**4.1 Production of Interleukin-6 in supernatant of human multiple myeloma cells**

Objectives: (a) spontaneous production, (b) production after treatment with VAQuFrF or Vincristine.

**Table 1** presents the values of IL-6 in myeloma cell line MOLP-8, LP-1, RPMI-8226, OPM-2, COLO-677. None of the five multiple myeloma cell lines produced Interleukin-6 spontaneously. This means that all the investigated cell lines are IL-6 independent or have **autocrine/paracrine** regulation mechanisms. In case of an **autocrine** regulation mechanism the cytokine is produced endogenously and affects its membrane receptor directly. In case **of paracrine** regulation mechanism the exogenous cytokine also affects the membrane receptor. In two cell lines (RPMI-8226 and OPM-2) exogenous IL-6 led to a high expression of membrane IL-6R and enhanced levels of sIL-6R in the supernatant (Kovacs, 2003; and results are not shown) indicating a paracrine regulation mechanism.


Maeasurements at 24 and 48 hours after tretment with VAQufrF extractor Vincristine. Dose=50 µg/106 cells in both cases.ND = not detectable.

Table 1. Production of Interleukin-6 in human multiple myeloma cell lines.

Treatment with VAQuFrF extract (dose: 50 µg/106 cells) and with Vincristine (dose: 50 µg/106 cells) also did not lead to IL-6 production in the five multiple myeloma cell lines. These results confirm the findings of previous studies (Kovacs et al., 2006; Kovacs, 2010b).

#### **4.2 Production of interleukin-10 in supernatant of human multiple myeloma cells**

Objectives: (a) spontaneous production, (b) production after treatment with IL-6 (dose: 5ng/106 cells), (c) production after treatment with VAQuFrF or Vincristine (dose: 50 µg/106 cells for both substances), (d) after treatment with IL-6+VAQuFrF or IL-6+Vincristine (doses: 5 ng/106 cells +50 µg/106 cells in each case). For the combined treatment IL-6 was added 2 hours before the test substances.

**Table 2** presents the production of Interleukin-10 in five human multiple myeloma cell lines. Spontaneous IL-10 production was found in 4/5 cell lines: MOLP-8, LP-1, RPMI-8226, COLO-677, however the cell lines MOLP-8 and COLO-677 secreted IL-10 not every time confirming the findings of previous study (Kovacs, 2010a). IL-6 led to a marked increase of

Objectives: (a) spontaneous production, (b) production after treatment with VAQuFrF or

**Table 1** presents the values of IL-6 in myeloma cell line MOLP-8, LP-1, RPMI-8226, OPM-2, COLO-677. None of the five multiple myeloma cell lines produced Interleukin-6 spontaneously. This means that all the investigated cell lines are IL-6 independent or have **autocrine/paracrine** regulation mechanisms. In case of an **autocrine** regulation mechanism the cytokine is produced endogenously and affects its membrane receptor directly. In case **of paracrine** regulation mechanism the exogenous cytokine also affects the membrane receptor. In two cell lines (RPMI-8226 and OPM-2) exogenous IL-6 led to a high expression of membrane IL-6R and enhanced levels of sIL-6R in the supernatant (Kovacs, 2003; and

Maeasurements at 24 and 48 hours after tretment with VAQufrF extractor Vincristine. Dose=50 µg/106

Treatment with VAQuFrF extract (dose: 50 µg/106 cells) and with Vincristine (dose: 50 µg/106 cells) also did not lead to IL-6 production in the five multiple myeloma cell lines. These results confirm the findings of previous studies (Kovacs et al., 2006; Kovacs, 2010b).

