**4.4.6 KMS-12-BM**

598 Pharmacology

Fig. 2. The effects of IL-6, VAQuFrF extract and Vincristine on the proliferation and on the apoptosis/necrosis in human multiple myeloma cell lines OPM-2, COLO-677 and KMS-12- BM. The mean values of four independent experiments are expressed as percentage of untreated samples (100%). Proliferation=105 cells. Apoptosis/necrosis=106 cells. ●- - -● apoptotic cells, ●––● necrotic cells. +P<0.05, \*P<0.01 compared with untreated samples

(Mann-Whitney U-test).

Proliferation: IL-6 increased the proliferation on average between 108 and 135%. VAQuFrF inhibited the proliferation only in dose of 10 µg/105 cells after 48 and 72 hours. Vincristine inhibited the proliferation markedly however without dose dependence at each dose and at each investigated time point.

Apoptosis/necrosis: The values of apoptosis in untreated cells lay above the values of necrosis. IL-6 did not alter the apoptosis and necrosis. VAQuFrF did not impair either the apoptosis or the necrosis in this cell line. Vinristine was effective in KMS-12-BM: The number of apoptotic/necrotic cells was significantly higher (P<0.01) at each time point, but without dose dependence.

It was reported that that inhibition of cell proliferation is a stronger prognostic indicator than the apoptosis (Stokke et al., 1998). There is a quantitative correlation between the inhibition of proliferation and apoptosis in lymphoma cells (Leoncini, et al., 1993).

Chemotherapeutic agents influence apoptosis through a mitochondrial pathway (Oancea, et al., 2004). Multiple myeloma cells overexpress Bcl-2, a mitochondrial membrane protein which suppresses apoptosis (Chanen-Khan, 2004; Tsujimoto & Shimizu, 2007). VAQuFrF decreases the levels of Bcl-2 in B and T lymphocytes (Duong Van Huyen et al., 2001).

Summarised: In this study the apoptotic/necrotic effect of Vincristine was more marked than its proliferative effect in all cell lines. There was no dose dependence between 10, 50 or 100 µg/106 cells/ml in both parameters. It is possible that Vincristine impairs the proliferation and apoptosis/necrosis with dose dependence only in a lower dose range.

VAQuFrF first inhibits the proliferation and then the cells die by apoptosis and/or necrosis in the MM cell lines LP-1, RPMI-8226 and COLO-677, confirming the findings with RPMI-8226 presented in a previous study (Kovacs et al., 2006a). The inhibitory effect of VAQuFrF was markedly weaker than that of Vincristine in cell lines OPM-2 and KMS-12-BM at each dose and at investigated time point.

## **4.5 The effect of VAQuFrf on the proliferation of cells with high proliferation rate**

The effect of VAQuFrF with doses of 5 and 10µg/105 cells was investigated in cell line RPMI-8226 with high proliferation rate, which remained unaltered during 2-3 days. VAQuFrF was more effective in cells having high proliferation rates than in those with low proliferation rates (Kovacs et al., 2006a). Recently the same findings were observed in cell lines LP-1 and OPM-2 (results are not shown).

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

**4.7 Investigation of cell cycle phases in human multiple myeloma** 

accumulation of the tumour cells.

was carried out 24, 48 and 72 hours after treatment.

number (100%). Range of four independent experiments.

each time point.

at 48 and 72 hours after incubation.

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 601

Cell division consists of mitosis (M) and interphase, which divides into phases G1, G2, and S. Non dividing cells are in the stable resting phase, called the G0 phase. The blockade in the cell division leads to an arrest in the different cycle phases. This arrest appears as an

The following cell lines: were investigated: MOLP-8, LP-1, RPMI-8226, OPM-2, U-266, COLO-677, KMS-12-BM. The myeloma cells were treated (a) with IL-6 (dose: 5 ng/106 cells) (b) with VAQuFrF and with Vincristine (doses: 10, 50, 100 µg/106 cells). The investigation

**Table 4** presents the range of the values of untreated cells in different cell cycle phases in percentage in the total viable cell number (100%) 24 hours after treatment. **Table 5** presents the mean values resp. the accumulation of treated cells 24 hours after treatment. For the cell lines RPMI-8226, OPM-2 and U-266 the presented values signalize the effects 48 and 72 hours after treatment with VAQuFrF or Vincristine. For a significant increase or decrease, the percentage

of the cell number of treated samples was compared with those of untreated samples.

