Mistletoe extracts Iscador

Gene Expression Profiles of Different

Breast Cancer Cells Compared with
their Responsiveness to Fermented
Mistletoe (Viscum album L.) Extracts
Iscador from Oak (Quercus), Pine (Pinus),
White Fir (Abies) and Apple Tree
(Malus) in vitro

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Jenny Eggenschwiler 1, Andrea Patrignani2, Ulrich Wagner 2, Hubert Rehrauer 2, Ralph Schlapbach2,
Lukas Rist 3, Mac H. Ramos4, and Angelika Viviani1
1
HSW − Hochschule Wädenswil, Wädenswil (Switzerland)
2
fgcz − Functional Genomic Center Zürich, Zürich University and Eidgenössische Technische Hochschule Zürich,
Zürich (Switzerland)
3
Paracelsus-Spital, Richterswil (Switzerland)
4
Weleda AG, Arlesheim (Switzerland)

Corresponding author: Prof. Angelika Viviani, PhD, HSW − Hochschule Wädenswil, Einsiedlerstrasse 29b,
8820 Wädenswil (Switzerland); e-mail: a.viviani@hsw.ch

Summary
Cytotoxicity assays in vitro (MTT test)
showed that the different breast cancer
cell lines Kpl-1, MCF-7 and Mfm-223 re-
spond differently to the mistletoe (Vis-
cum album L.) preparations Iscador .
Quercus (Qu), Abies (A), Malus (M) and
Pinus (P). In order to determine the dif-
ferences in the responsiveness of the
cells more exactly, the gene expression
profiles were determined by cells, which
were treated with Mistletoe extracts, com-
pared with untreated control cells. Such
differences can be analysed in more de-
tail by looking at the gene expression us-
ing Human Whole Genome microarray
chips (41,000 genes). The results of the
transcriptome analyses suggested that Is-
cador preparations influenced the overre-
gulation of genes regarding immune de-
fense, stress response, apoptosis and
cell-cell adhesion pathways. Within the
Mfm-223-Zellen was the Genexpression
in all 9 metabolic pathways evident. The
Mfm-223 cells included all effects found
Key words
in MCF-7 and Kpl-1. The MCF-7 cells
were affected on the genes which are in- 䊏 Breast cancer cells, cDNA
volved in cell-cell contacts whereas Kpl-1 microarray analyses, gene
responded to the mistletoe extracts by expression
changing the mRNA levels of the im-
䊏 Immune defense, stress
mune and stress response pathways. Con-
response
cerning the effects of the mistletoe ex-
tract, we conclude that Iscador Qu and M 䊏 Iscador 
have a greater influence on the immune 䊏 Mistletoe extracts,
defense and stress response genes cytotoxicity tests
whereas Iscador A tends to affect the 䊏 Viscum album L.
cell-cell adhesion and cytoskeleton path-
ways. In summary, cDNA microarray Arzneim.-Forsch./Drug Res.
analyses give us information on whether 56, No. 6a, 483−496 (2006)
a cancer cell is sensitive to mistletoe ex-
tracts in relation to how many genes are
significantly overrepresented after mistle-
toe treatment, and whether a particular
mistletoe extract is more effective on a
specific cancer cell than the other pre-
paration.

Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)

Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells 483
Mistletoe extracts Iscador

Zusammenfassung

Genexpressionsprofile verschiedener
Brustkrebszelllinien verglichen mit deren
Ansprechbarkeit auf fermentierte Mistel
(Viscum album L.)-Extrakte Iscador von
der Eiche (Quercus), der Kiefer (Pinus),
der Weisstanne (Abies) und dem Apfel-
baum (Malus) in vitro
Zytotoxizitätstests ergaben bei In-vi-
tro- Experimenten (MTT-Test), dass ver-
schiedene Brustkrebszelllinien wie Kpl-1,
MCF-7 und Mfm-223 unterschiedlich auf
die Mistelpräparate Iscador  Quercus
(Qu), Abies (A), Malus (M) und Pinus (P)
reagierten. Um die Unterschiede in der
Ansprechbarkeit der Zellen genauer fest-
zustellen, wurden die Genexpressionspro-
file von Zellen ermittelt, die mit Mistelex-
trakten im Vergleich zu unbehandelten
Kontrollzellen behandelt wurden. Die
cDNA-Mikroarray-Analysen wurden mit
Human Whole Genome Microarray-Chips
(41 000 Gene) durchgeführt. Aus den Re-
sultaten geht hervor, dass Gene, die der
Immunreaktion, Stressantwort, Apopto-
sis und der Zell-Adhäsion zugeschrieben
werden, vermehrt durch die Behandlung
mit Mistelpräparaten betroffen waren.
Die MCF-7 -Zellen wurden in den Genen
beeinfluβt, die in die Zell-Zell-Kontakte
miteinbezogen werden, während Kpl-1-
Zellen auf die Mistelextrakte reagierten,
indem sie die mRNA-Niveaus der Im-
mun- und Stressantwort änderten. Bei
den Mfm-223-Zellen war die Genexpres-
sion in allen 9 betroffenen Stoffwechsel-
wegen nachweisbar. Die MCF-7-Zellen
hingegen zeigten vor allem Änderungen
in der Genregulation, die mit Zellkontak-
ten und Zytoskelettproteinen zusammen-
hingen, während bei Kpl-1 vor allem die
Immunantwort-bezogenen Gene aktiviert
oder deaktiviert wurden. Von den Mistel-
präparaten aktivierten Iscador Qu und M
vor allem Gene der Immunabwehr und
der Stressantwort, während Iscador A ten-
denziell auf Gene der Zell-Adhäsion und
des Zytoskeletts wirkte. Zusammenfas-
send kann gesagt werden, dass cDNA-Mic-
roarray-Daten Aufschluss geben, ob eine
Krebszelle resistent oder sensitiv gegen-
über bestimmten Mistelpräparaten ist
und welches Präparat die effektivste Wir-
kung zeigt.

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1. Introduction have begun to apply microarray analyses to predict the
sensitivity of a tumor to a certain cytostatic agent,
Many different mistletoe (Viscum album L.) prepara-
shown with docetaxel [17].
tions are available; the differences between them can be
Other microarray analyses evaluated for cancer pre-
attributed to the host trees and/or the manufacturing
diagnosis have been carried out on established cell
processes and can be defined chemically by the content
lines [11−14] or on cancer tissues [15, 16] and confirm
of important lead compounds. Because of the hetero-
the significance of gene-expression profiles as an im-
geneity of the extract composition, different mistletoe
portant bioanalytical tool.
preparations will elicit different responses from a par-
Pharmacogenomic analysis using microarrays is at-
ticular cancer cell. We use microarrays to analyse the
tracting increasing interest in conventional medicine
up- or down-regulation of affected genes in order to
for examining the action of mono-compounds in cells
describe the biological effectiveness of different mistle-
[18]. Until now, few efforts have been made to analyse
toe extracts.
the mRNA expression profile of cancer cells caused by
It is known that cancer cells behave in a different
plant extracts, which are mixtures of many different
manner in the presence of different mistletoe extracts
components. Two studies were found where Iizuka et
[1−10]. This is due to the cytotoxicity of each extract,
al. and Yin et al. [19, 20] stated that antitumor activity
which is mainly dependent on the content of the
can be diagnosed by microarrays even when phyto-
mistletoe lectins, but also other components. Toxicity
pharmaceutical extracts are used. They achieved good
tests were carried out on several cell lines which had
results with Coptidis rhizoma and Scutellaria barbata,
been established from different cancer types and sub-
two plants with the cytotoxic potential of ED50 lying be-
types. The common conclusion was that each cell line
tween 0.01 and 0.3 mg/ml.
responds in its own individual way. It is therefore very
The main advantage of microarray analysis to the
difficult for an oncologist to prescribe the most suitable
phyto-pharmaceutical sector will be that the results will
mistletoe preparation for a particular patient-related make it possible to describe the effectiveness of the
cancer because almost nothing is known about the re- plant extracts on the cells without knowing their exact
sponsiveness and sensitivity of the cancer cells to a cer- chemical composition. Such a characterisation of plant
tain mistletoe extract. extracts at gene-expression level could provide an im-
There is a general question in cancer therapy of portant control tool to complement the existing com-
whether a tumor will respond to a certain chemothera- mon chemical analysis describing particular com-
peutic agent or not. In traditional chemotherapy, more pounds and corresponding amounts.
than one chemotherapeutic agent is applied, assuming
these substances are suitable. Several scientific groups

Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)