Objectives: (a) spontaneous production, (b) production after treatment with IL-6 (dose: 5ng/106 cells), (c) production after treatment with VAQuFrF or Vincristine (dose: 50 µg/106 cells for both substances), (d) after treatment with IL-6+VAQuFrF or IL-6+Vincristine (doses: 5 ng/106 cells +50 µg/106 cells in each case). For the combined treatment IL-6 was added 2

**Table 2** presents the production of Interleukin-10 in five human multiple myeloma cell lines. Spontaneous IL-10 production was found in 4/5 cell lines: MOLP-8, LP-1, RPMI-8226, COLO-677, however the cell lines MOLP-8 and COLO-677 secreted IL-10 not every time confirming the findings of previous study (Kovacs, 2010a). IL-6 led to a marked increase of

**4.2 Production of interleukin-10 in supernatant of human multiple myeloma cells** 

Table 1. Production of Interleukin-6 in human multiple myeloma cell lines.

**4.1 Production of Interleukin-6 in supernatant of human multiple myeloma cells** 

results are not shown) indicating a paracrine regulation mechanism.

cells in both cases.ND = not detectable.

hours before the test substances.

**4. Results** 

Vincristine.

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 nondetectable amounts.

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 Vincristine.


Maesurements at 24 and 48 hors after tretment. IL-6:5ng/106 cells.VAQuFrF and Vincristine: 50 µg/106 cells. Range of 4 independent measurements. ND=not detectable


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.

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

MOLP-8, COLO-677 and KMS-12-BM additional experiments are planned.

**apoptosis and necrosis in multiple myeloma cells (cytocidal effect)** 

measured at 24, 48 and 72 hours after incubation with the test substances.

assessed versus untreated samples (100%).

between the substances in any dose.

were big differences between the tumour cell lines.

(P<0.05 and P<0.01). Vincristine had the same effect as VAQuFrF.

VAQuFrF.

bone marrow.

**4.4.1 MOLP-8** 

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 595

The surface expressions of IL-6R in untreated cells of all three cell lines were in the similar range (results are not shown). Exogenous IL-6 increased the membrane expression its receptor significantly (P<0.05). VAQuFrF reduced the membrane expression markedly in LP-1 and RPMI-8226 (P<0.05), it had no effect in OPM-2. With IL-6+VAQuFrF the values were lower than with IL-6 (P<0.05), but higher than after treatment with

In cell lines MOLP-8, COLO-677 and KMS-12-BM exogenous IL-6 led to down-regulation of its receptor, signalling the possible process of endocytosis (results are not shown). It is interesting that all three cell lines in which IL-6 upregulated its membrane receptor sourced from blood. To investigate of the membrane expression of IL-6 receptor in the cell lines

**4.4 Inhibition of proliferation of multiple myeloma cells (cytostatic effect). Induction of** 

**Figure 1** and **Figure 2** present the mean values of the proliferation and those of apoptosis/necrosis in six human multiple myeloma cell lines treated with IL-6 or VAQuFrF or Vincristine. The cell lines MOLP-8, LP-1, RPMI-8226, OPM-2 sourced from blood, COLO-677 which is a derivative of RPMI-8226 from lymph node, KMS-12-BM from

To measure the proliferation the following doses were applied (1) IL-6: 0.5 ng/105 cells, (2) VAQuFrF or Vincristine: 1, 5, 10 µg/105 cells. To measure apoptosis/ necrosis (1) IL-6: 5 ng/106 cells, (2) VAQuFrF or Vincristine; 10, 50, 100 µg/106 cells. The parameters were

Proliferation: The values of the treated samples are expressed as percentages of the untreated samples and are the average of four independent experiments. Significance was

Apoptosis/necrosis: The values are expressed as percentage of total cell numbers and are the average of four independent experiments. In the untreated samples the percentage of apoptotic cells lay in the range of 5-38%, that of necrotic cells 10-35% during 72 hours. There

Proliferation: IL-6 increased the proliferation on average up to 130-155%. Comparison of VAQuFrF with Vincristine: 24 and 48 hours after incubation VAQuFrF at the dose of 5 and 10 µg/105 cells was more effective than Vincristine. 72 hours after there was no difference

Apoptosis/necrosis: In the untreated tumour cells the values of apoptosis lay either in the range of necrosis or above them. IL-6 treatment did not impair either the apoptosis or necrosis. To measure the effects of the two substances on the apoptosis/necrosis we applied ten times less doses. VAQuFrF increased the apoptosis and necrosis at 5 and 10 µg/106 cells

