J Ethnopharmacol. 2019 May 23;236:100-107.

Antiproliferative potential from aqueous Viscum album L. preparations and their main constituents in comparison with ricin and purothionin on human cancer cells.

Felenda JE, Turek C, Stintzing FC.

WALA Heilmittel GmbH, Dorfstr. 1, 73087, Bad Boll/Eckwälden, Germany.

 

Abstract:

Ethnopharmacological relevance: Mistletoe has been used since ancient times in Europe mostly for medicinal purposes. Since 1917, mistletoe preparations have been applied in cancer therapy and today are the most frequently used complementary medicine in tumor treatment. The main cytotoxic constituents of Viscum album are lectins and viscotoxins.

Aim of the study: The aim of this in vitro study was to investigate the antiproliferative potential of Viscum album preparations from different host trees and to assess the impact of mistletoe lectin 1 (ML-1) and viscotoxin A (VTA) in comparison to a structurally similar lectin and thionin.

Materials and methods: By means of widely accepted 2D Alamar Blue Assay, based on population counting of living cells using a fluorescent cell viability dye, the potential impact to inhibit tumor cell of the mistletoe preparations (Iscucin®) and their single compounds (ML-1 and VT-A) on the cell growth of six human cancer cell lines were evaluated. Also the mixture of ML-1 and VT-A corresponding to the contents in the specific mistletoe preparations were monitored. Ricin and purothionin were used as reference lectin and reference thionin, respectively.

Results: The lung carcinoma cell line HCC827 was very sensitive to the Iscucin® preparations. Very strong antiproliferative effects were found with Iscucin® Salicis and Tiliae and a strong with Iscucin® Crataegi, Mali and Populi. The IC50 concentrations of the Iscucin® preparations correlated with their respective ML-1 contents, but the ML-1 levels were much lower than the IC50 concentration of isolated ML-1 (1 ng/ml – 56 ng/ml). ML-1 was much more effective than ricin. Iscucin® preparations, ML-1 and ricin showed antiproliferative activity on human tumor cells. VT-A and purothionin had no effect on cell viability in the concentration ranges tested.

Conclusion: The complete mistletoe extract is more potent to inhibit tumor cell proliferation than isolated ML-1 at an equivalent concentration level. Phenolic compounds found in all Iscucin® preparations might contribute to uphold the cytotoxic activity of ML-1 by antioxidative action. However, further studies are necessary to evaluate the role of VT-A and possible synergistic actions to the antiproliferative effect of aqueous mistletoe extracts.

https://doi.org/10.1016/j.jep.2019.02.047

 

Supplement:

Today complementary therapies, as part of a patient-oriented integrative medicine, become more and more important. Since 2000, tumor diseases are treated by complementary concepts with increasing tendency. Mistletoe preparations belong to the most common used complementary therapies in cancer patients. The most important entity is mamma carcinoma [1-3]. Traditionally mistletoe (Viscum album L.) has been used for treating hypertension, arteriosclerosis and other cardiovascular diseases in Europe since ancient times [4]. Rudolf Steiner (1861–1925) has inaugurated mistletoe preparations in cancer therapy in 1917 [5]. Crucial benefits of mistletoe application are improving quality of life, reducing side effects of chemotherapy and lowering cancer-related fatigue, all of which have been shown in several clinical studies [6-11]. Furthermore in vitro studies have proven cytotoxic, antiproliferative and apoptotic effects in tumor cells [11-13].

The generally accepted active constituents of Viscum album are lectins and viscotoxins. Like ricin and abrin, the mistletoe lectins belong to the ribosome inactivating proteins (RIPs) and induce apoptosis. Viscotoxins belong to the group of thionins like crambin, β-hordothionin and α-purothionin. Inter alia, the content of mistletoe lectin and viscotoxin depend on the host tree, where mistletoe host as a hemiparasite (Table 1). For example: mistletoe preparations from the lime tree (i.e., Iscucin® Tiliae) have a high mistletoe lectin content reaching 17,000 ng/ml whereas Iscucin® Pini has a low mistletoe lectin content reaching 900 ng/ml and contains no viscotoxin. Inversely, the highest viscotoxin content is found in Iscucin® Crataegi (49 µg/ml).

Our in vitro investigation showed antiproliferative effects of mistletoe preparations on selected human tumor cell lines. Also, the isolated mistletoe lectin I was active. The isolated viscotoxin A alone induced no antiproliferative effect, but we assume that viscotoxin A slightly pushed the effect in combination with high concentrations of mistletoe lectin I. Results showed, that antiproliferative effects on human tumor cells are not exclusively dependent on mistletoe lectin I content. Rather, the complete mistletoe extract as a multicompound mixture is more potent to inhibit tumor cell proliferation. Therefore, a concerted action of all mistletoe lectins and viscotoxins including ML II, ML III and VT B might play a role in the tumor cell growth inhibition is assumed. Moreover, in Iscucin® preparations the phenolic compounds such as syringin and syringenin-4’-0-apiosylglucoside are present [14 a, 14 b] and these components have been associated with anticancer effects [15-17].

The calculated correlation coefficient after Pearson (r) for IC50 values vs. mistletoe lectin contents showed a positive correlation between the mistletoe preparations with middle and high lectin content. Only mistletoes of the conifers, especially Iscucin® Pini, deviate slightly from the linear correlation (Fig. 1). DLD-1 (colon carcinoma cell line) showed a good correlation, but here no IC50 value could be determined for Iscucin® Pini. Interestingly the correlation for lung carcinoma cell line HCC827 showed a worse result, even though the antiproliverative effect was high for the Iscucin® preparations with middle and high mistletoe contents. This cannot be explain at this time. Perhaps other ingredients in the mistletoe preparations, such as the phenolic compounds, are responsible for the antiproliferative effect in this cell line.

In the correlation of IC50 values vs. viscotoxin contents the mistletoe extracts from the conifers, especially Iscucin® Pini, and the apple tree deviate from a linear relationship. The correlation coefficient was calculated ranging from -0.33 to -0.84 (Fig. 2). While the IC50 values more strongly correlate with the mistletoe lectin content than with the viscotoxin content, three cell lines showed values ≤ -0.80. This shows that the viscotoxin content might also display an antiproliferative effect.

Further research is necessary to evaluate the role of VT-A and to determine which specific components support the antiproliferative effect of mistletoe lectins in human tumor cells.

 

Table 1: Overview of mistletoe lectin and viscotoxin levels in aqueous mistletoe preparations (Iscucin®) from different host trees (internal data)

 

 

Figure 1: Results of correlation analysis with IC50 concentrations vs. mistletoe lectin content for (a) HCC827, (b) SK-N-SH, (c) HeLa, (d) Caki-2, (e) LN229 and (f) DLD-1 cell line. In all cases, the correlation analysis showed a negative correlation for mistletoe lectin contents and the IC50 concentrations. The mistletoe extracts from the deciduous trees match well together. The mistletoe preparations from the conifers, especially Iscucin® Pini, deviate slightly from the linear correlation.

 

 

Figure 2: Results of correlation analysis with IC50 concentrations vs. viscotoxin content for (a) HCC827, (b) DLD-1, (c) SK-N-SH, (d) HeLa, (e) LN229 and (f) Caki-2 cell line. A correlation of the viscotoxin contents and the IC50 concentrations is obvious, but was not as good as for the mistletoe lectin content. The mistletoe extracts from the conifers, especially Iscucin® Pini, and the apple tree deviate from linear correlation.

 

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