ANTICANCER RESEARCH 33: 3799-3806 (2013)

Multiple Anticancer Effects of Damsin

and Coronopilin Isolated from
Ambrosia arborescens on Cell Cultures
RODRIGO VILLAGOMEZ1,2, GLORIA C. RODRIGO3,4, ISIDRO G. COLLADO5,
MARCO A. CALZADO6, EDUARDO MUÑOZ6, BJÖRN ÅKESSON4,7,
OLOV STERNER2, GIOVANNA R. ALMANZA1 and RUI-DONG DUAN8

1Chemical
Research Institute and 3Molecular Biology and Biotechnology
Institute, Major University of San Andres, La Paz, Bolivia;
2Center of Analysis and Synthesis, 4Biomedical Nutrition, Pure and Applied Biochemistry, and
8Gastroenterology and Nutrition Laboratory, Institution of Clinical Sciences, Lund University, Lund, Sweden;
5Department of Organic Chemistry, Cadiz University, Cadiz, Spain;
6Maimónides Institute for Biomedic Research of Córdoba, Córdoba University, Córdoba, Spain;
7Department of Clinical Nutrition, Skåne University Hospital, Lund, Sweden

Abstract. Terpenoids in plants are important sources for The rates of colorectal cancer have increased significantly
drug discovery. In this study, we extracted damsin and during the past decades, reaching the third position of the
coronopilin, two sesquiterpene lactones, from Ambrosia most common types of cancer in the world. The progression
arborescens and examined their anticancer effects on cell of normal colonic epithelium to the malignant phenotype
cultures. Damsin and coronopilin inhibited cell proliferation, occurs stepwise through a series of genetic events, which
DNA biosynthesis and formation of cytoplasmic DNA histone involve mutations of adenomatous polyposis coli (APC)
complexes in Caco-2 cells, with damsin being more potent gene, Kirsten rat sarcoma virus oncogen (K-RAS), deleted in
than coronopilin. Further studies using the luciferase colorectal carcinoma (DCC) and p53 (1). The genetic events
reporter system showed that damsin and coronopilin also affect many signal transduction pathways such as β-catenin,
inhibited expressions of nuclear factor-κB (NF-κB) and cyclooxygenase-2 (COX-2) (2), phosphoinositol-3-kinase-
signal transducer and activator of transcription-3 (STAT3), Akt (PI3K/AKT) (3), nuclear factor-κB (NF-κB) and signal
indicating that these sesquiterpenes can interfere with NF- transducer and activator of transcription-3 (STAT3) pathways
κB and STAT3 pathways. Finally, we examined the effects of (4), which regulate cell proliferation and apoptosis.
two synthetic dibrominated derivatives of damsin, 11α,13- Disturbances of the balance of cell proliferation and
dibromodamsin and 11β,13-dibromodamsin. While apoptosis are considered as an important factor leading to
bromination appeared to weaken the antiproliferative effects cancer development. Many drugs exert their anticancer
of damsin, the β epimer had strong inhibitory effects on effects by inhibiting cell proliferation and/or promoting
STAT3 activation. In conclusion, the sesquiterpene lactones apoptosis via various signal transduction pathways. Among
damsin and coronopilin have inhibitory effects on cell these anticancer drugs, about 67% are of natural origin,
proliferation, DNA biosynthesis and NF-κB and STAT3 indicating that medicinal herbs are important sources for
pathways, thus being potentially important for discovery of discovery of drugs against cancer (5).
drugs against cancer. Terpenoids and their derivatives are phytochemicals. Due
to their wide distribution in nature and multiple biological
functions (6, 7), they may have particular importance for use
in developing novel drugs. For example, ursolic acid and
Correspondence to: Rui-Dong Duan, MD, Ph.D., Biomedical boswellic acids, two extensively studied triterpenoids, have
Center B11, Gastroenterology and Nutrition Lab, Institution of been shown to inhibit cell proliferation and stimulate
Clinical Sciences, Lund University, S-221 84 Lund, Sweden. Tel:
apoptosis of cancer cells (8, 9). Another group of natural
+46 462220709, e-mail: Rui-dong.duan@med.lu.se
terpenoids, sesquiterpene lactones (SLs), which are 15-
Key Words: Ambrosia arborescens, damsin, coronopilin, carbon compounds consisting of three isoprene units and a
chemoselective bromination, sesquiterpenes, cell proliferation, lactone group, were recently proposed as a new source of
STAT3, NF-κB, apoptosis. drugs with potential values in the treatment of inflammation

