Chemico-Biological Interactions 242 (2015) 415e421

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Cytotoxic and apoptotic effects of leptocarpin, a plant-derived

sesquiterpene lactone, on human cancer cell lines
Claudia Bosio a, Giacomo Tomasoni a, Rolando Martnez a, *, Andre

s F. Olea b,
He
ctor Carrasco b, Joan Villena c, **
a
Departamento de Qumica, Facultad de Ciencias Exactas, Universidad Andr
~ a del Mar, 2520000, Chile
es Bello, Quillota 910, Vin
b
Instituto de Ciencias Qumicas Aplicadas, Facultad de Ingeniera, Universidad Auto
noma de Chile, Llano Subercaseaux 2801, San Miguel, Santiago, Chile
c
Centro de Investigaciones Biom
edicas (CIB), Escuela de Medicina, Universidad de Valparaso, Av. Hontaneda 2664, Valparaso, 234000, Chile

a r t i c l e i n f o a b s t r a c t

Article history: Sesquiterpene lactones have attracted much attention in drug research because they present a series of
Received 3 July 2015 biological activities such as anticancer, antifungal, anti-inammatory, antimicrobial and antioxidant.
Received in revised form Leptocarpin (LTC) is a sesquiterpene lactone isolated from a native Chilean plant, Leptocarpha rivularis,
18 October 2015
which has been widely used in traditional medicine by Mapuche people. Previous work has demon-
Accepted 4 November 2015
Available online 10 November 2015
strated that LTC decreases cell viability of cancer cell lines. In this contribution, we analyze the mech-
anism of LTC cytotoxicity on different cancer cell lines. The results show that in all cases LTC induces an
apoptotic process and inhibition of NF-kB. Apoptosis has been conrmed by observing condensation of
Keywords:
Sesquiterpene lactones
chromatin, nuclear fragmentation, release of cytochrome c into the cytosol, and increasing of caspase-3
Leptocarpin activity. It has also been found that LTC is an effective inhibitor of NF-kB, which suggests that leptocarpin-
Cytotoxicity induced cytotoxicity involves in some degree the inhibition of NF-kB signaling pathway. The concen-
Apoptosis tration at which LTC inhibits NF-kB activity to the control level is similar or even lower than that found
Factor NF-kB for parthenolide and others sesquiterpene lactones. These results indicate that leptocarpine is a very
Cancer cell lines interesting molecule that could be considered as therapeutic agent for cancer treatment.
2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The number of chemotherapeutics drugs that are natural
products or chemical derivatives of them is increasing every day
[1]. Thus, the search of new products with potential biological ac-
tivity has been focused on medicinal plants that have been tradi-
tionally used for their various curative abilities. Plants synthesize
many chemicals both as constitutive products and as secondary
metabolites. In this context, the family of Asteraceas emerges as an
interesting alternative, since they are considered as the most
developed group of plants, and they have been used by many
indigenous people in folk medicine. Members of this family are rich
in bioactive compounds such as polyacetylenes, diterpenes and
* Corresponding author.
** Corresponding author.
E-mail addresses: samybrs@hotmail.com (C. Bosio), volgin_4@hotmail.com
(G. Tomasoni), rmartinez@unab.cl (R. Martnez), andres.olea@uautonoma.cl
(A.F. Olea), hector.carrasco@uautonoma.cl (H. Carrasco), juan.villena@uv.cl
(J. Villena).
sesquiterpene lactones (SQL). The latter are highly interesting since
they have demonstrated a number of clinical activities, such as
antitumor, antiulcer, anti-inammatory, neurocytotoxic and
cardiotonic activities [2]. This wide range of biological activities are
known to be due to inactivation of nuclear factor kappa B (NF-kB)
via a a-methylene g-butyrolactone group which is chemically
reactive [3,4]. Thus, SQL may be able to interfere with key biological
processes such as: cell signaling, cell proliferation, apoptosis and
mitochondrial respiration [5e7]. Apoptosis is a particularly inter-
esting process because it has been proposed as a new approach for
the treatment of cancer. Apoptosis is a programmed, physiological
mode of cell death that occurs naturally in cells and is characterized
by morphological changes such as chromatin condensation and
nuclear fragmentation with the formation of apoptotic bodies fol-
lowed by ordered cell demise through phagocytosis. Apoptosis is
triggered by signals involving the mitochondria (intrinsic) or death
receptors (extrinsic), which initiate activation of Caspases, a family
of intracellular proteases, that are responsible of the morphological
and biochemical events that characterize classical apoptosis. The
mitochondrial pathway involves release of cytochrome c and others

