Cytotoxicity

The most prominent effect of VA-E is their cytotoxic property. The first investigations of cytostatic and/or cytotoxic effects were conducted with plants and found that synergistic activity of the alkaloid colchicin and a dialysate of Viscum album (i.e., no viscalbin alkaloid present) resulted in greater growth-inhibition to roots and root-hairs of wheat germ than a comparable amount of dialysed VA-E or colchicum (Havas, 1937). Simultaneous administration of colchicum and a pressed sap of Viscum album that was rich in viscalbin, produced no such effects, however, the total weight of the shoots increased (Havas, 1937). After application of sap from Viscum album, Crown-Gall tumours normally induced by Agrobacterim tumefaciens were also rejected (Havas, 1939).

Animals

Intracutaneus administration of high drug concentrations (Koch, 1938a) to mice and rabbits was less toxic compared to an intravenous application. However, intra-or peritumoural injection in murine Ehrlich ascites carcinoma resulted in tumour necrosis without significant affections of the healthy tissue, and in several cases, complete remissions were observed (Koch, 1938b). Using a dried VA-E and an isolated compound termed "Toxomelin", Chernov (1954, 1955) observed decreased growth of tumour cell lines (i.e., M1 sarcoma, Sarcoma 180, Ehrlich ascites sarcoma). In rabbits, Toxomelin reduced the growth rate of Brown-Pearse tumours and slightly decreased incidence of metastases (Chernov, 1955). In a series of experiments with mice, 6 per group, subcutaneous injection of a mixture of tumour ascites combined with increasing concentrations of VA-E (Iscador and Plenosol) that were incubated for 30 minutes at 4°C, prevented the development of Ehrlich ascites carcinoma in a dose-dependent manner (Seeger, 1965a-c). Histological analyses revealed lymphocytosis, demarcation, encapsulation of necrotic tumours, and necrosis of the tumour in several cases. Although not confirmed, these effects were hypothesized to be the result of increased activity of the reticulo-endothelial system induced by a proposed phytotoxin present in the drug extracts (Seeger, 1965a-c). This antigenic phytotoxin resembled acetylcholin, but might be mistletoe lectin (ML).

Several experiments using tumour-bearing animals showed impressive reduction of tumour growth and/or increased survival with the application of mistletoe therapy (Selawry et al., 1959, 1961; Franz, 1986; Nienhaus and Leroi, 19 70; Nienhaus et al., 1970; Beuth et al., 1991; Drees et al., 1996; Mengs et al., 1998). Intraperitoneal injection of a saline VA-E (2 h at 40°C) and the fermented VA-E (Iscador) effectively inhibited development of several tumour types (Carcinoma 755, Leukemia 1210, Ehrlich carcinoma, Sarcoma 180) inoculated subcutaneously in mice (Selawry et al., 1959). Tests for tumour inhibition of several fractions from Viscum album found that the most active fraction was present in the native protein fraction (Selawry et al., 1961). Prophylactic subcutaneous injections of a fermented VA-E (Iscador Q i.p.) or selected basic protein fractions from Viscum album prior to transplantation of Sarcoma 180 cells into mice significantly reduced or prevented tumour growth (Nienhaus and Leroi, 1970; Nienhaus et al., 1970). Encapsulation of the tumour in firm connective tissue was accompanied by an increased weight of thymus, indicating an activation of immunocompetent cells. However, other studies on the subcutaneous treatment with the fermented drug extract Iscador did not reveal significant inhibition of subcutaneous transplants of B16 melanoma or leukemia L1210 in BDF1 mice (Khwaja et al., 1986) and leukemia L5222 in BDIX rats (Berger and Schmahl, 1983). These conflicting results were thought to be related to differences in tumour strains, mode of drug administration, and/or age of animals (Khwaja et al., 1986); however, one may also suggest that the results may be related to differences in the biological activity of the compounds present in a given drug extract.