0250-7005/2013 $2.00+.40 3799

 ANTICANCER RESEARCH 33: 3799-3806 (2013)

and cancer (10-12). The SLs are well-known alkylating

agents of cysteine residues in proteins through a Michael
addition of their characteristic α-methylene-γ-lactone moiety
(11-13). The NF-κB pathway is considered to be one of the
most interesting targets of SLs (13, 14).
In this study we extracted two SLs, damsin and coronopilin
(Figure 1) isolated from Ambrosia arborescens (15-17), and
examined their biological effects on colon cancer cell lines and
their influence on expression of NF-κB and STAT3. To evaluate
the role of the α-methylene-γ-lactone moiety in the
compounds, the effects of these two natural products were
compared against those of two synthetic brominated
derivatives, 11α,13-dibromodamsin and 11β,13-
dibromodamsin (Figure 1).

Materials and Methods

Materials. Caco-2 and HeLa cells were purchased from the

American Tissue Culture Collection (Rockville, MD, USA). A
luciferase activity assay kit was obtained from Berthold
Technologies (Bad Wildbad, Germany). Cell death detection kit and
the cell proliferation reagent (WST-1) were obtained from Roche Figure 1. Sesquiterpene lactones from Ambrosia arborescens and
Diagnostics GmbH (Mannheim, Germany). [3H]Thymidine was brominated derivatives. Damsin (1), coronopilin (2), 11α,13-
purchased from American Radiolabeled Chemicals (St. Louis, MO, dibromodamsin (3) and 11β,13-dibromodamsin (4).
USA). The cell culture mediums used were obtained from Sigma-
Aldrich (Stockholm, Sweden). Luciferase activity was measured
using an Autolumat LB 9501 (Berthold Technologies, Bad Wildbad,
Germany). All other chemicals were obtained from commercial and Na2CO3 (855 mg, 8.07 mmol) was added. After 20 h, 30 ml of
suppliers of analytical grade. High resolution mass spectrometry diethyl ether was added and the resulting white precipitate was
(HMRS) electrospray ionization (ESI) spectra were recorded with a filtered, and then the solvent was evaporated under vacuum and the
micromass quadrupole-time of flight (Q-TOF) micro spectrometer. crude reaction products were separated by column chromatography
Nuclear magnetic resonance (NMR) spectra (in CDCl3) were with a gradient of petroleum ether (40-60˚):ethyl acetate yielding
recorded with a Varian Gemini at 300 Mhz (1H) and at 75 MHz 11α,13-dibromodamsin (122 mg, 74%) and 11β,13-dibromodamsin
(13C). Chemical shifts are given in ppm relative to the residual (20 mg, 12%).
CHCl3 in CDCl3 (7.25 ppm 1H and 77.00 ppm 13C). All flash
chromatography was performed with 60 Å 30-75 μm Silica gel. Culture of Caco-2 cells. The Caco-2 cells were cultured in
Thin layer chromatography (TLC) analyses were carried out on Dulbecco’s minimal essential medium (DMEM) with L-glutamine,
Silica Gel 60 F254 plates (Merck, Darmstadt, Germany). containing 100 IU/ml penicillin, 10 μg/ml streptomycin and 10%
(v/v) heat-inactivated fetal calf serum (FCS) as previously described
Isolation of damsin and coronopilin. Damsin and coronopilin were (18). They were maintained at 37˚C in a humidified incubator
isolated from A. arborescens. The dry plant (164 g) was extracted containing 95% air and 5% CO2. For assays of cell proliferation,
in a Soxhlet extractor with petroleum ether (20-40˚C). The crystals DNA replication and apoptosis, the cells were detached with 0.02%
of damsin were filtered directly from the petroleum ether extract EDTA/0.05% trypsin resuspended to a density of 1×105/ml and
and purified using a silica gel 60 column chromatography by elution seeded into 96-well plates. The phytochemicals tested were
with petroleum ether (40-60˚C): ethyl acetate (7:3 v/v), yielding dissolved in dimethyl sulfoxide (DMSO). The concentration of
1.17 g (0.7%) of pure damsin in the fractions 6-10. The remaining DMSO in the cell culture was 0.1%, and medium containing 0.1%
plant material was extracted with dichloromethane in the Soxhlet DMSO only was used as the control.
extractor, producing 6.35 g of crude extract. Then 1.04 g of the
extract were fractionated using Sephadex LH-20 column Cell proliferation assay. The cell proliferation of Caco-2 cells was
chromatography by elution with methanol:dichloromethane (1:1 v/v) assayed by use of the reagent WST-1 (4-[3-84-iodophenyl)-2-(4-
followed by a silica gel 60 column chromatography with petroleum nitrophenyl)-2H-5-tetrazoliol]-1,3-benzene disulfonate), which is
ether (40-60˚C):ethyl acetate (3:2 v/v) as eluent, yielding 0.20 g metabolized by mitochondrial dehydrogenases to a formazan
(0.7%) of coronopilin in the fractions 11-20. The purity of the staining dark red (19). The formation of formazan is proportional
compounds was confirmed by TLC and NMR. to the number of viable cells. Briefly, 2×104 Caco-2 cells in 200 μl
medium were incubated with the compounds tested at
Bromination of damsin with trimethylammonium perbromide concentrations from 25 to 100 μM for 24 h, followed by adding
(TMPAP)/Na2CO3. To a solution of damsin (100 mg, 0.40 mmol) in 20 μl WST-1 reagent. After incubation for 1 h, the optical density
dioxane (10 ml), a solid mixture of TMPAP (191 mg, 0.51 mmol) was read at 405 nm using 655 nm as background. The cell

