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Galium verum aqueous extract strongly inhibits the motility of head and neck cancer cell lines and protects mucosal keratinocytes against toxic DNA damage

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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 influence of a Galium verum decoction 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 influenced by expression of p-glycoprotein (MDR-1) in the carcinoma cell lines. Mucosal keratinocytes, although void of MDR-1 expression, showed only 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 significantly more pronounced in the invasive HLaC78 cell line. mRNA expression analysis of matrix metalloproteinases MMP-2 and MMP-9 and their inhibitors TIMP-1/-2 revealed significant TIMP-1 upregulation after an 8-h Galium exposition in FADU cells. Gelatinolytic activity, however, was not influenced 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.
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ONCOLOGY REPORTS 32: 1296-1302, 2014
129 6
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 inuence 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 inuenced 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 signicantly 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 signicant TIMP-1
upregulation after an 8-h Galium exposition in FADU cells.
Gelatinolytic activity, however, was not inuenced 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.
Introduction
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
ca ncer.
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-scientic
publications and in internet platforms as an anticancer medici ne.
On the scientic level, a variety of bioactive substances
have been identied 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 inuence 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
DOI: 10.3892/or.2014.3316
Correspondence to: Dr Marianne Schmidt, Department of
Otorhinolaryngology, University of Wuerzburg, Josef-Schneider-
Strasse 11, D-97080 Wuerzburg, Germany
E-mail: schmidt_m2@klinik.uni-wuerzburg.de
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 modied 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 dened 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. Identication 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 quantication 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 magnication 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). Quantication of migrated cells was carried
out using ImageJ software [National Institutes of Health
(NIH) USA].
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).
Results
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
129 8
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 amplication 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 quantication 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
------------------------
Area t=0
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;
Fig. 4).
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 signicantly
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 signicant 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 signicant 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 signicant
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).
Discussion
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
cystitis (3).
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 signicant 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 signicant 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 signicant values; unpaired
t-test. Co, control; Ga, Galium decoction.
ONCOLOGY REPORTS 32: 1296-1302, 2014
130 0
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 signicant 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
decoction.
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 signicant 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 detoxication 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.
Acknowledgements
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.
References
1. Guntinas-Lichius O, Wendt T, Buentzel J, et al: Head and neck
cancer in Germany: a site-specic analysis of survival of the
Thuringian cancer registration database. J Cancer Res Clin
Oncol 136: 55-63, 2010.
2. Pfreundner L, Hoppe F, Willner J, Preisler V, Bratengeier K,
Hagen R, Helms J and Flentje M: Induction chemotherapy with
paclitaxel and cisplatin and CT-based 3D radiotherapy in patients
with advanced laryngeal and hypopharyngeal carcinomas - a
possibility for organ preservation. Radiother Oncol 68: 163-170,
20 03.
3. Blaschek W, Ebel S, Hilgenfeldt U, Holzgrabe U, Reichling J and
Schulz V: Hagers Enzyklopädie der Arzneistoffe und Drogen
(Hager ROM; elektronische Buch-Version). Springer Verlag,
Berlin, 2009 (In German).
4. Böjthe-Horváth K, Hetényi F, Kocsis Á, Szabó L, Varga-Balázs M,
Máthé I Jr and Tétényi P: Iridoid glycosides from Galium verum.
Phytochemistry 21: 2917-2919, 1980.
5. Zhao C, Shao J, Cao D, Zhang Y and Li X: Chemical constituents
of Galium verum. Zhongguo Zhong Yao Za Zhi 34: 2761-2764,
2009 (In Chinese).
6. Corrigan D, Timoney RF and Donnelly D: Iridoids and alkanes
in twelve species of Galium and Asperula. Phytochemistry 17:
1131-1133, 19 78.
7. Shafaghat A, Salimi F, Aslaniyan N and Shoaei Z: Flavonoids
and an ester derivative isolated from Galium verum L. World
Appl Sci J 11: 473-477, 2010.
8. Zhao CC, Shao JH, Li X, Kang XD, Zhang YW, Meng DL and
Li N: Flavonoids from Galium verum L. J Asian Nat Prod Res 10:
613- 617, 2 0 0 8.
9. Zhao CC, Shao JH, Li X, Xu J and Wang JH: A new anthraqui-
none from Galium verum L. Nat Prod Res 20: 981-984, 2006.
