Abstract. Background: Oral squamous cell carcinoma
(OSCC) is a challenging disease with a high mortality rate.
Natural products represent a valuable source for the
development of novel anticancer drugs. We investigated the
cytotoxic potential of essential oil from the leaves of a
medicinal plant, Levisticum officinale (lovage) on head and
neck squamous carcinoma cells (HNSCC). Materials and
Methods: Cytotoxicity of lovage essential oil was investigated
on the HNSCC cell line, UMSCC1. Additionally, we
performed pharmacogenomics analyses. Results: Lovage
essential oil extract had an IC50 value of 292.6 μg/ml. Genes
involved in apoptosis, cancer, cellular growth and cell cycle
regulation were the most prominently affected in microarray
analyses. The three pathways to be most significantly
regulated were extracellular signal-regulated kinase 5
(ERK5) signaling, integrin-linked kinase (ILK) signaling,
virus entry via endocytic pathways and p53 signaling.
Conclusion: Levisticum officinale essential oil inhibits
human HNSCC cell growth.
Oral squamous cell carcinoma (OSCC) is among the top ten
most commonly occurring carcinomas worldwide with a high
mortality rate. It was estimated that 35,720 people (25,240
men and 10,480 women) were diagnosed with cancer of the
oral cavity and pharynx in the United States of America in
2009 and that 7,600 would die of it (1). In spite of advances
in therapy, the 5-year survival rate for oral cancer patients has
remained at 50% over the past five decades (2). Various
multimodal therapy strategies including surgery, radiation and
chemotherapy determine the standard treatments for patients
with OSCC. However, the treatment of advanced-stage OSCC
is associated with morbidity and poor patient outcomes (3).
Therefore, alternative therapeutic strategies are called for.
As the majority of anticancer drugs are of natural origin,
natural products represent a valuable source for the
identification and development of novel treatment options for
cancer (4). During the past few decades, research has
focused on the health effects of phytochemicals and plant-
derived extracts. Plants of the genus Levisticum have been
attributed with anticancer activity (5, 6). Levisticum
officinale W. D. J. Koch (lovage) belongs to the family of
Apiaceae. The name lovage originates from the Latin word
ligusticus (meaning from Liguria, as the herb used to grow in
the Liguria region of northwest Italy).
Apiaceae is a large plant family with about 3,000 species.
This family includes many species with medicinal properties
which are frequently used in traditional medicine. A
common characteristic of this family is the presence of
bioactive secondary metabolites in all plant parts: essential
oils, polyphenols (flavonoids, phenolic acids), coumarins
(furano- and pyranocoumarins), saponins, alkaloids and
L. officinale has been used as a medicinal plant for
centuries due to its carminative, spasmolytic and diuretic
effect (8, 9). Clinically it is a potent diuretic (9). It is
approved by the German Commission E for use in lower
urinary tract infections and urinary gravel (10). The
antimycobacterial activity of L. officinale is rooted in its
Correspondence to: Professor Dr. Thomas Efferth, Department of
Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,
University of Mainz, Staudingerweg 5, 55128 Mainz, Germany. Tel:
+49 61313925751, Fax: +49 61313923752, e-mail: efferth@uni-
Key Words: Levisticum officinale lovage, oral cavity squamous cell
carcinoma OCSCC, head and neck squamous cell carcinoma
HNSCC, natural product, pharmacogenomics.
ANTICANCER RESEARCH 31: 185-192 (2011)
Chemical Composition and Antiproliferative Activity of
Essential Oil from the Leaves of a Medicinal Herb,
Levisticum officinale, against UMSCC1
Head and Neck Squamous Carcinoma Cells
SERKAN SERTEL1,2,3, TOLGA EICHHORN2,3, PETER K. PLINKERT1and THOMAS EFFERTH2,3
1Department of Otorhinolaryngology, Head and Neck Surgery, University of Heidelberg, Heidelberg, Germany;
2Pharmaceutical Biology (C015), German Cancer Research Center, Heidelberg, Germany;
3Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,
University of Mainz, Mainz, Germany
There are no published data concerning the clinical
application of L. officinale in otorhinolaryngology or
experimental data of cytotoxic activity in head and neck
squamous cell carcinoma (HNSCC). For this reason, the
objective of the present study was to investigate the
cytotoxicity of L. officinale leaf essential oil towards the
human HNSCC cell line, UMSCC1. To gain insight into the
molecular mode of action of Levisticum officinale leaf
essential oil towards the cancer cells, microarray-based mRNA
expression profiling was applied. Differentially expressed
genes were subjected to signaling pathway analysis.