484 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells

2. Methods
2.1. Materials
The mistletoe extracts Iscador  Quercus (Qu, lot no. 41011),
Iscador Malus (M, lot no. 30611), Iscador Pinus (P, lot no.
40511) and Iscador Abies (A, lot no. 41111) were provided by
Hiscia AG (Arlesheim, Switzerland); their current lectin content
is listed in Table 1. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-di-
phenyltetrazolium bromide) and FCS (fetal calf serum) were
purchased from Sigma Chemical Co., Aldrich (Switzerland),
DMSO and trypan blue from Fluka (Buchs, Switzerland), Dul-
becco’s Modified Eagle’s Medium high glucose (DMEM), RPMI
1640, Dulbecco’s phosphate buffered saline (PBS) and trypsin-
EDTA solution (0.05 % trypsin/0.02 % EDTA) from AMIMED
BioConcept (Allschwil, Switzerland). The cell lines MCF-7 (ACC
115), Kpl-1 (ACC 317) and Mfm-223 (ACC 422) were supplied
by DSMZ (Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Braunschweig, Germany).
2.2. Cultivation of cancer cell lines
The breast cancer cells MCF-7, Kpl-1 and Mfm-223 were all
estrogen-receptor positive. Kpl-1 and Mfm-223 are described
as ductal carcinoma and MCF-7 as adenocarcinoma (www.
dsmz.de).
MCF-7, Kpl-1 and Mfm-223 were maintained in the media
recommended by the suppliers and with their suggested serum
concentration. The cells were cultured at 37 °C in a humidified
atmosphere with 5 % CO2. The adherent cells were subcultured
twice a week by washing with PBS and digesting the monolayer
with trypsin/EDTA at 37 °C for 5 min.
2.3. Cytotoxity assay
Cells were seeded at the density of 105 cells/ml (10 000 cells/
well) in a 96-well polycarbonate plate and incubated for 24 h
at 37 °C and 5 % CO2 before being exposed to different concen-
trations of appropriate dilutions of mistletoe extracts or blank
medium (control) for 48 h. At the end of the time period, 20 µl
of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) solution prepared in Dulbecco’s phosphate buffered
saline at the concentration of 5 mg/ml were added to each
well. The cells were incubated for 4 h at 37 °C. The purple for-
mazan crystals were solubilized with 200 µl DMSO per well.
The plates were gently shaken for 10 min and the absorbance
was measured at 570 nm on a microplate reader (MRX, Dyna-
tech Laboratories, Frankfurt/Main, Germany). The absorbance
value provides a direct indication of viable cells. The cytotoxic
response was graded by the percentage of surviving cells,
which were treated with mistletoe extract and compared to un-
treated control cells.
The toxic effect of each mistletoe treatment concentration
was calculated by taking the OD mean value (± standard devi-
ation) of 16 treatment wells relative to the OD mean value (±
standard deviation) of 24 control wells (100 % = no toxicity).
The blank, cell-free medium was subtracted from the absorp-
tion values. Statistical differences between control and treated
groups were evaluated using Student’s t test in Microsoft Excel.
P value < 0.05 was considered to represent significant differ-
ences between group values. P value of < 0.01 represents high
significance between group values. For this prediction, the
variance of treated wells (n = 48) and control wells (n = 48)
were compared with the F-test to choose the right Student’s t-
test. If the p value from the F-test was > 0.05, then we used the
Student’s t-test for two samples with identical variance. If the
Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)
Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
Mistletoe extracts Iscador
Table 1: Lectin concentration of the currently used Iscador pre-
parations analysed by Institute Hiscia Arlesheim using ELISA.
The values represent the mean of 4 measurements ± standard de-
viation.
Mistletoe Lectin content Lectin
preparation (ng/ml) (ng/mg extract)
Iscador Qu, 20 mg 1070 ± 29 50
Iscador M, 20 mg 493 ± 6 25
Iscador P, 20 mg 26 ± 0.4 1.25
Iscador A, 20 mg 20 ± 0.5 1
p value from F-test was < 0.05, we used the Student’s t-test for
two samples with no identical variance.
Dose-response curves were generated by determining the
effective mistletoe concentration where 50 % of the cells were
dead (EC50). The tested concentrations ranged between 2, 1,
0.5, 0.1, 0.01 to 0.001 mg/ml. The evaluation was performed
graphically.
2.4. mRNA isolation for microarray experiments
Downloaded by: University of Liverpool. Copyrighted material.
Cell lines were grown over night in 75 cm2 culture flasks to 70−
80 % confluence and then treated for 24 h with 0.1 mg Iscador
preparation. Then, the cells were detached by trypsinization
and collected by centrifugation at 400 g for 5 min. The mRNA
was isolated from lysates of 1−2 · 106 cells using the Qiagen
RNEasy Mini Kit (Qiagen GmbH, Hilden, Germany). The total
RNA was inspected by its quality and integrity with a nanodrop
ND-1000 spectrophotometer and an Agilent Bioanalyzer 2100.
Only those samples with a 260 nm/280 nm ratio between 1.8−
2.1 and a 28S/18S ratio within 1.5−2 were further processed.
2.5. Target preparation and hybridization
RNA amplification and labeling were done using the Agilent’s
low RNA input fluorescent linear amplification kit (P/N 5184-
3523, Agilent Technologies, Palo Alto, CA, USA). RNA samples
(2.5 µg) were reverse-transcribed into double-stranded cDNA
in presence of a T7 Promotor Primer. Labeled cDNA from
treated and untreated samples was prepared by in vitro tran-
scription incorporating Cyanine 3-CTP (P/N NEL580, Perkin
Elmer, Boston, MA, USA). A commercially available Human
Reference RNA (P/N 740000, Stratagene, Amsterdam, The
Netherlands). was used as a control and labeled with Cyanine
5-CTP (P/N NEL581, Perkin Elmer) according to the Agilent low
RNA input fluorescent linear amplification protocol (www.Agi
lent.com). The labeled cDNA was purified using an RNeasy
Mini Kit (P/N 74104, Qiagen, Hilden, Germany) and its
quantity and quality was determined using NanoDrop ND 1000
and Agilent Bioanalyzer 2100. Labeled RNA was fragmented for
30 min at 60 °C in the presence of a fragmentation buffer (P/N
5184-3568, Agilent Technologies). Each Cy-3 labeled sample (1
µg) was combined with the Cy-5 labeled control (1µg) and hy-
bridized onto Agilent Human Whole Genome 41k arrays (P/N
G4112A, Agilent Technologies) for 17 h at 65 °C. Arrays were
then washed according to the Agilent 60-mer oligo microarray
processing protocol (www.agilent.com).
2.6. Evaluation of microarray experiments
Hybridized arrays were scanned using the Agilent DNA Micro-
array (P/N G2565BA, Agilent Technologies) and the scanned
images were quantified with the Agilent Feature Extraction
Software 7.1 which provided the gProcessedSignal and rProces-
485
Mistletoe extracts Iscador

sedSignal as expression values. Data were excluded for genes Table 2: Effective concentrations (EC50) of the Iscador prepara-
that did not have an expression signal above 200 in either tions when breast cancer cells Mfm-223, Kpl-1 or MCF-7 were
incubated in the presence of the corresponding mistletoe extracts
channel. Each gene’s measured intensity was divided by its for 48 h.
control channel value in each sample; if the control channel
was below 1 then 1 was used instead. If the control channel Mfm-223 Kpl-1 MCF-7
(mg/ml) (mg/ml) (mg/ml)
and the signal channel were both below 1 then no data was re-
ported. Iscador Qu 0.01−0.1 0.01−0.1 0.01−0.1
For each cell line, the cells incubated with the four different Iscador M 0.1 0.1 0.1
Iscador P 0.5 0.5−1 0.5−1
Iscador preparations were compared with untreated ones to Iscador A 0.1 0.5 0.1
obtain lists of mistletoe affected genes using Genespring. Then,
the obtained gene lists were used to find the pathways where
the regulated genes were significantly overrepresented. Over-
representation was declared as significant if the p-value for
overrepresentation was below 0.05 and if at least three regu-
lated genes were present on a pathway.
extracted from MTT results with 48 replicates. The EC50
of Iscador Qu ranged between 0.01 and 0.1 mg extract/
ml for Kpl-1, MCF-7 and Mfm-223; that of Iscador M
3. Results was 0.1 mg/ml. Iscador P inhibited 50 % of cell prolif-
3.1. Effective concentration (EC50) of Iscador eration between 0.1 and 0.5 mg extract/ml. Finally, 0.1
preparations on the breast cancer cell lines Kpl-1, mg Iscador A/ml was needed to stop 50 % growth of the
MCF-7 and Mfm-223 Mfm-223 or MCF-7 cells or 0.5 mg/ml for Kpl-1.

Downloaded by: University of Liverpool. Copyrighted material.