10. Borisov MI, Zaitsev KV and Zaitsev VG: The chemical composi-
tion of Galium verum. Chem Nat Compd 7: 511, 1971.
11. Mavi A, Terzi Z, Ozgen U, Yildirim A and Coşkun M: Antioxidant
properties of some medicinal plants: Prangos ferulacea
(Apiaceae), Sedum sempervivoides (Crassulaceae), Malva
neglecta (Malvaceae), Cruciata taurica (Rubi ace a e), Rosa pimp-
inellifolia (Rosa cea e), Galium verum subsp. verum ( Ru bia cea e),
Urtica dioica (Urticaceae). Biol Pharm Bull 27: 702-705, 2004.
12. Khaliq Jan A1, Raza Shah M, Anis I and Khan Marwat I: In vitro
antifungal and antibacterial activities of extracts of Galium
tricornutum subsp. longipedunculatum. J Enzyme Inhib Med
Chem 24: 192-196, 2009.
13. Morimoto M, Tanimoto K, Sakatani A and Komai K: Antifeedant
activity of an anthraquinone aldehyde in Galium aparine L.
against Spodoptera litura F. Phytochemistry 60: 163-166, 2002.
14. Tzakou O, Mylonas P, Vagias C and Petrakis PV: Iridoid gluco-
sides with insecticidal activity from Galium melanantherum.
Z Naturforsch C 62: 597-602, 2007.
15. Hartwell JL: Plants Used Against Cancer: A Survey. Quarterman
Publications, Lawrence, MA, pp438-439, 1982.
16. Amirghofran Z, Bahmani M, Azadmehr A and Javidnia K:
Anticancer effects of various Iranian native medicinal plants on
human tumor cell lines. Neoplasma 53: 428-433, 2006.
17. Zhao R, Chen Z, Jia G, Li J, Cai Y and Shao X: Protective effects
of diosmetin extracted from Galium verum L. on the thymus of
U14-bearing mice. Can J Physiol Pharmacol 89: 665-673, 2011.
18. Zenner HP, Lehner W and Herrmann IF: Establishment of
carcinoma cell lines from larynx and submandibular gland. Arch
Otorhinolaryngol 225: 269-277, 1979.
19. Imaizumi F, Asahina I, Moriyama T, Ishii M and Omura K:
Cultured mucosal cell sheet with a double layer of keratinocytes and
broblasts on a collagen membrane. Tissue Eng 10: 657-664, 2004.
20. Schmidt M, Scholz CJ, Gavril GL, et al: Effect of Galium verum
aqueous extract on growth, motility and gene expression in
drug-sensitive and -resistant laryngeal carcinoma cell lines. Int
J Oncol 44: 745-760, 2014.
21. Livak KJ and Schmittgen TD: Analysis of relative gene expres-
sion dat a usi ng real-time quantitative PCR and the 2-ΔΔCT method.
Methods 25: 402-408, 2001.
22. Buehrlen 1, Harréus UA, Gamarra F, Hagen R and Kleinsasser NH:
Cumulative genotoxic and apoptotic effects of xenobiotics in a
mini organ culture model of human nasal mucosa as detected by
the alkaline single cell microgel electrophoresis assay and the
annexin V-afnity assay. Toxicol Lett 169: 152-161, 2007.
23. Olive PL and Banáth JP: Growth fraction measured using the
comet assay. Cell Prolif 25: 447-457, 1992.
24. Vinci M, Gowan S, Boxall F, et al: Advances in establishment
and analysis of three-dimensional tumor spheroid-based func-
tional assays for target validation and drug evaluation. BMC
Biol 10: 29, 2012.
25. Heussen C and Dowdle EB: Electrophoretic analysis of
plasminogen activators in polyacrylamide gels containing
sodium dodecyl sulfate and copolymerized substrates. Anal
Biochem 102: 196-202, 1980.
26. Denissenko MF, Pao A, Tang M and Pfeifer GP: Preferential
formation of benzo[a]pyrene adducts at lung cancer mutational
hotspots in P53. Science 274: 430-432, 1996.
27. Willfort R: Gesundheit Durch Heilkräuter. Trauner Verlag, 1982
(In German).
28. Treben M: Gesundheit Aus Der Apotheke Gottes. Ennsthaler
Verlag, Steyr, 1980 (In German).
29. Sahai E: Mechanisms of cancer cell invasion. Curr Opin Genet
Dev 15: 87-96, 2005.