Materials and Methods
Plant material. L. officinale was cultivated in Ross-on-Wye,
Herefordshire, UK. Lovage leaf oil was obtained from the leaves by
steam distillation (T=100˚C). The procedure was carried out by
Norfolk Essential Oils (Norfolk, UK).
The L. officinale leaf essential oil used in this study was provided
by REHAU AG + Co. (Rehau, Germany).
Cell culture. UMSCC1 cells were originally derived from a male
patient with a T2 N0 M0 squamous cell carcinoma of the oral cavity
(12). The UMSCC1 cells were cultured in McCoy’s medium
containing 10% fetal bovine serum (FBS) supplemented with 1%
antibiotic-antimycotic (100×), liquid (containing 10,000 units of
penicillin (base), 10,000 μg of streptomycin (base) and 25 μg of
amphotericin B/ml utilizing penicillin G (sodium salt), streptomycin
sulfate and amphotericin B as Fungizone®antimycotic in 0.85%
saline) purchased from Invitrogen GmbH (Karlsruhe, Germany).
The cells were maintained as monolayers in plastic culture flasks at
37˚C in a humidified atmosphere containing 5% CO2.
Gas chromatography. The oil extract was analyzed by gas
chromatography using an Agilent Technologies 6890N GC
instrument and a HP-5 capillary column (0.32 mm × 30 m; 0.25 μm
film thickness; Santa Clara, CA, USA). Gas chromatography was
performed by Dr. Otto GmbH (Wittenberge, Germany). The oil was
diluted with hexane (1:10).
XTT cytotoxicity assay. Cytotoxicity was assessed using the
5-carbo-xanilide inner salt (XTT) assay kit (Roche, Indianapolis,
USA), which measures the metabolic activity of viable cells (13,
14). The cytotoxicity of L. officinale leaf essential oil was
determined using the Cell Proliferation Kit II (Roche Diagnostics,
Mannheim, Germany). This test is based on the cleavage of the
XTT salt by ubiquitous dehydrogenases leading to the formation of
an orange formazan dye. The intensity of dye is commensurate to
the number of metabolic active cells. A stock solution of L.
officinale leaf essential oil was prepared in DMSO. A dilution
series ranging from 0.54 μg/ml to 18 mg/ml was prepared using
DMEM medium to perform the XTT test. The cells were suspended
to a final concentration of 1×105cells/ml. 100 μl of the cell
suspension were placed into the wells of a 96-well culture plate
(Costar, Corning, NY, USA). The marginal wells were filled with
100 μl of McCoy´s medium, in order to minimize evaporation
effects. Besides, wells filled with medium were required to
determine the background absorbance caused by non-metabolized
XTT. A row of wells containing cells was left untreated and another
row of wells containing cells was treated with 1 μl DMSO and this
served as the solvent control. Each concentration was tested in at
least two independent plates containing different batches of cells.
After incubation for 72 h with lovage leaf oil at 37˚C, 5% CO2in
a humidified atmosphere, XTT reagent was freshly prepared and
added to each well as specified by the manufacturer. XTT-labeling
reagent and electron-coupling reagent were mixed in a ratio of 50:1
and 50 μl of this mixture were added to each well of the 96-well
plate. The plates were incubated for about 3 h at 37˚C, 5% CO2in
humidified a atmosphere and read out after incubation.
Quantification of cell cytotoxicity was performed in an ELISA plate
reader (Bio-Rad, München, Germany) at 490 nm with a reference
wavelength of 655 nm. The mean absorbance values of the blank
wells were subtracted from the experimental values. The cytotoxic
effect of the treatment was determined as percentage of viability and
compared to untreated cells (13). The toxicity of compounds was
determined by means of the formula:
Absorbance of sample cells
Cell viability (%) = ×100
Absorbance of untreated cells
The simple ligand-binding module of Sigma plot software (version
10.0; Systat, San Jose, CA, USA)) was used for the analysis.
RNA isolation. The total RNA of the UMSCC1 cells was extracted
from the test samples using the RNeasy®Mini Kit (Qiagen Inc.,
Valencia, CA, USA) according to the manufacturer’s instructions to
obtain highly pure RNA. The isolated total RNA was re-suspended
in sample buffer provided by the manufacturer. The concentration
and quality of the total RNA were verified by electrophoresis using
the total RNA Nanochip assay on an Agilent 2100 Bioanalyzer
(Agilent Technologies, Santa Clara, CA, USA). Only the samples
with RNA index values greater than 8.5 were selected for expression
profiling. RNA concentrations were determined using the NanoDrop
spectrophotometer (NanoDrop Technologies, Wilmington, DE,
USA). All of the RNA samples were stored at –80˚C until used for
Probe labeling and Illumina Sentrix BeadChip array hybridization.
