Natural daucane esters induces apoptosis in leukae

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Natural daucane esters induces apoptosis in leukaemic cells through ROS
production
Stefano Dall’Acqua a,⇑, Maria Antonella Linardi b, Roberta Bortolozzi b, Maria Clauser a, Sara Marzocchini a,
Filippo Maggi c, Marcello Nicoletti d, Gabbriella Innocenti a, Giuseppe Basso b, Giampietro Viola b,⇑
a
Dipartimento di Scienze del Farmaco, Università di Padova, Padova, Italy
Dipartimento di Salute della Donna e del Bambino – SDB, Laboratorio di Oncoematologia, Università di Padova, Padova, Italy
Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino, Camerino, Italy
d
Dipartimento di Biologia Ambientale, Università ‘‘La Sapienza’’ Roma, Italy
b
c
a r t i c l e
i n f o
Article history:
Received 12 April 2014
Received in revised form 1 September 2014
Available online xxxx
Keywords:
Natural compounds
Ferula communis
Ferulago campestris
Coumarins
Daucane sesquiterpenes
Reactive oxygen species
Apoptosis
a b s t r a c t
Continuing our research on antiproliferative agents from plants, we extended our interest on further
compounds isolated from Ferula communis and Ferulago campestris. One new daucane (DE-20) and one
new phenol derivative (PH-3) were isolated and characterized in addition to six daucane, three coumarins and four simple phenolics. The cytotoxic activity was evaluated against a panel of six human tumor
cell lines. The derivative DE-17 that resulted moderately active on all the studied cell lines was studied to
evaluate its possible mechanism of action. DE-17 was able to induce apoptosis in a time and concentration-dependent manner in SEM and Jurkat cell lines. We observed that DE-17 just after 1 h of treatment
increased the reactive oxygen species (ROS) production and that the co-incubation of DE-17 with ROS
scavengers signiﬁcantly increased cell viability suggesting that ROS-mediated downstream signaling is
essential for the antiproliferative effects of DE-17. At later times of incubation DE-17 induced mitochondrial depolarization, as well as caspase-3 and -9 activation suggesting that apoptosis follow the
mitochondrial pathway. Concomitantly to ROS induction, a remarkable decrease of mRNA expression
of several antioxidant enzymes and intracellular GSH content was detected in treated cells compared
to controls further indicative of oxidative stress. Taken together our results showed for the ﬁrst time that
daucane esters induces apoptotic cell death through a ROS-mediated mechanism in human leukemia
cells.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
In the proceeding of our work on natural antiproliferative
compounds we continue to investigate Ferula communis (FCO)
and Ferulago campestris (FCP) constituents. In a previous paper,
we described the antiproliferative and proapoptotic effects of
Abbreviations: SAR, structure–activity relationships; PI, propidium iodide; PS,
phospholipid
phosphatidylserine;
JC-1,
5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine; ROS, reactive oxygen species; HE, hydroethidine;
H2DCFDA, 2,7-dichlorodihydroﬂuorescein; PARP, poly-ADP-ribose polymerase;
SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
⇑ Corresponding authors at: Dipartimento di Scienze del Farmaco, University of
Padova, Via Marzolo 5, 35128 Padova, Italy. Tel.: +39 49 8275344; fax: +39 49
8275366 (S. Dall’Acqua). Dipartimento di Salute della Donna e del Bambino – SDB,
Laboratorio di Oncoematologia, University of Padova, Via Giustiniani 3, 35128
Padova, Italy. Tel.: +39 49 8211451; fax: +39 49 8211462 (G. Viola).
E-mail addresses: [email protected] (S. Dall’Acqua), Giampietro.viola.
[email protected] (G. Viola).
fourteen natural daucane derivatives (Dall’Acqua et al., 2011).
The rational for the evaluation of such compounds started from
the observation that some natural sesquiterpene lactones (SQLs)
as cynaropicrin, parthenolide and dehydrocostuslactone were previously studied as possible antitumor agents showing promising
results. Furthermore recent ﬁndings indicated that the presence
of the a-methylene-c-lactone functionality has a signiﬁcant role
in the mechanism by which SQLs exert their biological activities.
Daucane derivatives present C-15 nucleus but lacks for the amethylene-c-lactone functionality being further potential scaffolds
for the development of new antiproliferative agents. More importantly, despite many papers describing the antiproliferative effects
of daucane esters, only one report address in detail the potential
mechanism(s) responsible of this activity. In this paper, Macho
et al. (2004) showed that ferutinin act as a calcium ionophore in
the T-leukemia cell line Jurkat and this event lead to a collapse
of the mitochondrial membrane potential with consequent
http://dx.doi.org/10.1016/j.phytochem.2014.09.001
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
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S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
generation of reactive oxygen species (ROS) that ultimately lead to
apoptosis.
ROS, the key mediators of cellular oxidative stress are involved
in cancer initiation and progression, have recently emerged as
promising targets for anticancer drug discovery. In cancer cells
the various process of cell growth, genetic instability and evasion
of senescence are considered to be at least in part related to high
levels of ROS. Thus a strategy for killing cancer cells would be to
expose cancer cells to compounds able to induce ROS generation
by itself or by inhibiting the cell protective mechanisms such as
enzymes like superoxide dismutase or NADPH oxidase, or compounds that are able to reduce the intracellular GSH levels, the
major determinant of the cellular redox status.
