ONCOLOGY REPORTS 32: 1296-1302, 2014
Abstract. Galium verum, also known as Lady's Bedstraw, is an
herbaceous plant native to Europe and Asia, and has been used in
traditional medicine as an anticancer medicine applied in most
cases as a decoction. The inuence of a Galium verum decoc-
tion on the head and neck cancer cell lines HLaC78 and FADU
was analyzed and proved to be toxic in high doses on both cell
lines. Cytotoxicity appeared to be inuenced by expression of
p-glycoprotein (MDR-1) in the carcinoma cell lines. Mucosal
kerat i no cy t es, although void of MDR-1 exp r ession, showe d on ly
low sensitivity against high Galium concentrations. Sublethal
doses of Galium extract acted as strong inhibitors of motility,
as shown by a spheroid-based invasion analysis on Matrigel-
coated surfaces. Inhibition of invasion was signicantly more
pronounced in the invasive HLaC78 cell line. mRNA expres-
sion analysis of matrix metalloproteinases MMP-2 and MMP-9
and their inhibitors TIMP-1/-2 revealed signicant TIMP-1
upregulation after an 8-h Galium exposition in FADU cells.
Gelatinolytic activity, however, was not inuenced by Galium
extract in HLaC78, in the FADU cells MMP-2/-9 activity was
slightly increased after incubation with Galium extract. In
primary mucosal keratinocytes, Galium decoction protected
DNA against benz[a]pyrene, one of the most DNA toxic agents
in cigarette smoke. In conclusion Galium extract may be useful
as a preventive and/or a concomitant therapeutic approach in
head and neck cancer.
According to an analysis in 2009 of over 3,000 cases of
primary head and neck tumours in Germany, the outcome
of this disease did not significantly improve from 1995 to
2006, despite new treatment strategies. Particularly the 5-year
overall survival rate for carcinomas of hypopharyngeal origin
is extremely low at 27.2% (1). Moreover, in advanced laryn-
geal and hypopharyngeal cancer, the functional and cosmetic
deformations produced by surgery can be very disabling for
patients. Chemoradiation is meanwhile commonly used for
advanced head and neck cancer in order to preserve laryngeal
and/or pharyngeal structures. Paclitaxel is one of the agents
used with high response rates; however, it failed to reach
local-regional tumour control in 12% of patients according to
a previously published study (2).
Galium verum, also known as Lady's Bedstraw, is an
herbaceous perennial plant of the family Rubiaceae, native
to Europe and Asia. Studies on Galium verum predominantly
originate from the Asian continent, where traditional medicine
is more frequently embedded in culture. However, in industrial
nations traditional phytomedicine has gained more and more
attention, especially with respect to alternative treatments of
The cut and dried aerial parts of Galium verum have been
used for exogenous treatment of psoriasis or delayed wound
healing or as a tea with diuretic effect for the cure of pyelitis or
cystitis (3). Today the use of Galium verum is considered to be
obsolete, although it is still mentioned in popular non-scientic
publications and in internet platforms as an anticancer medici ne.
On the scientic level, a variety of bioactive substances
have been identied in Galium verum plants such as iridoid
glycosides (4-6), avanoids (5,7,8), anthraquinones (9) and
chlorogenic acid (10). Galium species are known to have
antioxidant [Galium verum (11)], antimicrobial/antifungal
[Galium tricornutum (12)], antifeedant [Galium aparine (13)]
and insecticidal [Galium melantherum (14)] properties.
According to a detailed survey by Hartwell (15) Galium
verum has been traditionally used in Europe and Northern
America for the treatment of cancerous ulcers or breast cancer.
Amirghofran et al (16) showed a cytotoxic effect of Galium
mite methanolic extracts on K561 and Jurkat cells. Zhao et al
isolated diosmetin from Galium verum plants and showed
protective effects on the thymus of U14-bearing mice (17).
In the present study, we tested the inuence of a Galium
verum ‘tea’ (decoction) on the growth and behaviour of head
and neck cancer cell lines and primary mucosal keratinocytes.