http://dx.doi.org/10.1016/j.cbi.2015.11.006
0009-2797/ 2015 Elsevier Ireland Ltd. All rights reserved.
416 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421
pro-apoptotic factors into the cytoplasm, through pores formed in
the mitochondrial membrane, leading to activation of Caspase-9.
This process is produced by change of mitochondrial membrane
permeability (MMP), which leads to loss of mitochondrial mem-
brane integrity. The changes in MMP are considered a determinant
factor in the execution of death cell.
The ability of cancer cells of resisting cell death has been
considered a hallmark in cancer [8]. The signal that triggers the
apoptosis process is product of a delicate balance between
apoptotic and antiapoptotic proteins. On the other hand, anticancer
studies have shown that constitutive activation of NF-kB is involved
in the tumor initiation, progression, and therapeutic resistance in
most of the major forms of human cancer [9,10]. Thus, a hallmark-
targeting cancer drug could be any natural or synthetic compound,
which by inhibiting NF-kB increases effectively apoptosis in cancer
cells. In other words, the inhibition of this transcription factor can
increase the efciency of cancer therapy by killing cancer cells
directly or render them more vulnerable to chemotherapeutic
agents [11e14]. Therefore, the concept in this new approach to
cancer treatment is to use naturally occurring chemical compounds
that can trigger or enhance cell death through the regulation of the
above factors. For example, it has been proposed that compounds
able of inhibiting NF-kB can be used in a combinatory therapy with
chemoagents for which cancer cells have developed chemo-
resistance. Escin a natural mixture of triterpene saponins, aug-
ments the efcacy of gemcitabine through down-regulation of
nuclear factor-kB and nuclear factor-kB-regulated gene products in
pancreatic cancer both in vitro and in vivo [15]. In the case of
sesquiterpene lactones there have been a number of studies where
their anticancer activity has been associated to some of these fac-
tors. Some examples of active SQL are parthenolide obtained from
the herb feverfew, which has shown potent antitumor activity
against cancer stem cells [16,17] and synergistic effect on treatment
of pancreatic adenocarcinoma and a xenograft model of breast
cancer when used in combination with docetaxel or sulindac,
respectively [18,19]; cynaropicrin that induces cytotoxicity of
leukocyte cancer cell lines such as U937 and Jurkat T cells [20]. In
both cases, it has been suggested that these molecules triggers
apoptosis in cancer cells by inhibition of NF-kB.
On the other hand, our research group has been engaged for
some time in the study of an autochthonous plant that has been
widely used as folk remedy by Mapuche people. This plant, called
palo negro and whose scientic name is Leptocarpha rivularis,
belongs to the family of Asteraceae, and it has been shown that
contains a high percentage of SQL, with leptocarpin (LTC) as the
Fig. 1. Chemical structure of leptocarpin, extracted from Leptocarpha rivularis.
major component [21e24] (Fig. 1). In a previous study, conducted
on cell lines SK-Hep-1 (adenocarcinoma of the liver), and epithelial
cell line HeLa, we have shown that LTC exhibits a signicant cyto-
toxic effect [23]. In this work, we have studied the effect of lep-
tocarpin on the apoptosis mechanism. The results show that this
compound triggers programmed cell death by acting on the NF-kB.
2. Material and methods
2.1. Materials
LTC (Fig. 1, MW 362 g/mol) was obtained from Leptocarpha riv-
ularis as previously described [24]. Structural identity of lep-
tocarpin was determined by using spectroscopic techniques (1H
and 13C NMR, IR, MS).
Staurosporin, 5-uorouracil 5-FU, sulforhodamine B (SRB),
rhodamine 123, Hoechst 33342 were purchased from Sigma-
eAldrich (St. Louis, MO) and used without further purication.
Fetal bovine serum, penicillin, streptomycin were obtained from
Hyclone (Santiago, Chile) and used as received.
HT-29 cells (colon cancer cell line), PC-3 and DU-145 (prostate
cancer cell lines), MDA-MB-231 and MCF7 (breast cancer cell lines),
CCD 841 CoN (human colon epithelial cells) and HDF (human
dermal broblasts) were obtained from the American Type Culture
Collection (Rockville, MD, USA).
2.2. Cell culture
All tested cell lines were maintained in a 1:1 mixture of Dul-
becco's modied Eagle's medium (DMEM) and Ham's F12 medium,
containing 10% heat-inactivated fetal bovine serum (FBS), ampho-
tericin (2.5 mg/mL), penicillin (100 U/mL) and streptomycin (100 mg/
mL).
2.3. In vitro cytotoxicity screening by using sulforhodamine B assay
[25,26]
Stock cells were incubated at 37
C under humid atmosphere
with 5% CO2 for 24 h before the test. The cell suspension was set up
at 3000 cells per well of a 96-at-bottomed 200 mL well microplate.
Leptocarpin was dissolved in dimethylsulfoxide (DMSO) at a con-
centration of 1 mg/mL and diluted with the growth medium to the
desired concentrations (0e25 mM). Negative control cultures were
prepared by adding just 0.1% DMSO. All culture microplates were
incubated at 37
C in a CO2 incubator with humidied 5% CO2 for
72 h. At the end of drug exposure, cells were xed with 50% tri-
chloroacetic acid at 4
C. After washing with water, cells were
stained with 0.1% sulforhodamine B (SRB) dissolved in 1% acetic
acid (50 mL/well) for 30 min, and subsequently washed with 1%
acetic acid to remove unbound stain. Protein-bound stain was
solubilized with 100 mL of 10 mM unbuffered Tris base, and the cell
density was determined using an ELISA uorescence plate reader at
an emission wavelength of 540 nm using the program Gen5 1.07.
The obtained data were expressed as percentages of viability cells
versus solvent control, whose viability was considered 100%. Values
shown are the mean SD of three independent experiments in
triplicate. The software used to calculate the IC50 values was Prism
6 version 6.0d.
2.4. Morphological assessment of cell apoptosis
Cell and nuclear morphology were analyzed, after 48 h of
treatment with LTC (0, 7 and 25 mM), using phase contrast micro-
scopy and immune uorescence microscopy, respectively. As a
positive control of apoptosis was used 5-FU at 25 mM.
 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421
Morphological changes in the nuclear chromatin of cells un-
dergoing apoptosis were revealed by a nuclear uorescent dye,
Hoechst 33342. Briey, CCD 841 CoN, HT-29, PC-3 and MCF7
(1  104 cells/mL) were cultured on 24-well chamber slides, and
exposed to LTC or 5-FU (positive control) for 48 h. The control group