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 Villagomez et al: Anticancer Effects of Damsin and Coronopilin

proliferation rate was expressed as a percentage of the control. All compositions were confirmed by 1H NMR and 13C NMR
determinations were carried out in triplicate in three different with comparison of authentic samples and data in the
experiments. literature (15, 22, 23). The chemoselective bromination of
damsin was carried out with TMPAP in basic medium, only
DNA replication. DNA synthesis was measured by the incorporation
of [3H]thymidine as described previously (20). Briefly Caco-2 cells affecting the double bond of the lactonic ring, giving the
were pre-incubated for 20 h with the compounds at different epimers 11α,13-dibromodamsin and 11β,13-dibromodamsin
concentrations. [3H]Thymidine (0.5 μCi/well) was added, followed with an overall yield of 86% with an epimeric excess of 62%
by incubation for 4 h. Cells were washed with phosphate buffered for 11α,13-dibromodamsin (Figure 1). Other brominating
saline, treated with 5% trichloroacetic acid for 20 min at 4˚C, and conditions using TMPAP/p-toluenesulphonic acid, bromine
fixed with methanol. The cells were lysed in 0.5 M sodium and N-bromosuccinimide did not have the desired selectivity
hydroxide and 0.1% sodium dodecyl sulphate for 15 min and the
and the α-carbonyl position was also affected. The 1H NMR
radioactivity in the lysate was counted by liquid scintillation. DNA
synthesis was expressed as a percentage of the non-treated cells. spectrum showed the nucleophilic 1,2 addition of bromine
by the perbromide reagent to the double bond (Table I), the
Assay of apoptosis. Apoptosis was assayed by a cell death detection signals of the exocyclic double bond of damsin (24-26) were
ELISAPLUS kit (Roche Diagnostics GmbH) (9) according to the shifted to high fields in both products as doublets. This was
instructions. Briefly, 1×104 cells were seeded in 96-well plates and confirmed by HRMS with an ion (M+Na)+ at 428.9677 for
treated with damsin and coronopilin at different concentrations. The 11α,13-dibromodamsin and an ion (M+H)+ at 406.9857 for
cells were lysed and centrifuged and the formation of cytoplasmic
11β,13-dibromodamsin. The absolute stereochemistry of
histone-associated DNA fragments was measured by a plate reader
at 415 nm using 490 nm as background. The enrichment factor was each epimer was established through 1H NMR analysis
calculated as fold increase comparing with that of control (cells (Table I), nuclear overhauser effect (NOE) difference
without treatment). experiments and comparison with computational models
(Figure 2). In the case of 11α,13-dibromodamsin, the α
NF-κB and STAT3 luciferase assays. To study the NF-κB-dependent disposition of bromine at C-11 was inferred from the
transcription, the 5.1 Jurkat cell line (21) was used. The cells are deshielded chemical shift (+0.45 ppm) observed in the signal
stably transfected with luciferase gene driven by the HIV-1-LTR
of H-6 (Table I), with respect to the same signal in
promoter, which contains two NF-κB-binding sites that are required
