Biological Activities

Aqueous VA-E has been used in adjuvant cancer therapy with both immunostimulatory and cytostatic/cytotoxic properties (Hajto et al., 1989; Stein and Berg, 1997; Beuth, 1997; Büssing et al., 1996). VA-E stimulate the immune system non-specifically, as it increases the number and activity of natural killer cells and neutrophils, induces cytokines such as tumour necrosis factor alpha (TNF-a), interferon-gamma (IFN-y), interleukin-1 (IL-1) and IL-6 (Hajto et al., 1990; Müller and Anderer, 1990; Stein et al., 1998a and 1998b). While the aqueous VA-E has been studied intensively, the biological studies mainly with V. album L. var. C. have been carried out recently.

The extract of V. album L. var. C. did not exhibit mutagenic properties as measured by Salmonella typhimurium TA98 and Ames test. Further, the extract inhibited mutagenesis induced by mitomycin C and N-methyl-N9-nitro-N-nitrosoguanidine by spore rec-assay (Ham et al., 1998). The ethanol and heated aqueous extracts inhibited mutagenesis induced by benzo-a-pyrene [B(a)P] and Trp-P-1 by SOS chromotest (Ham et al., 1998).

The aqueous extract of the plant was cytotoxic (ID50:8 Ug/ml) to both, nontumorigenic A31 and tumorigenic MSV cells, while the heat-treated extract was less cytotoxic (ID50, 300 ^g/ml) (Park et al., 1997b and 1998b). A fresh extract of Korean mistletoe was reported to be more active (ID50:0.1 Ug/ml) than fermented one (ID50:9.6 jUg/ml) (Khwaja et al., 1986), but the activity of the heat-treated extract increased by fermentation. In fermented Korean mistletoe, the lectins decreased significantly, while the presence of a new lectin was recognised (Park et al., 1994b, 1995). It is supposed that the heat-denatured lectins in the heat-treated extract were digested by fermentation process and some fragments of soluble cytotoxic peptide were produced. The extract was also cytotoxic against 6 human tumour xenografts (i.e. ovarian cancer, small cell and large cell lung carcinoma, colon, renal and melanoma xenografts) (Choi et al., 1996).

Similar effects of VA-E have been reported (Ribereau-Gayon et al., 1986): The unfermented VA-E was approximately 10 times more cytotoxic to leukemic Molt 4 cells than fermented one; and the fermented one contained lower amount of lectin, while unfermented preparation contained about 10 times more.

The aqueous extract of the Korean mistletoe also inhibited tumour metastasis and angiogenesis caused by hematogenous and non-hematogenous tumour cells in mice

[Yoon et al., 1995]. The extract significantly inhibited lung metastasis produced by highly metastatic murine tumour cells (Yoon et al., 1995). In vivo analysis for tumour-induced angiogenesis revealed that the extract suppressed tumour growth and inhibited the number of blood vessels oriented towards the tumour mass (Yoon et al., 1995). The culture supernatants of murine peritoneal macrophages treated with the aqueous mistletoe extract showed tumour necrosis factor-a (TNF-a) activity, and the supernatant used inhibited the growth of rat lung endothelial cells in vitro. The antimetastatic effect may result from the suppression of tumour growth and the inhibition of tumour-induced angiogenesis by inducing TNF-a (Yoon et al., 1994, 1995). Further, the extract induced the secretion of IL-1, IL-6, and INF-y from macrophage; these cytokines were found to be cytotoxic against carcinoma cells (Yoon et al., 1997).

The prophylactic effect of the extract on tumour metastasis produced by highly metastatic tumour cells, colon 26-M3.1 carcinoma, B16-BL6 melanoma and L5178Y-ML25 lymphoma cells, was recognised using experimental models in mice. Intravenous administration of the extract 2 days before tumour cell inoculation significantly inhibited lung metastasis of B16-BL6 and colon 26-M3.1 cells, and liver and spleen metastasis of L5178Y-ML25 cells (Yoon et al., 1994, 1995). Furthermore, mice administered the extract 2 days before tumour cell inoculation showed significantly prolonged survival rates compared with the untreated mice. The administration of the extract significantly augmented NK cytotoxicity to Yac-1 tumour cells after the treatment (Yoon et al., 1994, 1995). Furthermore, depletion of NK cells by injection of rabbit anti-asialo GM1 serum completely abolished the inhibitory effect of the extract on lung metastasis of colon 26-M3.1 cells. These results suggest that the extract possesses immunopotentiating activity which enhances the hose defence system against tumours, and that its prophylactic effect on tumour metastasis is mediated by NK cell activation.

