Withaferin A-related steroids from Withania aristata exhibit potent antiproliferative activity by inducing apoptosis in human tumor cells

Article abstract of DOI:10.1016/j.ejmech.2012.05.032

Six new withanolides (1-6) along with eleven known ones (7-17) were isolated from the leaves of Withania aristata. Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D NMR techniques. Semisynthesis of the minority metabolites 7 and 15 from compounds 6 and 9, respectively, as starting material, was performed. The isolated compounds as well as three derivatives (7a, 9a and 9b) of withaferin A were evaluated for cytotoxicity against HeLa (carcinoma of the cervix), A-549 (lung carcinoma) and MCF-7 (breast adenocarcinoma) human cancer cell lines, and against normal Vero cells (African green monkey kidney). Five compounds from this series (8, 9a, 9b, 11 and 13) exhibited potent antiproliferative effects on the tumor cells, even higher than the well known anticancer agent, withaferin A (9). Phosphatidylserine externalization, chromatin condensation, and caspase-3 activation clearly indicated apoptosis as a mechanism of action. The structure-activity relationship revealed valuable information on the pharmacophore for withanolide-type compounds.

Full text of DOI:10.1016/j.ejmech.2012.05.032

European Journal of Medicinal Chemistry 54 (2012) 499e511
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Withaferin A-related steroids from Withania aristata exhibit potent
antiproliferative activity by inducing apoptosis in human tumor cells
,
Gabriel G. LLanos a, Liliana M. Araujo b, Ignacio A. Jiménez a, Laila M. Moujir b, Isabel L. Bazzocchi a
*
a Instituto Universitario de Bio-Orgánica “Antonio González” and Departamento de Química Orgánica, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2,
38206 La Laguna, Tenerife, Spain
b Departamento de Microbiología y Biología Celular, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Spain
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
< A series of new withanolides were isolated from Withania aristata.
< First examples of silyl etherewithaferin A derivatives.
< Five compounds of this series exhibited higher antiproliferative effects than withaferin A.
< Hoechst, Annexin V/PI and caspase-3 studies revealed that compounds induce apoptosis in HeLa cells.
< This study sheds more light on the potential of withanolides as anticancer agents.
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new withanolides (1e6) along with eleven known ones (7e17) were isolated from the leaves of
Withania aristata. Their structures were elucidated on the basis of spectroscopic analysis, including 1D
and 2D NMR techniques. Semisynthesis of the minority metabolites 7 and 15 from compounds 6 and 9,
respectively, as starting material, was performed. The isolated compounds as well as three derivatives
(7a, 9a and 9b) of withaferin A were evaluated for cytotoxicity against HeLa (carcinoma of the cervix), A-
549 (lung carcinoma) and MCF-7 (breast adenocarcinoma) human cancer cell lines, and against normal
Vero cells (African green monkey kidney). Five compounds from this series (8, 9a, 9b, 11 and 13)
exhibited potent antiproliferative effects on the tumor cells, even higher than the well known anticancer
agent, withaferin A (9). Phosphatidylserine externalization, chromatin condensation, and caspase-3
activation clearly indicated apoptosis as a mechanism of action. The structureeactivity relationship
revealed valuable information on the pharmacophore for withanolide-type compounds.
Received 20 January 2012
Received in revised form
18 May 2012
Accepted 23 May 2012
Available online 1 June 2012
Keywords:
Withania aristata
Withanolides
Antiproliferative
Human cancer cell lines
Apoptosis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Caspase-3
Structureeactivity relationship
* Corresponding author. Tel.: þ34 922 318594; fax: þ34 922 318571.
E-mail address: ilopez@ull.es (I.L. Bazzocchi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.032

500
G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
1. Introduction
In the course of our search for bioactive metabolites isolated
from species which grow in the Canary Islands, we focused our
attention in Withania aristata (Ait.) Pauq. (Solanaceae), an endemic
plant from Canary Islands, popularly known as “orobal”. This
species has long been used in traditional medicine as an antitu-
moral, antirheumatic and antispasmodic, as well as for insomnia,
constipation and urinary pathologies [17] Previous phytochemical
studies on W. aristata reported the isolation of six withanolides
[18e22], and the cytotoxic [21] and diuretic activities [22] of
withaferin A. Recently, we reported the isolation of seven new
withanolides and their cytotoxicity against several human tumor
cell lines [23].