was exposed to 0.1% DMSO. The cells were washed twice with
phosphate buffer solution, xed with 3.7% formaldehyde and
washed again with phosphate buffer solution. Following the addi-
tion of Hoechst 33342 solution (1 mM diluted with PBS) cells were
incubated in a dark room at room temperature for 30 min. After
washing, they were examined under an immunouorescence mi-
croscope at 465 nm (Olympus IX 81 model inverted microscope).
2.5. Determination of mitochondrial potential by ow cytometry
Rhodamine 123, a cationic voltage-sensitive probe that revers-
ibly accumulates in mitochondria, was used to detect changes in
mitochondrial membrane potential [27,28]. Cells were incubated
with leptocarpin (0, 7 and 25 mM) for 48 h, and subsequently were
stained with rhodamine 123 (1 mM) and incubated in darkness for
1 h at 37
C. Then, the medium was removed and cells were washed
twice with PBS. Later the cells were trypsinized and collected by
centrifugation (10 min at 1500 g). The supernatant was discarded
and the cells pellets were resuspended in PBS and analyzed by ow
cytometry using the lter FL1. Results are expressed as percentage
of cells stained with rhodamine 123.
2.6. Evaluation of cytochrome c release
The release of cytochrome c from the mitochondria into the
cytoplasm was assessed by using the FlowCellect cytochrome c
kit (Millipore Corp.). Briey, cells were plated in 5 mL of culture
medium into 60 mm Petri dishes. After treatment with LTC (0, 7,
25 mM for 24 h), cells were collected and centrifuged at 300 g for
5 min at 4
C. Subsequently, cells were submitted to per-
meabilization, xation and blocking buffers according to supplier's
instructions. Immunolabeling for cytochrome c is achieved by
incubating permeabilized cells with anti-cytochrome c-FITC anti-
body (10 mL) for 30 min at room temperature. Cells were centri-
fuged and the pellets were resuspended in blocking buffer and
analyzed by ow cytometry. Results are expressed as percentage of
cells with reduced uorescence of FITC. This percentage is a mea-
sure of the number of apoptotic cells that have already released
their cytochrome c from the mitochondria to the cytoplasm.
2.7. Determination of caspases activation
Analysis of Caspase-3 Activity. The activity of Caspases was
determined by using a uorescent inhibitor of caspases tagged with
uorescein isothiocyanate, FITC-VAD-FMK. The CaspACE FITC-
VAD-FMK In Situ Marker was obtained from Promega. Briey,
cells were treated with leptocarpine (0, and 25 mM) for 48 h. The
cells were incubated with CaspACE FITC-VAD-FMK in darkness
for 20 min at room temperature. Then, the medium was removed
and cells were washed twice with PBS. Exposed cells were collected
by tripsinization and centrifugation (10 min at 1500 g). The su-
pernatant was discarded and the cells were re-suspended in PBS
and analyzed by ow cytometry using the lter FL3. Results are
expressed as percentage of cells stained with CaspACE FITC-VAD-
FMK [29].
2.8. NF-kB analysis
2.8.1. Detection of the DNA-binding activity of NF-kB factor
Cells previously stimulated with TNF-a (10 ng/mL, 1 h) [30,31]
417
were submitted to different treatments: LTC (7 mM) for 48 h; 0.1%
DMSO (for 48 h); and caffeic acid phenethyl ester (CAPE) (25 mg/mL,
24 h), an inhibitor of NF-kB that was used as negative control [32].
After these treatments, cells were collected and centrifuged at
300 g for 5 min at 4
C. Nuclear fractions were isolated and sub-
sequently analyzed for NF-kB activity following manufacturer's
instructions for detecting specic transcription factor DNA binding
assay, NF-kB (p65) transcription factor assay kit (Cat# 10011223,
Cayman).
2.9. Statistical analysis
Data are reported as mean values SD. Experiments were
repeated three times, with triplicate samples for each. Data were
analyzed by Prism 6 version 6.0d. Statistical signicance was
dened as p < 0.05.
To analyze the normality in the distribution of the data, the test
ShapiroeWilk was used. While for the statistical analysis of data
with no normal distribution, the non-parametric test of Wilcoxon
with designed range was utilized.
3. Results
The cytotoxicity of leptocarpin was evaluated in vitro against
different cancer cell lines: HT-29 colon cancer; PC-3 and DU-145
human prostate cancer; MCF7 and MDA-MB-231 breast cancer
and two non tumoral cell lines; CCD 841 CoN human colon
epithelial cells (CoN) and human dermal broblasts (HDF). The
sulforhodamine B colorimetric assay was set up to estimate the IC50
values; nine serial dilutions (from 1 to 25 mM) for each sample were
evaluated in triplicate. The results obtained from these assays are
shown in Table 1. The IC50 values obtained for cancer cell lines are in
the range of 2.0e6.4 mM, being the lowest value (highest cytotox-
icity) that observed in cancer cell line DU-145. On the other hand,
LTC is much less cytotoxic for broblasts with an IC50 value of
18.7 mM. This result indicates that LTC acts selectively on cancer cell
lines. The inhibitory effect of LTC on cancer cells viability is similar
to that found for other sesquiterpene lactones [20,33,34].
In order to determine whether the effect of LTC on the reduction
in cell viability involves cell death by triggering apoptosis, changes
in cell and nuclear morphology were assessed with phase contrast
microscopy and by Hoechst 33342 staining using uorescence
microscopy, respectively. In Fig. 2 are shown some representative
images obtained for MCF-7.
Images obtained with phase contrast microscope of control cells
(0.1% DMSO) (Ctrl) and cells treated with LTC (7 mM) for 48 h are
given in the left side. These images show clearly that MCF-7 cells
treated with LTC are smaller in size and they lose their character-
istic shape appearing as round mass. In addition, the number of
cells is also reduced indicating a progression to cell death. On the
other hand, the nuclear morphology changes of MFC-7 cells were
examined on images obtained by uorescence microscopy after
Hoechst 33342 staining (Fig. 2, right side). It can be seen that cells
treated with LTC (7 mM) for 48 h show signicant chromatin
condensation and fragmentation, compared to control cells.
The results obtained for different cells lines were quantied by
Table 1
IC50 values (mM) of LTC for different cancer cell lines and human dermal broblasts.
DU-145 PC-3 HT-29 MCF7 MDA CoN HDF
2.0 0.8 4.5 0.3 3.8 0.2 3.1 0.3 6.4 0.5 5.2 0.3 18.7 3.5
Data are reported as mean values SD of three different experiments with samples
in triplicate.
418 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421