Our aim was to describe the biological response of
cancer cells to different Viscum album extracts using
microarrays. First of all we had to take four important
aspects into consideration: i) there must be detectable
differences between extract-treated and untreated cells,
ii) the differences can refer to the different composition
of the mistletoe extracts, or iii) to different sensitivities
of the three cell lines used, iv) the in vitro concentration
of the mistletoe extract should be as close as possible
to in vivo applications.
Initially, the effective extract concentration where
50 % of the cells died within 48 h (EC50) had to be deter-
mined. The data reported in Table 2 were graphically
The EC50 ranged between 0.01 and 1 mg mistletoe
extract/ml for Kpl-1, MCF-7 and Mfm-223. For the fol-
lowing cDNA microarray experiments, designed to ex-
plore the gene-expression of mistletoe treated com-
pared with untreated cells, 0.1 mg Iscador preparation/
ml was used. At this concentration a cytotoxic effect of
Iscador Qu, M, A and P on Kpl-1, MCF-7 and Mfm-223
cells was detected (Fig. 1). MCF-7 cells responded
weakly to the Iscador preparations with growth reduc-
tions of between 12 % for Iscador Qu and P to 27 and
31 % for Iscador A and M, respectively. Kpl-1 was re-
sponsive to Iscador Qu, with a proliferation reduction
of 63 %, followed by Iscador M and A with 29 and 23 %.

120 60

100 50

80 40
ng lectin/mg extract

Mfm-223
MTT (%)

Kpl-1
60 30
MCF-7
Lectin

40 20

20 10

0 0
Iscador Qu Iscador M Iscador P Iscador A

Fig. 1: Relative mitochondrial activity (MTT − %) of mistletoe-treated breast cancer cells Kpl-1, MCF-7 and Mfm-223 compared to
untreated control cells. The cells were incubated with 0.1 mg Iscador preparation/ml for 48 h (n = 48; mean ± standard deviation). The
lectin concentration (ng/mg mistletoe extract) of the corresponding Iscador preparation is shown at the y-axis on the right.

Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)

486 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
 Mistletoe extracts Iscador

Iscador P had no effect on Kpl-1 at the concentration
and incubation time used. The Mfm-223 cells were af-
fected more strongly by the preparations than MCF-7
and Kpl-1. Iscador Qu reduced their cell proliferation
by 70 %, Iscador A by 46 %, Iscador M by 37 % and Is-
cador P by 18 %.
3.2. Lectin content and the strength of cytotoxicity
The cytotoxicity of Viscum album extracts is mainly at-
tributed to the mistletoe lectins [7, 23, 28, 31] and it
often correlates directly with the lectin concentration.
The content of the currently used Iscador prepara-
tions is shown in Table 1 (kindly analysed by M. Werner,
Institute Hiscia Arlesheim, using ELISA). The lectin con-
centration was highest in Iscador Qu with 1070 ng/ml,
half the amount in Iscador M and 40−50 times lower in
Iscador A and P, respectively. Therefore Iscador Qu
should produce the strongest effect on the cancer cell
lines and Iscador A the weakest. From Fig. 1 we con-
M, A and P/ml, using Whole Human Genome chips
(41 000 genes).
Initial incubation experiments were carried out with
Kpl-1 and MCF-7 cells to determine whether 4 or 24 h
incubation time were necessary to obtain overregu-
lation of genes at a suitable intensity. In both cell lines
no changes in gene regulation were found after 4 h.
After 24 h 652 genes were differently expressed in Is-
cador Qu treated Kpl-1 cells compared to untreated
ones (Table 3). Therefore, all the subsequent experi-
ments were carried out using 24 h treatment with 0.1
mg/ml Iscador preparations before isolating mRNA.
After amplification and hybridization, lists of the mis-
tletoe extract-affected genes in comparison with the
untreated control samples were made using Gene-
spring. These lists were used to extract the number of
commonly regulated genes in regard to treatments and
cell lines using Venn diagram (Tables 3 and 4).
In Kpl-1 cells 652 genes were affected by Iscador Qu,
358 by Iscador M, 317 by Iscador A and 193 by Iscador

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clude that MCF-7 cells were not sensitive to changes in
lectin concentrations because the cytotoxic effect of all
the preparations was similarly weak. However, Kpl-1
and Mfm-223 responded most strongly to the lectin-
rich Iscador Qu as expected, but the sensitivity of both
cell lines to the lectin-poor Iscador A cannot be explai-
ned by its lectin concentration because the measured
effect of this mistletoe extract on Kpl-1 and Mfm-223 is
too strong.
3.3. Microarray data: gene-expression changes after
mistletoe treatment
As summarized from our cytotoxicity test results, it is
not possible to interpret the efficacy of a mistletoe
preparation only in regard to its chemical composition.
Therefore in order to gain a clearer and more complex
understanding of the differences in the cytotoxicity be-
tween the Iscador preparations, we analysed the gene-
expression patterns of the three cell lines Kpl-1, MCF-7
and Mfm-223 after treatment with 0.1 mg Iscador Qu,
P compared to untreated cells; this order of overrepres-
ented genes corresponded well to the cytoxicity data of
the MTT tests (Fig. 1): Iscador Qu>M>A>P. Only few
genes were overregulated in MCF-7 cells by Iscador Qu
(101), Iscador M (71) and Iscador P (101); this means
that MCF-7 cells are not sensitive to the tested mistletoe
extracts under the given conditions, as was seen with
the cytotoxicity tests. The 360 genes overrepresented in
MCF-7 after treatment with Iscador A did not corres-
pond with the results of the in vitro assay. The same
phenomenon was observed with Mfm-223 cells, where
832 genes were affected by Iscador A, and only 154 and
172 by Iscador M and P, respectively.
It might be concluded that the higher the number of
affected genes, the more sensitive is the cell line to Is-
cador preparations if the criterion for sensitivity is cyto-
toxic activity.
For medical purposes it is also important to know
whether the different mistletoe preparations cause the
same changes in gene overregulations in a specific cell

Table 3: Lists of genes which were affected by Iscador Qu, A, M or P in Kpl-1, MCF-7 or Mfm-223 cells, obtained using Venn diagram.
The numbers in bold italics show the total number of common genes overrepresented in the corresponding cell line after treatment
with the specific mistletoe extract compared to untreated control cells. The other numbers represent the common genes which occur
in two different experiments comparing different treatments, different cell lines or both.

Cell line Kpl-1 MCF-7 Mfm-223

Iscador Qu A M P Qu A M P A M P

Kpl-1 Qu 652 6 18 8 32 24 20 10 41 13 9
A 6 317 218 93 3 16 4 31 23 10 18
M 18 218 358 106 7 13 7 34 21 12 19
P 8 93 106 193 2 8 4 6 16 9 13

MCF-7 Qu 32 3 7 2 101 10 14 5 6 4 4
A 24 16 13 8 10 360 37 48 33 9 14
M 20 4 7 4 14 37 71 32 4 2 3
P 10 31 34 6 5 48 32 101 8 3 8

Mfm-223 A 41 23 21 16 6 33 4 8 832 62 76
M 13 10 12 9 4 9 2 3 62 154 73
P 9 18 19 13 4 14 3 8 76 73 172

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Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells 487
Mistletoe extracts Iscador

Table 4: The number of genes in overrepresented pathways was listed from the experiments with Mfm-223, Kpl-1 and MCF-7 cells
which were treated with 0.1 mg Iscador Qu, M, P or A/ml for 24 h. The number after the pathway name in parentheses () represents
the total number of genes within the Keggs pathway. To be significant the corresponding “Gene list vs pathway overlap p-value” should
be lower than 0.05 and at least three genes within one experiment should be affected or the same gene should be found in at least
three different experiments. Italic printing indicates the significant results.
Qu, M, P, A: Iscador Qu, Iscador M, Iscador P, Iscador A.

Mfm-223 Kpl-1 MCF-7

Pathway
M P A Qu M P A Qu M P A

Apoptosis (95 genes) Affected genes 4 2 5 8 1 1 1 1 0 0 0

P value 1.8E-08 3.5E-03 3.9E-11 3.9E-19 1.6E-02 1.6E-02 1.6E-02 1.6E-02

Calcium signaling Affected genes 1 1 3 3 2 1 0 1 3 0 2

(193 genes) P value 1.6E-02 1.6E-02 6.8E-05 6.7E-05 1.6E-02 1.6E-02 4.7E-02 6.8E-05 1.4E-02

Complement and coagulation Affected genes 3 0 2 2 0 0 0 2 1 0 0

cascade (68 genes) P value 3.4E-06 2.1E-02 2.1E-03 2.1E-03 1.9E-02

Cytokine-cytokine receptor Affected genes 5 3 7 14 5 2 2 1 0 0 1

interaction (242 genes) P value 4.7E-09 1.4E-04 1.5E-13 2.8E-29 4.6E-09 2.3E-02 2.3E-02 1.6E-02 1.6E-02

Focal adhesion Affected genes 3 2 7 5 2 1 1 6

(157 genes) P value 3.8E-05 1.0E-02 9.6E-20 5.3E-10 0 0 0 1.0E-02 1.9E-02 1.9E-02 2.0E-12

MAPK signaling pathway Affected genes 3 3 7 9 2 0 1 8 0 0 4

(239 genes) P value 3.7E-05 1.4E-04 1.5E-13 4.6E-18 2.3E-02 1.9E-02 7.1E-19 7.9E-07