In the present work, and as a continuation of our research
directed at the discovery a novel drugs for treatment of cancer, we
report herein on the isolation, structure elucidation and anti-
proliferative activity of six new (1e6) and eleven known (7e17)
withanolides. In addition, the semisynthesis of the natural
compounds 7 and 15 was carried out. The isolated compounds
(1e17) and derivatives 7a, 9a and 9b were evaluated against
a representative panel of cancer cell lines, HeLa (carcinoma of the
cervix), A-549 (lung carcinoma), and MCF-7 (breast adenocarci-
noma), together with normal cells, Vero (African green monkey
kidney) searching for selectivity. The present study was also
undertaken to broaden the structural elements that are key for the
efficient anticancer activity of this withanolide series. In order to
investigate a possible mechanism for their antiproliferative effects,
a series of experiments evaluating the induction of apoptosis in
HeLa cells of the most active compounds 8, 11, 13, 9a, 9b and
withaferin A (9), was performed.
Cancer still belongs to the second-leading cause of death
worldwide. Because for many types of cancer no curative therapy is
available, there is an ongoing need for novel leads for chemo-
therapy. Natural products represent a rich source of drug leads [1]
and they have played a significant role in the discovery and
development of new anticancer agents [2].
Withanolides are structurally diverse C28-steroidal compounds
built on an ergostane skeleton in which C-22 and C-26 are oxidized
to form a d-lactone ring on the nine-carbon side chain [3]. This class
of steroids has attracted much interest due to their complex struc-
tural features, but also their broad range of biological activities [4].
Apoptosis is a fundamental and complex biological process in
which cells play an active role in their own death. Dysregulation of
apoptosis is the hallmark of all cancer cells and agents that activate
programmed cell death could be valuable anticancer therapeutics [5].
Withaferin A, a prototype of the withanolides class of natural
products, exerts its anticancer effect, through induction of
apoptosis in a variety of human cancer cells, including prostate [6],
colon [7], breast [8], leukemia [9], pancreatic [10], renal [11], head
and neck [12], among others. It was shown that withaferin A actives
p38 MAP kinase, inhibits the Notch signalling pathway and nuclear
factor-lB activation [7,13], induces Akt inactivation, death receptor
5 (DR5) up-regulation and down-regulation of c-FLIP [11,14] as well
as induces apoptosis through its ability to generate reactive oxygen
species [15,16]. However, despite these advances, the precise
mechanism by which withaferin A mediated cell death is not fully
understood.
28
27
R
OR2
OH
21
22 26
O
20
18
O
O
O
O
O
O
O
17
15
O
19
13
11
OH
1
4
10
8
6
R1
O
OH
O
OH
O
OH
4
R1= OH, R2= Ac
1
2
3
8
9
R= OAc
R= OC(CH3)2OCHO
R= H
10 R1= H, R2= H
R= OH
9a R= OTBDMS
OH
R
R
O
O
O
O
O
O
O
O
O
HO
O
OH
OH
OH
OH
OH
CHO
16
2
11
12
13
R= OH
R= H
R= H
5
6
7
R= OH
16
14
R= OH
7a R= H
OH
OH
OR
O
OH
O
O
O
O
O
O
O
O
O
O
OH
O
OH
OH
R
14
15 R= H
9b R= TBDMS
16 R= OH
17 R= Cl
Fig. 1. Structure of natural withanolides (1e17) and derivatives (7a, 9a and 9b).

G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
, CDCl3, J values in Hz in parentheses) data of compounds 1e4.
501
Table 1
1H (400 MHz), and 13C (100 MHz) NMR (
d
No.
