**3. Materials and methods**

## **3.1 Test substances**

Viscum album QuFrF (VAQuFrF) extract was obtained from the Hiscia Institute (Arlesheim, Switzerland). According to the manufacturer the aqueous unfermented solution of extract 10 mg/ml contains 2 µg lectin and 10 µg viscotoxin. Vincristine sulfate salt was obtained from Sigma GmbH (Schnelldorf Germany, No 8879). Recombinant human interleukin-6 (rh IL-6) was obtained from R & D Systems (No. 206-IL, United Kingdom) and reconstituted in phosphate-buffered saline with 0.18% bovine serum albumin.

## **3.2 Cells and culture condition**

Human myeloma cell lines (MOLP-8, LP-1, RPMI-8226, OPM-2, U-266, COLO-677, KMS-12- BM, were obtained from DSMZ (Braunschweig, Germany). Five cell lines derived from blood, COLO-677 from lymph node, KMS-12-BM from bone marrow. The cells were cultivated in RPMI 1640 supplemented with 10-20% foetal calf serum, 2mM L-glutamine and 1 % gentamicin in a humidified atmosphere with 5% C02 at 37°C. The doubling times of tumour cell lines were between 35 and 96 hours. For the measurement of the parameters the cell cultures were used within 4-6 weeks after thawing.

#### **3.3 Treatment of cells with VAQuFrF extract or vincristine**


#### **3.4 Treatment of cells with Interleukin-6**


#### **3.5 Measurement of viability**

The viabilities of the cultivated tumour cells were determined by 7-aminoactinomycin D (7- AAD), to exclude the non-viable cells in flow cytometric assays. The values are given in %.

#### **3.6 Measurement of cytokine production**

590 Pharmacology

Viscum album QuFrF (VAQuFrF) extract was obtained from the Hiscia Institute (Arlesheim, Switzerland). According to the manufacturer the aqueous unfermented solution of extract 10 mg/ml contains 2 µg lectin and 10 µg viscotoxin. Vincristine sulfate salt was obtained from Sigma GmbH (Schnelldorf Germany, No 8879). Recombinant human interleukin-6 (rh IL-6) was obtained from R & D Systems (No. 206-IL, United Kingdom) and reconstituted in

Human myeloma cell lines (MOLP-8, LP-1, RPMI-8226, OPM-2, U-266, COLO-677, KMS-12- BM, were obtained from DSMZ (Braunschweig, Germany). Five cell lines derived from blood, COLO-677 from lymph node, KMS-12-BM from bone marrow. The cells were cultivated in RPMI 1640 supplemented with 10-20% foetal calf serum, 2mM L-glutamine and 1 % gentamicin in a humidified atmosphere with 5% C02 at 37°C. The doubling times of tumour cell lines were between 35 and 96 hours. For the measurement of the parameters

a. To measure viability, cytokine production, membrane expression of IL-6 receptor, cell cycle phases and apoptosis /necrosis the cells were cultured at a density of 0.5-0.7x106 cells/ml, except for COLO-677 (0.2x106 cells/ml). After 24 hours the cells were incubated with VAQuFrF extract or Vincristine (doses: 0, l0, 50 or 100 µg/106 cells/ml).

b. To measure proliferation the cells were cultured at a density of 0.5-0.7x105 cells/100 µl, except for COLO-677 (0.2x105 cells/100 µl). After 24 hours the cells were incubated with VAQuFrF extract or Vincristine (doses: 0, 1, 5, 10 µg/105 cells/100l). The parameter

a. To measure viability, cytokine production, membrane expression of IL-6 receptor, cell cycle phases and apoptosis /necrosis the cells were cultured at a density of 0.5-0.7x106 cells/ml, except for COLO-677 (0.2x106 cells/ml). After 24 hours the cells were incubated with IL-6 (dose: 5 ng/106cells/ml). The parameters were measured after 24,

b. To measure proliferation the cells were cultured at a density of 0.5-0.7x105 cells/100 µl, except for COLO-677 (0.2x105 cells/100 µl). After 24 hours the cells were incubated with IL-6 (dose: 0.5 ng/105cells/100l). The parameter was measured after 24, 48 and 72 hours.

The viabilities of the cultivated tumour cells were determined by 7-aminoactinomycin D (7- AAD), to exclude the non-viable cells in flow cytometric assays. The values are given in %.

phosphate-buffered saline with 0.18% bovine serum albumin.

the cell cultures were used within 4-6 weeks after thawing.