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Regulation of actin Affected genes 3 2 9 5 0 0 0 1 1 0 5
cytoskeleton (204 genes) P value 8.6E-05 1.7E-02 1.1E-18 2.0E-09 1.7E-02 2.1E-09

TGF-beta signaling pathway Affected genes 1 1 4 4 1 0 1 2 0 0 2

(80 genes) P value 1.9E-02 1.9E-02 9.6E-09 9.6E-09 1.9E-02 1.9E-02 2.7E-03 2.7E-03

Toll-like receptor signaling Affected genes 2 2 2 8 0 0 0 2 1 1 2

pathway (90 genes) P value 3.5E-03 3.5E-03 3.5E-03 2.8E-19 3.4E-03 1.8E-02 1.8E-02 3.5E-03

Wnt signaling pathway Affected genes 1 0 6 6 3 3 4 1 3 1 6

(143 genes) P value 1.8E-02 1.2E-12 1.2E-12 3.1E-05 3.1E-05 1.0E-07 3.5E-02 3.1E-05 1.8E-02 1.2E-12

line or even in different cell lines. Using Venn diagram
the co-occurence of affected genes in MCF-7, Kpl-1 and
Mfm-223 after treatment with Iscador Qu, A, M, P was
quantified (Table 3). We deduce that in general only
small numbers of common genes were affected,
whether two different treatments on one specific cell
line or one particular Iscador treatment on two cell
lines or both were analysed. 218 genes represented the
highest coincidence found when Iscador A-treated Kpl-
1 cells were compared with Iscador M-treated ones. The
small numbers of overrepresented genes between dif-
ferent treatments and/or cell lines may suggest that the
different mistletoe extracts produced individual effects
on a particular cancer cell.
3.4. Pathways affected by Iscador preparations
on the different breast cancer cell lines
The effects of mistletoe extracts on cancer cells can be
described by characterizing a particular, temporary, in-
tracellular phenomenon which can occur in a cell in
vitro using different analytical methods (cytotoxicity
tests, ELISA, PCR, RT-PCR). No overall picture of cell
activity is obtained. By analysing the gene-expression
on the whole genome it is possible to gain an overview
of changes at mRNA levels of a certain cell under given
conditions (cell type, drug concentration, incubation
time). The affected genes had to be listed in signifi-
cantly regulated, mistletoe-affected pathways. The ob-
tained gene lists (Table 3) were compared with the
genes of the selected Keggs pathways (160 Homo sapi-
ens pathways in the Genespring program) by means of
Venn diagram. The condition was that at least three
genes per pathway had to be up- or down-regulated
and the p-value should not exceed 0.05. Under these
restrictions 101 genes from a total number of 4870 were
found to be overrepresented in 9 different pathways in
the experiments with Kpl-1, MCF-7 and Mfm-223 after
mistletoe treatment (Table 4).
None of the metabolic pathways were involved in the
Viscum album treatments. The overregulated pathways
can roughly be subdivivded into inflammation-related
pathways such as MAPK signaling, TGF-beta signaling,
Toll-like receptor signaling, cytokine-cytokine receptor
interaction, apoptosis, and cell interaction/cytoskele-
ton-related pathways such as ECM-receptor interac-
tion, calcium signaling, regulation of actin cytoskeleton,
Wnt signaling and focal adhesion.
From Tables 4 and 6 it is evident that more signifi-
cant results were obtained with Mfm-223 in the pres-
ence of any of the Viscum album extracts and with Is-
cador Qu-treated Kpl-1 cells than with MCF-7 cells,
which were rather resistant to 0.1 mg of all Iscador pre-
parations/ml in the previous cytotoxicity tests. Further-
more it can be concluded that affinities of a certain cell

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488 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
 Mistletoe extracts Iscador

Table 5: Number and name of the genes which were overrepresented in the Iscador-treated cancer cells Mfm-223, Kpl-1 and MCF-7.

Gene number Gene name (Homo sapiens, mRNA)

A−23−P102113 Wingless-type MMTV integration site family, member 10A (WNT10A)

A−23−P106002 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA)
A−23−P106194 v-fos FBJ murine osteosarcoma viral oncogene homolog (FOS)
A−23−P110712 Dual specificity phosphatase 1 (DUSP1)
A−23−P111132 Heat shock 70kDa protein 1A (HSPA1A)
A−23−P120933 Activating transcription factor 4 (tax-responsive enhancer element B67) (ATF4), transcript variant 1
A−23−P122924 Inhibin, beta A (activin A, activin AB alpha polypeptide) (INHBA)
A−23−P128230 Nuclear receptor subfamily 4, group A, member 1 (NR4A1), transcript variant 1
A−23−P130429 Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)
A−23−P135769 Actin, beta (ACTB)
A−23−P138352 Wingless-type MMTV integration site family, member 2B (WNT2B), transcript variant WNT-2B2
A−23−P142361 Phosphoinositide-3-kinase, regulatory subunit, polypeptide 2 (p85 beta) (PIK3R2)
A−23−P142872 Transcription factor 7-like 1 (T-cell specific, HMG-box) (TCF7L1)
A−23−P145844 Met proto-oncogene (hepatocyte growth factor receptor) (MET)
A−23−P147431 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN)
A−23−P156687 B-factor, properdin (BF)
A−23−P157495 Protein phosphatase 3 (formerly 2B), (calcineurin A gamma) (PPP3CC)
A−23−P161727 Heat shock 27kDa protein 2 (HSPB2)
A−23−P163166 Leukotriene B4 receptor 2 (LTB4R2)
A−23−P167096 Vascular endothelial growth factor C (VEGFC)

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A−23−P167692 Mitogen-activated protein kinase 9 (MAPK9), transcript variant 1
A−23−P169331 TNF receptor-associated factor 2 (TRAF2)
A−23−P170857 Interleukin 1 receptor accessory protein (IL1RAP), transcript variant 1
A−23−P171054 ELK1, member of ETS oncogene family (ELK1)
A−23−P17814 Phospholipase A2, group III (PLA2G3)
A−23−P19624 Bone morphogenetic protein 6 (BMP6)
A−23−P201538 v-jun sarcoma virus 17 oncogene homolog (avian)
A−23−P204564 Protein phosphatase 1, regulatory (inhibitor) subunit 12A (PPP1R12A)
A−23−P206022 Integrin, alpha 11 (ITGA11)
A−23−P207319 Mitogen-activated protein kinase kinase kinase 14 (MAP3K14)
A−23−P209689 Rho-associated, coiled-coil containing protein kinase 2 (ROCK2)
A−23−P215701 Caspase 2, apoptosis-related cysteine protease (CASP2)
A−23−P215956 v-myc myelocytomatosis viral oncogene homolog (avian) (MYC)
A−23−P24104 Plasminogen activator, urokinase (PLAU)
A−23−P256334 Integrin, alpha subunit
A−23−P256724 TNF receptor superfamily, member 10c, decoy without an intracellular domain (TNFRSF10C)
A−23−P26325 Chemokine (C-C motif ) ligand 17 (CCL17)
A−23−P29495 Catenin (cadherin-associated protein), beta 1, 88kDa (CTNNB1)
A−23−P29953 Interleukin 15, transcript variant 1
A−23−P304897 Bradykinin receptor B2 (BDKRB2)
A−23−P30655 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon (NFKBIE)
A−23−P337242 Transforming growth factor, beta receptor II (70/80kDa) (TGFBR2)
A−23−P337800 Interleukin 29 (interferon, lambda 1) (IL29)
A−23−P356070 fms-related tyrosine kinase 4 (FLT4), transcript variant 2
A−23−P35916 Ataxia telangiectasia mutated (includes complementation groups A, C and D) (ATM)
A−23−P365610 jun D proto-oncogene (JUND)
A−23−P36611 Apoptotic protease activating factor (APAF1), transcript variant 3
A−23−P376488 Tumor necrosis factor (TNF superfamily, member 2) (TNF)
A−23−P385690 Wingless-type MMTV integration site family, member 3A (WNT3A)
A−23−P392457 Low density lipoprotein receptor-related protein 6 (LRP6)
A−23−P397999 Frizzled homolog 5 (Drosophila) (FZD5)
A−23−P399201 Thymosin, beta 4, X-linked (TMSB4X)
A−23−P404494 Interleukin 7 receptor (IL7R)
A−23−P40693 E1A binding protein p300 (EP300)
A−23−P40718 Parvin, beta (PARVB)
A−23−P42882 Calcium/calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B)
A−23−P45140 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog (KRAS2), transcript variant a
A−23−P49338 Tumor necrosis factor receptor superfamily, member 12A (TNFRSF12A)
A−23−P51754 RAP1A, member of RAS oncogene family (RAP1A)
A−23−P51856 Dual specificity phosphatase 10 (DUSP10), transcript variant 1
A−23−P62021 Thrombospondin 2 (THBS2) mRNA, complete cds
A−23−P64873 Unknown
A−23−P70213 Adenomatosis polyposis coli (APC)
A−23−P71037 Interleukin 6 (interferon, beta 2) (IL6)