Measurements at 24 hour after incubation. Values are expressed in percentage of total viable cell

*Phases GO/G1:* IL-6 did not affect this cell cycle phase. VAQuFrF led to an accumulation of cells in cell line MOLP-8, LP-1 and in KMS-12-BM (p < 0.05 and p < 0.01). Vincristine had effect in the cell lines MOLP-8 and LP-1 (p < 0.05). The both substances were effective at

*Phase S:* With IL-6 the number of cells was increased markedly in all cell lines during 72 hours except with OPM-2 and KMS-12-BM. In these cell lines there was no effect at 72 hours. VAQuFrF increased the cell number (p < 0.01) in RPMI-8226 and U-266 (p<0.05), Vincristine in RPMI-8226, OPM-2 and U-266 (p < 0.01 and p < 0.05). Both substances were effective only

Table 4. Values of untreated human multiple myeloma cells in different cell cycle phases.

#### **4.6 The effect of combined treatment with Interleukin-6+VAQUFrF on the proliferation in human multiple myeloma cells**

The cell lines MOLP-8, LP-1, RPMI-8226 and COLO-677 were treated with Interleukin-6+VAQuFrF for 24, 48 and 72 hours. To measure the proliferation the following doses were used: 0.5 ng/105 cells + 1, 5, 10 µg/105 cells. For the combined treatment IL-6 was added to the cell cultures 2 hours before VAQuFrF. For comparison the cell lines were treated only with IL-6 or only with VAQuFrF.

**Figure 3** presents the range of values expressed in percentage compared with untreated samples (100%). As expected, VAQuFrF inhibited the spontaneous proliferation markedly in all cell lines. The effect was dose-dependent. IL-6 led to enhanced proliferation in each case. We expected that with the combined treatment the values will be lower than after single treatment of IL-6, but higher than after single treatment of VAQuFrF. This situation has been found only in cell lines MOLP-8 and LP-1 for dose 1µg/105 cells. It is suggested that the 2 hours pre-treatment with IL-6 is too short.

Fig. 3. The effect of combined treatment with IL-6 + VAQuFrF on the proliferation in human multiple myeloma cells. IL-6=0.5 ng/105 cells.

#### **4.7 Investigation of cell cycle phases in human multiple myeloma**

600 Pharmacology

**4.6 The effect of combined treatment with Interleukin-6+VAQUFrF on the proliferation** 

The cell lines MOLP-8, LP-1, RPMI-8226 and COLO-677 were treated with Interleukin-6+VAQuFrF for 24, 48 and 72 hours. To measure the proliferation the following doses were used: 0.5 ng/105 cells + 1, 5, 10 µg/105 cells. For the combined treatment IL-6 was added to the cell cultures 2 hours before VAQuFrF. For comparison the cell lines were treated only

**Figure 3** presents the range of values expressed in percentage compared with untreated samples (100%). As expected, VAQuFrF inhibited the spontaneous proliferation markedly in all cell lines. The effect was dose-dependent. IL-6 led to enhanced proliferation in each case. We expected that with the combined treatment the values will be lower than after single treatment of IL-6, but higher than after single treatment of VAQuFrF. This situation has been found only in cell lines MOLP-8 and LP-1 for dose 1µg/105 cells. It is suggested that the 2

Fig. 3. The effect of combined treatment with IL-6 + VAQuFrF on the proliferation in human

**in human multiple myeloma cells** 

with IL-6 or only with VAQuFrF.

hours pre-treatment with IL-6 is too short.

multiple myeloma cells. IL-6=0.5 ng/105 cells.

