Омела белая Viscum album Mistletoe
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- 2.2. Cell Culture
- 2.3. Cell Growth Assay
- 2.4. Morphological Colchicine Tumor Assay
2. Materials and Methods2.1. Mistletoe Extracts and Pharmaceutical ProcessingTo produce the constituents for the investigated mistletoe extracts, mistletoe plants (Viscum album L. ssp. album) growing on either apple trees (Malus domestica Borkh.) or pine trees (Pinus sylvestris L.) were harvested shortly before midsummer and end of the year, respectively. The one- to two-year-old leaves, the stems, the generative organs, and in the winter harvest the ripened berries were mechanically opened in a roll mill and extracted by fermentation with mistletoe derived lactobacillus in distilled water. After three-day fermentation, the extract was obtained by pressing and sterile filtration. The basic winter and summer mistletoe extracts possess a drug-extract ratio of 1  : 5 (extract of 200 mg mistletoe fresh plant in 1 mL fluid).
To produce anthroposophically processed Viscum album extract (APVAE), winter mistletoe extract is fed into the center of a 1 This specific blending process for winter and summer mistletoe extract was developed at the Hiscia Institute of the Society for Cancer Research (Arlesheim, Switzerland), with emphasis on realizing Steiner's original suggestions as precisely as possible [11, 20]. This process is being used for the production of Iscador. As comparison sample relative to APVAE, we used a Viscum album extract (VAE) consisting of the same constituents as the APVAE sample (winter and summer mistletoe extracts of the same batch as used for production of APVAE). For this purpose, a container was successively filled with winter and summer mistletoe extracts that were mixed together homogenously by upending the container 10 times. For the experiments, APVAE and VAE were sterile filtered immediately after mixing and filled into 1 mL glass ampoules under aseptic conditions for storage at 4°C. Two sorts of mistletoe extracts were investigated: extracts from mistletoe plants growing on the host tree apple (APVAE/VAE Mali) and extracts from mistletoe plants growing on the host tree pine (APVAE/VAE Pini).
2.2. Cell CultureThe human carcinoma cell lines HCC1143 (breast carcinoma), PA-TU-8902 (pancreas adenocarcinoma), DU-145 (prostate carcinoma), and NCI-H460 (lung carcinoma) were obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Germany). MV3 cells (human metastatic melanoma) were kindly provided by Anka Dahl, University Hospital, Hamburg-Eppendorf (Germany). All cell lines except PA-TU-8902 were cultivated under standard cell culture conditions (37°C, 95% relative humidity, 5% CO2) in RPMI-1640 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, Switzerland), 2 mM L-Glutamine (Sigma-Aldrich, Switzerland), and Penicillin-Streptomycin (Sigma-Aldrich, Switzerland; Penicillin 10,000 units/mL and 10 mg/mL Streptomycin). Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, Switzerland) supplemented with the ingredients mentioned above was used to cultivate PA-TU-8902 cells. The cells were maintained in adherent, exponential growth in 25 cm2 culture flasks (TPP, Faust Laborbedarf AG, Switzerland) and cells from subconfluent monolayer were harvested by brief exposure to trypsin-EDTA solution (Sigma-Aldrich, Switzerland).
2.3. Cell Growth AssayCells were seeded at a density of 2500 cells in 90 μL/well in a 96-well plate (TPP, Faust Laborbedarf AG, Switzerland) and incubated under standard conditions for 4 hours to attain adherence to the bottom. Thereafter, the cells were exposed during 48 hours to different concentrations of appropriate dilutions of Viscum album extract (VAE Mali, lot 1210/2338), anthroposophically processed Viscum album extract (APVAE Mali, lot 1210/2339), or blank medium (control) by adding a volume of 90 μL/well resulting in a total volume of 180 μL/well. After 2-day incubation, none of the cell lines had reached full confluence.
In this assay, only VAE/APVAE Mali was used due to the selective toxicity of the cancer cell lines used towards mistletoe lectins (VAE/APVAE Pini is almost void of mistletoe lectins). VAE Mali (lot 1210/2338) had a concentration of 10,400 ± 283 Cell growth was assessed by using a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 (Roche Diagnostica, Rotkreuz, Switzerland) to formazan by the mitochondrial dehydrogenase in viable cells [22]. For this purpose, after 48 Possible effects depending on the position of the samples within the 96-well plate were eliminated by systematic exchange of the sample positions. All manipulations by the experimenter were performed with coded (blinded) VAE and APVAE samples. VAE and APVAE samples were diluted to obtain the following final concentrations of the extracts: 800, 600, 400, 200, 100, 50, 25, and 12.5 Viability of the cells was calculated by setting the OD values of the cell free background control to 0% and the OD values of the VAE/APVAE-untreated cells to 100%. ED50 was determined for each experiment by quadratic regression of viability versus concentration, including the concentrations with viability > 15% (12.5–200 μg/mL for NCI-H460, DU-145, HCC1143, and MV3 and 12.5–800 μg/mL for PA-TU-8902). Mean ED50 values ± standard deviation/error were calculated based on the eight ED50 values obtained in the eight independent experiments for each cell line. ED50 values for VAE and APVAE were compared using a t-test for independent samples. Calculations were carried out with Microsoft Excel for Mac 2011 (version 14.2.2) and Statistica 4 (Statsoft Inc., Tulsa, USA).
2.4. Morphological Colchicine Tumor AssaySeedlings of Lepidium sativum L. (Ekkharthof, Lengwil, Switzerland) were cultivated in the dark at room temperature on chromatography paper (2043 A, Schleicher and Schuell, Dassel, Germany) in hanging LD-PE plastic bags (minigrip, Semadeni, Ostermundigen, Switzerland). Per bag, 16 sorted seeds were put on chromatography paper that had been soaked with 3 mL of fluid, namely, either (i) distilled water (Büchi, Fontavapor 250, Flawil, Switzerland), (ii) VAE Mali (lot 0404/4141) 2 mg/mL, (iii) APVAE Mali (lot 0404/4142) 2 mg/mL, (iv) VAE Pini (lot 0404/4143) 2 mg/mL, or (v) APVAE Pini (lot 0404/4144) 2 mg/mL. Mistletoe extracts were diluted 1 : 100 in distilled water (from 200 mg/mL to 2 mg/mL). Cultivation fluids were double coded and randomized. To all cultivation fluids, colchicine (Calbiochem, EMD Chemicals, San Diego, USA) was added at a fixed concentration within one experiment (see below).
Growth status was documented by photocopying the seedlings after 93 ± 4 A total number of seven independent experiments were carried out. Within one experiment, each experimental group comprised 20 bags, with each containing 16 seeds, amounting to 320 seeds per parameter per experiment or 11,200 seeds in total. Concentration of colchicine in the cultivation fluid was 17 Application of colchicine to Lepidium sativum led to dose-dependent shortening and thickening of the shoot (Figure 2) and to lengthening of the root. Hence, the ratio of root to shoot length increased due to colchicine. We therefore defined the ratio of root to shoot length as main outcome parameter for this morphological bioassay. All data were analyzed with the statistics software Statistica 6 (Statsoft Inc., Tulsa, USA). Statistical evaluation was based on ANOVA (analysis of variance) procedures. Planned comparisons were evaluated with the LSD test only if the preceding global F-test was significant (P < 0.05) (protected Fisher's LSD). This procedure constitutes a good safeguard against type I as well as type II errors [23]. Statistical analysis was performed with data from n = 10,612 seedlings (data from 588 seedlings (5.25%) were missing, either due to failing germination or due to exclusion during length measurement according to the criteria defined above). Treatment parameters were decoded only after length measurement was accomplished.
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