**3.3 Treatment of cells with VAQuFrF extract or vincristine** 

The parameters were measured after 24, 48 and 72 hours.

was measured after 24, 48 and 72 hours.

**3.4 Treatment of cells with Interleukin-6** 

48 and 72 hours.

**3.5 Measurement of viability** 

**3. Materials and methods** 

**3.2 Cells and culture condition** 

**3.1 Test substances** 

The IL-6 production or IL-10 production in the supernatant of the cultured cells was determined by chemiluminescent immunometric assay. The lowest detectable level was 2 pg/ml or 5 pg/ml.

#### **3.7 Measurement of membrane expression of IL-6 receptor**

For immunofluorescence staining 3x105 cells/100 l were incubated with 20 l phycoerithrin (PE) conjugated monoclonal antibody (CD 126, Immunotech, France) for 30 min at 4 ◦C. Then the cells were washed, sedimented and analysed in the FACSCalibur flow cytometer. For the expression of the membrane IL-6R (CD 126) the signal intensity (geometric mean of the fluorescence intensity x counts) was used as parameter. The signal intensity of the treated samples was compared with that of untreated samples, which were taken as 100%.

#### **3.8 Measurement of the cell cycle phases**

The cell cycle phases GO/GI, S, G2/M were assessed using the cycle test plus DNA reagent kit on a flow cytometer (BD, BioSciences, San Jose, USA No 340242). Briefly: 5x10**5** cells were incubated at room temperature with trypsin buffer and additionally with trypsin inhibitor+RNAse buffer. The values are expressed in percentage of total viable cell number (100%).

#### **3.9 Measurement of apoptosis and necrosis**

Apoptosis was measured using Annexin V-FITC (BD Biosciences Pharmingen, San Diego, USA No 556 570). Necrosis was measured using propidium iodide (PI). Briefly: 1x105 cells were incubated with Annexin V-FITC or PI at room temperature in the dark. Thereafter the samples were analysed in a flow cytometer. Apoptotic cells: Annexin V-FITC positive and PI negative. Necrotic cells: Annexin V-FITC positive and PI positive. The values are given in percent of total cell number.

#### **3.10 Measurement of the proliferation**

The proliferation was assessed using cell proliferation reagent WST-1 (Roche, Mannheim, Germany, No 1644 807). The colorimetric assay is based on the reduction of the tetrazolium salt WST-1 by viable cells. The reaction produces the soluble formazan salt. The quantity of the formazan dye is directly correlated to the number of the metabolically active cells. The proliferation rate was measured 1, 2 and 4 h after incubation with the reagents at time points 24, 48 and 72 h. The upper limit of the absorbance was 2.0–2.1. The intra-sample variance of the untreated cells was <10% (3–8%).

#### **3.11 Statistical analysis**

Three to four independent measurements were carried out. For the evaluation of the parameters the Mann-Whitney U-test was used. The limit of significance was taken as P<0.05.

The Effects of *Viscum album* (Mistletoe) QuFrF Extract and Vincristine in Human Multiple

detectable amounts.

Vincristine.

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 593

IL-10 production (up to 946 pg/ml) in 5/5 cell lines. VAQuFrF extract and Vincristine reduced the spontaneous IL-10 production in MOLP-8, LP-1 and COLO-677 to non-

With IL-6+VAQuFrF or IL-6+Vincristine the values were markedly lower after addition of IL-6 but higher than without IL-6 treatment. VAQuFrF and Vincristine reduced the induced IL-10 production to the same degree in cell lines RPMI-8226 and LP-1. In the cell lines OPM-2, MOLP-8, COLO-677 the extract of VAQuFrF inhibited the IL-10 production weaker than

Maesurements at 24 and 48 hors after tretment. IL-6:5ng/106 cells.VAQuFrF and Vincristine: 50 µg/106

Table 2. Production of Interleukin-10 (pg/ml)in human multiple myeloma cell lines.

The values are presented in percentage. Range of four independent measurements. Table 2.A. Viability of human multiple myeloma cells (cell lines see Table 2).

The IL-10 production was measured at 24 and 48 after incubation with IL-6. The results show that in tumour cell lines MOLP-8 and RPMI-8226 the IL-10 production was high during the two days. In the other three cell lines the production decreased slightly at 48 h.

cells. Range of 4 independent measurements. ND=not detectable