Cell division consists of mitosis (M) and interphase, which divides into phases G1, G2, and S. Non dividing cells are in the stable resting phase, called the G0 phase. The blockade in the cell division leads to an arrest in the different cycle phases. This arrest appears as an accumulation of the tumour cells.

The following cell lines: were investigated: MOLP-8, LP-1, RPMI-8226, OPM-2, U-266, COLO-677, KMS-12-BM. The myeloma cells were treated (a) with IL-6 (dose: 5 ng/106 cells) (b) with VAQuFrF and with Vincristine (doses: 10, 50, 100 µg/106 cells). The investigation was carried out 24, 48 and 72 hours after treatment.

**Table 4** presents the range of the values of untreated cells in different cell cycle phases in percentage in the total viable cell number (100%) 24 hours after treatment. **Table 5** presents the mean values resp. the accumulation of treated cells 24 hours after treatment. For the cell lines RPMI-8226, OPM-2 and U-266 the presented values signalize the effects 48 and 72 hours after treatment with VAQuFrF or Vincristine. For a significant increase or decrease, the percentage of the cell number of treated samples was compared with those of untreated samples.


Measurements at 24 hour after incubation. Values are expressed in percentage of total viable cell number (100%). Range of four independent experiments.

Table 4. Values of untreated human multiple myeloma cells in different cell cycle phases.

*Phases GO/G1:* IL-6 did not affect this cell cycle phase. VAQuFrF led to an accumulation of cells in cell line MOLP-8, LP-1 and in KMS-12-BM (p < 0.05 and p < 0.01). Vincristine had effect in the cell lines MOLP-8 and LP-1 (p < 0.05). The both substances were effective at each time point.

*Phase S:* With IL-6 the number of cells was increased markedly in all cell lines during 72 hours except with OPM-2 and KMS-12-BM. In these cell lines there was no effect at 72 hours. VAQuFrF increased the cell number (p < 0.01) in RPMI-8226 and U-266 (p<0.05), Vincristine in RPMI-8226, OPM-2 and U-266 (p < 0.01 and p < 0.05). Both substances were effective only at 48 and 72 hours after incubation.

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

IL-10 production, (c) the proliferation, (d) the apoptosis/necrosis.

chemotherapeutic substances in the therapy of multiple myeloma.

**5. Summary and conclusion** 

test substances.

**5.1 Key results** 

lines.

necrosis.

**to the in vivo situation.** 

myeloma.

apoptosis/ necrosis.

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 603

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

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

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

a. Interleukin-6 leads to a markedly increased IL-10 production. Interleukin--6 upregulates markedly the expression of its membrane receptor (IL-6R). IL-6 increases the proliferation and it is effective in the S cell cycle phase. IL-6 does not affect the

b. Neither VAQuFrF nor Vincristine produce IL-6 or lead to an enhanced IL-10 production in any cell line. VAQuFrF and Vincristine inhibits the spontaneous IL-10 production. Both substances **counteract** the increased IL-10 production induced by IL-6. The effects of the two substances are comparable. The results indicate that the effect of the both test

d. The **cytocidal effect** of Vincristine is more marked than its **cytostatic** effect in all cell

e. VAQuFrF inhibits the proliferation in cells with **high proliferation** rate **more** 

f. **Cell cycle phases:** VAQuFrF extract has the same effect as Vincristine in five out of

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

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

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 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

substances on the IL-10 production is due to their apoptotic/necrotic effects. c. VAQuFrF **inhibits** the membrane expression of IL-6 receptor. VAQuFrF **counteracts** the

enhanced membrane expression of this receptor induced by IL-6.

**effectively** than in those with **low growth** rate.

seven tumour cell lines, however in a higher dose range.


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.

*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 hours after treatment in some cell lines.

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 a different sensitivity to Vincristine or VAQuFrF.

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 cell proliferation.