Materials and methods
Cell lines and cell culture. The cell line FADU originating
from a hypopharyngeal carcinoma was grown in RPMI-1640
Galium verum aqueous extract strongly inhibits the motility
of head and neck cancer cell lines and protects mucosal
keratinocytes against toxic DNA damage
MARIANNE SCHMIDT, CHRISTINE POLEDNIK, JEANETTE ROLLER and RUDOLF HAGEN
Department of Otorhinolaryngology, University of Wuerzburg, D-97080 Wuerzburg, Germany
Received April 22, 2014; Accepted May 28, 2014
Correspondence to: Dr Marianne Schmidt, Department of
Otorhinolaryngology, University of Wuerzburg, Josef-Schneider-
Strasse 11, D-97080 Wuerzburg, Germany
Key word s: Galium verum, cancer, carcinoma, head and neck,
paclitaxel, herbal drug, metastasis, in vitro, HNSCC
SCHMIDT et al: Galium verum AQUEOUS EXTRACT ON MOTILITY OF HEAD AND NECK CANCER CELL LINES 129 7
medium (Seromed, Munich, Germany), supplemented with
10% fetal calf serum (FCS). The HLaC78 cell line originated
from a larynx carcinoma (18) and was maintained similar to
FADU cells in RPMI-1640 medium. Mucosal keratinocytes
were prepared from tonsillar tissue according to standard
protocols (19). In brief, the mucosa was cut into small pieces
and incubated overnight with 0.2% dispase (Sigma-Aldrich,
Steinheim, Germany) in Dulbecco's modied Eagle's medium
(DMEM; Seromed). The epithelium was separated with
sterile forceps and digested with 0.1% trypsin (Seromed) for
20 min at 37˚C. Residual trypsin was inactivated by addition
of FCS. Mucosal keratinocytes were collected by centrifuga-
tion and cultured in dened keratinocyte serum-free medium
(Keratinocyte-SFM; Invitrogen, Karlsruhe, Germany).
Galium verum d ecoction. Dried and cut Galium verum L. leaves
(Herba galii lutei) were kindly provided by Dr Ivo Pischel,
PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany).
Tea was prepared as follows: 100 ml boiling water was poured
over 15 g of dried and powdered Galium leaves. After cooling,
the supernatant was cleared by centrifugation and sterile
ltration. Aliquots were frozen at -80˚C. One batch of frozen
Galium extract was used for all experiments. Identication of
the extract ingredients is presented elsewhere (20).
Real-time PCR. To measure gene expression rates, real-time
TaqMan® PCR (Applied Biosystems) was performed. RNA
was isolated from cell lines and primary cells with the RNeasy
kit (Qiagen, Hilden, Germany) according to the manufacturer's
instructions. The High Capacity RNA-to-cDNA Master Mix
(Applied Biosystems, Darmstadt, Germany) was used for
cDNA reverse transcription. Real-time PCR was performed
in triplicates on a real-time PCR cycler (Applied Biosystems)
using the TaqMan gene expression assays for MDR-1,
MMP-9/MMP-2 and TIMP-1/-2. Relative quantication was
calculated according to the 2-ΔΔCT method (21). Expression
values were normalised to the expression of GAPDH as an
endogenous control which proved to be expressed most stably
throughout the cell lines.
Cell viability and proliferation assay. Cells were seeded at
5,000 cells/well in 96-well plates. Cells were treated with
increasing concentrations of Galium verum aqueous extract
(50 and 100 µl/ml) for 48 h. Controls were kept in medium
supplemented with 100 µl/ml water. Cell proliferation was
measured after 48 h by replacing the culture medium with
medium containing 1 mg/ml MTT. After a 4-h incubation,
MTT staining solution was replaced by isopropanol, and the
cells were incubated at 37˚C for 45 min. The colour conversion
of MTT to a blue formazan dye was measured with an ELISA
reader at a wavelength of 570 nm. The amount of formazan
dye is in direct proportion to the number of metabolically
active cells in the culture. Relative toxicity was calculated
as the percentage of surviving cells by setting control cells
treated with vehicle as having 100% surviving cells.
Alkaline single-cell microgel electrophoresis assay. The
alkaline single-cell microgel electrophoresis technique (comet
assay) was applied to detect DNA strand breaks and alkali
labile plus incomplete excision repair sites in single cells.
Slide preparation was performed as previously described by
Buehrlen et al (22). The evaluation of the slides was carried out
on a DMLB uorescence microscope (Leica Microsystems,
Wetzlar, Germany) with a lter system incorporating a green
excitation lter (515-560 nm band pass), a dichromatic beam
splitter (580 nm long pass), and an emission lter (590 nm
long pass) at a magnication of x4,003. For every sample,
two slides with 50 randomly selected cells each were counted
(total of 100 cells). For analysis of the DNA fragmentation the
Comet 5.5 Image System (Kinetic Imaging, Liverpool, UK)
was used. For analysis, the olive tail moment (OTM) as a
product of the median migration distance and the percentage
of DNA in the tail was used (23).