1
2
3
4
a
a
a
a
dH
dC
dH
dC
dH
dC
dH
dC
1
2
201.9 s
132.0 d
202.1 s
132.1 d
202.4 s
132.3 d
209.8 s
42.5 t
6.21 d (10.0)
6.21 d (10.0)
6.22 d (9.7)
2.53 dd (5.0, 15.4)
3.21 dd (6.4, 15.4)
4.34 dd (4.2, 10.3)
3.46 d (3.7)
3
4
5
6
7
7
8
9
6.95 dd (5.9, 10.0)
3.77 d (5.9)
141.8 d
69.6 d
63.7 s
62.2 d
30.8 t
6.95 dd (5.9, 10.0)
3.76 d (5.9)
141.7 d
69.6 d
63.6 s
62.3 d
30.9 t
6.95 dd (5.2, 9.7)
3.79 d (5.2)
141.9 d
69.9 d
63.9 s
62.7 d
31.2 t
68.5 d
76.3 d
64.2 s
60.1 d
30.9 t
3.26 br s
1.40 m
2.21 m
1.64b
3.24 br s
1.29 m
2.15 m
1.53 m
1.02 m
3.26 br s
1.28 m
2.16 m
1.54 m
1.05 m
3.31 br s
1.36 m
2.20 m
1.44 m
1.33 m
a
b
28.4 d
43.8 d
47.5 s
22.0 t
36.6 t
44.2 s
52.4 d
36.0 t
71.5 d
150.9 s
16.1 q
17.1 q
129.5 s
12.0 q
77.6 d
34.5 t
29.5 d
43.9 d
47.4 s
21.9 t
39.1 t
42.3 s
55.8 d
24.0 t
27.0 t
51.7 d
11.4 q
17.2 q
38.5 d
13.1 q
78.0 d
29.8 t
29.4 d
44.1 d
47.7 s
22.2 t
39.4 t
42.6 s
56.1 d
24.3 t
27.3 t
52.0 d
11.6 q
17.5 q
38.8 d
13.4 q
78.8 d
30.0 t
29.0 d
42.3 d
50.2 s
21.8 t
39.2 t
42.7 s
56.0 d
24.2 t
27.3 t
52.0 d
11.6 q
16.1 q
38.8 d
13.3 q
78.2 d
30.1 t
1.11 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
23
24
25
26
27
28
1.51, 1.93 m
1.50b, 2.29 m
1.48, 1.83 m
1.13, 2.00 m
1.52, 1.87 m
1.14, 1.98 m
1.34, 1.53 m
1.19b, 1.94 m
1.50b
0.95 m
0.96 m
1.01 m
1.64b
1.18, 1.65 m
1.38, 1.70 m
1.10 m
0.70 s
1.41 s
1.20, 1.67 m
1.40, 1.68 m
1.10 m
0.73 s
1.44 s
1.19b, 1.69 m
1.40, 1.70 m
1.13 m
4.73 br s
0.89 s
1.42 s
0.70 s
1.35 s
2.03 m
1.03 d (6.4)
4.44 dt (3.0,13.3)
2.05 m
2.00 m
2.04 m
1.81 s
5.37 dd (3.5, 12.9)
2.23 m
1.00 d (6.6)
4.41 dt (3.3,13.2)
2.01 m
1.02 d (6.2)
4.48 br d (13.4)
2.03 m
a
b
2.72 m
2.53 m
2.57 m
2.55 m
153.5 s
125.1 s
166.7 s
57.2 t
156.8 s
121.6 s
165.2 s
57.8 t
20.4 q
170.7 s
20.7 q
156.7 s
123.0 s
167.9 s
54.5 t
20.5 q
104.9 s
22.4 q
22.7 q
161.5 d
157.0 s
121.9 s
165.3 s
58.0 t
20.6 q
170.9 s
20.9 q
4.38, 4.43 dAB (12.6)
2.02 s
4.88, 4.92 dAB (11.9)
2.08 s
4.27, 4.32 dAB (10.0)
2.10 s
4.89, 4.92 dAB (12.0)
2.11 s
19.6 q
2.06 s
1.46 s
1.48 s
10.62 s
2.09 s
a
Data are based on DEPT, HSQC and HMBC experiments.