to induce the transactivation. The cells were preincubated for 30 min compound 11β,13-dibromodamsin. The NOE correlations
with the compounds and stimulated with tumor necrosis factor-α and the interatomic distances are shown in Figure 2.
(TNFα) (5 ng/ml) for 6 h. Then the cells were lysed in 25 mM
Tris–phosphate, pH 7.8, containing 8 mM MgCl2, 1 mM Inhibitory effect of damsin and coronopilin on the growth of
dithiothreitol (DTT), 1% Triton X-100, and 7% glycerol. Luciferase Caco-2 cells. After incubation of Caco-2 cells with damsin
activity was measured following the instructions of the assay kit. and coronopilin at different concentrations, we found that
Basal luciferase activity was subtracted from that of TNFα-induced
damsin dose-dependently inhibited the cell proliferation,
activity. To determine STAT3 transcriptional activity, HeLa cells
were transiently transfected with the plasmid p4xM67-tk-Luc, in with 36% inhibition occurring at 50 μM and 45% at 100
which the luciferase gene is driven by a promoter containing four μM. Compared to damsin, coronopilin was less effective and
copies of the STAT binding site. Twenty-four hours after 100 μM coronopilin only led to 19% inhibition (upper panel
transfection, the cells were stimulated with interferon gamma of Figure 3). We further examined the effects of the
(IFNγ) (10 μM) for 6 h. The luciferase activity was measured as compounds on DNA replication and found that both damsin
described above. Basal luciferase activity was subtracted from and coronopilin strongly reduced [3H]thymidine
IFNγ-induced activity. The inhibitory activity of the compounds is
incorporation into DNA in a dose-dependent manner (lower
expressed as a percentage of those treated with only TNFα and
IFNγ, respectively. Half maximal inhibitory concentration (IC50) panel of Figure 3). The effect of damsin was also greater
activity was calculated using SigmaPlot software (Systat Software than that of coronopilin as 50 μM damsin almost completely
GmbH, Germany). blocked DNA biosynthesis, whereas a higher concentration
(100 μM) of coronopilin was required to achieve similar
Results and Discussion effects. Since DNA synthesis occurs in the S phase and
mitosis in the M phase of the cell cycle, our results indicate
Isolation and derivatization of SLs from Ambrosia that both damsin and coronopilin have inhibitory effects on
arborescens. The dried leaves of Ambrosia arborescens were the transition of the cells from the S to the M phase, thus
extracted continuously with petroleum ether. Crystallized blocking cell proliferation. It is well-known that cyclins and
damsin was purified using silica gel chromatography. different cyclin-dependent kinases (CDKs) are key
Coronopilin was isolated from the dichloromethane extract molecules that regulate the transition of the cell cycle.
using molecular exclusion chromatography (Sephadex LH- Whether damsin and coronopilin inhibit the expression of
20) and purified by silica gel chromatography. Both damsin cyclins and activities of CDKs is an interesting question for
and coronopilin were obtained as white crystals, and the further investigation.