In this context it is worthwhile to note that many natural products and some of antiproliferative sesquiterpene were reported to
act by increasing the ROS levels in tumor cell lines (Wen et al.,
2002; Cho et al., 2004; Hung et al., 2010). More important some
compounds has also showed a partial selectivity toward cancer
cells in comparison to normal cells as recently reported for the natural compound piperlongumine (Raj et al., 2011).
In this paper we describe the isolation of a new daucane ester
and the investigations about the ROS-mediated mechanism of
action of pro-apoptotic antiproliferative daucane esters.
2. Results and discussion
2.1. Structures of isolated compounds
The isolated compounds were obtained from the ethyl acetate
soluble fractions FCO and FCP, on the basis of extensive chromatographic separations, as described in Section 3. Seven daucane
derivatives (DE-17DE-23) and ﬁve phenolic constituents (PH1PH-5) were obtained from the FCO extract while the three coumarins (CU-5CU-7) were obtained from the FCP extract (Chart 1).
The structures of isolated compounds were determined on the
basis of 1D and 2D NMR (HMQC, HMBC, COSY, NOESY) experiments as well as on MS experiments. The structure elucidation of
compounds DE-20 and PH-3, isolated for the ﬁrst time from natural source, are here described.
Compound DE-20 was isolated as a clear solid. The MS spectrum revealed [M + Na]+ m/z at 439.2098 (calculated 439.2097)
that suggest a molecular formula of C24H32O4Na. 1H NMR spectrum
allow to observe typical signal for daucane derivatives (Miski and
Mabry, 1985) characterized by the two doublets at d 0.85 (J = 6.5)
and 1.03 (J = 6.7) each integrating for 3 protons, supporting the
presence of isopropyl unit, the singlets at d 1.05 and 1.86 ascribable
to methyl groups 15 and 14 respectively. Furthermore the doublet
of doublet of doublets at d 5.32 (J = 2.0, 10.0, 6.5) and the multiplet
at d 5.10 both integrating for one proton corresponding to positions
H-6 and H-2 respectively. The compound was characterized on the
basis of HSQC, HMBC, COSY and NOESY spectra. The spectral data
are mostly similar to the 2a-acetoxy-6a-p-methoxybenzoyl-10bhydroxy-jaeschkeanadiol (Miski and Mabry, 1985, 1986b; Miski
and Jakupovic, 1990) except for signals corresponding to the aromatic ester in position 6. Compound DE-20 presents a benzoyl substituent instead of p-methoxy benzoate that was present in the
previously reported derivative. Thus the structure of DE-20 was
established as 2a-acetoxy-6a-benzoyl-10b-hydroxy-jaeschkeanadiol. This compound was not previously reported from natural
sources.
Compound PH-3 was isolated as a light powder. Its MS spectrum revealed a molecular ion [M + H]+ at m/z 223.0608 and
[M + Na]+ at m/z 245.0456. The 1H NMR spectrum revealed the
presence of a CH3CH2– chain due to the triplet at d 1.19 and the
quartet at d 2.91 integrating for 3 and 2 protons respectively. Further signals were a singlet at d 6.11 (2H) and two meta coupled
doublets at d 7.14 and 7.26 (J = 1.8 Hz). Careful reading of HSQC
and HMBC spectra allowed to establish the presence of an aromatic
conjugated keto group revealed by the correlation observed from
the CH3 and CH2 signals as well as from the two aromatic protons
(d 7.14 and 7.26) with the carbon resonance at d 200.0. Further
diagnostic HMBC from the proton signal at d 7.14 with a carbon
at d 167.0 support the presence of a carboxylic acid. Thus the
structure of PH-3 was assigned to the 6-propionyl-benzo[1,3]dioxole-4-carboxylic acid. At our knowledge this compound was not
previously reported from a natural source.
2.2. Identiﬁcation of known compounds
The structures of the following constituents, 2a-acetyl ferutinin
(DE-17) (Miski and Mabry, 1985), kuhistanicaol G (DE-18)
(Tamemoto et al., 2001; Chen et al., 2000), 10a-angeloyl ferutinin
(DE-19) (Lhuillier et al., 2005), and fercolide (DE-21) (Miski and
Mabry, 1986a), decursin (CU-6) (Rosselli et al., 2009), egelinol benzoate (CU-5) (Rosselli et al., 2009; Basile et al., 2009), samarcandin
(CU-7) (Basile et al., 2009), latifolon (PH-1) (Miski et al., 1985),
vanillic acid (PH-4) were identiﬁed by comparison of their spectral
data with the literature, ferulic acid (PH-5) structure was assigned
also by comparison with authentic sample.
All the isolated compounds were studied in order to assess their
ability to induce cytotoxicity against different human tumor cell
lines.
2.3. Antiproliferative activity of isolated compounds
The cytotoxic activity of all the isolated compounds was evaluated against different human tumour cell lines of both solid carcinoma (HeLa, MCF-7, HT-29) and leukemia (Jurkat, RS 4;11, SEM).
The activity of the isolated compounds are reported in Table 1
and some preliminary evaluation of structure activity relationships
can be traced considering the measured GI50 values and comparing
the values with the one obtained in our previous work (Dall’Acqua
et al., 2011). Other authors reported the antileukemic activity of
other daucane derivatives, and the 6-anthraniloyljaeschkeanadiol
(elaeochytrin A), isolated from Ferula elaeochytris was active
against K562R (imatinib-resistant) human chronic myeloid leukaemia and DA1-3b/M2(BCR-ABL) (dasatinib-resistant) mouse leukemia cell line, and HL-60 cell line and was non toxic against
normal peripheral blood mononuclear cells (Alkhatib et al., 2008).