Overlapping signals.
b
2. Results and discussion
carbons [dC 150.9 (C-17), 129.5 (C-20)], three oxygenated methines
dC 69.6 (C-4), 71.5 (C-16), 77.6 (C-22)], an epoxide [dC 63.7 (C-5),
[
2.1. Chemistry
62.2 (C-6)], and one oxygenated methylene at dC 57.2 (C-27). These
data indicated that compound 1 is an oxo-witha-trienolide, which
presents, along with the usual unsaturations at C-2 and C-24, an
additional one at C-17(20) suggested by the high chemical shifts of
H-22 (dH 5.37) and Me-21 (dH 1.81) [24], and a hydroxyl group at C-
16. The 16-hydroxy-17(20)-ene functional group is extremely
uncommon in the withasteroid skeleton, and there are only two
such withanolides found in nature [25]. These structural features
The dichloromethane extract of the leaves of W. aristata was
subjected to a series of column chromatography on silica gel and
Sephadex LH-20, as well as preparative TLC and HPTLC, affording
six new withanolides (1e6), along with the known ones 7e17
(Fig. 1).
Compound 1 was isolated as a white amorphous solid and
showed the molecular formula C28H36O7 by HREIMS. The EIMS
displayed peaks at m/z 484 [M]þ, m/z 466 [Mþ ꢀ H2O], m/z 448
[Mþ ꢀ 2ꢁ H2O] and m/z 343 [Mþ ꢀ side chain]. The UV spectrum
exhibited a strong absorption at 215 nm, indicating the presence of
2
8
21
a
,b
-unsaturated carbonyl systems, and absorption bands for
hydroxyl (3415 cmꢀ1), -unsaturated carbonyl (1685 cmꢀ1) and
a
,b
24
18
22
epoxide (1180 cmꢀ1) groups were observed in its IR spectrum.
The 1H NMR spectrum (Table 1) displayed an AMX system with
signals at dH 6.21 (JAM ¼ 10.0 Hz), 6.95 (JMA ¼ 10.0 Hz, JMx ¼ 5.9 Hz)
and 3.77 (JxM ¼ 5.9 Hz), three oxymethine protons at dH 3.26 (br s)
4.73 (br s) and 5.37 (dd, J ¼ 3.5, 12.9 Hz), and an oxymethylene at dH
4.38, 4.43 (dAB, J ¼ 12.6 Hz) as the most downshift signals. In
addition, two singlet methyl groups at dH 2.02 and 1.81 attached to
olefinic carbons, and two methyl groups at dH 0.89 and 1.42 were
observed. Its 13C NMR spectrum and DEPT experiments (Table 1)
CH2OH
20
OH
O
2
7
6
2
17
13
14
H
11
8
10
O
H
O
O
1
HO
H
6
4
H
H
H
revealed 28 carbon signals, including an -unsaturated ketone [dC
201.9 (C-1), 132.0 (C-2), 141.8 (C-3)], an a,b-unsaturated lactone [dC
153.5 (C-24), 125.1 (C-25), 166.7 (C-26)], two olefinic quaternary
a
,
b
Fig. 2. 1H-13C long-range correlations (solid line) and ROE effects (dashed line) for
compound 1.

502
G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
Table 2
1H (400 MHz), and 13C (100 MHz) NMR (
d
, CDCl3, J values in Hz in parentheses) data of compounds 5e7.
No.
