**4.4.2 LP-1**

596 Pharmacology

Fig. 1. The effects of IL-6, VAQuFrF extract and Vincristine on the proliferation and on apoptosis/necrosis in human multiple myeloma cell lines MOLP-8, LP-1 and RPMI-8226. 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: With IL-6 the proliferation rate lay on average between 128-162% during 72 hours. VAQuFrF at the dose of 10 µg/105 cells inhibited the proliferation more effectively (P<0.01) than Vincristine.

Apoptosis/necrosis: In the untreated cell the values of apoptosis lay in the range of necrosis. There was no difference between the values of untreated and with IL-6 treated cells. VAQuFrF did not greatly alter the apoptosis during the investigation time. There was a necrotic effect with a dose dependence from 50 up to 100 µg/106 cells (P<0.05). Vincristine increased the number of apoptotic cells and that of necrotic cells (P<0.05), however without dose dependence. The number of necrotic cells was higher than that of apoptotic cells at each dose after 48 and 72 hours (P<0.05 and P<0.01).

## **4.4.3 RPMI-8226**

Proliferation: IL-6 increased the proliferation on average up to 105-141%.

The test substances inhibited the proliferation: After 24 and 48 hours VAQuFrF in dose of 10 µg/105 cells was more effective than Vincristine (P<0.01). In lower doses (5 µg/105 cells and 1 µg/105 cells) VAQuFrF had the same effect as Vincristine.

Apoptosis/necrosis: The values of apoptosis in untreated cells lay below the necrosis. There were no differences between the values of cells treated with IL-6 and that those of untreated cells.

VAQuFrF and Vincristine did not alter the apoptosis. At 72 hours after treatment with both substances he numbers of necrotic cells was higher than those of apoptotic cells at each dose (P<0.05 and P<0.01).

## **4.4.4 OPM-2**

Proliferation: IL-6 increased the proliferation on average between 110-130%. Comparison of VAQuFrF with Vincristine: The inhibitory effect of VAQuFrF was weaker than that of Vincristine at each dose and at investigated time point. Additional investigation indicate that higher doses increase the effect of VAQuFrF (results are not presented).

Apoptosis/necrosis: In the untreated cells the values of apoptosis lay in the range of necrosis. IL-6 did not impair either the apoptosis or necrosis of the cells. None of the test substances altered the apoptosis. Vincristine increased markedly the number of necrotic cells between 10 and 100 µg/106 cells without dose dependence after 72 hours of treatment. VAQuFrF was ineffective.

#### **4.4.5 COLO-677**

Proliferation: With IL-6 the values of proliferation lay on average between 110-115%.

The inhibitory effect of VAQuFrF was weaker than that of Vincristine in doses of 1 and 5 µg/105 cells. At the dose of 10 µg/105 cells the anti-proliferative effects of VAQuFrF and Vincritine was the same at each investigated time point.

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

dose-dependent.

**4.4.6 KMS-12-BM** 

each investigated time point.

without dose dependence.

dose and at investigated time point.

OPM-2 (results are not shown).

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 599

Apoptosis/necrosis: In the untreated tumour cells the values of apoptosis lay either in the range of necrosis or below them. There was no alteration after treatment with IL-6. VAQuFrF did not alter the apoptosis. There was a necrotic effect with a dose dependence (from 50 up to 100 µg/106 cells) (P<0.05). Vincristine increased the number of apoptotic cells (P<0.05). The number of necrotic cells was higher than that of apoptotic at each dose and at each time point (P<0.05 and P<0.01). The apoptotic /necrotic effects of Vincristine were not

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

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

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

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

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

**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

inhibition of proliferation and apoptosis in lymphoma cells (Leoncini, et al., 1993).

decreases the levels of Bcl-2 in B and T lymphocytes (Duong Van Huyen et al., 2001).

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).

Apoptosis/necrosis: In the untreated tumour cells the values of apoptosis lay either in the range of necrosis or below them. There was no alteration after treatment with IL-6. VAQuFrF did not alter the apoptosis. There was a necrotic effect with a dose dependence (from 50 up to 100 µg/106 cells) (P<0.05). Vincristine increased the number of apoptotic cells (P<0.05). The number of necrotic cells was higher than that of apoptotic at each dose and at each time point (P<0.05 and P<0.01). The apoptotic /necrotic effects of Vincristine were not dose-dependent.