In vitro motility assays. Tumour spheroids were generated
by seeding 5,000 cells/well of HLaC78 and FADU cells on
ultra-low attachment (ULA) 96-well round-bottomed plates
(Corning, Amsterdam, The Netherlands) (24). The surface of
the at-bottomed 96-well plates was coated with 125 µg/ml
Matrigel® (Becton-Dickinson, Heidelberg, Germany) for 2 h
at room temperature. Wells were washed twice with phos-
phate-buffered saline (PBS) and subsequently blocked with
1% bovine serum albumin in PBS for 1 h. For 3 days on the
ULA (see above) plates, pre-cultivated spheroids (see above) of
HLaC78 and FADU cell lines were transferred to the coated
wells with a multichannel pipette. Spheroids were incubated
with or without the Galium decoction (33.3 µl/ml). Migration
was recorded by photographing spheroids after 1 and 18 h with
a Leica DMI 4000 inverted uorescence microscope (Leica
Microsystems). Quantication of migrated cells was carried
out using ImageJ software [National Institutes of Health
Gelatin zymography. Cell lines were treated with 33.3 µl/
ml Galium verum extract for 48 h. After 48 h, the cells were
seeded after incubation with Galium extract at equal cell
numbers in multi-wall plates. Complete medium (MEM
or RPMI) was replaced after attachment by Opti-MEM
(Invitrogen, Karlsruhe, Ger many), which is a complete, serum-
free medium. Conditioned medium was collected after 18 h
and concentrated using Amicon® Ultra-4 Centrifugal Filters
(Merck Millipore, Darmstadt, Germany). Five microliters
of the concentrated medium was subjected to electropho-
resis on 10% SDS-polyacrylamide gels under non reducing
conditions (25), containing 1 mg/ml gelatin (Sigma-Aldrich,
Traunstein, Germany). After electrophoresis, gels were
renatured two times for 30 min in 2.5% Triton X-100 and
developed overnight in developing solution (50 mM Tris-HCl,
pH 6.8, 0.2 M NaCl, 10 mM CaCl2, 0.02% Brij-35) at 37˚C.
Subsequently they were stained with Coomassie brilliant blue,
destained and dried.
Statistical analysis. All statistical analyses and graphs were
performed with GraphPad Prism 4 (Graphpad Software,
La Jolla, CA, USA).
Expression of p-glycoprotein (p-gp; MDR-1). To de t e r m i ne
detoxification capacities of HLaC78 and FADU cells, and
ONCOLOGY REPORTS 32: 1296-1302, 2014
mucosal keratinocytes, quantitative RT-PCR was performed.
Expression of p-gp in the HLaC78 and FADU cells, and
mucosal keratinocytes (MKs) was tested by TaqMan qRT-PCR.
qRT-PCR revealed distinctly increased MDR-1 expression in
FADU cells, when compared to HLaC78 cells (Fig. 1). There
was no amplication detectable in primary MKs.
Cytotoxicity. The two cell lines (FADU and HLaC78) and
primary MKs were treated with increasing concentrations of
Galium aqueous extract (Fig. 2). Cell viability and cytotoxicity
of the used drug were assessed with the MTT assay. Mean
percent inhibition was calculated from at least three indepen-
dent experiments. Galium extract significantly suppressed
the growth of both cell lines (Kruskal-Wallis test, p<0.05). In
HLaC78 and FADU cells growth inhibition corresponded to
the expression rate of MDR-1 (Fig. 2).
Primary keratinocytes, however, were less affected by
high Galium concentrations than HLaC78 cells, although no
MDR-1 transcript was detectable in these cells.
There was no obvious correlation between the sensitivity to
high Galium concentrations and the proliferation rates of the
cell lines/primary cells.
Cell motility on extracellular matrix (ECM) proteins.
Investigation of invasion and motility was carried out using
spheroid-based experiments. First, these experiments better
reflect the solid tumour-microenvironment interaction.
Second, the widely used Boyden chamber assay proved to be
not reproducible in the actual system.
Spheroids of both cell lines were grown in ultra-low
attachment plates (ULA plates) wells and were subsequently
transferred manually to wells coated with Matrigel. Images of
the cells were captured after attachment to ECM (1 h, t=0) and
after 18 h (t=18).
For quantication of the cells migrating out of the spheroids,
the areas of the spheroids at t=0 and t=18 were photographed,
and the images were examined using Image J area calculation.
Areas at t=0 were subtracted from the areas measured after
18 h. For each condition (with or without Galium, HLaC78 or
FADU cells) at least 10 spheroids were measured.
For evaluation of cell motility, the area at t=0 was set at
100%. The percent of the migrated area was calculated using
the following formula:
% Migrated area = 100 x Δ Area
whereas Δ Area = Area t=18 - Area t=0.
