and their interest in cancer treatment.

Identity of the N-terminal sequences of the three A chains of mistletoe (Viscum album L.) lectins: homology with ricin-like plant toxins and single-chain ribosome-inhibiting proteins.

Mistletoe lectin (ML) I increases the production of cytokines by mononuclear cells and has been proposed as a useful biological response modifier in the treatment of cancer. Two other lectins, ML II and ML III, have been identified in mistletoe. We report that the N-terminal sequences of the three A chains of ML I, ML II and ML III are identical, and have interesting homology with the N-terminal sequences of the A chain of ricin-like toxins and of single-chain ribosome-inhibiting proteins. In addition, the three mistletoe lectins inhibit the growth of the human tumor cell line Molt 4, ML III being the most potent. followed by ML II and ML I. This inhibition is suppressed by addition of rabbit anti-ML I antibodies to the cultured cells. The data obtained suggest that the three lectins have amino acid sequences which show extensive homology and exert very similar biological effects. They may be derived from the same precursor.

Characterization of cytotoxic proteins from mistletoe (Viscum album L.).

Proteins from a laboratory-made oak mistletoe extract and from the commercial mistletoe preparation Iscador Quercus were cytotoxic for leukemia Molt 4 cells in culture. A 50% growth inhibition was obtained with 0.1 microgram/ml proteins for the mistletoe extract and 0.025 microgram/ml for Iscador. On cation exchange chromatography, cytotoxic proteins from the mistletoe extract were mainly eluted at the same positions as purified lectins, while those of Iscador were eluted at the positions of viscotoxins. The data are discussed in relation to the pharmacological activities of the mistletoe protein complexes described in the literature.

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells.

Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.

Are lectins of Viscum album interesting tools in lung diseases? A review of recent results.

The data presented provide evidence that lectins isolated from different sources such as Abrus and Ricinus seeds and Viscum leaves are structurally related proteins and are probably homologues. The lectins have overall similarities in physical, chemical and biological properties. These proteins appear to be immunologically closely related and possess a high degree of conservation in their primary structures, anticipating a role in fundamental pathways common to all plants. Methods for the preparation, purification and characterization of the Viscum album lectins and comparative crossreactions with closely related lectins of the "toxic lectin" group are summarized. The biological properties of Viscum album lectins, especially their reaction with phagocytes, (phagocytosis), basophil granulocytes (histamine release) and tumour cells (inhibition of tumour growth) are presented and compared with those obtained with an anti-tumoral preparation derived from Viscum album, ISCADOR.

Comparison of the effects of fermented and unfermented mistletoe preparations on cultured tumor cells.

The bacterially fermented mistletoe preparation Iscador, used in cancer therapy for 30 years, and the recently prepared unfermented preparation, have been tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. As observed by phase-contrast microscopy, treatment of HTC cells with fermented or unfermented Iscador, at a concentration corresponding to 1 mg of fresh plant per milliliter culture, led to rapid lysis of cellular membranes. At a lower concentration, 0.1 mg/ml, unfermented Iscador led to the formation of polynucleated cells. On Molt 4 cells, fermented Iscador also produced cytolysis but after a longer time of action. Unfermented Iscador showed a much stronger cytotoxic effect on these cells than on HTC cells. Fermented Iscador was slightly more potent than unfermented Iscador in inhibiting the growth of HTC cells, but on Molt 4 cells fermented Iscador was less active than unfermented Iscador. DNA synthesis, measured by [3H]thymidine incorporation in HTC and Molt 4 cells, was inhibited by fermented and unfermented Iscador with the same type of differences of action as on cell growth. Fermented Iscador contained a low amount of lectins, approximately 100 ng/ml, while unfermented Iscador contained about 10 times more. A purified mistletoe lectin produced effects on HTC and Molt 4 cells similar to those of unfermented preparations. HTC cells were 100 times less sensitive to this lectin than Molt 4 cells. These results are discussed in relation to the known biological effects of lectins.

Photoinactivation of aldolases by pyridoxal phosphate and its analogues.

Pyridoxal phosphate can act as a specific photosensitizer for amino acid residues in rabbit muscle and spinach leaf aldolases, but the residues affected depend on the pH of the reaction. Below pH 8 one histidine residue per enzyme subunit is destroyed; above pH 8.5 there is little loss of histidine, and photoinactivation is associated with the destruction of specific tyrosine residues, particularly the COOH-terminal residues. Pyridoxal and 4-pyridinecarboxaldehyde are much less effective than pyridoxal phosphate at neutral pH, but are similar to pyridoxal phosphate in their photosensitizing activity at the higher pH. Compounds lacking the aldehyde group or the pyridine ring show little or no activity. A number of other enzymes, including alpha-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, and yeast hexokinase, were also photoinactivated in the presence of pyridoxal phosphate; however, rabbit liver aldolase and two isomerases tested were completely resistant. The results suggest that certain enzymes, including rabbit muscle and spinach aldolases, but not rabbit liver aldolase, contain a specific site which interacts with pyridoxal phosphate, and that the conformation of this site changes in the pH range between 8.0 and 8.5