Fig. 2. Effect of leptocarpin on cellular morphology, chromatin condensation and fragmentation, of MCF-7. Ctrl: cells exposed to 0.1% DMSO; Lepto: cells treated with LTC (7 mM) for
48 h. Arrow heads and arrows indicate not condensed nuclei and condensed and/or fragmented nuclei, respectively.

measuring the percentages of condensed and/or fragmented nuclei Table 3

observed after treatment with leptocarpin (7 and 25 mM for 48 h). Changes induced by treatment with leptocarpin, in mitochondrial membrane
permeability of HDF, CoN, MCF7, HT-29 and PC-3 cells. Cells were stained with
Cells exposed to 0.1% DMSO were used as control (Ctrl), and 5-
rhodamine 123, and then analyzed by ow cytometry. Permeability changes are
uorouracil (5-FU), an apoptosis inductor compound, was used as measured as percentage of rhodamine 123 stained cells found in cell lines treated
positive control. The obtained results (*p < 0.01) are summarized in with leptocarpin (7e25 mM) against control cells (0.1% DMSO). As positive control,
Table 2. FCCP (10 mM) was used.
As shown in Table 2, in cancer cell lines the number of cells with Cell lines Leptocarpin Control FCCP, 10 mM
condensed and/or fragmented nuclei increases signicantly by
7 mM 25 mM
treatment with LTC, reaching percentages values even higher than
those obtained with 5-FU. Interestingly, almost no effect is HDF 99.9 1.1 73.4 1.1 98.9 6.5 8.7 1.4**
MCF7 53.8 2.3* 21.5 0.4* 97.1 3.9 10.4 0.9**
observed for CoN cells exposed to LTC 7 mM. These results suggest PC-3 57.3 6.7* 46.5 7.2* 96.8 6.4 6.9 1.0**
the occurrence of a LTC-induced apoptosis that acts selectively on HT29 61.5 5.7* 53.4 2.9* 101.3 4.9 8.0 1.7**
cancer cells [35]. CoN 100.1 2.7 23.1 0.2* 100.5 6.5 7.5 0.6**
Mitochondria play a crucial role in the apoptotic cascade by *p < 0.05, **p < 0.001.
serving as a convergent center of apoptotic signals originating from
both the extrinsic and intrinsic pathways [36]. Changes induced in
the mitochondrial membrane permeability (MMP) have been re- reduced by treatment with LTC (7e25 mM for 24 h) as compared to
ported previously to represent a determinant effect in the execu- control cells (0.1% DMSO). FCCP (10 mM) was used as positive
tion of cell death [37]. Thus, the effect of leptocarpin on MMP has control. The effect of LTC on mitochondrial membrane permeability
been analyzed using ow cytometry with rhodamine 123 stain [27]. is higher in cancer cells than in non-tumor cells. Actually, LTC
The results, given as percentage of rhodamine 123 stained-cells, are (25 mM) has almost no effect on HDF cells when compared to cancer
collected in Table 3, and show that these values are signicantly cells. Thus, LTC-induced loss of mitochondrial membrane potential
correlates quite well with decreased cell viability (see Table 1).
It has been proposed that depletion of mitochondrial membrane
Table 2 potential leads to release of apoptogenic factors, such as cyto-
Percentages of condensed and/or fragmented nuclei of different cell lines treated chrome c in the apoptotic cascade and activation of caspases [38].
with LTC (7 and 25 mM) or with 5-FU (25 mM) for 48 h. Cells exposed to 0.1% DMSO To conrm that LTC-induced apoptosis involves the mitochondria-
were used as control.
dependent pathway both the release of cytochrome c and caspases
Cell lines Leptocarpin Control 5-FU, 25 mM activity was evaluated next. The release of cytochrome c was
7 mM 25 mM assessed by ow cytometry analysis of permeabilized-cells labeled
with anti-cytochrome c-FITC antibody. Cells that have not released
MCF7 33.1 5.3* 52.1 6.3* 5.1 0.6 30.4 4.5*
PC-3 28.2 3.6* 47.3 6.5* 6.2 2.4 36.9 5.1*
their cytochrome c are highly uorescent, whereas apoptotic cells
HT29 21.5 5.7* 43.4 2.9* 7.3 1.9 33.0 4.9* that have already released their cytochrome c exhibit a reduced
CoN 8.0 1.2 13.6 0.3* 5.5 1.0 21.5 0.6* uorescence. Thus, from ow cytometry data the percentage of
 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421
cells that have released their cytochrome c can be determined. The
results obtained for PC3, HT29 and HDF cells treated with LTC (0, 7
and 25 mM) for 24 h are shown in Fig. 3. These results indicate that
cytochrome c is released from all cell lines treated with LTC, and
that this effect is higher in cancer cell lines than in normal cells.
These results follow the same trend found for the LTC-induced
decrease of MMP.
Regarding caspases activity, the focus was put on Caspase-3 that
is a main executor of apoptosis playing a central role in its biological
processing. The effect of treatment with LTC (7 mM) on Caspase-3
activation on normal and cancer cells is shown in Table 4.
As shown in Table 4, the activation of Caspase-3 is higher in cells
exposed to LTC than in control cells (0.1% DMSO), except for HDF
cells. In other words, LTC increases the activity of Caspase-3 in
cancer cells but has no effect on normal cells. It is worth to mention
that these results were obtained by using a caspases inhibitor
tagged with uorescein isothiocyanate FTC-VAD-FMK, and it was
assumed that binding to apoptotic cells is due mainly to activation
of caspase-3, even though other caspases could be contributing to
this binding [29].
To determine if the observed apoptotic effect induced by lep-
tocarpin is consequence of inhibition of NF-kB, we have analyzed
the effect of LTC on the NF-kB activity using a DNA binding assay. All
studied cells were previously treated with TNF-a, a tumor necrosis
factor that actives NF-kB. The results given in Table 5 indicate that
stimulation of different cancer cells with TNF-a increases signi-
cantly the activity of NF-kB, and this activity is almost 2e3 times
higher than in control cells. On the other hand, cells pretreated with
TNF-a that are exposed to LTC show a signicant reduction of NF-kB
activation. The magnitude of this inhibition is similar to that
observed in cells treated with CAPE (25 mg/mL), a specic and
potent inhibitor of NF-kB [32]. The treatment with LTC or CAPE
alone has no effect on NF-kB activation.
The present data demonstrates that LTC is an effective inhibitor
of NF-kB in different cancer cell lines, suggesting that the mecha-
nism of cell death induced by LTC involves in some degree the in-
hibition of NF-kB signaling pathway.
Fig. 3. Percentage of cells which have released their cytochrome c from the mito-
chondria into the cytosol: PC-3 (black bars), HT-29 (white bars) and HDF (grey bars).
Cell lines were treated with leptocarpin (0, 7, 25 mM) for 24 h. The percentage of cells
that releases cytochrome c was obtained by measuring FITC uorescence by ow
cytometry. Results are presented as means SD of three independent experiments. (*)
Values statistically signicant in comparison to control cells, p < 0.05; (#) Statistically
signicant differences observed between the cancer cell lines and non-tumoral cell
HDF, p < 0,05.
419
4. Discussion
Leptocarpin is a sesquiterpene lactone that has been isolated as
one of the major components of palo negro a member of the
Asteraceae family [24]. In a previous work we have shown that LTC
exhibit high cytotoxicity on human liver adenocarcinoma (SK-Hep-
1) and HeLa cells. It has also been shown that acid hydrolysis of the
oxirane ring reduces signicantly the activity against these cell
lines [21,23]. There are other sesquiterpene lactones such as cos-
tunolide, cynaropicrin, parthenolide, with chemical structures that
are very similar to LTC, which have anticancer and antitumoral
effects [20,34,39]. For example, parthenolide has shown potent
antitumor activity against cancer stem cells [16,17], breast [19],
gastric [40], and renal cancer lines [41]. Therefore, in this study we
have explored more deeply the molecular mechanism by which LTC
affects the viability of cancer cells. The results indicate that LTC has
inhibitory and proapoptotic effects on cell viability in a dosage and
time dependent manner.
Cytotoxicity results show that, under the same conditions, LTC is
more cytotoxic for cancer cells than for broblasts. The IC50 values
obtained for cancer cell lines are in the range of 2.0e6.4 mM,
whereas an IC50 value of 18.7 mM was measured for broplast cells.
Cho et al. have also shown that cynaropicrin inhibited efciently
proliferation of leukocyte cancer cells lines, whereas human
broblast or Chang cells were slightly affected [20]. These results
indicate that LTC and other similar SQL inhibit selectively the pro-
liferation of cancer cell lines. The cytotoxic property of SQL has been
attributed to the presence of a a-methylene-g-lactone moiety in
the molecule regardless of other differences in structure [3,34]. The
mechanism of action associated to the activity of these molecules
involves a covalent bond with sulfhydryl groups present in the
cysteine aminoacid of proteins or enzymes through a Michael type
reaction [33,42], which would alter the functions of these
macromolecules.
SQL are known to induce cytotoxicity by triggering apoptosis,
and therefore changes in cell and nuclear morphology were used to
demonstrate the occurrence of apoptosis. The results shown in
Fig. 2 and Table 3 indicate that after treatment with LTC, cells
change their morphology becoming rounded, and besides chro-
matin condensation and nuclear fragmentation are observed. These
results are clear evidence that LTC induces an apoptosis process
[35]. Similar effects have been reported in the apoptosis induced by
parthenolide in multiple myeloma and prostate cancer [43].
Mitochondria play a crucial role on apoptosis initiation, and
therefore induction of mitochondrial apoptotic activity is consid-
ered a potential therapeutic approach. Proapoptotic drugs that act
on mitochondrial pores can affect the mitochondrial membrane
permeability enhancing the release of cytochrome c into the cyto-
plasm, where it forms a complex polymer with apoptotic enzyme
activators [36,38]. This complex activates and starts a caspase
cascade reaction, in which the downstream caspase 3 and other
members of the caspase family are activated. Caspase 3 is the ter-
minal factor in the enzymatic cascade reaction in mammalian cells
and it is also an effector leading to apoptosis [38]. This process is the
mitochondrial pathway and is one of the caspase cascade activation
pathways. Our results show that treatment with LTC increases
MMP, induces release of cytochrome c, and activation of caspase 3.
These effects indicate that LTC induces apoptosis through the
mitochondrial pathway, and its effect is higher in cancer cells than
in HDF cells. The activity of downstream effector caspase 3 is also
upregulated in cancer cells treated with parthenolide [44].
On the other hand, the extrinsic pathway involves death re-
ceptors and is activated by TNF. However, the apoptotic effect of
TNF is counter-balanced by simultaneous activation of NF-kB, an
anti-apoptotic factor that is over expressed in most cancer types. It
420 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421