The purified ML I, administered subcutaneously twice and four times per week, significantly decreased the number of lung (L-1 sarcoma) and liver colonies (RAW 117-H 10 lymphosarcoma) in BALB/c mice after tumour cell inoculation (Beuth et al., 1991). ML I used either subcutaneously (0.014, 0.14 and 1.4 pg/kg BW) or orally (70 pg/kg BW) reduced the number of lung metastases in mice (C57 Bl-6) injected with Lewis lung tumor cells, and raised the level of serum TNF-a (Kubasova et al., 1998). However, ML I treatment of mice receiving radiation prior to or after the injection of tumor cells strongly increased the number of lung metastases (Figure 1). In another study (Kunze et al., 1997, 1998), ML I at 1 ng/kg BW applied twice week subcutaneously for 15 month was ineffective to inhibit N-methyl-N-nitrosurea-induced urinary bladder carcinogenesis in rats.

Intravenous application of VA-E in allogenic BALB/c mice (n=5 per group) receiving intravenously immunogenic B16 melanoma cells reveiled a significant reduction of melanoma cells in the pulmonary lavage at necropsy 3 weeks later (Mengs et al., 1998), while, however, the decreased number of melanoma cells in the lung tissue did not significantly differ from the number of melanoma cells in placebo-treated mice (Weber et al., 1998). Anyway, the number of lung macrophages in bronchoalveolar fluid and thymocytes in the thymus gland increased in response to VA-E (Mengs et al., 1998; Weber et al., 1998). Thus, it is tempting to speculate that intravenous VA-E-treatment may prevent the development of injected melanoma cells in the lung by an activation of macrophages.

Recent reports indicate that even an oral uptake of high ML I concentrations may have an inhibitory effect on tumours. In NMRI mice injected subcutaneously Krebs II lymphosarcoma cells and prefed on diets containing 0.42, 0.83 and 1.67 mg ML I per g diet (6 g/mouse average daily food intake), the tumour growth decreased in a dose-dependent manner within 10 d (Pryme et al., 1998b). ML I stimulated a hyper-plastic growth of the small intestine as observed also in mice fed on PHA (Pryme et al., 1998a). The ML did bind strongly to the surface of jejunal villi with a moderate degree of endocytosis, while the lumenal surface of the crypts was strongly stained and endocytosis was apparent; apoptotic bodies were detected usually in the proximal half of the villi (Ewen et al., 1998). Furthermore, rats fed on diet containing 67 or 200 mg ML I per kg BW for 10 d induced hypertrophy of the pancreas (with depression of circulating insulin level), lungs, and small intestine, and increased systemic catabolism (Pusztai et al., 1998). These animals exhibited higher TNF-a and IL-1^ level as compared to the controls (Pusztai et al., 1998), indicating that immunomodulation by the ML may not be restricted to parenteral application alone. The lymphosarcoma-bearing mice fed on lactalbumin and ML I at 10 mg showed a complete histological dissappearance of the transplanted tumour cells in 3 of 5 animals (Ewen et al., 1998). Thus, in contrast to its parenteral toxicity, ML when applied orally did not display overt toxicity symptoms but was capable of affecting tumour growth, probably by depriving the tumour of nutritional and growth factors by maximal crypt hyperplasia.

Taken together, VA-E and defined components were reported to inhibit tumour development in the animals after adequate application of the drug. However, in most cases it is unclear, whether the remission of highly immunogenic tumours after injection of tumor cells reflects a clinical relevant anti-tumour activity or not. Effectiveness may also depend on the mode application and composition of the drug.

Figure 1 Effects of ML I applied subcutaneously (A) and orally (B) 4xwith 5-day intervals in female C57 Bl-6 mice receiving Lewis lung tumor cells (LLT, 5x105) intramuscularly and radiation (2 Gy), as indicated. The number of lung metastases (mean value of 3-7 mice) was measured in animals sacrified after 19 d. Modified according to Kubasova et al. (1998).

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