Biotin-labeled cRNA samples for hybridization on Illumina Mouse
Sentrix-8 BeadChip arrays (Illumina Inc., San Diego, CA, USA)
were prepared according to Illumina’s recommended sample
labeling procedure based on a previously published protocol (13).
In brief, 250 ng total RNA were used for complementary DNA
(cDNA) synthesis, followed by an amplification/labeling step (in
vitro transcription) to synthesize biotin-labeled cRNA according to
the MessageAmpII aRNA Amplification kit (Ambion, Inc., Austin,
TX, USA). Biotin-16-UTP was purchased from Roche Applied
Science, Penzberg, Germany. The cRNA was column purified
according to the TotalPrep RNA Amplification Kit, and eluted in 60
μl of water. The quality of cRNA was controlled using the RNA
NanoChip Assay on an Agilent 2100 Bioanalyzer and
spectrophotometrically quantified (NanoDrop).
Hybridization was performed at 58˚C, in GEX-HCB buffer
(Illumina Inc.) at a concentration of 50 ng cRNA/μl in an unsealed
wet chamber for 20 h. Spike-in controls for low, medium and
highly abundant RNAs were added along with mismatch control
and biotinylation control oligonucleotides. The microarrays were
ANTICANCER RESEARCH 31: 185-192 (2011)
washed twice in E1BC buffer (Illumina Inc.) at room temperature
for 5 min. After blocking for 5 min in 4 ml of 1% (w/v) blocker
casein in phosphate-buffered saline Hammarsten grade (Pierce
Biotechnology Inc., Rockford, IL, USA), array signals were
developed by 10 min incubation in 2 ml of 1 μg/ml Cy3-
streptavidin (Amersham Biosciences, Buckinghamshire, UK)
solution and 1% blocking solution. After a final wash in E1BC, the
arrays were dried and scanned.
Scanning and data analysis. For microarray scanning, a bead
station array scanner (Illumina, Inc.) was adjusted to a scaling
factor of 1 and the photomultiplier set to 430. Data extraction was
carried out for all the beads individually, and outliers are removed
when >2.5 MAD (median absolute deviation). All the remaining
data points were used for the calculation of the mean average
signal for a given probe, and the standard deviation for each
probe was calculated. The RNA from the UMSCC1 cells was
subjected to microarray analysis at least twice. The normalized
data obtained from the duplicated hybridizations were averaged
to obtain a final dataset. Reproducibility of the data was assessed
by calculating a percent error (standard deviation/mean ×100) for
each gene element.
The data were cropped to a final set of 678 elements by
eliminating genes with a differential expression under a single
standard deviation. Next, statistical significance was verified by
means of empirical Bayes t-test and the false discovery rate was
corrected with the Benjamini–Hochberg method. Ultimately, genes
with p>0.05 were discarded after allocation of p-values.
Data analysis was carried out with the Chipster analysis software
(http://chipster.csc.fi) for DNA microarray expression data by
normalization of the signals using the cubic spline algorithm after
background subtraction. Differentially regulated genes were defined
by calculating the standard deviation differences of a given probe
in a one-by-one comparison of samples or groups. For all the genes
scored, the fold change and p-values were determined.
Signaling pathway analysis. The data obtained from Chipster were
analyzed using the Ingenuity Pathways Analysis software (version
6.5) from Ingenuity Systems (Redwood City, CA, USA). This
software utilizes the raw image from Chipster in order to align the
image and determine expression values for all of the elements.
Statistical analysis. For statistical evaluations, the SPSS 10.0 for
Windows software package (SPSS Inc., Chicago, USA) was used.
The results were expressed as mean±S.E.M. (standard error of
means) of five independent experiments. In the Student’s t-test, p-
values <0.05 were considered to be significant.
Essential oil composition. The chemical composition of the
oil as assessed by gas chromatography is shown in Table I. L.
officinale leaf essential oil was characterized by 9
constituents (73.74 % of the total oil). Monoterpenes were
the major fraction, of which α-terpinyl acetate was the most
abundant compound (48.15 %).