Table 4
Activity of Caspase-3 in HDF, MCF7, PC-3, HT-29 and CoN cells treated with LTC (7 mM). Data is reported as ratio of activities of treated cells and control cells (0.1% DMSO), which
were arbitrarily assigned a unitary value.

HDF MCF7 PC-3 HT29 CoN

LTC 1.10 0.13 2.51 0.31** 5.5 0.45** 3.45 0.23** 1.32 0.30
Control 1.12 0.16 0.98 0.15 0.91 0.16 1.11 0.10 1.07 0.10

*p < 0.05 or **p < 0.001.

Table 5 changes, signicant chromatin condensation and fragmentation in

Inhibitory effect on NF-kB activity of LTC (7 mM, 24 h), in MCF-7, CoN and PC-3 cells, cancer cells as compared to control cells. As further evidence of
which were previously exposed to TNF-a. Values are mean S.D. (n 3). All data
values are reported as percentage in comparison with control cells (0.1% DMSO),
apoptosis, it has been shown by ow cytometry that LTC induces
which were arbitrarily assigned a 100% value. depletion of the mitochondrial membrane potential and, conse-
quently release of cytochrome c and an increase of caspase-3 ac-
MCF7 PC-3 HT29 CoN
tivity has also been observed. Finally, the effect of LTC on NF-kB
Ctrl 101.9 4.3 101.5 6.7 102.1 5.7 100.1 5.7 activation has been assessed. The results indicate that cells treated
Leptocarpin 71.4 4.9 97.1 5.5 79.8 5.2 97.7 7.2
with LTC exhibit an important reduction of NF-kB activity at con-
CAPE 89.2 7.7 104.1 3.8 99.7 5.4 92.9 6,3
TNF-a 323.1 8.9* 301.3 11.1* 279.8 15.3* 231.7 24.0* centrations as low as 7 mM.
TNF-a Lepto 81.4 10.9# 93.7 7.2# 86.0 4.1# 105.1 8.5# In summary, our results suggest that leptocarpin decreases cell
TNF-a CAPE 112.1 10.4# 85.9 6.6# 98.3 5.4# 81.4 5.3# viability of cancer cell lines by inducing caspase-dependent
*p < 0.05, signicantly different from control; #p < 0.05, signicantly different from apoptosis and by inhibition of the NF-kB factor.
cells treated with TNF-a.
Acknowledgments