ONCOLOGY REPORTS 32: 1296-1302, 2014
130 2
30. Yagasaki K, Miura Y, Okauchi R and Furuse T: Inhibitory effects
of chlorogenic acid and its related compounds on the invasion of
hepatoma cells in culture. Cytotechnology 33: 229-235, 2000.
31. Hwang YP, Yun HJ, Choi JH, et al: 3-Caffeoyl, 4-dihydrocaf-
feoylquinic acid from Salicornia herbacea inhibits tumor cell
invasion by regulating protein kinase C-δ-dependent matrix
metalloproteinase-9 expression. Toxicol Lett 198: 200-209,
2010.
32. Tsai CM, Yen GC, Sun FM, Yang SF and Weng CJ: Assessment
of the anti-invasion potential and mechanism of select cinnamic
acid derivatives on human lung adenocarcinoma cells. Mol
Pharm 10: 1890-1900, 2013.
33. Jin UH, Lee JY, Kang SK, et al: A phenolic compound, 5-caffeoyl-
quinic acid (chlorogenic acid), is a new type and strong matrix
metalloproteinase-9 inhibitor: isolation and identification from
methanol extract of Euonymus alatus. Life Sci 77: 2760-2769, 2005.
... W lecznictwie tradycyjnym G. verum znajduje zastosowanie w chorobach skóry, np. w łuszczycy oraz w trudno gojących się ranach [1][2][3]. Jako diuretyk jest zalecana w leczeniu dny moczanowej, odmiedniczkowego zapalenia nerek, zapalenia pęcherza moczowego i w chorobach reumatycznych. Ziele stosuje się także jako środek żółciopędny, napotny i oczyszczający. ...
... Ziele stosuje się także jako środek żółciopędny, napotny i oczyszczający. Napary z przytulii rekomenduje się w postaci płukanek do jamy ustnej lub herbatek do picia, w nowotworach języka i krtani, w owrzodzeniach nowotworowych i raku piersi [1,2]. W piśmiennictwie opisano także korzystny wpływ rośliny na choroby sercowo-naczyniowe, nadpobudliwość i stany lękowe [3]. ...
... Tradycyjne stosowanie przytulii właściwej w chorobach nowotworowych skłoniło Schmidta i wsp. do badań, w których wykazano cytotoksyczny wpływ odwarów z G. verum na linie komórkowe raka głowy i szyi (HLaC78 i FADU) [2], a także na komórki raka krtani, zarówno wrażliwe (Hep2 i HLaC79), jak i oporne (Hep2-Tax, HLaC79--Tax) na chemioterapię [1]. ...
Article
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Subject of research. Galium verum L. (lady’s bedstraw, yellow bedstraw) (Rubiaceae) is used in folk medicine in dermatoses, bladder and kidneys diseases as well as a choleretic agent. So far, scientific studies have shown as main constituents: iridoids, flavonoids, phenolic acids, and triterpenes. Previous studies confirmed antioxidant, cytotoxic, antimicrobial, hepatoprotective, and immunostimulating activities of bedstraw. Aim. The aim of this work was to investigate the chemical profile of essential oil isolated from G. verum collected in Poland. Material and methods. The essential oil was extracted from the herb of G. verum by hydrodistillation in Clevenger-type apparatus and the volatile constituents were identified by gas chromatography coupled with mass spectrometry (GC-MS). Results. 2.60 ml/kg of essential oil was obtained from the herb of G. verum, in which 71 volatile ingredients were identified (100%). According to GC-MS analysis for palmitic acid (10.87%) and anethole (8.39%) as well for menthol (5.28%) and linoleic acid (4.91%), a significant amount was noted. In a smaller number were present carvone, β-ionone, phytol, menthone, estragole, linalool and β-farnesene. Terpene compounds represented 31.34% of essential oil, including 9 sesquiterpenes (5.51%), 15 monoterpenes (24.04%), 1 diterpene (1.79%) and 3 phenylpropanoids (10.21%). Among fatty acids, the fraction was dominated by palmitic acid (10.87%) and linoleic acid (4.91%), in the oil were also methyl palmitate (0.41%) and methyl salicylate (0.14%) present. The other active ingredients of the essential oil include eucalyptol, carvacrol, thujone, camphor, isopulegone, caryophyllene oxide, farnesyl acetate, terpinen-4-ol, γ-terpineol, α-terpineol, myrtenol, methyl salicylate, α-ionone, β-ionone 5,6-epoxide, cis-α-bisabolene, shiobunon, nerolidol, and spatulenol. The ingredients of the essential oil are multi-directional activity such as anti-inflammatory, antimicrobial, antiproliferative, proapoptotic, anti-itching, and also affecting the wound healing process. Conclusions. Quantify and a number of ingredients of the essential oil have proven that it may be one of the bioactive important fractions of the bedstraw herb and partly justify the traditional use of G. verum.