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Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells 489
Mistletoe extracts Iscador

Table 5 cont‘d

Gene number Gene name (Homo sapiens, mRNA)

A−23−P7144 Chemokine (C-X-C motif ) ligand 1 (melanoma growth stimulating activity, alpha) (CXCL1)
A−23−P7313 Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I, early T-lymphocyte activation 1) (SPP1)
A−23−P7582 Transcription factor 7 (T-cell specific, HMG-box) (TCF7), transcript variant 1
A−23−P887 KIAA0151 gene, partial cds
A−23−P88767 Phospholipase A2, group X (PLA2G10)
A−23−P89431 Chemokine (C-C motif ) ligand 2 (CCL2)
A−23−P91390 Thrombomodulin (THBD)
A−23−P92754 Unknown
A−23−P93348 Lymphotoxin beta (TNF superfamily, member 3) (LTB)
A−23−P9402 Ciliary neurotrophic factor receptor (CNTFR)
A−23−P94230 Lymphocyte antigen 96 (LY96)
A−23−P94879 Coagulation factor II (thrombin) (F2)
A−23−P96144 Bone morphogenetic protein receptor, type II (serine/threonine kinase) (BMPR2)
A−23−P98350 Baculoviral IAP repeat-containing 3 (BIRC3)
A−24−P114604 Likely ortholog of mouse IRA1 protein (IRA1)
A−24−P120934 Growth arrest and DNA-damage-inducible, gamma (GADD45G)
A−24−P122137 Leukemia inhibitory factor (cholinergic differentiation factor) (LIF)
A−24−P131589 CD86 antigen (CD28 antigen ligand 2, B7-2 antigen) (CD86)
A−24−P141707 Inhibin, beta E (INHBE)

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A−24−P148811 RuvB-like 1 (E. coli) (RUVBL1)
A−24−P158089 Serine proteinase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), (SERPINE1)
A−24−P243329 Integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) (ITGA2)
A−24−P253003 Wingless-type MMTV integration site family, member 11 (WNT11)
A−24−P256764 Nonmuscle myosin heavy chain-B mRNA, partial cds
A−24−P278747 Cyclin D2 (CCND2)
A−24−P285522 Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3)
A−24−P287664 Phospholipase C, beta 2 (PLCB2)
A−24−P347946 ARF GTPase-activating protein GIT1 mRNA
A−24−P398572 Insulin-like growth factor 1 (somatomedin C) (IGF1)
A−24−P402438 Transforming growth factor, beta 2 (TGFB2)
A−24−P405298 Protein phosphatase 1, catalytic subunit, beta isoform (PPP1CB)
A−24−P412156 Chemokine (C-X-C motif ) ligand 12 (stromal cell-derived factor 1) (CXCL12)
A−24−P51855 Dual-specificity phosphatase 7 PYST2-L (PYST2)
A−24−P62708 Protein kinase, cAMP-dependent, catalytic, beta (PRKACB)
A−24−P63380 Bone morphogenetic protein receptor, type IB (BMPR1B)
A−24−P766204 HeLa mRNA isolated as a false positive in a two-hybrid-screen
A−32−P87013 Interleukin 8 (IL8)

line to a particular mistletoe extract produced specific
gene-expression profiles. The Kpl-1 cells were mainly
involved in the inflammation-related pathways under
the influence of Iscador Qu: 14 genes of cytokine-cyto-
kine receptor interaction, 9 of MAPK signaling, 8 of Toll-
like receptor signaling and 8 apoptosis genes were up-
or down-regulated, respectively. In contrast, Iscador A
treated MCF-7 cells were more affected in focal adhe-
sion (6 genes) and regulation of actin cytoskeleton (5
genes). Finally, activated genes in Mfm-223 cells were
found in all the pathway categories mentioned, espe-
cially after treatment with Iscador M or A.
From these preliminary results we also deduce that
Iscador preparations trigger certain pathways preferen-
tially. Iscador M and Qu influenced the immune and
stress response genes, Iscador A more the cell adhesion
genes (Table 4).
All experiments were carried out only once; therefore
the results should be seen as preliminary. However, the
co-occurrence of overrepresented genes in only 9 out of
160 Keggs pathways among the different experiments
strongly suggests that the results were reliable.
3.5. Up- and down-regulated genes of the breast
cancer cells after mistletoe extract treatment
The 101 selected genes (Table 5) were assigned to path-
ways, as obtained from Venn diagram. Their up- or
down-regulation is listed in Tables 6 a and b. A gene
was defined as significantly up-regulated if the fold-
change is higher than 2 and as down-regulated if it was
below 0.5.
The majority of genes found in the inflammation-
related pathways were overregulated: 77 % in apoptosis,
73 % in cytokine-cytokine receptor interaction, 63 % in
MAPK signaling, 50 % in TGF-beta and 70 % in Toll-like
receptor signaling pathway. Genes categorized as cell
interaction- and cytoskeleton-related were down-regu-
lated: 54 % in focal adhesion, 58 % in regulation of actin
cytoskeleton and 55 % in Wnt signaling. The only ex-
ception is the calcium signaling pathway with 74 % up-
regulated genes; these are involved in a large number

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490 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
 Mistletoe extracts Iscador

Table 6a: Overview of the most affected genes in the inflammatory pathways after treatment of Mfm-223, Kpl-1 and MCF-7 cells with
Iscador Qu, M, P or A. A fold change above 2 is defined as up-regulation and below 0.5 as down-regulation. The numbers in italics
represent genes defined as significant within the pathway because three or more genes were affected in the same experiment or the
same gene is overrepresented in at least three different experiments in the same direction. The second column lists pathways where
the gene occurs additionally.

Inflammation related pathways Fold change of gene expression

Coinciding pathways Mfm-223 Kpl-1 MCF-7

Overrepresented genes
Gene number M P A Qu M P A Qu M P A

Apoptosis
A−23−P106002 Toll-like receptor 2.84 2.96 3.11 3.21
A−23−P142361 Focal adhesion, cytoskeleton, 0.48
Toll-like receptor
A−23−P157495 Calcium signalling 2.09
MAPK, Wnt signalling
A−23−P169331 MAPK 2.20
A−23−P170857 Cytokine 3.01
A−23−P207319 3.66
A−23−P256724 Cytokine 2.01 2.26
A−23−P35916 Toll-like receptor 0.49
A−23−P36611 0.40 0.48
A−23−P376488 Cytokine, MAPK, TGF-beta, 2.04 2.12 2.14
Toll-like receptor
A−23−P98350 Focal adhesion 2.60 2.26 2.18 9.80 4.54
A−24−P62708 Calcium signalling 0.37

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MAPK, Wnt signalling
Cytokine-cytokine receptor
interaction
A−23−P122924 TGF-beta 2.01
A−23−P128503 0.44 0.49 0.47
A−23−P145844 Focal adhesion 0.45
A−23−P167096 Focal adhesion 2.18
A−23−P170857 Apoptosis 3.01
A−23−P256724 Apoptosis 2.00 2.26
A−23−P26325 2.27
A−23−P29953 0.39
A−23−P337242 MAPK, TGF-beta 2.15
A−23−P337800 4.68
A−23−P356070 Focal adhesion 0.37
A−23−P376488 Apoptosis, MAPK, 2.04 2.12 2.14
TGF-beta, Toll-like receptor
A−23−P404494 2.10
A−23−P49338 3.27
A−23−P71037 Toll-like receptor 6.35
A−23−P7144 3.13
A−23−P89431 4.11 3.51 2.20
A−23−P93348 4.30 3.67 5.29 4.77 0.46 0.39
A−23−P9402 2.11 2.58 3.00
A−23−P96144 TGF-beta 0.46
A−24−P122137 2.23
A−24−P141707 TGF-beta 0.03
A−24−P402438 MAPK, TGF-beta 3.05
A−24−P412156 2.61
A−24−P63380 TGF-beta 0.47
A−32−P87013 Toll-like receptor 2.24

MAPK signaling
A−23−P106194 Toll-like receptor 2.47
A−23−P110712 2.20 2.23
A−23−P111132 0.44
A−23−P120933 2.10
A−23−P128230 2.00 3.03 2.41
A−23−P157495 Calcium signalling, 2.09
MAPK, Wnt signalling
A−23−P161727 2.44
A−23−P167692 Focal adhesion, Toll- 0.45
like receptor, Wnt signalling
A−23−P169331 Apoptosis 2.20
A−23−P171054 Focal adhesion 0.50
A−23−P17814 2.09
A−23−P201538 Focal adhesion, Toll- 2.29
like receptor, Wnt signalling
A−23−P207319 Apoptosis 3.66
A−23−P215701 0.28
A−23−P215956 TGF-beta, Wnt signalling 0.47
A−23−P337242 Cytokine, TGF-beta 2.15
A−23−P365610 2.20
A−23−P376488 Apoptosis, cytokine, TGF-beta, 2.04 2.12 2.14
Toll-like receptor