has been demonstrated that NF-kB inhibits the induction of

apoptosis by stimulating the production of antiapoptotic proteins
as Bcl-2 and Bcl-XL [15,45]. Thus, the inhibition of this transcription
factor can increase the efciency of cancer therapy by killing cancer
cells directly or render them more vulnerable to chemotherapeutic
agents [46e49]. Some SQL, such as parthenolide, helenalin, costu-
nolide and cynaropicrin, that are important inhibitors of NF-kB
[4,50e53] have been evaluated as potential anticancer drugs. Thus,
we have determined the effect of LTC on NF-kB activation in
different cancer cells, where apoptosis was induced with TNF-a.
The results indicate that cancer cells treated with TNF-a increase
their NF-kB activity in almost 2e3 times over the level measured in
control cells. However, cells pretreated with TNF-a that have been
exposed to LTC show a signicant reduction of NF-kB activation.
The magnitude of this inhibition is similar to that observed in cells
treated with CAPE (25 mg/mL), a specic and potent inhibitor of NF-
kB [32]. The treatment with LTC or CAPE alone has no effect on NF-
kB activation. It is interesting to mention that the concentration at
which leptocarpin reduces NF-kB activity to the control level (7 mM)
is similar or even lower than that found for parthenolide and others
sesquiterpene lactones [54,55]. It has also been shown that par-
thenolide inhibit completely NF-kB activation mainly by alkylation
of p65, independent of the cell type used, at concentrations ranging
between 10 and 20 mM [54]. Our results suggest that the effect of
LTC on NF-kB activity is associated to a decreased DNA binding
capacity of this transcription factor; however the specic mecha-
nism is still unknown.
Thus, in this study it has been proved that LTC can effectively
induce apoptosis in different cancer cell lines by activating the
mitochondrial pathway, and by inhibiting NF-kB. These results
suggest that leptocarpin has a great potential for application in
treating cancer cells.
5. Conclusions
Leptocarpin signicantly decreases cell viability of cancer cell
lines, such as HT-29, PC-3, DU-145, MCF7 and MDA MB-231. This
inhibitory effect is dose-dependent with IC50 in the range of
2.0e6.4 mM. However, the effect of LTC on human dermal bro-
blasts is much lower with IC50 of 18.7 mM. This effect has being
linked to apoptosis by showing that LTC induces morphological
The authors are grateful to DIUV of Universidad de Valparaso
(J.V.) and FONDECYT for nancial support of this work under Grants
50/2011 and1130742, respectively.
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2015.11.006.
References
[1] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the
30 years from 1981 to 2010, J. Nat. Product 75 (2012) 311e335.
[2] M. Robles, M. Aregullin, J. West, E. Rodriguez, Recent studies on the zoo-
pharmacognosy, pharmacology and neurotoxicology of sesquiterpene lac-
tones, Planta Med. 61 (1995) 199e203.
[3] P.M. Bork, M.L. Schmitz, M. Kuhnt, C. Escher, M. Heinrich, Sesquiterpene
lactone containing Mexican Indian medicinal plants and pure sesquiterpene
lactones as potent inhibitors of transcription factor NF-kB, FEBS Lett. 402
(1997) 85e90.
[4] S.P. Hehner, M. Heinrich, P.M. Bork, M. Vogt, F. Ratter, V. Lehmann, K. Schulze-
Osthoff, W. Droge, M.L. Schmitz, Sesquiterpene lactones specically inhibit
activation of NF-kB by preventing the degradation of IkB-a and IkB-b, J. Biol.
Chem. 273 (1998) 1288e1297.
[5] A. Modzelewska, S. Sur, S.K. Kumar, S.R. Khan, Sesquiterpenes: natural prod-
ucts that decrease cancer growth, Curr. Med. Chem. Anticancer Agents 5
(2005) 477e499.
[6] S. Zhang, Y.K. Won, C.N. Ong, H.M. Shen, Anti-cancer potential of sesquiter-
pene lactones: bioactivity and molecular mechanisms, Curr. Med. Chem.
Anticancer Agents 5 (2005) 239e249.
[7] C. Wu, F. Chen, J.W. Rushing, X. Wang, H.J. Kim, G. Huang, V. Haley-Zitlin,
G. He, Antiproliferative activities of parthenolide and golden feverfew extract
against three human cancer cell lines, J. Med. Food 9 (2006) 55e61.
[8] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144
(2011) 646e674.
[9] C. Van Waes, Nuclear factor-kB in development, prevention, and therapy of
cancer, Clin. Cancer Res. 13 (2007) 1076e1082.
[10] C. Van Waes, M. Yu, L. Nottingham, M. Karin, Inhibitor-kB kinase in tumor
promotion and suppression during progression of squamous cell carcinoma,
Clin. Cancer Res. 13 (2007) 4956e4959.
[11] A.S. Baldwin, Control of oncogenesis and cancer therapy resistance by the
transcription factor NF-kB, J. Clin. Investig. 107 (2001) 241e246.
[12] J. Wang, H. An, M.W. Mayo, A.S. Baldwin, W.G. Yarbrough, LZAP, a putative
tumor suppressor, selectively inhibits NF-kB, Cancer Cell 12 (2007) 239e251.
[13] D. Basseres, A. Baldwin, Nuclear factor-kB and inhibitor of kB kinase pathways
in oncogenic initiation and progression, Oncogene 25 (2006) 6817e6830.