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 ANTICANCER RESEARCH 33: 3799-3806 (2013)

Table I. Nuclear magnetic resonance (NMR) spectroscopic data for 11α,13-dibromodamsin and 11β,13-dibromodamsin (300 MHz for 1H NMR and
75 MHz for 13C NMR) in CDCl3.

Pos 11α,13-dibromodamsin 11β,13-dibromodamsin

δ 1H (J in Hz) δ 13C NOE δ 1H (J in Hz) δ 13C NOE

1 2.04 m 45.3 d 1.92 m 48.6 d

2a 2.07 m 24.1 t 2.03 m 23.7 t
2b 1.86 m 1.86 m
3a 2.53 ddd (19.1, 7.4, 1.5) 34.7 t 2.48 ddd (18.9, 8.4, 1.0) 35.9 t
3b 2.24 dd (19.5, 10.2) 2.24 m
4 220.5s 216.9
5 53.8 s 54.3
6 4.96 dd (5.1, 1.0) 80.8 d 4.51 d (9.6) 83.4 d
7 2.72 ddd (1.5, 10.0, 5.0) 56.1 d H-6, H-13a, 3.29 ddd (12.0, 9.7, 5.1) 44.8 d H-6, H-13a,
H-13b, H-1, H-13b, H-8α,
H-8α, H-8β H-9α, H-8β,
H-9β
8β 1.78 m 19.2 t 1.73 m 25.9 t
8α 1.65 m 2.11 m
9a 1.93 m 36.8 t 1.64 m 33.8 t
9b 1.68 m 1.93 m
10 2.23 m 34.4 d 2.22 m 34.1 d
11 61.8 s 62.3
12 170.6 s 172.0
13a 3.92 d (12.6) 35.4 t H-13b 4.22 d (103) 35.7 t H-13b
13b 3.68 d (12.6) H-13a, 3.80 d (10.3) H-7, H-8α
H-8α, H-8β
14 1.07 d (7.6) 16.6 q 1.12 d (7.5) 14.8 q
15 1.18 s 16.0 q 1.21 s 13.9 q

Pos: Position; NOE: Nuclear Overhauser Effect.

Figure 2. Nuclear Overhauser Effect (NOE) correlations used to confirm the relative stereochemistry of compounds 11α,13-dibromodamsin (3) and
11β,13-dibromodamsin (4). The interatomic distances are specified in parentheses.

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 Villagomez et al: Anticancer Effects of Damsin and Coronopilin

Figure 4. Effect of damsin (1) and coronopilin (2) on apoptosis. Caco-

2 cells were incubated with the compounds for 6 h and the formation
of DNA histone complexes was determined. The results are the means
(±SD) of three separate experiments performed in triplicate.

shown). The results do not indicate a strong pro-apoptotic

effect of damsin and coronopilin, and their anticancer effects
may mainly be mediated by their inhibitory effects on cell
proliferation.

