**1.1 Multiple myeloma**

**Multiple myeloma** (MM) is a haematological disorder of malignant plasma cells. B lymphocytes start in the bone marrow and move to the lymph nodes.

When they are activated to secrete antibodies, they are known as plasma cells, which are crucial part of the immune system. Due to the fundamental nature of the system affected, multiple myeloma manifests systemic symptoms that make it difficult to diagnose. Multiple myeloma is characterised by slow proliferation of the tumour cells, mainly in the bone marrow, by production of large amounts of immunoglobulins and osteolytic lesions. Multiple myeloma is a generally incurable disease at present, but remissions may be induced with stem cells transplants, steroids, chemotherapy and treatment with vincristine + doxorubicin + dexamethasone or thalidomide + dexamethasone or bortezomib based regimens or lenalidomide. The different therapeutic modalities have different "target location".

#### **1.2 Epidemiology of multiple myeloma**

Multiple myeloma mainly affects older adults, but its causes and other risk factors are unknown. Yearly incidence is 3-6/100 000 worldwide, accounts for 1-2 % of all human cancer. Median survival is 50–55 months.

#### **1.3 The role of cytokines in the growth, progression and dissemination of multiple myeloma**

**Cytokines** are soluble proteins, peptides or glycoproteins that are released by cells. Cytokines can affect the cells via an autocrine and/or paracrine regulation mechanisms. In case of an autocrine regulation mechanism the endogenous produced cytokine affects the same type of cell. In case of a paracrine regulation mechanism the target cell is near to the

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

factor for these tumour cells (Kovacs, 2010a).

**1.4 Cytostatic effect and cytocidal effect** 

**1.5 Viscum album (VA) extract** 

treatment of tumour patients.

**1.6 Vincristine** 

**2. Aim** 

to the in vivo situation.

used in the treatment of tumour patients.

spindle formation (Harmsma et al., 2004).

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 589

Interleukin-10 enhances the survival and proliferation of B cells. IL-10 is a growth factor for myeloma cells (Kovacs, 2010a), enhances the proliferation of freshly explanted myeloma cells in a short-term bone marrow culture (Lu et al., 1995). Three out of seven human myeloma cell lines produce IL-10. Elevated IL-10 levels were detected in serum from about 50% of patients having multiple myeloma showing a relation to the clinical manifestation (Otsuki et al., 2000; 2002). Interleukin-6 leads to a marked production of Interleukin-10 in several human multiple myeloma cells. Interleukin-10 is an Interleukin-6 related growth

**Cytocidal effect**: It is known that there are two important pathways against tumours: To inhibit the tumour cell proliferation (**cytostatic effect**) and/or to induce the death of the

The apoptosis is a physiological process in the life of healthy cells, whereas necrosis is a pathological process for tumour cells. **Cytotoxicity** is the quality of being toxic to cells. There are a **direct** and an **indirect** (cell-mediated) **cytotoxicity.** In case of direct cytotoxicity the

Viscum album (VA) extract from **European mistletoe** plants has fermented and nonfermented preparations. Active components of VA extracts include mistletoe lectins (I, II, III) and viscotoxin, additionally aminoacids, polysaccharides and lipids. The fermented preparations are used either alone or in combination with chemo/radiotherapy in the

The Viscum album QuFrF (VAQuFrF) is an aqueous and unfermented extract of mistletoe plants growing in the oak tree. It contains 1 µg lectin and 6 µg viscotoxin in 5 mg/ml or 2 µg lectin and 10 µg viscotoxin in 10 mg/ml. The extract is an experimental drug that is not yet

Vincristine is a vinca alkaloid. As a chemotherapeutic agent is used mainly in combination with other chemotherapeutic substances in the therapy of multiple myeloma. Vincristine inhibits the proliferation of these tumour cells and as a CCS blocker (El Alaoui, 1997; Lin et al., 1998; Mastberger et al., 2000) arrests the cell cycle phase G2/M by blocking the mitotic

**2.1** Comparison of the effects of Viscum album QuFrF extract with those of Vincristine.

**2.3** To assess the effective doses of Viscum album QuFrF extract and to transfer these doses

**2.2** Mode of action of Viscum album QuFrF extract and Vincristine.

tumour cells **(cytocidal effect**). **Cytocidal effect**: apoptosis or necrosis.

cells are treated with cytotoxic compounds leading to necrosis.

cytokine produced cell. Cytokine binds to a specific receptor and causes a change in function or in development (differentiation) of the target cell. In both cases, i.e. autocrine and paracrine regulation mechanisms the expression of the membrane receptor is altered.