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Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells 491
Mistletoe extracts Iscador

Table 6a cont‘d

Inflammation related pathways Fold change of gene expression

Coinciding pathways Mfm-223 Kpl-1 MCF-7

Overrepresented genes
Gene number M P A Qu M P A Qu M P A

MAPK signaling
A−23−P45140 Focal adhesion, cytoskeleton 0.49
A−23−P51754 Focal adhesion 0.48
A−23−P51856 2.50 2.30
A−23−P887 Toll-like receptor 0.44
A−23−P88767 2.06 2.01
A−23−P92754 Cytoskeleton 0.45
A−24−P120934 2.32
A−24−P285522 0.49
A−24−P402438 Cytokine, TGF-beta 3.05
A−24−P51855 0.48
A−24−P62708 Apoptosis, Calcium 0.37
signalling, Wnt signalling
A−24−P766204 0.48

TGF-beta signalling pathway

A−24−P402438 Cytokine, MAPK 3.05
A−23−P122924 Cytokine 2.01
A−23−P130429 Focal adhesion, cytoskeleton, 0.44
Wnt signalling

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A−23−P19624 2.11
A−23−P209689 Focal adhesion, cytoskeleton 2.15
A−23−P215956 MAPK, Wnt signalling 0.47
A−23−P337242 Cytokine, MAPK 2.15
A−23−P376488 Apoptosis, cytokine, 2.04 2.12 2.14
MAPK, Toll-like receptor
A−23−P40693 Calcium signalling, Wnt signalling 0.28
A−23−P62021 0.34
A−23−P64873 0.46
A−23−P96144 Cytokine 0.46
A−24−P141707 Cytokine 0.03
A−24−P63380 Cytokine 0.47

Toll-like receptor signalling

A−23−P71037 Cytokine 6.35
A−23−P106002 Apoptosis 2.84 2.96 3.11 3.21
A−23−P106194 MAPK 2.47
A−23−P142361 Apoptosis, focal adhesion, 0.48
A−23−P167692 Focal adhesion, MAPK 0.45
Wnt signalling
A−23−P201538 Focal adhesion, MAPK 2.29
Wnt signalling
A−23−P35916 Apoptosis 0.49
A−23−P376488 Apoptosis, cytokine, 2.04 2.12 2.14
MAPK, TGF-beta
A−23−P887 MAPK 0.44
A−23−P94230 0.47
A−24−P131589 0.49 2.96 3.62 4.24
A−32−P87013 2.24

of cellular responses induced by hormones, neuro- (A−23−P29953), interleukin 29 (A−23−P337800), the

transmitters and other signalling and should therefore
be classified between the two categories.
Most of the genes were only weakly overregulated
due to the chosen culture conditions. On the basis of
the current results it was not possible to describe af-
fected pathways step by step. However, the data indic-
ate that the gene expressions found are based on reli-
able facts, because not only were pathways of the simi-
lar functions affected, but also genes in clusters of the
same function were detected. The inflammatory cyto-
kines were very well represented; they were up-regu-
lated in different experiments. The most important
members were tumor necrosis factor α (A−23−P376488),
lymphotoxin-β (A−23−P93348), interleukin 6 (A−23−
P71037), interleukin 8 (A−32−P87013), interleukin 15
chemokine ligands CCL2 (A−23−P89431), CCL17 (A−23−
P26325), CXCL1 (A−23−P7144) and CXCL12 (A−24−
P412156), or receptors of inflammatory signal factors
such as bradykinin receptor (A−23−P304897), TNF re-
ceptor 10C and 12A (A−23−P256724, A−23−P49338), in-
terleukin 7 receptor (A−23−P404494) and interleukin 1
receptor associated protein (A−23−P170857). Most of
these genes were overexpressed in Kpl-1 cells after
treatment with Iscador Qu: these were TNFα
lymphotoxin-β, interleukin 6, interleukin 8, interleukin
29, the chemokine ligands CCL2, CXCL1, CXCL12,
bradykinin receptor and TNFα receptor 12A.
Lymphotoxin-β was found in all three experiments with
Mfm-223; moreover, TNFα, the TNF receptor 10C and
the chemokine ligand CCL2 were up-regulated in Mfm-

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492 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
 Mistletoe extracts Iscador

Table 6b: Overview of the overregulated genes in calcium signalling, cell adhesion-, and cytoskeleton-related pathways when Mfm-223,
Kpl-1 and MCF-7 cells were cultured in the presence of 0.1 mg/ml Iscador Qu, M, P or A for 24 h. The numbers in italics represent
genes defined as significant within the pathway because three or more genes were affected in the same experiment or the same gene
is overrepresented in at least three different experiments in the same direction.

Cell interaction and cytoskeleton Fold change of gene expression

related pathways
Coinciding pathways Mfm-223 Kpl-1 MCF-7
Overrepresented genes M P A Qu M P A Qu M P A
Gene number

Calcium signalling
A−23−P147431 2.74 2.74
A−23−P157495 Apoptosis, MAPK, 2.09
Wnt signalling
A−23−P163166 0.45
A−23−P304897 Cytoskeleton 2.46 2.19 2.43 2.02
A−23−P30655 2.69
A−23−P40693 TGF-beta, Wnt signalling 0.28
A−23−P42882 Wnt signalling 0.41 0.48 2.16 2.02
A−24−P287664 Wnt signalling 2.61 2.22 2.89
A−24−P62708 Apoptosis, MAPK, 0.37
Wnt signalling

Focal adhesion
A−23−P130429 Cytokskeleton, TGF-beta, 0.44
Wnt signalling
A−23−P135769 Cytoskeleton 2.12

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A−23−P142361 Apoptosis, cytoskeleton, 0.48
Toll-like rec.
A−23−P145844 Cytokine 0.45
A−23−P167096 Cytokine 2.18
A−23−P167692 MAPK, Toll-like receptor, Wnt sign. 0.45
A−23−P171054 MAPK 0.50
A−23−P201538 MAPK, Toll-like receptor, Wnt sign. 2.29
A−23−P204564 Cytoskeleton 0.36 0.42
A−23−P209689 Cytoskeleton, TGF-beta, 2.15
Wnt signalling
A−23−P256334 Cytoskeleton 8.67
A−23−P29495 Wnt signalling 3.37
A−23−P356070 Cytokine 0.37
A−23−P40718 0.49
A−23−P45140 MAPK, cytoskeleton 0.49
A−23−P51754 MAPK 0.48
A−23−P7313 2.13
A−23−P98350 Apoptosis 2.60 2.26 2.18 9.80 4.54
A−24−P243329 Cytoskeleton 0.32 0.38 0.37
A−24−P278747 Wnt signaling 2.17
A−24−P398572 12.87

Regulation of actin cytoskeleton

A−23−P130429 Focal adhesion, TGF-beta, 0.44
Wnt signalling
A−23−P135769 Focal adhesion 2.12
A−23−P142361 Apoptosis, focal adhesion, 0.48
Toll-like receptor
A−23−P204564 Focal adhesion 0.36 0.42
A−23−P206022 2.05 2.37
A−23−P209689 Focal adhesion, TGF-beta, 2.15
Wnt signalling
A−23−P256334 Focal adhesion 8.67
A−23−P304897 Calcium Signalling 2.46 2.19 2.43 2.02
A−23−P399201 0.44
A−23−P45140 Focal adhesion, MAPK 0.49
A−23−P70213 Wnt signalling 0.43
A−23−P92754 MAPK 0.45
A−23−P94879 2.10
A−24−P243329 Focal adhesion 0.32 0.38 0.37
A−24−P256764 0.48 0.46
A−24−P347946 0.48 0.38
A−24−P405298 2.39

Wnt signalling
A−23−P102113 0.31 0.46 0.32
A−23−P130429 Focal adhesion, cytoskeleton, 0.44
TGF-beta
A−23−P138352 2.20
A−23−P142872 0.49
A−23−P157495 Apoptosis, calcium signalling, 2.09
MAPK
A−23−P167692 Focal adhesion, MAPK, Toll-like 0.45
receptor

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Mistletoe extracts Iscador

Table 6b cont‘d

Cell interaction and cytoskeleton Fold change of gene expression

related pathways
Coinciding pathways Mfm-223 Kpl-1 MCF-7
Overrepresented genes M P A Qu M P A Qu M P A
Gene number