Representative examples for FADU and HLaC78 cells
migrating on Matrigel with or without treatment of Galium
are shown in Fig. 3.
Comparing the percentage of the migrated areas of HLaC78
and FADU cells, HLaC78 cells turned out to be highly invasive,
compared to the FADU cell line (unpaired t-test, p<0.0001;
In both cell lines, Matrigel invasion was inhibited signi-
cantly by Galium (<0.0001; Fig. 5) decoction at sublethal
doses of 33.3 µl/ml (unpaired t-test, p<0.0001). Comparison of
the percent reduction of the migrated areas caused by Galium
in the two cell lines revealed a stronger invasion inhibition in
the aggressively invading HLaC78 cells (5104±287.3%) when
compared to FADU cells (723.3±48.79%).
Expression of matrix metalloproteinase MMP-2 and MMP-9
and their inhibitors. FADU and HLaC78 cell lines were culti-
vated with or without Galium extract for 4 or 8 h, respectively.
Expression levels of MMP-2 and MMP-9 as well as TIMP-1
and TIMP-2 RNA were measured using qRT-PCR. Results are
displayed in Fig. 6.
In both cell lines, MMP-9 and TIMP-1 were signicantly
upregulated after a 4-h incubation with Galium decoction.
Figure 2. Cytotoxicity of Galium decoction on FADU and HLaC78 cell lines, deter mined using the MT T assay. *p<0.0001, statistically signicant values;
one-way analysis of variance. Co, control; Ga, Galium decoction; MKs, mucosal keratinocy tes.
Figure 1. Expression of MDR-1 mRNA, measured by TaqMan qRT-PCR.
y-axis values were calculated according to the 2-ΔΔCT method. Values a re
mean ± SE and wer e normalized to the expression of GAPDH.
SCHMIDT et al: Galium verum AQUEOUS EXTRACT ON MOTILITY OF HEAD AND NECK CANCER CELL LINES 129 9
After 8 h however only TIMP-1 expression remained increased
in FADU cells, when compared to untreated controls. HLaC78
cells displayed no signicant changes in gelatinase A and B
and TIMP mRNA expression after 8 h.
MMP-2/-9 activity. To test the actual proteolytic activities,
conditioned media of HLaC78 and FADU cells incubated with
or without Galium aqueous extract for 18 h were applied to
gelatin zymographic gels.
FADU cells showed higher overall gelatinolytic activity
than HLaC78 cells. Gelatin zymography revealed no signi-
cant changes in MMP-2/-9 activity after treatment with Galium
decoction in the HLaC78 cell line. Galium-treated FADU
cells showed even higher MMP-9 and MMP-2 activity, when
compared with the untreated control cells (Fig. 7).
DNA protection. The Olive Tail Moment (OTM) was used to
evaluate DNA damage. Benzo[a]pyrene, found in tobacco smoke
(including cigarette smoke), has been shown to cause genetic
damage in lung cells that was identical to the damage observed
in the DNA of most malignant lung tumours (25). After a 1-h
treatment of MKs with 200 mM benzo[a]pyrene, a signicant
increase in the mean olive tail moment was observed (Fig. 8). A n
overnight preincubation with Galium decoction (50 µl/ml) signi -/ml) signi -ml) signi -
cantly decreased benzo[a]pyrene-induced DNA damage (Fig. 8).
Galium verum is a traditional medicinal plant commonly used
for the exogenous cure of psoriasis, delayed wound healing
or as a tea with diuretic effect for the cure of pyelitis or
Some popular compendia for herbal medicine recommend
Galium verum for the therapy of mouth/neck cancer (27,28).
According to detailed survey by Hartwell (15), Galium verum
was traditionally used in Europe and Northern America for the
treatment of cancerous ulcers or breast cancer.
In the present study the effect of a simple decoction of
Herba galii lutei on two different head and neck cancer cell
lines, differing in cell motility and chemoresistance were
tested, and signicant growth inhibition was noted at higher
doses in both cell lines, albeit somewhat extenuated in the
stronger MDR-1-expressing FADU cells. On sensible primary
mucosal cells, showing no p-glycoprotein expression, however,
Galium decoction proved to be less toxic than in the HLaC78
laryngeal cancer cell line.
Figure 4. Percentage of invasion of HLaC78 or FADU cells on Matrigel.
*p<0.0001, statistically signicant value; unpaired t-test.
Figure 3. Invasion patterns of HLaC78 and FADU cells on Matrigel-coated surfaces at t=0 (0 h after transfer to substrate) and t=18 (18 h later) with Galium
verum (Ga) aqueous extract or without [control (Co)].