The daucane derivatives are based on a central bicycle hydrocarbon skeleton, whose geometry depends by the stereochemistry
of the junction positions thus inﬂuencing the ﬂatness of the molecule. The bicyclic core is surrounded by a crown of oxygenated
functions and a series of acylating moieties, responsible of the differences in products polarity. Cytotoxic activity of this series of
derivatives conﬁrms that all the most active compounds, such as
DE-17, possess ring fusion with trans geometry. Comparing the
activity of the present compounds with the series of daucane esters
we previously reported (Dall’Acqua et al., 2011) we could observe
that the DE-21 is inactive as DE-10 (siol anisate). Modiﬁcation of
the double bond in the hepta-atomic ring appears critical as compound DE-20 presents low activity. The presence of hydroxyl or
methoxy residue in para in the aromatic ester of position 6 is
important for the biological effect. Compound DE-20 is inactive
while its hydroxyl or methoxy derivatives present moderate
activity on several cell lines (Dall’Acqua et al., 2011).
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
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S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
O
O
14
O
15
10 9
2 1
3
5
4
HO
8
7
H
6
OH
O
OH
H
O
11 13
O
O
O
O HO
H
H
O
O HO
O
O HO
O
12
HO
DE-17
O
HO
HO
DE-18
DE-19
OH
O
O
DE-20
OH
H
O
O
HO
H
O
HO
O
O
DE-21
OH
DE-22
DE-23
H2 C
O
O
O
O
3
O
O
O
HO
4
HO
5
O
PH-1
OH
CU-7
CU-6
O
O
O
H
CU-5
O
O
O
O
O
2
6
OH
O
7
O
OH
O
PH-3
PH-2
O
OH
O1
O
O
O
HO
O
O
O
O
PH-4
OH
PH-5
Chart 1. Structures of isolated compounds.
2.4. Effects of compound DE-17 on drug resistant cell lines
Drug resistance is an important therapeutic problem caused by
the emergence of tumor cells possessing different mechanisms
that confer resistance against a variety of anticancer drugs. Among
the more common mechanisms are those related to the overexpression of a cellular membrane protein called P-gp that mediates
the efﬂux of various structurally unrelated drugs (Szakács et al.,
2006; Baguley, 2010). In this context, we evaluated sensitivity of
DE-17 one of the most active compounds, in two multidrug-resistant cell lines, one derived from a colon carcinoma (LoVoDoxo)
(Toffoli et al., 1991), the other derived from a lymphoblastic
leukemia (CEMVbl-100) (Dupuis et al., 2003). Both these lines
express high levels of the P-gp (Toffoli et al., 1991; Dupuis et al.,
2003). As shown in Table 2, DE-17 was slight less potent toward
cells resistant to doxorubicin or vinblastine showing a resistance
index (RI), which is the ratio between GI50 values of resistant cells
and sensitive cells, of 1.6 and 2.9 respectively, while doxorubicin in
LoVoDoxo and vinblastine in CEMVbl100 showed a high RI of 68 and
109 respectively. Altogether these results suggest that these compounds might be useful in the treatment of drug refractory tumors.
2.5. Compound DE-17 induced apoptosis in leukemic cell lines
To characterize the mode of cell death, we performed a biparametric cytoﬂuorimetric analysis using propidium iodide (PI) and
annexin-V-FITC which stain DNA and phosphatidylserine (PS) residues, respectively (Vermes et al., 1995). After drug treatment for
24 h, SEM and Jurkat cells were labeled with the two dyes, washed
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
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S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
Table 1
Antiproliferative activity of isolated compounds in human tumor cells.
IC50a (lM)
Compound
HeLa
MCF-7
HT29
Jurkat
RS 4;11
SEM
CU-5
CU-6
CU-7
DE-17
DE-18
DE-19
DE-20
DE-21
DE-23
PH2
PH5
52.6 ± 6.2
>100
>100
31.4 ± 1.4
47.5 ± 4.5
>100
>100
>100
>100
42.5 ± 2.3
>100
45.9 ± 4.5
>100
>100
44.5 ± 4.9
>100
27.3 ± 5.6
>100
>100
>100
37.0 ± 2.6
>100
61.7 ± 5.6
>100
>100
27.9 ± 2.5
26.0 ± 2.8
39.4 ± 5.8
>100
94.0 ± 2.1
>100
56.9 ± 6.8
>100
41.6 ± .3.2
>100
>100
68.6 ± 0.9
>100
>100
>100
>100
>100
63.5 ± 5.3
>100
29.2 ± 2.3
>100
>100
36.6 ± 3.9
33.9 ± 2.2
21.9 ± 1.9
>100
>100
>100
18.8 ± 1.2
>100
29.7 ± 1.8
>100
34.1 ± 2.9
18.6 ± 2.6
29.6 ± 6.1
24.2 ± 2.7
>100
>100
>100
25.9 ± 2.7
>100
Daunorubicin hydrochloride
0.27 ± 0.07
0.47 ± 0.15
0.35 ± 0.1
0.025 ± 0.009
0.40 ± 0.12
0.18 ± 0.07
Values are the mean ± SEM for four separate experiments.
a
Compound concentration required to reduce cell growth inhibition by 50%.
Table 2
In vitro cell growth inhibitory effects of DE-17 on drug resistant cell lines.