Wnt signalling
A−23−P201538 Focal adhesion, MAPK, Toll-like 2.29
receptor
A−23−P209689 Focal adhesion, cytoskeleton, 2.15
TGF-beta
A−23−P215956 MAPK, TGF-beta 0.47
A−23−P29495 Focal adhesion 3.37
A−23−P385690 2.17
A−23−P392457 0.30 0.29
A−23−P397999 2.29
A−23−P40693 Calcium signalling, TGF-beta 0.28
A−23−P42882 Calcium signalling 0.48 2.16 2.02
A−23−P70213 Cytoskeleton 0.43
A−23−P7582 2.17 0.30 0.35 0.33 2.61
A−24−P114604 0.33
A−24−P148811 2.15
A−24−P253003 0.42
A−24−P278747 Focal adhesion 2.17
A−24−P287664 Calcium signalling 2.61 2.22 2.89
A−24−P62708 Apoptosis, calcium signalling, 0.37
MAPK

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223 cells treated with Iscador M and A. The bradykinin
receptor was also significantly expressed in Mfm-223 in
the presence of Iscador A and in MCF-7 in the presence
of Iscador Qu and M. CCL17 and interleukin 7 receptor
was detected in Kpl-1 after Iscador M-treatment.
Other remarkable gene clusters were the different
wingless-type MMTVs in the Wnt signaling pathway
(Wnt2B = A−23−P138352, Wnt3A = A−23−P385690,
Wnt10 A = A−23−P102113 and Wnt11 = A−24−P253003)
and the three types of integrin-α (A−23−P206022, A−23−
P256334, A−24−P243329). In Kpl-1 cells Wnt11 was
down-regulated after treatment with Iscador Qu and
Wnt10A after treatment with Iscador A, M and P. Wnt3A
was up-regulated in Kpl-1 cells and Wnt2B in MCF-7;
both cell lines were treated with Iscador A.
The in-depth analyses of the 12 overrepresented
apoptosis genes according to the Keggs pathways
(http://www.genome.jp/kegg/) imply that the up-regu-
lated TNFα binds to its receptor in the cell membrane.
The activated receptor interacted with TRAF2, which is
involved in stress kinase and NF-κB activation. However
the clear up-regulation of the NF-κB inhibitor in several
experiments (A−23−P106002 = Nuclear factor of kappa
light polypeptide gene enhancer in B-cells inhibitor, al-
pha (NFKBIA) and A−23−P30655 = Nuclear factor of
kappa light polypeptide gene enhancer in B-cells inhib-
itor, epsilon (NFKBIE)) suggests that the NF-κB activity
was inhibited and DNA degradation could therefore be
induced. This explanation is currently the most plaus-
ible because the clearly up-regulated BIRC3 (A−23−
P98350) inhibits the downstream caspases 6, 3, 7 and 9
so that the caspase-cascade is blocked. The fact that
Apaf-1 (A−23−P36611) and ATM (A−23−P35916) are
down-regulated further support this assumption.
The apoptosis pathway, which includes 95 genes, is
closely connected to all other pathways involved in
mistletoe treatments. The cytokine and chemokine
stimulation can also be combined with cytokine-cytok-
ine receptor interaction (242 genes) (Fig. 2), MAPK sig-
naling (239 genes), TGFβ signaling (80 genes) and cal-
cium signaling (193 genes) pathways. However the in-
terpretation of the distinct steps within the pathways is
difficult because mainly the inducers or the end prod-
ucts of the pathways were affected by Viscum album ex-
tracts.
Wnt signaling (143 genes) describes the signal trans-
duction triggered by Wnt family members which bind
to the cell surface receptor Frizzled. The stimulation of
Frizzled leads over a series of steps to DNA synthesis or
changes in the cytoskeleton. Interestingly, Frizzled (A−
23−P397999) was up-regulated in Kpl-1 cells treated
with Iscador Qu whereas in those treated with Iscador
M and A the co-receptor of Frizzled, the low density
lipoprotein receptor-related protein 6 (A−23−P392457),
was down-regulated. Wnt 11 (A−24−P253003), which
binds to Frizzled, was down-regulated in Iscador Qu-
treated Kpl-1 cells. Also APC (A−23−P70213) and the
transcription factor 7 (A−23−P7582, A−23−P142872)
were down-regulated in Kpl-1 cells so that DNA syn-
thesis was not stimulated. However changes in cytoske-
leton interactions and in cell contact could probably
occur because integrin expression was clearly affected:
integrin α2 was down-regulated and integrin α11 was
up-regulated, not only in Kpl-1 treated with Iscador Qu,
but also in Mfm-223 treated with Iscador A.
One gene is of special interest: A−24−P348118, cod-
ing for galectin 7, a galactoside-binding lectin, is not
integrated in the pathways. With regard to mistletoe ex-
tract-induced effects on cancer cells, this gene is inter-
esting because it is down-regulated by Iscador A and M
in Kpl-1 and by Iscador P in MCF-7 cells. The mistletoe-
lectins I and II themselves are galactoside-recognizing
lectins [21−30] and therefore they seem to replace the
function of the cells’ own lectins.

Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)