[14] A. Loercher, T.L. Lee, J.L. Ricker, A. Howard, J. Geoghegen, Z. Chen, J.B. Sunwoo,
R. Sitcheran, E.Y. Chuang, J.B. Mitchell, A.S. Baldwin, C. Van Waes, Nuclear
factor-kB is an important modulator of the altered gene expression prole and
malignant phenotype in squamous cell carcinoma, Cancer Res. 64 (2004)
 C. Bosio et al. / Chemico-Biological Interactions 242 (2015) 415e421
6511e6523.
[15] Y.W. Wang, S.J. Wang, Y.N. Zhou, S.H. Pan, B. Sun, Escin augments the efcacy
of gemcitabine through down-regulation of nuclear factor-kB and nuclear
factor-kB-regulated gene products in pancreatic cancer both in vitro and
in vivo, J. Cancer Res. Clin. 138 (2012) 785e797.
[16] M.L. Guzman, L. Karnischky, X.J. Li, S.J. Neering, R.M. Rossi, C.T. Jordan, Se-
lective induction of apoptosis in acute myelogenous leukemia stem cells by
the novel agent parthenolide, Blood 104 (2004) 697A.
[17] M.L. Guzman, R.M. Rossi, L. Karnischky, X.J. Li, D.R. Peterson, D.S. Howard,
C.T. Jordan, The sesquiterpene lactone parthenolide induces apoptosis of
human acute myelogenous leukemia stem and progenitor cells, Blood 105
(2005) 4163e4169.
[18] M.T. Yip-Schneider, H. Nakshatri, C.J. Sweeney, M.S. Marshall, E.A. Wiebke,
C.M. Schmidt, Parthenolide and sulindac cooperate to mediate growth sup-
pression and inhibit the nuclear factor-kB pathway in pancreatic carcinoma
cells, Mol. Cancer Ther. 4 (2005) 587e594.
[19] C.J. Sweeney, S. Mehrotra, M.R. Sadaria, S. Kumar, N.H. Shortle, Y. Roman,
C. Sheridan, R.A. Campbell, D.J. Murry, S. Badve, H. Nakshatri, The sesquiter-
pene lactone parthenolide in combination with docetaxel reduces metastasis
and improves survival in a xenograft model of breast cancer, Mol. Cancer Ther.
4 (2005) 1004e1012.
[20] J.Y. Cho, A.R. Kim, J.H. Jung, T. Chun, M.H. Rhee, E.S. Yoo, Cytotoxic and pro-
apoptotic activities of cynaropicrin, a sesquiterpene lactone, on the viability
of leukocyte cancer cell lines, Eur. J. Pharmacol. 492 (2004) 85e94.
[21] R. Martinez, V. Kesternich, E. Gutierrez, H. Dolz, H. Mansilla, Conformational-
Analysis and biological activity of leptocarpin and leptocarpin acetate, Planta
Med. 61 (1995) 188e189.
[22] R. Martinez, V. Kesternich, H. Carrasco, C. Bustos, S. Fernandez, Structure,
conformation and biological activity studies on rivularin, a new heliangolide
isolated from Leptocarpha rivularis, Bol. Soc. Chil. Quim. 43 (1998) 7e12.
[23] R. Martinez, V. Kesternich, H. Carrasco, C. Alvarez-Contreras, C. Montenegro,
R. Ugarte, E. Gutierrez, J. Moreno, C. Garcia, E. Werner, J. Carcamo, Synthesis
and conformational analysis of leptocarpin derivatives. Inuence of modi-
cation of the oxirane ring on leptocarpin's cytotoxic activity, J. Chil. Chem. Soc.
51 (2006) 1010e1014.
[24] R. Martinez, B. Ayamante, J.A. Nunez-Alarcon, A.R. de Vivar, Leptocarpin and
17,18-dihydroleptocarpin, two new heliangolides from Leptocarpha rivularis,
Phytochemistry 18 (1979) 1527e1528.
[25] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. Mcmahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity
assay for anticancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107e1112.
[26] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity
screening, Nat. Protoc. 1 (2006) 1112e1116.
[27] R.K. Emaus, R. Grunwald, J.J. Lemasters, Rhodamine 123 as a probe of trans-
membrane potential in isolated rat-liver mitochondria: spectral and meta-
bolic properties, BBA Bioenerg. 850 (1986) 436e448.
[28] J. Villena, M. Henriquez, V. Torres, F. Moraga, J. Diaz-Elizondo, C. Arredondo,
M. Chiong, C. Olea-Azar, A. Stutzin, S. Lavandero, A.F. Quest, Ceramide-
induced formation of ROS and ATP depletion trigger necrosis in lymphoid
cells, Free Radic. Biol. Med. 44 (2008) 1146e1160.
[29] P. Pozarowski, X. Huang, D.H. Halicka, B. Lee, G. Johnson, Z. Darzynkiewicz,
Interactions of uorochrome-labeled caspase inhibitors with apoptotic cells: a
caution in data interpretation, Cytom. Part A 55A (2003) 50e60.
[30] S. Miyamoto, M. Maki, M.J. Schmitt, M. Hatanaka, I.M. Verma, Tumor necrosis
factor a-induced phosphorylation of IkBa is a signal for its degradation but not
dissociation from NF-kB, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 12740e12744.
[31] S. Dhingra, A.K. Sharma, R.C. Arora, J. Slezak, P.K. Singal, IL-10 attenuates TNF-
a-induced NF kB pathway activation and cardiomyocyte apoptosis, Car-
diovasc. Res. 82 (2009) 59e66.
[32] K. Natarajan, S. Singh, T.R. Burke, D. Grunberger, B.B. Aggarwal, Caffeic acid
phenethyl ester is a potent and specic inhibitor of activation of nuclear
transcription factor NF-kappa B, Proc. Natl. Acad. Sci. U. S. A. 93 (1996)
9090e9095.
[33] T.J. Ha, D.S. Jang, J.R. Lee, K.D. Lee, J. Lee, S.W. Hwang, H.J. Jung, S.H. Nam,
K.H. Park, M.S. Yang, Cytotoxic effects of sesquiterpene lactones from the
owers of Hemisteptia lyrata B, Arch. Pharm. Res. 26 (2003) 925e928.