Compound
GI50a (lM)
LoVoDoxo
Resistance ratiob
47.4 ± 5.2
10.2 ± 210
1.9
68
CEM
CEMVbl100
Resistance ratiob
27.8 ± 2.5
0.002 ± 0.0005
71.2 ± 3.7
0.21 ± 0.08
2.6
105
LoVo
DE-17
Doxorubicin
DE-17
Vinblastine
25.1 ± 2.3
0.15 ± 0.05
a
IC50 = compound concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean ± SE from the dose–response curves of at least
three independent experiments.
b
The values express the ratio between IC50 determined in resistant and nonresistant cell lines.
and the resulting red (PI) and green (FITC) ﬂuorescence was monitored by ﬂow cytometry.
Fig. 1 (panel A), shows as representative, two biparametric histograms in which the effect of DE-17 in SEM cells after 24 h of
incubation is depicted. It is quite evident that DE-17 induced, an
accumulation of both annexin-V (A+/PI) and annexin-V/propidium iodide (A+/PI+) positive cells in comparison to the non treated
cells, in both cell lines. A quantitative analysis of the results is presented in Fig. 1 (panels B and C) for the compound, evaluated both
in SEM (panel B) and Jurkat (panel C) at different concentrations
ranging from 12.5 to 100 lM. It can be observed a concentration
dependent increase of annexin-positive cells both in two cell lines
evaluated.
2.6. Compound DE-17 induced cell cycle arrest in G1 phase
To investigate the effects of DE-17 on the cell cycle, SEM cells
were treated with the test compound at different concentrations.
After 24 h incubation, the cells were ﬁxed and labeled with propidium iodide. The different phases of the cell cycle were analyzed
by ﬂow cytometry. As can be observed from Fig. 2, DE-17 induced,
in a concentration-dependent manner, a G1 arrest along with a
decrease of the S phase, whereas the G2/M remain practically
unchanged. These results are in agreement with that of Poli et al.
(2005) which reported that daucane esters induced antiproliferative activity in three colon cancer cell lines and this effect was preceded by cell cycle arrest in the G1 phase. In this context it is also
worthwhile to note that our previous work (Dall’Acqua et al., 2011)
indicates that other daucane esters may induce a cell cycle arrest in
G1 or in G2/M phase depending on the different structure.
Fig. 1. Determination of the mode of cell death using annexin-V and PI staining and
ﬂow cytometric analysis. (Panel A) Representative histograms of SEM cells treated
with the indicated concentration of DE-17 after 24 h of incubation. The cells were
collected and stained with annexin-V-FITC and propidium iodide (PI) and analyzed
by ﬂow cytometry. (Panels B and C) Percentage of cells found in the different region
of the biparametric histograms showed in panel A for SEM cells (panel B) or Jurkat
cells (panel C).
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
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Fig. 2. Effects of DE-17 on the cell cycle in SEM cells. Cells were treated with
different concentrations ranging from 1.25 to 100 lM for 24 h. Then the cells were
ﬁxed and stained with PI to analyze DNA content by ﬂow cytometry. Data are
presented as mean ± SEM of three independent experiments.
2.7. Compound DE-17 induced ROS production
To better understand the mode of daucane-induced cell death
we analyze ROS production. SEM cells were treated for different
times and the level of intracellular ROS were monitored using
two ﬂuorescent probes 20 ,70 -dichlorodihydroﬂuorescein diacetate
(H2DCFDA) and hydroethidine (HE) (Rothe and Valet, 1990) respectively. As shown in Fig. 3 (panel A) ﬂow cytometric analysis
showed an early and rapid increase (1–6 h) in the H2DCFDA-positive cells that occur in a concentration-dependent manner.
Using HE, that mainly detect superoxide anion (Cossarizza et al.,
2009), we did not detected any increase until 6 h of treatment,
whereas a signiﬁcant increase occur only after 12 h of incubation
with the daucane derivative following apoptotic stimuli. This effect
could be due to a production of superoxide anion by depolarized
mitochondria (Cai and Jones, 1998; Nohl et al., 2005). Indeed a
time-course in which the mitochondrial potential was monitored
by ﬂow cytometry with the ﬂuorescent probe JC-1 (Fig. 3, panel
C), clearly indicated a signiﬁcant mitochondrial depolarization
starting from 12 h. Since the mitochondrial membrane depolarization has been associated with mitochondrial production of ROS
(Zamzami et al., 1995), these ﬁndings suggest that ROS, detected
by H2DCFDA, prior to the onset of apoptosis were not produced
as a consequence of mitochondrial damage. However, at the same
time these results suggest that the apoptosis induced by DE-17 follow the mitochondrial pathway. It is worthwhile to note that
another daucane derivative the 2a-acetoxy-6a-p-methoxybenzoyl-10b-acetoxy–jaeschkeanadiol (DE-8) (Dall’Acqua et al.,
2011) produced analogous effects (Fig. S9, Supporting information)
suggesting a common mechanism for the antiproliferative effects
shared by daucane esters. In this context our ﬁndings are different
from that of Macho et al. (2004) who found an increase of ROS
(measured with HE) after 6 h of incubation with ferutinin in Jurkat
cells. In fact, this effect occurs concomitantly with mitochondrial
depolarization indicating that ROS generation are only a consequence of mitochondrial collapse.
To prove that ROS are involved in the mechanism of cell death
of our compound, we analyze the cell viability in the presence of
different antioxidants such as tocopherol acetate (TOC), 2,6-ditert-butylhydroxyanisole (BHA) and N-acetylcysteine (NAC). As
showed in Fig. 4 all three scavengers signiﬁcantly increase the cell
viability, indicating that ROS mediated downstream signaling is
essential for the antiproliferative effects observed. In well agreement, the same antioxidants protect SEM cells from the cell death
induced by DE-8 (Fig. S10, Supporting information).