... Several researches have been recently conducted on the apoptotic effects of the Rubiaceae family plants on cancer cells. However, research in this area is still challenging for scientists [30,31]. Galium (G.) verum is a well-known medicinal plant used for cancer treatment [7,13,24]. ...
... Galium (G.) verum is a well-known medicinal plant used for cancer treatment [7,13,24]. Many studies have shown that G. verum contains chemical compounds with high cytotoxic properties [30,31]. Also, several studies have reported the cytotoxic effects of G. verum extract on several cancer types, including breast, larynx [31], head and neck [30], and liver [31] cancers. ...
... Many studies have shown that G. verum contains chemical compounds with high cytotoxic properties [30,31]. Also, several studies have reported the cytotoxic effects of G. verum extract on several cancer types, including breast, larynx [31], head and neck [30], and liver [31] cancers. A recent study on G. verum extract confirmed its cytotoxic effects on colon cancer cell lines [8,27,31]. ...
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The study reports the effects of methanol extract of Gallium verum on the cell proliferation of HT-29 (colon cancer) and AGO (fibroblast) cell lines. The cell lines were exposed to the G. verum extracts at the concentrations 12.5-400 μg/mL for a period of 72 h. Cell viability was evaluated by MTT assay. Apoptosis and cycle distribution were evaluated using flow cytometry, and RT-PCR was utilized for evaluating gene expression of the apoptotic cells. The results showed that the highest concentration (400 μg/mL) induced a significant decrease of cell viability in both HT29 and AGO cell lines. The percentage of apoptotic cells in the experimental group increased significantly when compared to the control group. The cell cycle was affected by an increased distribution of cells at G0/G1 phase and decreased occurrence of cells at S phase. The generation of reactive oxygen species (ROS) and the BAX/BCL2 ratio was found to be higher in the experimental group than that of the control group, respectively. Gallium verum at 400 μg/mL was found to decrease cell viability, induce apoptosis, increase BAX/BCL2 ratio and ROS production, and arresting cells at the G0/G1 phase.
... In another study, Schmidt indicated that the G. verum aqueous extract decrease the cellular rate of neck cancer cells at 48 hours [19]. Using the MTT method, Harika Atmaca reported the highest reduction in the survival rate of the methanolic extract of G. aparine on the normal cells in the range of 600-800 μg/mL at 72 hours [20]. Findings of this research also indicated that the methanolic extract of G. verum significantly induces apoptosis. ...
... Moreover, the results confirmed a significant increase in BAX/BCL2 gene expression and ROS production. In line with this finding, the results of a study demonstrated that the methanolic extract of the G. aparine plant at a concentration of 250 μg/mL caused primary and final apoptosis in normal cells [20]. ...
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Objectives: Although many studies have suggested the anticancer properties of Galium verum, there is still no accurate information regarding its side effects on normal cells. Accordingly, this study aimed to investigate the dual effects of the whole Galium verum methanolic extract on the normal human fibroblast cell line (AGO) cell line at different concentrations. Methods: The cell line was randomly divided into a control group and groups exposed to concentrations of 12.5 to 400 μg/mL. Extraction was performed by the maceration method. In addition, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was applied to measure cytotoxicity and flow cytometry. Further, BCL2 associated X (BAX) and B-cell lymphoma 2 (BCL2) genes were expressed by the real-time polymerase chain reaction to evaluate apoptosis and reactive oxygen species (ROS). Finally, data were compared between groups using a one-way analysis of variance. Results: A significant reduction was observed in the cell viability of 90% at a concentration of 400 μg/mL compared to the control. In comparison, a significant increase was reported in cell viability at concentrations of 25–200 μg/mL (P < 0.0001). Furthermore, there was a significant 2.87-time increase in apoptosis compared to the control group (P < 0.0001), but no significant differences were reported in cellular phases. ROS increased significantly by 5.7 times (P < 0.05), and a significant 80-fold increase was found in the BAX/BCL2 gene ratio (P < 0.05). Conclusion: The whole methanolic extract could lower the viability of human fibroblasts at 400 μg/mL and more by increasing apoptosis, thereby increasing BAX/BCL2 gene expression and ROS production. However, the extract exerted an increased effect on cell viability in a concentration-dependent manner on AGO and increased cell growth at concentrations less than 400 μg/mL, highlighting different effects of the whole extract on the AGO cell line
... In another study, Schmidt indicated that the G. verum aqueous extract decrease the cellular rate of neck cancer cells at 48 hours [19]. Using the MTT method, Harika Atmaca reported the highest reduction in the survival rate of the methanolic extract of G. aparine on the normal cells in the range of 600-800 μg/mL at 72 hours [20]. Findings of this research also indicated that the methanolic extract of G. verum significantly induces apoptosis. ...