[34] K.-Y. Lee, E.-S. Huang, C. Piantadosi, J.S. Pagano, T.A. Geissman, Cytotoxicity of
sesquiterpene lactones, Cancer Res. 31 (1971) 1649e1654.
[35] P.D. Allen, A.C. Newland, Apoptosis detection by DNA analysis, in: F.E. Cotter
(Ed.), Molecular Diagnosis of Cancer, Humana Press, New Jersey, 1996, pp.
207e213.
421
[36] A. Fleischer, A. Ghadiri, F. Dessauge, M. Duhamel, M.P. Rebollo, F. Alvarez-
Franco, A. Rebollo, Modulating apoptosis as a target for effective therapy, Mol.
Immunol. 43 (2006) 1065e1079.
[37] J. Villena, A. Madrid, I. Montenegro, E. Werner, M. Cuellar, L. Espinoza,
Diterpenylhydroquinones from natural ent-labdanes induce apoptosis
through decreased mitochondrial membrane potential, Molecules 18 (2013)
5348e5359.
[38] R. Kim, M. Emi, K. Tanabe, Role of mitochondria as the gardens of cell death,
Cancer Chemother. Pharmcol. 57 (2006) 545e553.
[39] M. Taniguchi, T. Kataoka, H. Suzuki, M. Uramoto, M. Ando, K. Arao, J.J. Magae,
T. Nishimura, N. Otake, K. Nagai, Costunolide and dehydrocostus lactone as
inhibitors of killing function of cytotoxic T-lymphocytes, Biosci. Biotechnol.
Biochem. 59 (1995) 2064e2067.
[40] L.J. Zhao, Y.H. Xu, Y. Li, Effect of parthenolide on proliferation and apoptosis in
gastric cancer cell line SGC7901, J. Dig. Dis. 10 (2009) 172e180.
[41] D. Oka, K. Nishimura, M. Shiba, Y. Nakai, Y. Arai, M. Nakayama, H. Takayama,
H. Inoue, A. Okuyama, N. Nonomura, Sesquiterpene lactone parthenolide
suppresses tumor growth in a xenograft model of renal cell carcinoma by
inhibiting the activation of NF-kappa B, Int. J. Cancer 120 (2007) 2576e2581.
[42] V.B. Mathema, Y.S. Koh, B.C. Thakuri, M. Sillanpaa, Parthenolide, a sesquiter-
pene lactone, expresses multiple anti-cancer and anti-inammatory activities,
Inammation 35 (2012) 560e565.
[43] R. Shanmugam, V. Jayaprakasan, Y. Gokmen-Polar, S. Kelich, K.D. Miller,
M. Yip-Schneider, L. Cheng, P. Bhat-Nakshatri, G.W. Sledge, H. Nakshatri,
Q.H. Zheng, M.A. Miller, T. DeGrado, G.D. Hutchins, C.J. Sweeney, Restoring
chemotherapy and hormone therapy sensitivity by parthenolide in a xeno-
graft hormone refractory prostate cancer model, Prostate 66 (2006)
1498e1511.
[44] Y.C. Chen, S.C. Shen, W.R. Lee, F.L. Hsu, H.Y. Lin, C.H. Ko, S.W. Tseng, Emodin
induces apoptosis in human promyeloleukemic HL-60 cells accompanied by
activation of caspase 3 cascade but independent of reactive oxygen species
production, Biochem. Pharmacol. 64 (2002) 1713e1724.
[45] J.g. Sun, C.y. Chen, K.w. Luo, C.l.A. Yeung, T.y. Tsang, Z.z. Huang, P. Wu,
K.p. Fung, T.t. Kwok, F.y. Liu, 3,5-Dimethyl-H-7-furo[3,2-g]chromen-7-one as
a potential anticancer drug by inducing p53-dependent apoptosis in human
hepatoma HepG2 cells, Chemotherapy 57 (2011) 162e172.
[46] A.S. Baldwin, Control of oncogenesis and cancer therapy resistance by the
transcription factor NF-kappa B, J. Clin. Investig. 107 (2001) 241e246.
[47] J. Wang, H. An, M.W. Mayo, A.S. Baldwin, W.G. Yarbrough, LZAP, a putative
tumor suppressor, selectively inhibits NF-kappa B, Cancer Cell 12 (2007)
239e251.
[48] D. Basseres, A. Baldwin, Nuclear factor-kappa B and inhibitor of kappa B ki-
nase pathways in oncogenic initiation and progression, Oncogene 25 (2006)
6817e6830.
[49] A. Loercher, T.L. Lee, J.L. Ricker, A. Howard, J. Geoghegen, Z. Chen, J.B. Sunwoo,
R. Sitcheran, E.Y. Chuang, J.B. Mitchell, A.S. Baldwin, C. Van Waes, Nuclear
factor-kappa B is an important modulator of the altered gene expression
prole and malignant phenotype in squamous cell carcinoma, Cancer Res. 64
(2004) 6511e6523.
[50] G. Lyss, A. Knorre, T.J. Schmidt, H.L. Pahl, I. Merfort, The anti-inammatory
sesquiterpene lactone helenalin inhibits the transcription factor NF-kB by
directly targeting p65, J. Biol. Chem. 273 (1998) 33508e33516.
[51] S.P. Hehner, T.G. Hofmann, W. Droge, M.L. Schmitz, The antiinammatory
sesquiterpene lactone parthenolide inhibits NF-kB by targeting the I kB kinase
complex, J. Immunol. 163 (1999) 5617e5623.
[52] T.H. Koo, J.H. Lee, Y.J. Park, Y.S. Hong, H.S. Kim, K.W. Kim, J.J. Lee,
A sesquiterpene lactone, costunolide, from Magnolia grandiora inhibits NF-
kappa B by targeting I kappa B phosphorylation, Planta Med. 67 (2001)
103e107.
[53] Y.T. Tanaka, K. Tanaka, H. Kojima, T. Hamada, T. Masutani, M. Tsuboi, Y. Akao,
Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by
inhibiting the transcription activity of nuclear factor-kappa B, Bioorganic Med.
Chem. Lett. 23 (2013) 518e523.
[54] A.J. Garcia-Pineres, M.T. Lindenmeyer, I. Merfort, Role of cysteine residues of
p65/NF-kappa B on the inhibition by the sesquiterpene lactone parthenolide
and-N-ethyl maleimide, and on its transactivating potential, Life Sci. 75 (2004)
841e856.
[55] P. Rungeler, V. Castro, G. Mora, N. Goren, W. Vichnewski, H.L. Pahl, I. Merfort,
T.J. Schmidt, Inhibition of transcription factor NF-kappa B by sesquiterpene
lactones: a proposed molecular mechanism of action, Bioorganic Med. Chem.
7 (1999) 2343e2352.