Fig. 3. DE induces ROS accumulation in SEM cells. Cells were treated with the
indicated concentrations of DE-17, and at different times cells were collected,
stained with H2-DCFDA (panel A) or HE (panel B) and analyzed by ﬂow cytometry.
Data are presented as mean ± SEM of three independent experiments. (Panel C)
Induction of mitochondrial depolarization by DE-17. SEM cells were treated as
above stained with the ﬂuorescent probe JC-1 and analyzed by ﬂow cytometry. The
graph shows the percentage of cells with low mitochondrial potential. Data are
presented as mean ± SEM of three independent experiments.
2.8. DE-17 induces alterations of several intracellular antioxidant
enzyme
To maintain ROS below their harmful levels several antioxidant
enzymes such as catalase, Cu–Zn superoxide dismutase (SOD1),
Mn–superoxide dismutase (SOD2), glutathione peroxidase (GPX),
Nrf2, peroxiredoxin (PRDX1), Thioredoxin (TRX) work as ROS scavengers in the cells (Trachootham et al., 2009).
Thus to further analyze the effects of DE-17 on cellular oxidative stress we evaluated at gene expression the levels of these antioxidants enzymes. As showed in Fig. 5, the compound rapidly
decrease between 6 and 12 h the mRNA levels of catalase, SOD2,
NRF2, PRDX1 and TRX, while on the contrary, the levels of SOD1
increase indicating that DE-17 promotes oxidative stress in SEM
cells.
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
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S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
*
*
*
Fig. 4. Effect of ROS scavengers on cell death induced by DE-17. SEM cells were
treated with DE at the concentration of 25 lM for 48 h in the presence of BHA
(10 lM) TOC (100 lM) and NAC (100 lM). Cell viability was measured by MTT test.
Data is represented as mean ± S.E.M. for four independent experiments. ⁄p < 0.01 vs
DE-17 alone.
2.9. DE-17 induced intracellular GSH depletion
Reduced glutathione (GSH) is the most abundant low molecular
weight thiol in animal cells and is involved in many cellular processes including antioxidant defense, drug detoxiﬁcation, cell signaling, and cell proliferation (Circu and Aw, 2008; Lu, 2009).
Intracellular GSH loss is an early feature in the progression of cell
death in response to different apoptotic stimuli and because of
its action as a primary intracellular antioxidant in the cells, a
reduction in the intracellular GSH content has principally been
thought to reﬂect the generation of ROS (Pompella et al., 2003).
Therefore we analyze the change of GSH levels in SEM cells using
a ﬂuorescent probe 5-chloromethylﬂuorescein diacetate (CMFDA),
which is a membrane permeable dye for determining levels of intracellular GSH (Hedley and Chow, 1994). DE-17 signiﬁcantly reduced
in a time and concentration-dependent manner the ﬂuorescence of
CMFDA indicating the depletion of GSH content (Fig. 5, panels A and
B). In this context, it is also worthwhile to note that some key
enzymes involved in glutathione synthesis such as TRX and PRDX1
are downregulated following treatment with DE-17 and this could
contribute to make cells more susceptible to oxidative stress. To further conﬁrm the GSH depletion in treated cells HPLC-MS measurement of GSH were performed. As shown in Fig. 5 (panel C), we
observed a signiﬁcative decrease of the intracellular GSH content
that occur in a time-dependent manner. Altogether these ﬁndings,
reveal a novel mechanism of daucane esters-mediated apoptosis,
that involve ROS production. As a sign of intracellular oxidative
stress, we detected profound alterations of several antioxidant
enzymes along with a decreased GSH content upon DE-17 treatment. Accordingly, the action of various natural compounds has
been linked to induction of oxidative stress, depletion of intracellular GSH, and consequent apoptosis induction (Porter and Jänicke,
1999; Fernandez-Capetillo et al., 2004; Bratton and Salvesen,
2010). At the present stage, the speciﬁc source of ROS and components of the signaling pathway connecting daucane-induced ROS
with apoptosis in leukemic cells remain to be elucidated.
2.10. DE-17 induced caspases activation and DNA damage
Caspases, which are proteolytic enzymes, are the central executioners of apoptosis, and their activation is mediated by various
inducers. Synthesized as proenzymes, caspases are themselves
activated by speciﬁc proteolytic cleavage reactions. Caspases-2, 8, -9, and -10 are termed apical caspases and are usually the ﬁrst
to be activated in the apoptotic process. Following their activation,
they in turn activate effector caspases, in particular caspase-3
(Porter and Jänicke, 1999). Following treatment of SEM cells with
DE-17 for 24 or 48 h, we observed activation of caspase-3 and of
the caspase-3 substrate poly (ADP-ribose) polymerase (PARP)
(Fig. 6, panel A) in well agreement with the data of Macho et al.
(2004). Consistent with the Dwmt results described above, DE17
treatment induced activation of caspase-9, the major initiator caspase of the intrinsic (mitochondrial) apoptosis pathway (Fig. 7). In
addition, because DE17 exposure induced the generation of ROS,
we subsequently investigated whether DE17 induced DNA damage
by examining the expression of phosphorylated histone H2AX at
Ser139 (cH2A.X). cH2A.X phosphorylation occurs shortly after
DNA double strand breaks (DSBs) induction, thus cH2A.X has been
identiﬁed as an early sensitive indicator of DSBs induced by ionizing radiation, oxidative stress and chemical agents (FernandezCapetillo et al., 2004). As depicted in Fig. 6 it can be observed that
DE17 induces a remarkable increase of the phosphorylation of
cH2A.X, suggesting that the compound is able to induce DNA
damage through oxidative stress.
