**5. Summary and conclusion**

602 Pharmacology

Treatment with IL-6(5ng/106 cells) or VAQuFrF or Vincristine(10,50,100 µg/106 cells); The investigation was carried out 24, 48 and 72 hours after treatment. Evaluation of three or four independent experiments =accumulation. The numbers present the mean values in percentage. Table 5. Accumulation of human multiple myeloma cells in different cell cycle phases.

hours after treatment in some cell lines.

a different sensitivity to Vincristine or VAQuFrF.

cell proliferation.

*Phases G2/M:* IL-6 treatment led to accumulation of cells in LP-1, RPMI-8226 and OPM-2 after 48 and 72 hours. VAQuFrF was effective in COLO-677 (p < 0.05). Vincristine led to marked increase of the cell numbers in cell lines RPMI-8226, COLO-677 and KMS-12-BM (p < 0.01).

With IL-6 the cell number of each cell line was enhanced in the S phase and in some cell lines in the G2/M phase too. This means that either the DNA synthesis of the cells is increased or the cells are arrested in these cell cycle phases. In this investigation IL-6 led to high proliferation in all cell lines indicating an increased DNA synthesis. This could lead with to arrest in the cycle phase G2/M. In fact we found the accumulation of cells 48 and 72

Vincristine blocks the mitotic process by binding to tubulin leading to an arrest of the cycle phase in G2/M (El Alaaoui et al., 1997; Lin et al., 1998). In this study Vincristine led to an accumulation of the cells in cycle phase G2/M in only three out of seven multiple myeloma cell lines. It was effective in S and in G0/G1 phases of five cell lines, indicating that Vincristine also affects these cycle phases. It is interesting that it was effective both in the S and in G2/M cycle phases of the cell line RPMI-8226. VAQuFrF extract had the same effects as Vincristine in five out of seven tumour cell lines; however in a higher dose range. We postulated that different tumour cell lines from the same disorder (multiple myeloma) show

The inhibition of the G0/G1 phases in different malignancies correlates with antiproliferative substances (El-Sherbiny et al., 2000; Pellizaro et al., 2008). In fact VAQuFrF blocked the cells in the G0/G1 phases in cell lines MOLP-8 and LP-1 and also inhibited the In this experimental study we compared **Viscum album (Mistletoe) extract** and **Vincristine** in several human multiple myeloma cell lines using the parameters: (a) the IL-6 production, (b) the IL-10 production, (c) the expression of membrane IL-6 receptor, (d) the proliferation, (e) the apoptosis/necrosis, (f) the cell cycle phases. The following parameters were measured in a "package" i.e. they measured simultaneously: (a) the IL-6 production, (b) the IL-10 production, (c) the proliferation, (d) the apoptosis/necrosis.

The parameters were measured at different times (24, 48 and 72 hours) after incubation with VAQuFrF and Vincristine. Interleukin-6 is a major proliferative factor for the malignant plasma cells (multiple myeloma cells). Therefore this cytokines was measured parallel to the test substances.

Viscum album QuFrF (VAQuFrF) is an experimental drug that is not yet used in the treatment of tumour patients. For this reason it was necessary and important to compare with a well-known clinic-substance. Vincristine is used mainly in combination with other chemotherapeutic substances in the therapy of multiple myeloma.

#### **5.1 Key results**


The effect of VAQuFrF focuses on the inhibition of proliferation (**cytostatic effect**). VAQuFrF first inhibits the proliferation and then the cells die by apoptosis and/or necrosis.


The findings indicate that VAQuFrF extract could be a novel drug in the therapy of multiple myeloma.

#### **To assess the effective doses of Viscum album QuFrF extract and to transfer these doses to the in vivo situation.**

The Viscum album QuFrF (VAQuFrF) is an aqueous and unfermented extract of mistletoe plants growing in the oak tree. It contains 2 µg lectin and 10 µg viscotoxin in 10 mg/ml. It

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

**8. References** 

Supplement, 21-24.

47, 366-376.

243-249.

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 605

Chanen-Khan, AA. (2004) Bcl-2 antisense therapy in multiple myeloma. *Oncology*

Duong Van Huyen, JP., Sooryanarayana Delignat, S., Bloch, MF., Kacatchkine, MD., Kaveri,

DuVillard, L., Guiguet, M., Casasnovas RO., Caillot, D., Monnier-Zeller, V., et al. (1995)

E1 Alaoui, S., Lawry, J., Griffan, M. (1997) The cell cycle and induction of apoptosis in a

El-Sherbiny, YM., Cox, MC., Ismail, CA., Shamsuddin, AM., Vucenik, I. (2001) G0/G1 arrest

Gaillard, JP., Liautard, J., Klein, P.,Brochier, J. (1997) Major role of the soluble interleukin- 6,

Harmsma, M., Gromme, M., Ummelen, M., Dignef, W., Tusenius, KJ., et al. (2004)

Hata, H., Matsuzaki, H., Takeya, HM., Yoshida, M., Sonoki, T., et al. (1995) Expression of

Kovacs, E. (2003). How does interleukin-6 affect the membrane expressions of interleukin-6