Inhibitory effects on NF-κB and STAT3 activities by damsin

and coronopilin. NF-κB and STAT3 are two important
pathways involved in cell survival and proliferation of cells.
NF-κB signalling is important for expression of several
inflammatory genes and anti-apoptotic genes. NF-κB has
been considered to be an important link between
Figure 3. a: Antiproliferative activity of damsin (1) and coronopilin (2) inflammation and cancer. In order to gain insight into their
on Caco-2 cells. Results are the means (±SD) of three independent mechanisms of action, the effects of damsin and coronopilin
experiments performed in triplicate. The p-values (Friedman test) for on NF-κB and STAT3 transcriptional activities were
the effect of (1) is 0.022, and for that of 2, 0.048. b: Effects of 1 and 2
on DNA synthesis in Caco-2 cells are expressed as a percentage of the
investigated in Jurkat cells and HeLa cells using the luciferase
control. The results are the means (±SD) of three separate experiments reporter system. The expression of NF-κB and STAT3 were
performed with sextuplicate samples. The absolute thymidine induced by TNFα and IFNγ, respectively. We found that
incorporation in control cell was 0.4 fmol/h/106 cells. damsin and coronopilin significantly inhibited the TNFα-
induced expression of NF-κΒ (Figure 5) and IFNγ induced
expression of STAT3. The IC50 values for the two compounds
are shown in Table II. In both cases, the IC50 values of
Effects of damsin and coronopilin on apoptosis. Proliferation damsin were lower than those of coronopilin, indicating again
and apoptosis are two physiological processes affecting cell that damsin is more effective than coronopilin. NF-κB
fates. Disturbance of the balance of cell proliferation and signalling can be inhibited by multiple mechanisms, including
apoptosis predisposes for cancer development. We therefore inhibition of inhibitor of NF-κB kinase (IKK) activity,
examined whether damsin and coronopilin affected apoptosis inhibitor of NF-κB (IκB) phosphorylation, IκB degradation,
by measuring the formation of cytoplasmic DNA histone and DNA binding. Some SLs, such as ergolide, artemisinin,
complexes, a biological marker of apoptosis. After treating costunolide and zerumbone, have been reported to have
the cells with the compounds for 6 h, a mild increase (about inhibitory effects on the NF-κB pathway (13). Our findings
40%) of cytoplasmic DNA histone complexes by damsin was that both NF-κB and STAT3 could be inhibited by damsin
observed, but the dose-dependent pattern was poor (Figure and coronopilin are interesting, as NF-κB and STAT3 are
4). Coronopilin was less effective. Longer stimulation of the constitutively activated in neoplastic cells in response to
cells for 24 h did not lead to positive results (data not autocrine and paracrine factors that are produced within the

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 ANTICANCER RESEARCH 33: 3799-3806 (2013)

Table II. Half -maximal inhibitory concentration (IC50; μM) values of

damsin, coronopilin and dibrominated derivatives 11α,13-
dibromodamsin and 11β,13-dibromodamsin toward nuclear factor κB
(NF-κB) and signal transducer and activator of transcription 3 (STAT3)
activation.

Compound NF-κB STAT3

Damsin 7.2 12.4

Coronopilin 10.1 18.3
11α,13-dibromodamsin >25a >25a
11β,13-dibromodamsin >25a 9.7

aInhibition not detected at the concentration tested.

However, for the effects on STAT3 the different effects cannot

be explained in the same way, and rather highlight the
Figure 5. Inhibitory activity of damsin (1), coronopilin (2), 11α,13-
dibromodamsin (3) and 11β,13-dibromodamsin (4) on nuclear factor κB
importance of stereoselective bromidation of the α-methylene-
(NF-κB) expression in luciferase-transfected 5.1 cells. The cells were γ-lactone moiety for the specific biological activities.
stimulated with tumor necrosis factor-α (TNFα) in the presence or In summary, we identified two SLs, damsin and coronopilin,
absence (0*) of the compounds. 0 is the negative control. that have antiproliferative activity towards the Caco-2 cell line.
Damsin was the most active. The antiproliferative effects may
be related to its inhibitory effects on NF-κB and STAT3
pathways. In addition, the inhibitory effect of damsin on
tumour microenvironment (27). STAT3 and NF-κB in a STAT3 can be significantly enhanced by dibromination to form
cooperative manner regulate several pro-inflammatory genes, 11β,13-dibromodamsin.
such as interleukin 6 (IL-6), IL-11, chemokines, growth
factors and COX2, that are crucial for maintaining a pro- Acknowledgements
carcinogenic inflammatory environment (6, 7). Therefore,
STAT3 and NF-κB establish a feed-forward loop between The study was supported by the Swedish International Development
cancer cells and the tumour microenvironment. The inhibitory Agency to a joint project on Plant Biodiversity between the Major
University of San Andrés, La Paz, Bolivia and Lund University, Sweden.
effects of damsin and coronopilin on both NF-kB and STAT3
Support was also obtained from the EU NoE ECNIS (513943) and its
indicate that they could be candidates for developing potent successor ECNIS2. R. Villagomez is grateful to the Alpha Network
anticancer drugs. EULADIV supported by the EC for a grant to stay in Cadiz, Spain.

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