Kwaja et al. (1980) reported the presence of alkaloids in Korean mistletoe, and addressed the anticancer activity of V. album L. var. C. to the alkaloids. Experiments with extracts from Californian, European and Korean mistletoe (Phoradendron villosum, V. album and V. album var. C.) showed that Korean mistletoe was more active in inhibiting the growth of leukemia L1210 cells as compared to the other extracts. Similarly, alkaloidal fraction II isolated from Korean mistletoe extract was the most active (ID50, 0.17 ^g/ml) of all other fractions. The data suggest that like viscotoxins and mistletoe lectins, biologically active mistletoe alkaloids and other compounds may have a contribution to the mechanism of cytotoxicity of mistletoe extracts, and that all biologically active compounds of mistletoe may exert synergistically therapeutic activity (Khwaja et al., 1986). However, the structures of these compounds have not been elucidated as they are extremely labile (Khwaja et al., 1980); others were unable to clearly show the presence of alkaloids in European mistletoe (Becker and Pfuller, personal communications).

Cytotoxicity of an aqueous extract from Korean mistletoe was strongly related to the activity of VCA. Using human lymphocytes and leukaemic Molt-4 T cells treated for 24 h with the whole plant extract and the purified VCA, a dose-dependent induction of mitochondrial Apo2.7 molecules (Figure 2), active caspase-3 and degradation of

Figure 2 Induction of apoptosis by by VCA. A. Peripheral blood mononuclear cells (PBMC) from a healthy individual were treated for 24 with VCA at 0, 10, 50 and 100 ng/ml, and the toxic lectins from European mistletoe (ML I and ML III) at 50 ng/ml. Expression of mitochondrial apoptosis marker Apo2.7 was measured by flow cytometry as described [Büssing et al., 1999]. VCA was the same effective to induce apoptosis as compared to the galNAc-binding ML III. B. Apo2.7 expression in peripheral blood mononuclear cells (PBMC; •), IgE-producing myeloma cell line U-266 (o) and the leukaemic Molt-4 T cells (♦) treated for 24 h with VCA at 0, 1, 10, 50 and 100 ng/ml. As observed also for the toxic lectins from Viscum album L. (ML I, ML II, ML III), Molt-4 blast cells were more sensitive towards the toxin than normal PBMC. Results with kind permission of A. Büssing, Herdecke.

Figure 2 Induction of apoptosis by by VCA. A. Peripheral blood mononuclear cells (PBMC) from a healthy individual were treated for 24 with VCA at 0, 10, 50 and 100 ng/ml, and the toxic lectins from European mistletoe (ML I and ML III) at 50 ng/ml. Expression of mitochondrial apoptosis marker Apo2.7 was measured by flow cytometry as described [Büssing et al., 1999]. VCA was the same effective to induce apoptosis as compared to the galNAc-binding ML III. B. Apo2.7 expression in peripheral blood mononuclear cells (PBMC; •), IgE-producing myeloma cell line U-266 (o) and the leukaemic Molt-4 T cells (♦) treated for 24 h with VCA at 0, 1, 10, 50 and 100 ng/ml. As observed also for the toxic lectins from Viscum album L. (ML I, ML II, ML III), Molt-4 blast cells were more sensitive towards the toxin than normal PBMC. Results with kind permission of A. Büssing, Herdecke.

Table 3 The biological activities of Korean mistletoe.

Cell line

Effects of processed plant material

References

Leukemia L1210

Leukemia L1210

Leukemia L1210

B16-BL6 melanoma, colon 26-M3.1 carcinoma, lymphoma

Ovarian and lung cancer; colon, renal and melanoma xenografts

Non-tumourigenic A31

Tumourigenic MSV

Murine cell lines: Colon 26, B16-BL6, 3LL, Meth-A, L1210, Yac-1, L5178Y, L929, 3T3

Human cell lines; U937, HL60, K562, THP-1, Jurkat, Raji, Hs578T

Fermented extract (ID50 9.6 Aig/ml)

Alkaloid (ICj0 0.17 jug/ml)

inhibition of tumour metastasis and angiogenesis, induction of IL-1, IL-6, and IFN-y, TNF-a and NK cell activity

Fresh extract (IC70 1.2 yu-g/ml)

Non-tumourigenic A31

Heat-treated extract (IC50 300 /u-g/ml)

Aqueous extract (ICS0, 0.4-307 ¿ig/ml) Lectin (IC50, 1-210 ng/ml)

Aqueous extract (IC50, 0.5-42 /u.g/ml) Lectin (ICS0, 0.1-62 ng/ml)

Khwaja et al., 1986 Khwaja et al., 1986 Khwaja étal, 1986 Yoon et al, 1995

Choi et al, 1996

Park et al., 1997-b Park et al., 1997-b Yoon etal., 1999

Yoon etal, 1999

Bcl-2 proteins was observed (Büssing and Park, in preparation), indicating the onset of the apoptotic cell death. The biological effects of Korean mistletoe are summarised in Table 3.

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