In conclusion in this paper we described the isolation and structural characterization of daucane esters, coumarins and phenolics
from F. communis and F. campestris as possible antiproliferative
agents. Results showed a moderate antiproliferative activity for
some of the isolated daucane against a panel of human tumor cell
lines. The daucane ester DE-17, was able to induce antiproliferative
effects in all the considered cell lines, also included that expressing
high levels of glycoprotein Pg-p. The compound induces apoptosis
in leukemic cells, that could be mediated by ROS induction. On the
basis of the presented data our current hypothesis is that DE-17
may induce oxidative stress by directly inhibiting antioxidant
enzymes into the cytoplasm or by cellular thiol depletion. We also
could not exclude that the two mechanisms works synergistically
to deactivate the cellular antioxidant systems leading to ROS generation. Studies are underway to determine the molecular mechanism and the cellular target of daucane esters. Understanding may
result in development of new cancer therapeutic strategies and
possibly also new drugs using daucane esters as a lead compound.
3. Experimental
3.1. Plant material
Roots of F. campestris (FCP) were collected during the vegetative
rest in November 2010 in rocky places sited in Madonna di Val
Povera (Camerino, Marche, Central Italy, 900 m above sea level,
N 43°06’33’’ E 13°00’06’’), while those of F. communis (FCO) were
collected in the same period along a speedway in Foligno (Umbria,
central Italy, 240 m above sea level, N 42°58’13’’ E 12°42’59’’). Voucher specimens were deposited in the Herbarium Camerinensis
(included in the online edition of Index Herbariorum: http://sweetgum.nybg.org/ih/) of the School of Biosciences and Veterinary
Medicine, University of Camerino, Italy, under Accession Nos.
CAME 13399 (FCP), and CAME 25672(FCO); they are also archived
and published in the anArchive system (http://www.anarchive.it).
3.2. Extraction and isolation
Silica gel plates (cod 5171 Merck) and silica gel (60 mesh) were
from Sigma (Milan, Italy). Solvents from Carlo Erba (Milan, Italy).
Varian Intelli-ﬂash ﬂash chromatograph was used for preparative
chromatography. NMR (1D and 2D) spectra were obtained on a
Bruker Avance 400 spectrometer. Optical rotation power was
recorded on a Yasco 2000 digital polarimeter. ESI-MS measurements were obtained on a Varian 500 MS ion trap spectrometer.
HR-MS were measured on an API-TOF spectrometer (Mariner
Biosystems).
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
7
Fig. 5. Quantitative RT-PCR analysis of mRNA expression of antioxidant enzymes in SEM cells after treatment with DE-17 (50 lM) for 6 h and 12 h. Catalase, glutathione
peroxidase (GPX), superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2), NRF2,peroxiredoxin (PRDX), thioredoxin reductase (TRX).
HPLC-DAD was obtained on an Agilent 1100 chromatographic
system with Diode Array (1100 series).
Roots of FCO (520 g fresh material) were chopped with ethyl
acetate (500 mL) and extracted at room temperature for 15 min
in an ultrasound bath (EA-extract). Extraction was repeated three
times. Solvent was anidriﬁed with sodium sulfate anhydrous and
removed under vacuum yielding a semi solid brown residue
(3.2% yield). A part of the solid residue (8 g) was dissolved in
acetone (50 mL) and was applied to a dry silica (Merck), acetone
was allowed to evaporate at 35 °C. This powder was charged on a
precolumn (DASI system of Intelliﬂash) and used for solid phase
charge of the ﬂash chromatography. A silica gel column (Agilent
Super Flash Analogix SF 25 120 g) was eluted using cyclohexane
(A) and ethyl acetate (B) starting from 100% A and gradually
increasing the B% up to 90% at a ﬂow of 30 mL/min. UV detection
was used at 254, 280 and 310 nm, and fractions of 12 mL were
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
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S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
A
B
C
Fig. 6. DE-17 induces GSH depletion in SEM cells. (Panel A) Representative histograms SEM cells treated with the indicated concentration of DE after 6 h of incubation and
stained with the ﬂuorescent probe CMFDA and analyzed by ﬂow cytometry. (Panel B) The graph represents the percentage of CMFDA negative cells. Data represent
mean ± S.E.M. of three independent experiments. (Panel C) Analysis of intracellular GSH content by LC-MS. Cells were treated with DE (50 lM) for the indicated time and GSH
content was determined as described in material and methods. Data represent mean ± S.E.M. of three independent experiments. ⁄p < 0.01 vs untreated cells.
Fig. 7. DE-17 induces caspases activation and cH2A.X histone phosphorylation.
Western blot analysis of caspase-3, cleaved caspase-9, PARP and phosphohistone
cH2A.X after treatment of SEM cells with DE-17 at the indicated concentration and
for the indicated times. To conﬁrm equal protein loading, each membrane was
stripped and reprobed with anti-b-actin antibody.
collected. Fractions were collected and pooled on the basis of their
chromatographic behavior in 20 different fractions. Fractions 10
(907 mg), 12 (550 mg) 14 (455 mg) and 18 (990 mg) were used
for compound isolation. Further puriﬁcations were obtained with
semipreparative HPLC on a Licrosphere 100 C-18 (9.6 300 mm,
10 m) using as mobile phase acetonitrile and water 0.1% formic acid
in gradient elution. Gradient was the following starting from 10%
acetonitrile and increasing to 30% in 10 min, then at 80% of acetonitrile in 25 min. The ﬂow rate was of 2.5 mL/min. Compounds were
obtained from fraction 18 PH-1 (12.0 mg), PH-2 (5 mg), PH-3
(3.9 mg), PH-4 (10 mg), PH-5 (43 mg), from fraction 12 and 14
DE-17 (10.5 mg), DE-18 (7.3 mg), DE-21 (6.0 mg), DE-22 (3.9 mg),
from fraction 14 DE-20 (8.0 mg) and DE-23 (9.0 mg) respectively.