**1.3.1 Interleukins a**re a group of cytokines which are produced by a wide variety of body cells. The majority of interleukins are synthesized by helper CD4+ T lymphocytes, monocytes/macrophages and by endothelial cells. If the produced cytokine is released, then it is measurable in the supernatant, if not then the cytokine is measurable only intracellular.

**Interleukin-6 (**IL-6) originally defined as a B cell differentiation factor is produced by different cell types and certain tumour cells. Interleukin-6 acts as a pro-inflammatory and an anti-inflammatory cytokine. As a pro-inflammatory cytokine regulates inflammatory reactions either directly or indirectly. As an anti-inflammatory cytokine Interleukin-6 reduces the inflammatory reactions. IL-6 exits in three molecular weights, i.e. 21-30 kDa, 150-200 or 450 kDa. The biological activity of IL-6 depends on binding to its specific receptors. These membrane receptors composed the glycoprotein gp80 Interleukin-6 receptor alpha (IL-6R, also called CD126) and a signal-transducing component gp130 (also called CD 130). The complex IL-6+IL-6R+gp130 initiate a signal transduction cascade through JAKs (Janus kinases) and STATs (Signal Transducer-Activator of Transcription). The membrane receptors are released from the cells as soluble receptor proteins (sIL-6R and sgp130). As agonist, sIL-6R enhances the biological activity of IL-6 and sgp130 is an antagonist against the complex IL-6+sIL-6R.

Interleukin-6 is a major proliferative factor for the malignant plasma cells (multiple myeloma cells). This cytokine produces by the plasma cells and it affects the cells by an autocrine regulation mechanism with an additional paracrine signalling. IL-6 in a concentration of 2pg/ml can induce 50% proliferation in myeloma cells.

The multiple myeloma cells can be classified into three groups depending on exogenous IL-6: (a) both proliferation and survival of the cells are dependent on IL-6, (b) only proliferation of the myeloma cells is affected by IL-6, (c) the cells are dependent on IL-6 only for survival, but not for proliferation. However there are also some cell lines that are independent of IL-6 both for survival and proliferation. The serum values of IL-6 in 35% or in 97% or in 42% of multiple myeloma patients were significantly higher than in healthy persons (Nachbour et al., 1991; DuVillard et al., 1995; Wierzbowska et al., 1999).

Because about 70% of the secreted IL-6 forms a complex with sIL-6R (Gaillard et al., 1987), the amount of the free IL-6 in serum is low. Therefore the serum level of the sIL-6R is an important parameter in the evaluation and in the progression of multiple myeloma (Papadaki et al., 1997; Wierzbowska et al., 1999).

**Interleukin-10** (IL-10) is known as a human cytokine synthesis inhibitory factor (CSIF). It produces by Thelper2 cells, monocytes/macrophages, by B lymphocytes and some tumour cells. Interleukin-10 has (1) immunosuppressive effect and (2) immunostimulatory effect. It down-regulates the expression of Thelper1 cytokines, MHC class II antigens and costimulatory molecules on macrophages. Interleukin-10 is a pleiotropic cytokine which increases Bcl-2 levels and protects cells from steroid or doxorubicin-induced apoptosis.

How might the presence of IL-10 contribute to a poor prognosis for some cancer? One possibility: Interleukin-10 is a growth factor for tumour cells. Second possibility: Interleukin-10 suppresses the anti-tumour immune responses.

Interleukin-10 enhances the survival and proliferation of B cells. IL-10 is a growth factor for myeloma cells (Kovacs, 2010a), enhances the proliferation of freshly explanted myeloma cells in a short-term bone marrow culture (Lu et al., 1995). Three out of seven human myeloma cell lines produce IL-10. Elevated IL-10 levels were detected in serum from about 50% of patients having multiple myeloma showing a relation to the clinical manifestation (Otsuki et al., 2000; 2002). Interleukin-6 leads to a marked production of Interleukin-10 in several human multiple myeloma cells. Interleukin-10 is an Interleukin-6 related growth factor for these tumour cells (Kovacs, 2010a).