... Moreover, the results confirmed a significant increase in BAX/BCL2 gene expression and ROS production. In line with this finding, the results of a study demonstrated that the methanolic extract of the G. aparine plant at a concentration of 250 μg/mL caused primary and final apoptosis in normal cells [20]. ...
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eeived heemer HPD PHPI Y eepted tnury PPD PHPPY ulished online wrh QHD PHPP Abstract Galium verum has been reported to have cytotoxic eects, however, the mechanism of Galium verum extract action on cancer cells is unclear in many cases. This study aimed to evaluate the cytotoxic of the whole plant methanolic extract on colon cancer (HT29) cells. colon cancer cells were randomly divided into control group and groups treated with 12.5, 25, 50, 100, 200 1nd 400 µg/mL of the extract for 72h. Plant extract was obtained through maceration. MTT assay was applied to test viability of cells.Apoptosis and cell-cycle, and intracellular levels of ROS were determined by ow cytometry. Bax and Bcl-2 expression levels were evaluated using RT-PCR. Treatment of HT29 cells with 400 µg/mL of whole plant extract resulted in signicant decreased viability and increased late apoptosis (P<0.0001), and a notable increase in cell number stoped in G0/G1 phase (P<0.05) and a notable decrease in cell number in S phase (P<0.05), and increased intracellular ROS levels (P<0.01). In conclusion, the high concentration of methanolic whole plant extract of Galium verum induces apoptosis through Bax-dependent pathway and increases intracellular levels of ROS in colon cancer (HT29) cells in vitro suggesting that the whole plant extract of Galium verum can play a role in treatment of colon cancer.
... Galium aparine possessed anticancer effect against human breast cancer cells (MCF-7), human colon cancer cells (Caco-2) and MCF-7 cell line (397)(398)(399)(400) . Galium verum inhibited the growth of neck cancer cell lines HLaC78 and FADU (401)(402)(403) . Glossostemon bruguieri possessedantiproliferative effects against hepatocellular (HCC), HepG2 and Hep3B cell lines (404)(405) . ...
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Cancer is the second leading cause of death worldwide. The alternative natural therapies are required as they considered to have less toxic side effects compared to current chemotherapy. In the current review Web Science, PubMed, Scopus and Science Direct, were searched to provide information about medicinal plants that have shown anticancer activity against various forms of cancer.
... Berberine, phenethyl isothiocyanate, resveratrol, gypenosides, lycopene, evodiamine, gallic acid, nobiletin, tricetin salvianolic acid A, pinosylvin, and extracts of Eclipta prostrata, Selaginella tamariscina, Leucaena leucocephala, Duchesnea indica, and Rubus idaeus inhibited the MAPK/ERK pathway (Ho et al., 2009;Hseu et al., 2011;Hsin et al., 2013;Schmidt et al., 2014;Chien et al., 2015;Ye et al., 2016;Yu et al., 2016;Chung HH. et al., 2017;Pang et al., 2017;Fang et al., 2018;. Additionally, genistein, triptolide, and Physalis angulata extract downregulated VEGF expression (Myoung et al., 2003;Hseu et al., 2011;Zhang et al., 2016). ...