Roots of FCP (450 g fresh material) were chopped with ethyl
acetate (500 mL) and extracted at room temperature for 15 min
in an ultrasound bath (EA-extract). Extraction was repeated three
times. Solvent was anidriﬁed with sodium sulfate anhydrous and
removed under vacuum yielding a semi solid brown residue
(4.5% yield). A part of the solid residue (6 g) was dissolved in
acetone (50 mL) and was applied to a dry silica (Merck), acetone
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
was allowed to evaporate at 35 °C. This powder was charged on a
precolumn (DASI system of Intelliﬂash) and used for solid phase
charge of the ﬂash chromatography. A silica gel column (Agilent
Super Flash Analogix SF 25 120 g) was eluted using cyclohexane
(A) and ethyl acetate (B) starting from 95% A and gradually increasing the B% up to 50% at a ﬂow of 30 mL/min. UV detection was used
at 254, 280 and 310 nm, and fractions of 12 mL were collected.
Fractions were collected and pooled on the basis of their chromatographic behavior in 12 different fractions. Fractions 6 (950 mg), 7
(530 mg) and 8 (850 mg) were used for compound isolation. Further puriﬁcations were obtained with semipreparative HPLC on a
Licrosphere 100 C-18 (9.6 300 mm, 10 lm) using as mobile
phase methanol and water (60:40) at a ﬂow of 2.5 mL/min. Compounds were obtained from fraction 6 CU-5 (18.9 mg), CU-6
(12.0 mg), CU-5 (18.9 mg), CU-7 (18.3 mg) and from fraction 7
DE-17 (10.5 mg), DE-18 (7.3 mg) respectively. Purity of isolated
compounds was checked by HPLC analysis and was >97% by
software integration.
NMR assignments of new compounds are reported in
Supplementary information ﬁle.
3.3. Antiproliferative assays
Human T-leukemia (Jurkat), human promyelocytic leukemia
(HL-60), human chronic myelogenous leukemia (K562), acute Blymphoblastic leukemia SEM and RS 4; 11 cells, were grown in
RPMI-1640 medium, (Gibco Milano Italy). Human non-small lung
carcinoma (A549) and human cervix carcinoma (HeLa) cells were
grown in DMEM medium (Gibco, Milano, Italy), all supplemented
with 115 units/mL of penicillin G (Gibco, Milano, Italy), 115 lg/
mL streptomycin (Invitrogen, Milano, Italy) and 10% fetal bovine
serum (Invitrogen, Milano, Italy). CEMVbl-100 cells are a
multidrug-resistant line selected against vinblastine (Dupuis
et al., 2003). LoVoDoxo cells are a doxorubicin resistant subclone
of LoVo cells (Toffoli et al., 1991) and were grown in complete
Ham’s F12 medium supplemented with doxorubicin (0.1 lg/mL).
LoVoDoxo and CEMVbl-100 were a kind gift of Dr. G. Arancia (Istituto
Superiore di Sanità, Rome, Italy). Individual wells of a 96-well tissue culture microtiter plate were inoculated with 100 lL of complete medium containing 8 103 cells. The plates were incubated
at 37 °C in a humidiﬁed 5% CO2 incubator for 18 h prior to the
experiments. After medium removal, 100 lL of the drug solution,
dissolved in complete medium at different concentrations, was
added to each well and incubated at 37 °C for 72 h. Cell viability
was assayed by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test as previously described (Viola et al.,
2008). The IC50 was deﬁned as the compound concentration
required to inhibit cell proliferation by 50%.
3.4. Annexin-V assay
Surface exposure of phosphatidylserine on apoptotic cells was
measured by ﬂow cytometry with a Coulter Cytomics FC500
(Beckman Coulter) by adding annexin-V-FITC to cells according
to the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostic). Simultaneously the cells were stained with PI. Excitation
was set at 488 nm, and the emission ﬁlters were set at 525 nm
and 585 nm, respectively.
3.5. Flow cytometric analysis of cell cycle distribution
For ﬂow cytometric analysis of DNA content, 2.5 105 Jurkat
cells in exponential growth were treated with different concentrations of the test compounds for 24 and 48 h. After an incubation
period, the cells were collected, centrifuged and ﬁxed with ice-cold
ethanol (70%). The cells were then treated with lysis buffer
9
containing RNAse A and 0.1% Triton X-100, and then stained with
PI. Samples were analyzed on a Cytomic FC500 ﬂow cytometer
(Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems).
3.6. Assessment of mitochondrial changes and ROS production
The mitochondrial membrane potential was measured with the
lipophilic cation 5,50 ,6,60 -tetrachlo-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine (JC-1, Molecular Probes, Eugene, OR, USA), while the
production of ROS was followed by ﬂow cytometry using the ﬂuorescent dyes hydroethidine (HE, Molecular Probes, Eugene, OR, USA)
and
20 ,70 -dichlorodihydroﬂuorescein
diacetate
(H2DCFDA,
Molecular Probes, Eugene, OR, USA), as previously described
(Dupuis et al., 2003).