**Table 2/A.** presents the range of cell viability without treatment and after treatment with the test substances: L-6, VAQuFrF extract and Vincristine. The viability of the untreated MM cells was different: LP-1>RPMI-8226>COLO-677>MOLP-8>OPM-2. The both test substances impaired the viability to different degrees*.*

IL-6 does not alter the viability, confirming the findings of previous investigations (Kovacs, 2006b, 2010a). It was reported that IL-6 enhances survival of the myeloma cells because it inhibits apoptosis of induction of the anti-Fas (Nordan & Potter, 1986; Hata et al., 1995).

**Summarised:** The results indicate that the effect of the both test substances on the IL-10 production is due to their apoptotic/necrotic effects. It is possible that VAQuFrF and Vincristine could also impair the membrane expression of IL-10 receptor. To explain this hypothesis further experiments are necessary.

#### **4.3 The effect of Interleukin-6, VAQuFrF and IL-6+VAQuFrF on the membrane expression of Interleukin-6 receptor in human multiple myeloma cells**

Objectives: (a) in untreated cells, (b) after treatment with IL-6 (dose: 5 ng/106cells), (c) after treatment with VAQuFrF (dose: 50 µg/106 cells), (d) after treatment with IL-6+ VAQuFrF(dose: 5 ng/106 cells+50 µg/106 cells). For the combined treatment IL-6 was added 2 hours before the test substance. For the expression of the membrane IL-6R the signal intensity (geometric mean of the fluorescence intensity x counts) was used as parameter. This parameter was measured at 24 and 48 hours after incubation. The signal intensity of the treated samples, expressed in percentage was compared with that of untreated samples, which were taken as 100%.

Table 3 presents the mean values of the membrane expression of Interleukin -6 receptor in the cell lines LP-1, RPMI-8226 and OPM-2.


Measurements at 24 h after treatment. Measurements at 48 h after treatment.

The mean values of three independent measurementa are expressed in percentage of untreated samples (100). a=p<0.05 vs.untreated samples, b=p<0.05 vs. with IL-6 treated samples.

Table 3. Membrane expression of Interleukin-6 receptor in human multiple myeloma cell lines.

The surface expressions of IL-6R in untreated cells of all three cell lines were in the similar range (results are not shown). Exogenous IL-6 increased the membrane expression its receptor significantly (P<0.05). VAQuFrF reduced the membrane expression markedly in LP-1 and RPMI-8226 (P<0.05), it had no effect in OPM-2. With IL-6+VAQuFrF the values were lower than with IL-6 (P<0.05), but higher than after treatment with VAQuFrF.

In cell lines MOLP-8, COLO-677 and KMS-12-BM exogenous IL-6 led to down-regulation of its receptor, signalling the possible process of endocytosis (results are not shown). It is interesting that all three cell lines in which IL-6 upregulated its membrane receptor sourced from blood. To investigate of the membrane expression of IL-6 receptor in the cell lines MOLP-8, COLO-677 and KMS-12-BM additional experiments are planned.

#### **4.4 Inhibition of proliferation of multiple myeloma cells (cytostatic effect). Induction of apoptosis and necrosis in multiple myeloma cells (cytocidal effect)**

**Figure 1** and **Figure 2** present the mean values of the proliferation and those of apoptosis/necrosis in six human multiple myeloma cell lines treated with IL-6 or VAQuFrF or Vincristine. The cell lines MOLP-8, LP-1, RPMI-8226, OPM-2 sourced from blood, COLO-677 which is a derivative of RPMI-8226 from lymph node, KMS-12-BM from bone marrow.

To measure the proliferation the following doses were applied (1) IL-6: 0.5 ng/105 cells, (2) VAQuFrF or Vincristine: 1, 5, 10 µg/105 cells. To measure apoptosis/ necrosis (1) IL-6: 5 ng/106 cells, (2) VAQuFrF or Vincristine; 10, 50, 100 µg/106 cells. The parameters were measured at 24, 48 and 72 hours after incubation with the test substances.