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Cancer is a leading cause of death worldwide. A systematic and complicated mechanism is involved in the transformation of a normal cell into cancerous form. In human body, most cell functions are controlled by genes through cell growth, signal transduction, protein transcription, cell cycle, apoptosis and DNA repair. Proto-oncogenes are essential for the normal functioning of cells and generally found in all the cells that have distinct role in making proteins necessary for the cell growth, division and other functions. There are 40 different proto-oncogenes discovered to date including; RAS, HER2, Myc and cyclin D. When mutations occur, proto-oncogenes are converted into oncogenes, which lead to uncontrolled cell growth. Chemopreventive phytochemicals can block initiation or reverse the promotion stage of multistep carcinogenesis. They can also halt or retard the progression of precancerous cells into malignant ones. Plant-derived compounds have historically led to some of our most useful cancer drugs (e.g. paclitaxel, vincristine, etc). In recent days, the rate of plant-based drug discoveries is decreasing exponentially due to various factors such as lack of meticulous scientific evidences, dominance of other efficient therapies and other socio-economic issues. However, the efficacy of chemotherapy of cancer is limited by the development of drug resistance and metastatic disease. Plant-derived compounds have historically led to some of our most useful cancer drugs (e.g. paclitaxel, vincristine, etc). Moreover, plant bioactives are gaining increased attention lately for their therapeutic activity against many cancers (nearly 2000 publications per year) as they possess biological properties to inhibit the initiation, promotion and progression of cancer, therefore, culminate in the overall protection. Since advanced, recurrent and metastatic tumors are practically lethal and cannot be cured by any therapy, cancer chemoprevention of earlier lesions should be the definitive goal if we are to eradicate this deadly disease. To fulfil the unmet needs of cancer treatments, a scrupulous repurposing of plant-based phytochemicals such as chemical modification of the pharmacophore, development of delivery strategies including nanoformulations, and their uses in adjuvant settings need to be considered. The goal of this Research Topic is to highlight research on phytochemicals that show great potential not only in prevention but also in controlling the cancer by modulating the oncogenes. With the major theme of “phytochemicals for cancer treatments”, this Topic will feature articles on potential anti-cancer phytochemicals and agents against various cancers. The Research Topic will also highlight the articles on the molecular targets and mechanism of actions of chemopreventives. However, to maintain the stringent guidelines of journal and quality of publications, manuscripts on i) uncharacterized crude plant extracts, ii) anti-cancer activity demonstration in single cancer cell line, and iii) data without cytotoxic and cytostatic effects against non-malignant normal cells will not be considered. We welcome articles on delivery of chemopreventives to enhance efficacy and overcome dose related toxicity. We will also invite articles highlighting enhanced activity using structural modifications as well as uses of potent agents to chemosensitize drug resistant cells in adjuvant settings to combat cancer. This therefore comprehends new research articles and timely Reviews on all aspects of chemoprevention, mechanisms of plant bioactives, their uses in adjuvant settings and in secondary prevention, and finally their role as plant therapeutics.
... G. verum was found to be cytotoxic against all tested laryngeal carcinoma cell lines (Schmidt et al. 2014a). In additional study by Schmidt et al. (2014b), sublethal doses of G. verum aqueous extract acted as strong inhibitor on the motility of human head and neck cancer cell lines also, the fractional extract of petroleum ether had promising cytotoxic effects on colon cancer HT29 (Pashapour et al. 2020). Furthermore, Aslantürk et al. (2017) proved that G. aparine ethyl acetate and methanol extracts have cytotoxic and apoptotic inducing effect on MCF-7 and Caco-2 cancer cells. ...
... The ulcer index was assessed as follows: if the ulcer length is: ˂ 1.0 mm = 1point, between 1 and 2mm = 2point, ≥ 3mm = 3point. The sum of the scores was divided by 10 (magnification of the lens) to obtain the ulcer index in rats [28]. ...
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Gallium verum, lady's bedstraw is an herbaceous annual plant belonging to the genus Rubiaceae; it possessed antioxidant, cytotoxic for cancer cells, antimicrobial, protective and endocrine effects. This work aimed to investigate the curative effects of G. verum extract on gastric ulcers following absolute ethanol administration in the healthy rat. Eighteen rats were randomly divided into three main groups; rats were fastened for 24 hours before ethanol administration. All groups except control administered ethanol (5 ml/kg body weight; orally). The rats were administrated distilled water (Ulcer group) or G. verum extract (100mg/kg) one hour later. G. verum extract caused a significant decrease in ulcer index, gastric juice volume, malondialdehyde, and nitric oxide, while gastric juice pH, glutathione, glutathione-S-transferase, and catalase increased significantly. The histological lesion score showed a significant enhancement in group G. verum compared to the ulcer group that scored the highest pathological destruction score. Immunohistochemical markers of NF-κB p65 and TNF-α showed a significant decrease in G. verum group. G. verum extract is a promising treatment modality against gastric injury through its powerful antioxidant, acid neutralizing, healing promotion, and ant-inflammation effects.