3.7. Detection of the intracellular glutathione content (GSH)
Cellular GSH levels were analyzed using 5-chloromethylﬂuorescein diacetate (CMFDA, Molecular Probes) (Zamzami et al., 1995).
Cells were treated with the test compound for different times. Cells
were harvested centrifuged and incubated in the presence of a
solution of CMFDA 5 lM at 37 °C for 30 min. Cytoplasmic esterases
convert non-ﬂuorescent CMFDA to ﬂuorescent 6-chloromethylﬂuorescein which can then react with glutathione. Fluorescence
intensity was determined by ﬂow cytometry. Intracellular GSH
content was also measured by HPLC-MS modifying a literature protocol (Steghens et al., 2003). GSH, GSSG, c-glutamyl-glutamic acid
(c-glu-glu), EDTA, sulfosalicylic acid (SSA) dehydrate, N-ethylmaleimid (NEM) and ammonium acetate were obtained from Sigma.
The precipitating solution was made by mixing 150 ll–l00 ll of a
solution containing NEM, EDTA and c-glu-glu (in water/methanol,
85/15 (v/v)) with 50:l of SSA; the ﬁnal concentrations in the precipitating solution were 20 mM, 2 mM, 250 lM and 2% (w/v) for NEM,
EDTA, c-glu-glu and SSA, respectively. The solution was allowed to
react for 30 min. The sample was then centrifugate (5000 rpm) and
the supernatant was used for the HPLC-MS analysis. For the MS
analysis the X-Terra column (3.5 150 mm) was used. Water with
1% formic acid (A) and methanol (B) were used as mobile phase.
Gradient elution start with 98% A in 11 min 0% A. Ion were
monitored in positive and the retention times of internal standard
(c-glu-glu m/z 277) and GSNEM (m/z 434) were 1.85 and 6.3 min
respectively.
3.8. Quantitative RT-PCR
Total RNA was isolated using trizol (Life Technologies, Monza,
Italy) and retrotranscribed as previously described. Real TimePCR were performed on an ABI Prism 7900HT Fast Real Time PCR
system (Applied Biosystems, Foster City, CA, USA) using PlatinumÒ
SYBRÒ Green qPCR SuperMix-UDG (Invitrogen Life Technologies).
Relative quantiﬁcation was calculated by the DDCt method, normalizing to GUS mRNA. Primers used for real-time quantitative
PCR analysis: TRX1-fwr: 50 -GCCAAGATGGTGAAGCAGAT-30 and
TRX1-rev 50 -TTGGCTCCAGAAAATTCACC-30 ; CATALASE-fwr: 50 -GT
TACTCAGGTGCGGGCATTCTAT-30 and CATALASE-rev: 50 -GAAGTTCT
TGACCGCTTTCTTCTG-30 ; MSOD1-fwr: 50 -CTCTCAGGAGACCATTGCATCA-30 , MSOD1-rev: 50 -CCTGTCTTTGTACTTTCTTCATTTCCA-30 ;
MSOD2-fwr: 50 - GTGGAGAACCCAAAGGGGAGTT-30 and MSOD2rev: 50 -GTGGAATAAGGCCTGTTGTTCCTT-30 ; NRF2-fwr: 50 -GAGAGCCCAGTCTTCATTGC-30 and NRF2-rev: 50 -TGCTCAATGTCCTGTTGCAT-30 ;
PRDX1-fwr:
50 -TGGGGTCTTAAAGGCTGATG-30
and
PRDX1-rev: 50 -TCCCCATGTTTGTCAGTGAA-30 ; GPX1-fwr: 50 -TGGG
GAGATCCTGAATTG-30 and GPX1-rev: 50 -GATAAACTTGGGGTCGG
T-30 ; GUS-fwd: 50 -GAAAATATGTGGTTGGAGAGCTCATT-30 ; GUSrev: 50 -GGAGTGAAGATCCCCTTTTTA-30 .
Please cite this article in press as: Dall’Acqua, S., et al. Natural daucane esters induces apoptosis in leukaemic cells through ROS production. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.09.001
10
S. Dall’Acqua et al. / Phytochemistry xxx (2014) xxx–xxx
3.9. Western blot analysis
SEM cells were incubated in the presence of test compounds
and, after different times, were collected, centrifuged and washed
two times with ice cold phosphate-buffered saline (PBS). The pellet
was then resuspended in lysis buffer. After the cells were lysed on
ice for 30 min, lysates were centrifuged at 15,000g at 4 °C for
10 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal
amounts of protein (20 lg) were resolved using sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (4.0–20%
precast acrylamide gels) and transferred to PVDF Hybond-p
membrane (GE Healthcare). Membranes were blocked with a 5%
solution of bovine serum albumin (BSA Sigma–Aldrich), the membrane being gently rotated overnight at 4 °C. Membranes were
then incubated with primary antibodies against procaspase-3
(Alexis), PARP, cleaved caspase-9, phosphohistone cH2A.X (Cell
Signaling), or b-actin (Sigma–Aldrich) for 2 h at room temperature.
Membranes were next incubated with peroxidase-labeled secondary antibodies for 60 min. All membranes were visualized using
ECL select (GE Healthcare) and exposed to Hyperﬁlm MP (GE
Healthcare). To ensure equal protein loading, each membrane
was stripped and reprobed with anti-b-actin antibody.
3.10. Statistical analysis
Unless indicated otherwise, the results are presented as
mean ± S.E.M. The differences between different treatments were
analyzed, using the two-sided Student’s t test. P values lower than
0.05 were considered signiﬁcant.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
09.001.
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