494 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells

Kpl-1 MCF-7
A M P no A M P no
Fig. 2: Gene tree with the overrepresented genes of Kpl-1 and
MCF-7 cells in the pathway of cytokine-cytokine receptor interac-
tion after 24-h treatment with 0.1 mg/ml Iscador A, M and P; no =
untreated control cells.
4. Discussion
We conclude that different cancer cell types responded
differently to mistletoe extracts. A certain cancer cell
can be reponsive or resistant to all Viscum album pre-
parations or only to particular ones. Therefore the cyto-
toxicity of a mistletoe preparation depends not only on
the cell type but also on the extract composition, which
can be influenced by its host tree, its geographical ori-
gin or the manufacturing process used [31].
On comparing the results of cytotoxicity tests (MTT)
with those of the cDNA microarray analyses we found
a good correlation in the order of the response-grade of
the Iscador preparations Qu, M and P on a cancer cell
when the criteria were reduced a) to the percentage of
cells which died within a certain period of mistletoe
exposure, using MTT tests, or b) to the total number of
genes which were affected, using transcriptomic analy-
ses. However, Iscador A does not behave as expected:
this lectin-poor extract (containing the same amount of
lectin as Iscador P) had a strong cytotoxic effect on can-
cer cells and caused the transcription of many more
genes than Iscador Qu, M and P.
The results of microarray analyses confirm the well-
documented effects that mistletoe extracts are able to
induce apoptosis [32−39] in cancer cells and to activate
the immune defense pathways [6, 31, 36]. The genes of
these pathways were more strongly induced by Iscador
Qu and M (0.1 mg/ml for 24 h) than by Iscador P and
A. These results again correspond with those of the
MTT tests.
Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)
Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
Mistletoe extracts Iscador
At this time we conclude that apoptosis occurs but
instead of the caspase-cascade it follows the proteolytic
way initiated by TRAF2 and directed by the inhibitor of
Nf-kB, NFKBIA. No explanations of the other affected
pathways can be given at this time because the over-
expression of up- or down-regulated genes in series is
not currently available. The reasons are the following: i)
the chosen culture conditions do not allow very high
changes in gene regulation; but with regard to future
screening analyses we will keep analysis duration as
small as possible; ii) the pathway analysis using
Genespring was carried out only with genes whose p-
value was below 0.05 and three genes per pathway had
to be in that category. Further experiments are neces-
sary, especially replicates. The orientation in the differ-
ent pathways can then be analysed further and genes
which are overexpressed in several experiments, but
have higher p-values, can be taken into account.
New insights were gained by microarraying mistle-
toe-treated cells: we found that cell adhesion pathways
Downloaded by: University of Liverpool. Copyrighted material.
are affected by the presence of Iscador preparations.
MCF-7 and Mfm-223 cells in particular were highly sus-
ceptible to changes in the gene-expression of cell em-
bedding and cytoskeleton pathways after incubation
with Iscador A. It is not yet known how these changes
on the cellular level may inhibit cell proliferation.
In summary, we can conclude that mistletoe extracts
are potent antitumoral agents which cause significant
changes to the gene-expression level of immune and
stress response pathways. The response profile at the
gene expression level allows us to differentiate between
various mistletoe preparations with regard to their di-
rect effect on cell behaviour in vitro. The up- and down-
regulation of important growth and transcription fac-
tors, cytokines and chemokines, their inhibitors or re-
ceptors can be quantified by microarray analyses which
can illustrate how strongly the particular cancer cell is
affected by a certain mistletoe extract. This suggests the
possibility of using microarray data for prediagnosis in
personalized cancer therapy.
So far, these conclusions cover only the experience
obtained with Iscador Qu, M and P. Iscador A seems to
affect the cancer cells in other pathways, which need to
be better understood and further analysed.
Acknowledgements
We would like to thank Nick Bell for his help in checking the
English version and Weleda AG, Arlesheim (Switzerland) for
financial support.
Literature
[1] Knopfl-Sidler, F., Viviani, A., Rist, L. et al., Human cancer
cells exhibit in vitro individual receptiveness towards different
mistletoe extracts. Pharmazie 60, 448 (2005)
[2] Sidler, F., Wermelinger, T., Rist, L. et al., Influence of
mistletoe extracts and its components on in vitro physiology
of cancer cells. In: Animal cell technology meets genomics. Pro-
495
Mistletoe extracts Iscador
ceeding of the 18th ESACT meeting, Granada, Spain, May 11−
14, 2003 (2005)
[3] Zuzak, T., Rist, L., Viviani, A. et al., Das Mistelpräparat
Iscucin − Herstellung, Analytik, Wirkung in vitro. Merkurstab
6, 467 (2004)
[4] Ulrich, W., Mechelke, F., Reactions of in vitro cultures
of human fibroblasts, of HeLa-cells and of murine L-cells on
application of a drug from Viscum album L. Arzneim.-Forsch./
Drug Res. 30,1722 (1980)
[5] Hülsen, H., Doser, C., Mechelke, F., Differences in the in
vitro effectiveness of preparations produced from mistletoes of
various host trees. Arzneim.-Forsch./Drug Res. 36, 433 (1986)
[6] Lichtenstein, A., Kahle, J., Anti-tumor effect of inflamma-
tory neutrophils: characteristics of in vivo generation and in
vitro tumor cell lysis. International J. Cancer 35, 121 (1985)
[7] Doser, C., Doser, M., Hülsen, H. et al., Influence of carbo-
hydrates on the cytotoxicity of an aqueous mistletoe drug and
of purified mistletoe lectins tested on human T-leukemia cells.
Arzneim.-Forsch./Drug Res. 39, 647 (1989)
[8] Lieberman, M. D., Shou, J., Sigal, R. K. et al., Transfusion-
induced immunosuppression results in diminished host sur-
vival in a murine neuroblastoma model. J. Surg. Res. 48, 498
(1990)
[9] Lieubeau, B., Heymann, M. F., Henry, F. et al., Immuno-
modulatory effects of tumor-associated fibroblasts in colorec-
tal-tumor development. Int. J. Cancer 81, 629 (1999)
[10] Li, R., Sonik, A., Stindl, R. et al., Aneuploidy vs. gene
mutation hypothesis of cancer: recent study claims mutation
but is found to support aneuploidy. Proc. Natl. Acad. Sci. USA
97, 3236 (2000)
[11] Perou, C. M., Sorlie, T., Eisen, M. B. et al., Molecular
portraits of human breast tumors. Nature 406, 747 (2000)
[12] Troester, M. A., Hoadley, K. A., Sorlie, T. et al., Cell-type-
specific responses to chemotherapeutics in breast cancer. Can-
cer Res. 64, 4218 (2004)
[13] Shimizu, D., Ishikawa, T., Ichikawa, Y. et al., Current
progress in the prediction of chemosensitivity for breast cancer.
Breast Cancer 11, 42 (2004)
[14] Angrist, M., Breast cancer: integrating the patient with
her genome. Trends Biotechnol. 23, 3 (2005)
[15] Zhang, D. H., Salto-Tellez, M., Chiu, L. L. et al., Tissue
microarray study for classification of breast tumors. Life Sci. 73
3189 (2003)
[16] Kalloniemi, O. P., Wagner, U., Kononen, J. et al., Tissue
microarrays technology for high-throughput molecular profil-
ing of cancer. Human Molecular Genetics 10, 657 (2001)
[17] Chang, J. C., Wooten, E. C., Tsimelzon, A. et al., Gene
expression profiling for the prediction of therapeutic response
to docetaxel in patients with breast cancer. Lancet 263, 362
(2003)
[18] Sauter, G., Simon, R., Predictive molecular pathology.
N. Engl. J. Med. 347, 1995 (2002)
[19] Iizuka, N., Oka, M., Yamamoto, K. et al., Identification
of common or distinct genes related to antitumor activities of
a medicinal herb and its major component by oligonucleotide
microarray. Int. J. Cancer 107, 666 (2003)
[20] Yin, X., Zhou, J., Jie, C. et al., Anticancer activities and
mechanism of Scutellaria barbata on human lung cancer cell
line A549. Life Sci. 75, 2233 (2004)
[21] Endo, Y., Tsuguri, K., Franz, H., The site of action of the
A chain of mistletoe lectin on eukaryotic ribosomes. FEBS Lett.
231, 378 (1988)
[22] Luther, P., Becker, H., Die Mistel. Botanik, Lektine, med-
izinische Anwendung. Springer-Verlag, Berlin − Heidelberg
etc. (1987)
496 Eggenschwiler et al. − Gene expression profile and responsiveness of breast cancer cells
[23] Luther, P., Theise, H., Chatterjee, B. et al., The lectin
from Viscum album L. − isolation, characterization, properties
and structure. Int. J. Biochem. 11, 429 (1980)
[24] Franz, H., Mistletoe lectins and their A and B chains.
Oncology 43 (Suppl. 1), 23 (1986)
[25] Ziska, P., Gelbin, M., Franz, H., Interaction of mistletoe
lectins ML-1, ML-2, and ML-3 with carbohydrates. In: Lectins:
Biology, Biochemistry, Clinical Biochemistry, pp. 10−13, Textop,
Hellerup, Denmark (1993)
[26] Debray, H., Montreuil, J., Franz, H., Fine sugar specifi-
city of the mistletoe (Viscum album) lectin I. Glycoconj. J. 11,
550 (1994)
[27] Pfüller, U., Göckeritz, W., Pfüller, K. et al., Cell surface
and soluble receptors of mistletoe lectins. COST 98, Vol 6. Offi-
cial Report of the EEC, Brussels (1998)
[28] Pfüller, U., Mengs, T., Schwarz, K. et al., Natürliche Mis-
tellektine und das rekombinante U Mistellektin im Vergleich −
Biochemische und biologische Eigenschaften. Die Mistel in der
Tumortherapie − Grundlagenforschung und Klinik. R. Scheer
et al. (eds.), pp. 3−13, KCV Verlag, Essen (2001)
[29] Lee, R. T., Gabius, H. J., Lee, Y. C., The sugar-combining
area of the galactose-specific toxic lectin of mistletoe extends
Downloaded by: University of Liverpool. Copyrighted material.
beyond the terminal sugar residue: comparison with a homo-
logous toxic lectin, ricin. Carbohydrate Res. 17, 254, 269 (1994)
[30] Schaffrath, B., Mengs, U., Schwarz, T., Anticancer activ-
ity of rViscumin (recombinant mistletoe lectin) in tumor colon-
ization models with immunocompetent mice. Anticancer Res.
21, 3981 (2001)
[31] Kienle, G. S., Kiene, H., Die Mistel in der Onkologie.
Fakten und konzeptionelle Grundlagen. Schattauer Verlag,
Stuttgart − New York (2003)
[32] Van Huyen, J. P., Delignat, S. S., Bloch, M. F. et al., Vari-
able sensitivity of lymphoblastoid cells to apoptosis induced by
Viscum album Qu FrF, a therapeutic preparation of mistletoe
lectin. Exp. Chemother. 47, 366 (2001)
[33] Urech, K., Scheer, J. M., Hostanska, K. et al., Apoptosis
inducing activity of viscin, a lipophilic extract from Viscum
album L. Pharm. Pharmacol. 57, 101 (2005)
[34] Lavastre, V., Pelletier, M., Saller, R. et al., Mechanisms
involved in spontaneous and Viscum album agglutinin-I-in-
duced human neutrophil apoptosis: Viscum album agglutinin-
I accelerates the loss of antiapoptotic Mcl-1 expression and the
degradation of cytosceletal paxillin and vimentin proteins via
caspases. J. Immunol. 168, 1419 (2002)
[35] Harmsma, M., Gromme, M., Ummelen, M. et al., Differ-
ential effects of Viscum album extract Iscador Qu on cell cycle
progression and apoptosis in cancer cells. Int. J. Oncol. 25,
1521 (2004)
[36] Thies, A., Nugel, D., Pfüller, U. et al., Influence of mistle-
toe lectins and cytokines induced by them on cell proliferation
of human melanoma cells in vitro. Toxicology 207, 105 (2005)
[37] Choi, S. H., Lyu, S. Y., Park, W. B., Mistletoe lectin in-
duces apoptosis and telomerase inhibition in human A253 can-
cer cells through dephosphorylation of Akt. Arch. Pharm. Res.
27, 68 (2004)
[38] Hostanska, K., Vuong, V., Rocha, S. et al., Recombinant
mistletoe lectin induces p53-independent apoptosis in tumor
cells and cooperates with ionising radiation. Br. J. Cancer 88,
1785 (2003)
[39] Savoie, A., Lavastre, V., Pelletier, M. et al., Activation of
human neutrophils by the plant lectin Viscum album agglu-
tinin-I: modulation of de novo protein synthesis and evidence
that caspases are involved in induction of apoptosis. J. Leukoc.
Biol. 68, 845 (2000)
Arzneim.-Forsch./Drug Res. 56, No. 6a, 483−496 (2006)