Figure 5. Percentage of inhibition of Matrigel invasion by Galium aqueous
extract. *p<0.0001 and **p<0.05, statistically signicant values; unpaired
t-test. Co, control; Ga, Galium decoction.
ONCOLOGY REPORTS 32: 1296-1302, 2014
In general, high motility of tumour cells is frequently
correlated with increased chemoresistance, as previously
shown for the paclitaxel-resistant head and neck cancer cell
line Hep2-Tax (20). In HLaC78 cells, high cellular motility
is combined with a slow division rate and chemosensitivity.
Increased motility of cancer cells has been reported to be based
on higher expression rates of a variety of adhesion and motility
associated and proteolytic genes as well as transcription factors
(reviewed in ref. 29). In the present study we did not observe
striking effects of Galium decoction on mRNA expression of
the matrix metalloproteinases MMP-9 and MMP-2 or their
inhibitors. Gelatinolytic activity also remained unaffected by
preincubation with Galium verum decoction in both cell lines.
Potential main agents in Galium extracts, used for the
present and previous study are chlorogenic acid, Luteolin-
7-O-glucoside and rutoside (20). Chlorogenic acid has been
shown to be antimetastatic in vivo and in vitro in a variety of
tumour systems (30-32). The antimetastatic activity appeared
in combination with the downregulation of MMP-9 expres-
sion/activity (31-33). In the present study, gelatinolytic activity
was not affected by non-toxic Galium doses, indicating that
Figure 6. Gene expression of gelatinolytic matrix-metalloproteinases MMP-2 and MMP-9 and their inhibitors TIMP-1 and TIMP-2. *p<0.0001 and **p<0.05,
statistically signicant values; unpaired t-test. Co, control; Ga, Galium decoction.
Figure 7. Analysis of gelatinolytic activity using gelatin-zymography.
Molecular weight in kilo dalton (kDa) is indicated. Co, control; Ga, Galium
Figure 8. DNA fragmentation expressed by the olive tail moment (OTM)
in human mucosal keratinocytes after exposure to benzo[a]pyrene with or
without preexposure to aqueous Galiu m verum (Ga) extract. *p<0.0001, sta-
tistically signicant value; unpaired t-test. Co, control.
SCHMIDT et al: Galium verum AQUEOUS EXTRACT ON MOTILITY OF HEAD AND NECK CANCER CELL LINES 1301
motility inhibition by Galium extract is not necessarily caused
by chlorogenic acid (alone). HLaC78 spheroids formed very
tight, nearly indestructible spheroids. Video recordings (data
not shown) revealed that HLaC78 spheroids incubated with
Galium decoction needed more time to adhere to the Matrigel-
coated surface than the untreated spheroids. It seems likely
that the strong motility inhibition in HLaC78 cells is based on
changes in cell attachment and/or tight cell-cell contacts in the
close tissue-like formations.
In the second approach, it was demonstrated that primary
epithelial cells of the upper aerodigestive tract are protected
against genotoxic agents by an aqueous extract of Galium
verum. Antioxidative properties of Galium verum extract have
been described previously (11), suggesting DNA-protective
properties of the extract as well.
In summary, Galium verum aqueous extract revealed a
growth inhibitory effect on the cell lines HLaC78 and FADU,
as well as on primary mucosal keratinocytes at high doses. The
toxic effect appears to be modulated by detoxication capaci-
ties of the carcinoma cell lines, since the MDR-1-expressing
FADU cells were less sensitive to higher Galium doses. In
primary mucosal cells, void of p-glycoprotein expression,
however, Galium extract also exerted only low toxicity even
at high concentrations. At non-toxic concentrations, Galium
aqueous extract inhibited dispersion of HLaC78 and FADU
spheroidal cells on Matrigel-coated surfaces significantly,
even more pronounced in the highly motile cell line HLaC78.
The observed inhibition of motility was not caused by reduced
expression or activity of matrix-metalloproteinases.
Galium decoction protected DNA of primary mucosal
epithelial cells against the mutagenic action of benzo[a]pyrene,
one of the major DNA-damaging agents in cigarette smoke.
Galium verum aqueous extract, therefore, may be useful as an
effective and safe concomitant therapeutic approach in acces-
sible tumours of the mouth or upper aerodigestive tract.
We would like to thank Dr Ivo Pischel (PhytoLab GmbH &
Co. KG) for supplying the Herba gallii lutei. We further thank
Dr Johannes Gottfried Mayer and Dr Heike Will (University
of Wuerzburg, Forschergruppe Klostermedizin) for providing
historical data and for the stimulating discussion.
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