Kovacs, E., Link, S., Toffol-Schmidt, U. (2006a). Cytostatic and cytocidal effects of mistletoe (Viscum album L.) Quercus extract Iscador. *Drug Research* 56, 467-473. Kovacs, E. (2006b) Multiple myeloma and B cell lymphoma. Investigation of IL-6, IL-6

Kovacs, E. (2010a*)* Interleukin-6 leads to interleukin-10 production in several human

Kovacs, E. (2010b) Investigation of the proliferation, apoptosis/necrosis and cell cycle

Leoncini, L., Vecchio, MTD., Megha, T., Barbini, P., Galieni, P., et al. (1993) Correlation

Lin, Ch-KE., Nguyen, TT., Morgan, TL., Mei, RL., Kaptein, JS., et al. (1998) Apoptosis may be

solid tumour chemotherapy. *J Neurol* 31, 195-207.

human myeloma cell lines. *Eur J Immunol* 27, 332-3340.

and apoptosis in cancer cells. *Int J Oncol* 125, 1521-1529.

*Anticancer Research* 21, 2393-2403.

disorders. *Blood* 86, 1939-1945.

*Biomed Pharmacother* 57, 489–94.

*The Scientific World Journal* 6, 888-898.

cells? *Leukemia Research* 34, 912-916.

lymphoma*. Am J Pathol* 142, 755-762.

or anti-IgM. *Experimental Cell Research* 244, 1-13.

*Journal* 10, 311-320.

SV. (2001) Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album QuFrF, a therapeutic preparation of mistletoe lectin. *Chemotherap*y

Diagnostic serum level of IL-6 in monoclonal gammopathies. *Brit J Haematol* 89,

hamster fibrosarcoma cell line treated with anti-cancer drugs: its importance to

and Sphase inhibition of human cancer cell lines by inositol hexaphosphate (IP6).

interleukin-6 receptor complex for the proliferation of interleukin-6-dependent

Differential effects of Viscum album extract IscadorR Qu on cell cycle progression

Fas/Apo-1 (CD95) and apoptosis in tumor cells from patients with plasma cell

receptor and gp130 and the proliferation of the human myeloma cell line OPM-2?

receptor antagonist (IL-6RA) and gp130 antagonist (gp130A) in an in vitro model.

multiple myeloma cell lines. Does interleukin-10 enhance the proliferation of these

phases in several human multiple myeloma cell lines. Comparison of Viscum album QuFrF extract with Vincristine in an in vitro model. *The Scientific World* 

between apoptotic and proliferative indices in malignant non-Hodgkin's

either suppressed or enhanced with strategic combinations of anti-neoplastic drugs

was tested in dose range of 10, 50, 100 µg/106 cells. 10 µg extract contains 0.002 µg lectin + 0.01 µg viscotoxin, 50 µg extract contains 0.01 µg lectin + 0.05 µg viscotoxin, 100 µg extract contains 0.02 µg lectin + 0.1 µg viscotoxin.

Dosage of Vincristine in the therapy for multiple myeloma: In combination with other chemotherapeutic agents as a part of the VAD regimen 0.4 mg/day intravenously (400 µg/day). In these experimental studies Vincristine was applied in dose range of 10, 50, 100 µg/106 cells. The effects of Vincristine on the proliferation and the apoptosis/necrosis were in each cell line without dose dependence. This means that these doses lay in a saturation range. It is planned to investigate the effects of Vincristine in a lower dose range.

The efficient dose range for VAQuFrF lies between 50 and 100 µg/106 cells (0.01µg lectin + 0.05 µg viscotoxin and 0.02 µg lectin + 0.1 µg viscotoxin). These date concern the cell lines LP-1, RPMI-8226 and COLO-677. For cell lines OPM-2, KMS-12-BM the dose range lies higher. In MOLP-8 cell line VAQuFrF inhibits the proliferation more effectively than Vincristine.

Our findings suggest that the in vivo effective (active) dose for VAQuFrF will be about 10- 20 times higher than that of Vincristine.

## **6. Future research**

1. **Viscum album** (VA) QuFrF extract contains two active components: mistletoe lectins (I, II, III) and viscotoxins**.** 

**Question**: Which component is responsible for the effects of this extract? Is this the **lectin(s)** or **viscotoxin** or **both?** Further study is planned to clarify this **question.** 

The non-fermented preparation from VAQuFrF extract contains 2 µg lectin and 10 µg viscotoxin in 10 mg/ml **(ratio between lectin and viscotoxin: 0.2).**

The **ratio between lectin and viscotoxin from the fermented preparations is 0.06** respectively **0.08.** The fermented preparations are used either alone or in combination with chemo /radiotherapy in the treatment of tumour patients. The extract presented in this study is an experimental drug that is not yet used in the treatment of tumour patients.


#### **7. Acknowledgements**

The measurements of the parameters were carried out in the laboratory of the Society of Cancer Research (Arlesheim, Switzerland). The idea of this study is based on the findings of the author. As principal investigator she wrote the study protocol and co-ordinated the study. The evaluation of the results, the writing and the completion of this manuscript were not supported from the Society of Cancer Research and from any foundation.