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Introduction:The detection of viral ribonucleic acid using reverse transcription polymerase chain reaction (RT-PCR) within nasopharyngeal swabs is essential for the diagnosis of SARS-CoV-2 infection. Myelodysplastic syndrome (MDS) represents a heterogeneous group of myeloid neoplasms which are characterized by ineffective hematopoiesis, cytopenia, and risk of progression to acute myeloid leukemia. Hereby, we present a 62-year-old female patient with both MDS and Covid-19, who continued to have a positive nasopharyngeal RT-PCR test for 46 days until she passed away. Case Report: The patient, who was diagnosed with MDS in August 2020, was complaining of high fever. She had had two doses of Sinovac vaccine. Past medical history was remarkable for chemotherapy which was ended 1 month before. Bone marrow transplantation was planned within a week. Despite the i.v. ertapenem treatment, high fever persisted, and neutropenia occurred. White blood cell count (Wbc) was 0.43 x109/L. Blood and urinary cultures revealed extended spectrum beta-lactamase (ESBL) producing Escherichia coli. In addition, SARS-CoV-2 RT-PCR test on nasopharyngeal swab was performed, which was revealed to be positive. Antibiotic treatment was initiated. The patient was admitted to the intensive care unit. Afterwards, serial nasopharyngeal swabs were collected from the patient for 46 days until she passed away, and all were positive for Covid-19. Discussion: In the medical literature, long-term (longer than 3 weeks) SARS-CoV-2 RT-PCR positivity has been reported in mild or asymptomatic patients. There are a few number of publications dealing with MDS and Covid-19 in the English medical literature. Qing et al. reported MDS in a 30-year-old man with Covid-19, however, RT-PCR positive period was not mentioned. SARS-CoV-2 clearance period depends on immunity of the patients and Covid-19 infection may have a prolonged course in patients with hematological disorders such as MDS. References 1. Platzbecker U, Kubasch AS, Hom:er-Bouthiette C, Prebet T. Current challenges and unmet medical needs in myelodysplastic syndromes. Leukemia. 2021;35(8):2182-98. 2. Kim SM, Hwang YJ, Kwak Y. Prolonged SARS-CoV-2 detection and reversed RT-PCR results in mild or asymptomatic patients. Infect Dis (Lond). 2021;53(1):31-7. 3. Bhattacharya B, Kumar R, Meena VP, Soneja M, Singh A, Das R, et al. SARS-CoV-2 RT-PCR profile in 298 Indian COVID-19 patients: a retrospective observational study. Pathog Dis. 2021;79(1). 4. Xu W, Piper-Vallillo AJ, Bindal P, Wischhusen J, Patel JM, Costa DB, et al. Time to SARS-CoV-2 clearance among patients with cancer and COVID-19. Cancer Med. 2021;10(5):1545-9. 5. Qing X, Cai J, Rock A. Myelodysplastic syndrome in a 30-year-old man with coronavirus disease 2019 (COVID-19): a diagnostic challenge. Autops Case Rep. 2021;11:e2021274.