Cytotoxicity. The cytotoxicity of L. officinale leaf essential
oil towards the human cancer cell line UMSCC1 as
determined by the XTT assay is shown in Figure 1. The
dose–response curve showed a steady rise in viability to
272.1% compared to the untreated controls at 0.18 mg/ml
and a subsequent rapid decrease in viability to 4.7% of
control at 0.54 mg/ml. The IC50value calculated from this
dose–response curve was IC50=292.6 μg/ml.
Differential gene expression. As determined by microarray
hybridization, a total of 678 genes were differentially
expressed after treatment with the IC50concentration of
lovage leaf oil (292.6 μg/ml). The positive fold changes after
log 2-transformation varied in a range of 1.63 to 1.27 and
Sertel et al: Antiproliferative Activity of Levisticum officinale Essential Oil
Table I. Chemical composition of Levisticum officinale leaf essential oil
as analyzed by gas chromatography.
Component Proportion (%)
Figure 1. Cytotoxicity of Levisticum officinale leaf essential oil towards
UMSCC1 cells as determined by the XTT assay. Mean dose–response
curve of three independent experiments are shown (error bars showing
standard error of means).
the negative fold changes varied in a range of 0.53 to 0.78.
The seven highest positive (up-regulated genes) and seven
lowest negative (down-regulated genes) fold changes are
shown in Table II.
Signaling pathway profiling. Ingenuity Pathway Analysis
(version 6.5) revealed that out of 64 functional groups of genes
listed by the software, 50 were regulated upon lovage essential
oil treatment at a significance level of p<0.05. The four
functional groups of genes (cell death, cancer, cellular growth
and proliferation, and cell cycle regulation) with the lowest p-
values are shown in Figure 2A. Among the 186 signaling
pathways analyzed by the pathway analysis software, the four
most significantly regulated pathways by lovage leaf oil were
the ERK5 signaling, ILK signaling, virus entry via endocytic
pathways and p53 signaling (p<0.05; Figure 2B). The genes
associated with these signaling pathways regulated upon
lovage leaf oil treatment are shown in Table III.
L. officinale ethanol extracts have shown apoptosis-inducing
activity in leukemia cell lines (15), but no other cytotoxic
activities have been reported. The principle constituents of L.
officinale have been shown to be polyacetylenes, i.e. 3(R)-
falcarinol and 3(R)-8(S)-falcarindiol (11). Polyacetylenes of the
bioactivities including anti-inflammatory (16), antimyco-
bacterial activity (11), antiplatelet-aggregatory (7), cytotoxic
(17-19) and antitumor activities (20). 3(R)-8(S)-Falcarindiol
was cytotoxic against murine L1210 leukemia cells, only (21).
have demonstrated many interesting
However, in the lovage leaf essential oil, falcarinol was not
detected by gas chromatography in the present study.
Therefore, we assume that constituents other than falcarinol
must have been responsible for the cytotoxic activity in the
present investigation. Although α-terpinyl acetate was the
major constituent identified, no explicit data concerning its
anticancer activity could be found in the literature, but it is
reasonable to propose that the cytotoxic activity of L. officinale
leaf essential oil towards the UMSCC1 cells was mainly due
to this constituent. Nevertheless, other constituents of the
essential oil may additionally contribute to cytotoxicity.
A surprising and unexpected, but repeatedly observed effect
was that subtoxic concentrations of lovage essential oil
stimulated proliferation and viability. At higher concentrations,
dose-dependent cytotoxic effects were found. Comparable
effects have previously been reported for standard anticancer
agents such as doxorubicin (22, 23). A proliferation-
stimulating effect of otherwise cytotoxic compounds can be
interpreted as a rescue mechanism. At low subtoxic
concentrations, cancer cells escape detrimental stimuli by the
induction of proliferation, while at higher concentrations this
defense mechanism is overridden by cytotoxic effects. The
possibility that cytotoxic compounds such as doxorubicin or
lovage leaf essential oil may exert tumor-promoting activity at
low concentrations should also be considered.
The differentially regulated genes after lovage leaf essential
oil treatment might help explain the effects on cancer cell
growth. CDCA7 acts as a c-Myc (a transcription factor and
major proto-oncogene) responsive gene, and behaves as a direct
c-Myc target gene. Overexpression of this gene has been found
to enhance the transformation of lymphoblastoid cells, and
ANTICANCER RESEARCH 31: 185-192 (2011)
Table II. Genes down- or up-regulated after treatment of UMSCC1 cells with Levisticum officinale leaf essential oil.
