The Effects of Viscum album (Mistletoe) QuFrF Extract and Vincristine in Human Multiple Myeloma Cell Lines – A Comparative Experimental Study Using Several and Different Parameters

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”.


Introduction 1.1 Multiple myeloma
Multiple myeloma (MM) is a haematological disorder o f m a l i g n a n t p l a s m a c e l l s .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".

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.
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.
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).

Cytostatic effect and cytocidal effect
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 tumour cells (cytocidal effect).Cytocidal effect: apoptosis or necrosis.
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 cells are treated with cytotoxic compounds leading to necrosis.

Viscum album (VA) extract
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 treatment of tumour patients.
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 used in the treatment of tumour patients.

Vincristine
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 spindle formation (Harmsma et al., 2004).

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

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

2.3
To assess the effective doses of Viscum album QuFrF extract and to transfer these doses to the in vivo situation.

Measurement of viability
The viabilities of the cultivated tumour cells were determined by 7-aminoactinomycin D (7-AAD), to exclude the non-viable cells in flow cytometric assays.The values are given in %.

Measurement of cytokine production
The IL-6 production or IL-10 production in the supernatant of the cultured cells was determined by chemiluminescent immunometric assay.The lowest detectable level was 2 pg/ml or 5 pg/ml.

Measurement of membrane expression of IL-6 receptor
For immunofluorescence staining 3x10 5 cells/100 l were incubated with 20 l phycoerithrin (PE) conjugated monoclonal antibody (CD 126, Immunotech, France) for 30 min at 4 •C.Then the cells were washed, sedimented and analysed in the FACSCalibur flow cytometer.For the expression of the membrane IL-6R (CD 126) the signal intensity (geometric mean of the fluorescence intensity x counts) was used as parameter.The signal intensity of the treated samples was compared with that of untreated samples, which were taken as 100%.

Measurement of the cell cycle phases
The cell cycle phases GO/GI, S, G2/M were assessed using the cycle test plus DNA reagent kit on a flow cytometer (BD, BioSciences, San Jose, USA No 340242).Briefly: 5x105 cells were incubated at room temperature with trypsin buffer and additionally with trypsin inhibitor+RNAse buffer.The values are expressed in percentage of total viable cell number (100%).

Measurement of apoptosis and necrosis
Apoptosis was measured using Annexin V-FITC (BD Biosciences Pharmingen, San Diego, USA No 556 570).Necrosis was measured using propidium iodide (PI).Briefly: 1x10 5 cells were incubated with Annexin V-FITC or PI at room temperature in the dark.Thereafter the samples were analysed in a flow cytometer.Apoptotic cells: Annexin V-FITC positive and PI negative.Necrotic cells: Annexin V-FITC positive and PI positive.The values are given in percent of total cell number.

Measurement of the proliferation
The proliferation was assessed using cell proliferation reagent WST-1 (Roche, Mannheim, Germany, No 1644 807).The colorimetric assay is based on the reduction of the tetrazolium salt WST-1 by viable cells.The reaction produces the soluble formazan salt.The quantity of the formazan dye is directly correlated to the number of the metabolically active cells.The proliferation rate was measured 1, 2 and 4 h after incubation with the reagents at time points 24, 48 and 72 h.The upper limit of the absorbance was 2.0-2.1.The intra-sample variance of the untreated cells was <10% (3-8%).

Statistical analysis
Three to four independent measurements were carried out.For the evaluation of the parameters the Mann-Whitney U-test was used.The limit of significance was taken as P<0.05.

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.
Table 1.Production of Interleukin-6 in human multiple myeloma cell lines.
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.
The values are presented in percentage.Range of four independent measurements.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.IL-6 does not alter the viability, confirming the findings of previous investigations (Kovacs, 2006b(Kovacs, , 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.

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/10 6 cells), (c) after treatment with VAQuFrF (dose: 50 µg/10 6 cells), (d) after treatment with IL-6+ VAQuFrF(dose: 5 ng/10 6 cells+50 µg/10 6 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<0.05vs.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.

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.
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.

MOLP-8
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/10 5 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/10 6 cells (P<0.05 and P<0.01).Vincristine had the same effect as VAQuFrF.
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/10 6 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).
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).

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/10 6 cells without dose dependence after 72 hours of treatment.VAQuFrF was ineffective.
The inhibitory effect of VAQuFrF was weaker than that of Vincristine in doses of 1 and 5 µg/10 5 cells.At the dose of 10 µg/10 5 cells the anti-proliferative effects of VAQuFrF and Vincritine was the same at each investigated time point.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/10 6 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.

KMS-12-BM
Proliferation: IL-6 increased the proliferation on average between 108 and 135%.VAQuFrF inhibited the proliferation only in dose of 10 µg/10 5 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 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).
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/10 6 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.

The effect of VAQuFrf on the proliferation of cells with high proliferation rate
The effect of VAQuFrF with doses of 5 and 10µg/10 5 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 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/10 5 cells + 1, 5, 10 µg/10 5 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/10 5 cells.It is suggested that the 2 hours pre-treatment with IL-6 is too short.

Investigation of cell cycle phases in human multiple myeloma
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.
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.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.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.
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).

Table 2 .
A. Viability of human multiple myeloma cells (cell lines see Table2).

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.

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