Proliferation: The values of the treated samples are expressed as percentages of the untreated samples and are the average of four independent experiments. Significance was assessed versus untreated samples (100%).

Apoptosis/necrosis: The values are expressed as percentage of total cell numbers and are the average of four independent experiments. In the untreated samples the percentage of apoptotic cells lay in the range of 5-38%, that of necrotic cells 10-35% during 72 hours. There were big differences between the tumour cell lines.

#### **4.4.1 MOLP-8**

594 Pharmacology

**Table 2/A.** presents the range of cell viability without treatment and after treatment with the test substances: L-6, VAQuFrF extract and Vincristine. The viability of the untreated MM cells was different: LP-1>RPMI-8226>COLO-677>MOLP-8>OPM-2. The both test substances

IL-6 does not alter the viability, confirming the findings of previous investigations (Kovacs, 2006b, 2010a). It was reported that IL-6 enhances survival of the myeloma cells because it inhibits apoptosis of induction of the anti-Fas (Nordan & Potter, 1986; Hata et al., 1995).

**Summarised:** The results indicate that the effect of the both test substances on the IL-10 production is due to their apoptotic/necrotic effects. It is possible that VAQuFrF and Vincristine could also impair the membrane expression of IL-10 receptor. To explain this

Objectives: (a) in untreated cells, (b) after treatment with IL-6 (dose: 5 ng/106cells), (c) after treatment with VAQuFrF (dose: 50 µg/106 cells), (d) after treatment with IL-6+ VAQuFrF(dose: 5 ng/106 cells+50 µg/106 cells). For the combined treatment IL-6 was added 2 hours before the test substance. For the expression of the membrane IL-6R the signal intensity (geometric mean of the fluorescence intensity x counts) was used as parameter. This parameter was measured at 24 and 48 hours after incubation. The signal intensity of the treated samples, expressed in percentage was compared with that of untreated samples,

Table 3 presents the mean values of the membrane expression of Interleukin -6 receptor in

Measurements at 24 h after treatment. Measurements at 48 h after treatment. The mean values of three independent measurementa are expressed in percentage of untreated samples

Table 3. Membrane expression of Interleukin-6 receptor in human multiple myeloma cell

(100). a=p<0.05 vs.untreated samples, b=p<0.05 vs. with IL-6 treated samples.

**4.3 The effect of Interleukin-6, VAQuFrF and IL-6+VAQuFrF on the membrane** 

**expression of Interleukin-6 receptor in human multiple myeloma cells** 

impaired the viability to different degrees*.*

hypothesis further experiments are necessary.

which were taken as 100%.

lines.

the cell lines LP-1, RPMI-8226 and OPM-2.

Proliferation: IL-6 increased the proliferation on average up to 130-155%. Comparison of VAQuFrF with Vincristine: 24 and 48 hours after incubation VAQuFrF at the dose of 5 and 10 µg/105 cells was more effective than Vincristine. 72 hours after there was no difference between the substances in any dose.

Apoptosis/necrosis: In the untreated tumour cells the values of apoptosis lay either in the range of necrosis or above them. IL-6 treatment did not impair either the apoptosis or necrosis. To measure the effects of the two substances on the apoptosis/necrosis we applied ten times less doses. VAQuFrF increased the apoptosis and necrosis at 5 and 10 µg/106 cells (P<0.05 and P<0.01). Vincristine had the same effect as VAQuFrF.

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

**4.4.2 LP-1** 

(P<0.01) than Vincristine.

**4.4.3 RPMI-8226** 

(P<0.05 and P<0.01).

VAQuFrF was ineffective.

**4.4.5 COLO-677** 

**4.4.4 OPM-2** 

cells.

each dose after 48 and 72 hours (P<0.05 and P<0.01).

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

and 1 µg/105 cells) VAQuFrF had the same effect as Vincristine.

Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters 597

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

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

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

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

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

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

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.

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

Vincritine was the same at each investigated time point.

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

that higher doses increase the effect of VAQuFrF (results are not presented).

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).