Probe ID SymbolDescription Fold change
Ferritin, heavy polypeptide-like 2
Casein kinase 1, gamma 2
Signal recognition particle 14 kDa (homologous Alu RNA binding protein) pseudogene 1
RNA, small nucleolar
Chromosome 19 open reading frame 43
Coiled-coil domain containing 94
Dishevelled-associated activator of morphogenesis 1
ATP-binding cassette, sub-family A (ABC1), member 1
Cell division cycle associated 7
FERM domain containing 6
Myeloid-associated differentiation marker
Ubiquitin-like with PHD and ring finger domains 1
Pyruvate kinase, muscle
contributes to c-Myc-mediated tumorigenesis (24). In the
present case, the up-regulated gene CDCA7 might explain the
increase in viable cancer cells at low concentrations of lovage
leaf essential oil. ABCA1 was also an up-regulated gene,
encoding a protein belonging to the ATP-binding cassette
(ABC) transporters. They are involved in the active transport of
phospholipids, ions, peptides, steroids, polysaccharides, amino
acids, bile acids, pharmaceutical drugs and other xenobiotic
compounds (25). The protein encoded by ABCA1 is a lipid
transporter that plays an important role in cholesterol efflux and
thereby prevents toxicity associated with cholesterol overload
(24). Gene silencing of ABCA1, in resistant M14 melanoma
sensitized M14 cells to the apoptotic effect of curcumin.
Moreover, ABCA1 silencing alone also induced apoptosis and
reduced p65 expression (26). Thus, it is plausible that up-
regulation of ABCA1 might also explain the increase in viable
cancer cells at low concentrations of lovage leaf essential oil.
Furthermore, overexpression of ABCA1 might activate
mechanisms of drug resistance and this might contribute to the
more aggressive growth of multiple drug-resistant carcinomas.
UHRF1 is another up-regulated gene that might assist the
cytotoxicity induced by lovage leaf essential oil treatment.
UHRF1 encodes an ubiquitin ligase that plays a major role in
the G1/S transition and functions in the p53-dependent DNA
damage checkpoint, expression of this gene is induced in
response to mitogenic stimulation (24, 27).
THBS1 was also an up-regulated gene. THBS1 encodes
adhesive glycoproteins that mediate cell-to-cell and cell-to-
matrix interactions. It is a potent inhibitor of tumor growth and
angiogenesis (24). These data indicated that the effect of lovage
leaf essential oil treatment on the UMSCC1 cells was regulated
by multiple genes and that a complex regulatory network
contributed to the biological activity. The down-regulated
genes did not reveal any association with anticancer activity.
As yet, no data on possible mechanisms of lovage
anticancer activity have been reported. Within the present
signaling pathway analyses, ERK5 signaling, ILK signaling,
virus entry via endocytic pathways and p53 signaling were
the four most significantly regulated pathways by L.
officinale essential oil treatment. ERK5, belonging to the
MAPK family, is expressed in a variety of tissues and is
activated by a range of growth factors, cytokines and cellular
stresses. ERK5 signaling is important in endothelial cells for
preventing apoptosis, regulating tumor angiogenesis and cell
migration (28). The influence of L. officinale in ERK5
signaling might offer a novel target for anticancer therapy as
an anti-angiogenic agent.
The oncogenic protein kinase, ILK functions as a tumor
suppressor protein in vitro and in vivo in rhabdomyosarcoma
(29). It is therefore conceivable that involvement of L.
officinale might suppress tumorigenesis. No evidence of a link
to cancer was found for the virus entry via endocytic pathway.
Interestingly, the L. officinale essential oil treatment
significantly regulated one of the most important tumor
suppressor pathways, namely p53 signaling in the UMSCC1
cells. The tumor suppressor protein p53 is activated, e.g.
upon DNA damage, nucleotide depletion or hypoxia and
initiates transcription of pro-apoptotic and cell cycle arrest-
inducing target genes (30). Lovage might regulate the
activity of p53 by post-translational modifications.
In conclusion, L. officinale leaf essential oil has the ability
to inhibit tumor cell growth of the HNSCC cell line,
UMSCC1, at high concentrations, whereas it promotes
proliferation at subtoxic doses. Genes involved in ERK5
Sertel et al: Antiproliferative Activity of Levisticum officinale Essential Oil
Figure 2. A: Functional groups of genes and B: Signaling pathways
regulated upon Levisticum officinale leaf essential oil treatment in
UMSCC1 cells. The evaluation of differentially expressed genes was
performed using the Ingenuity Pathway Analysis software.