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Introduction: Faecal immunochemical test (FIT), faecal occult blood test (FOBT), flexible sigmoidoscopy (FS), Guaiac faecal occult blood test (gFOBT) and total colonoscopy (TC) can be used for colorectal cancer screening. However, Turkey and other European countries have different practices for colorectal screening tests, beginning-stopping ages and screening intervals. Method: World Cancer Report, which was published by World Health Organization (WHO) International Agency For Research on Cancer in 2020, has been throughly examined (1). Results: Colorectal cancer screening starting age is 40 in Bulgaria and Austria, whereas it is 50 in Belgium, Croatia, Denmark, France, Germany, Greece, Italy, Latvia, Lithuania, Portugal, Slovenia, Spain, Scotland, Serbia, Switzerland and Turkey, and it is 55 in Netherlands, Poland and Norway, whereas it is 60 in Estonia, Finland, Ireland, Sweden and United Kingdom. The stopping age for colorectal cancer screening is 60 in Bulgaria, 69 in Turkey, 80 in Austria and Switzerland. Interval period is 1 year in Bulgaria and Latvia, whereas it is 2 years in most of the European countries. Attendance to the colorectal screening programme is 1% in Hungary and Portugal, 2% in Poland, 8% in Greece and Spain,13% in Sweden, 30% in Turkey, 56% in United Kingdom and 61% in Austria. Conclusion: WHO reported no definitive colorectal cancer screening interval period for Turkey, however, Turkish Ministry of Health recommends FOBT every 2 years and TC in every 10 years (2). For gFOBT, dietary restrictions are required before testing (1). However, FIT is based on human haemoglobin antibodies, and thus it does not require a special diet before testing. This might explain why FIT is the preferred colorectal cancer screening test in most of the European countries. Keywords: cancer, colorectal, Europe, screening, Turkey References: 1- Wild CP, Weiderpass E, Stewart BW, editors (2020). World Cancer Report: Cancer Research for Cancer Prevention. Lyon, France: International Agency for Research on Cancer. Available from: http://publications.iarc.fr/586. Licence: CC BY-NC-ND 3.0 IGO. 2- Turkish Ministry of Health. Colorectal Cancer Screening Programme National Standards. Available from: https://hsgm.saglik.gov.tr/tr/kanser-tarama-standartlari/listesi/kolorektal-kanser-tarama-program%C4%B1-ulusal-standartlar%C4%B1.html
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A new ester derivative and 2 flavonoids have been isolated from the methanolic extract of aerial parts of Galium verum and purified by column chromatography, TLC and preparative TLC techniques. Those chemical structures were determined by spectroscopy techniques and were established as apigenin 4΄- O- rhamnoside(1), trans- 2΄, 3, 4, 4΄, 6΄- pentahydroxychalcone(2) and 2-ethoxypropyl 2-ethylhexanoate(3). The structure of the compounds was elucidated by UV, 1H- and 13C- NMR, HMBC, EI-MS and IR spectra. Key words Index: Galium verum, Rubiaceae, Methanolic extract, Flavonoid, apigenin 4΄- O- rhamnoside, trans- 2΄, 3, 4, 4΄, 6΄- pentahydroxychalcone.
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Galium verum, also known as Lady's Bedstraw, is a herbaceous perennial plant of the family Rubiaceae, native to Europe and Asia and used in traditional medicine as an anticancer medicine. It is used as a decoction in most traditional recipes, applied externally as well as internally. We produced a Galium verum decoction and applied it in vitro to chemosensitive (Hep-2 and HLaC79) and chemoresistant, P-glycoprotein-overexpressing (Hep2-Tax, HLaC79-Tax) laryngeal carcinoma cell lines. It could be demonstrated that Galium aqueous extract is cytotoxic for all cell lines. A detailed spheroid-based 3D invasion analysis of Hep2 and Hep2-Tax in semisolid collagen gels and on different extracellular matrix coatings was performed, which showed an inhibition of invasion by sublethal concentrations of Galium decoction and proved to be even more pronounced in the more aggressively invading chemoresistant Hep2-Tax cell line. Gelatinolytic activity of MMP-2 was downregulated in three of the four cell lines. Angiogenesis (endothelial tube formation) in contrast, was not affected by Galium aqueous extract. Gene expression array on HLaC79 and Hep2 cell lines treated with Galium decoction vs. untreated controls revealed no unique pathway activation patterns in these cells. Results are discussed with respect to the use of herbal drugs as a preventive and/or a concomitant therapeutic approach in head and neck cancer.
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For many techniques in the oncology of head and neck tumors large amounts of pure tumor cells are required. Although larynx and salivary gland tumors are common in man, no report exists on isolation and purification of tumor cells of which malignancy was proved. The present paper describes in vitro cultivation of living human malignant tumor cells from a larynx and a submandibular gland carcinoma. Carcinoma cells were freed from all non-tumor cells and cloned thus indicating that cultures contained only a single cell type. Transplantation of grown cells s.c. into athymic (nu/nu) mice induced rapidly growing tumors of which malignancy was demonstrated by histology.
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The results of an analysis of the iridoid and n-alkane patterns in twelve species of Asperula and Galium revealed similarities rather than differences between and within the genera. The ease with which artefacts can be produced from asperuloside limits the taxonomic usefulness of asperuloside-type iridoids. A sub-division of the genus Galium based on the dominant alkanes was not completely in accord with the existing division of the genus into sections. The alkane patterns are largely unaffected by plant age or geographical location. They do support some of the taxonomic views currently held concerning the inter-relationships of different species in the genus Galium.