5
6
7
a
a
a
dH
dC
dH
dC
dH
dC
1
2
3
4
5
6
7
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
209.8 s
41.4 t
208.9 s
136.8 d
152.5 d
199.7 d
70.9 s
216.2 s
32.5 t
22.5 t
204.3 d
60.5 s
67.4 d
33.9 t
2.53b, 2.69 d (18.5)
4.38 m
6.55 d (5.8)
6.97 d (5.8)
8.79 s
2.39, 2.57 m
1.83b, 2.03 m
9.60 s
72.9 d
77.7 d
4.46b
76.3b
76.3b
31.8 t
s
d
4.14 d (7.9)
1.22b
2.08 m
1.58 m
1.52 m
4.58 br s
1.00, 1.96 m
67.0 d
35.2 t
4.31 t (2.8)
1.49, 1.87 m
a
b
30.9 d
40.6 d
54.6 s
20.8 t
39.2 t
42.7 s
57.8 d
24.2 t
27.4 t
51.6 d
11.6 q
14.7 q
38.8 d
13.3 q
78.8 d
29.8 t
153.3 s
125.6 s
167.2 s
57.4 t
20.0 q
1.89 m
1.12 m
30.1 d
47.0 d
55.5 s
20.7 t
39.1 t
42.9 s
55.8 d
23.9 t
27.1 t
51.7 d
11.7 q
12.1 q
38.5 d
13.1 q
78.4 d
29.5 t
153.3 s
125.5 s
166.7 s
57.2 t
19.8 q
1.83b
1.09 m
28.9 d
41.9 d
52.4 s
22.4 t
39.1 t
43.0 s
55.7 d
23.9 t
27.1 t
51.7 d
11.7 q
13.1 q
38.5 d
13.2 q
78.5 d
29.5 t
152.5 s
125.5 s
166.8 s
57.3 t
19.8 q
1.54 m
1.11, 2.02 m
1.43 m
1.06 m
1.27b, 1.67 m
1.12, 2.02 m
1.01 m
0.94 m
1.08 m
1.22b, 1.70 m
1.41, 1.72 m
1.15 m
1.23, 1.64 m
1.40, 1.66 m
1.09 m
0.77 s
1.22 s
1.27, 1.7b
1.43, 1.7b
1.16 m
0.72 s
1.26 s
2.05 m
0.77 s
1.19 s
2.04 m
1.03 d (6.6)
4.45 dt (3.5, 13.0)
2.00, 2.54 m
2.00 m
1.02 d (6.5)
1.01 d (6.7)
4.41 dt (3.6, 13.3)
1.97 m, 2.51 t (12.4)
4.46b
2.00, 2.53b
m
4.36, 4.42 dAB (12.4)
2.07 s
4.34, 4.40 dAB (12.5)
2.01 s
4.37, 4.42 dAB (12.3)
2.06 s
a
Data are based on DEPT and HSQC experiments.
Overlapping signals.
b
were confirmed by 2D NMR spectra. Thus, in its 1He1H COSY
spectrum, H-3 correlated with both H-2 and the oxygenated
methine proton H-4, confirming the presence of a 4-hydroxy-2-en-
1-one unit in the molecule. Also cross-peaking was found between
H-6/H-7, H-15/H-16 and H-22/H-23.
The regiosubstitution of 1 was determined by an HMBC exper-
iment (Fig. 2), showing as the most relevant three-bond correla-
tions those of the proton resonance at dH 3.77 (H-4) with the signals
at dC 132.0 (C-2), 62.2 (C-6) and 47.5 (C-10), correlation of the
proton resonance at dH 4.73 (H-16) with the signals at dC 42.2 (C-
13), 52.4 (C-14), 150.9 (C-17) and 129.5 (C-20), and those of the
signal at dH 1.81 (Me-21) with the resonances at dC 150.9 (C-17) and
129.5 (C-20). The relative configuration of 1 was established on the
basis of the coupling constants, molecular mechanic calculation
using the PC Model [26] and confirmed by a ROESY experiment
iochromolide [28], whose structure was confirmed by X-ray anal-
ysis. In the ROESY experiment a cross-peak of H-4 with H-9 and
another of H-16 with H-14 and H-22 confirmed the -stereo-
a
a
a
b
chemistry of the hydroxyl groups at C-4 and C-16, while correlation
of Me-21 with Me-18 and H-12 indicated a Z configuration of the
double bond
,16 ,27-trihydroxy-1-oxo-witha-2,17(20),24-trienolide.
Compound 2 showed a molecular formula of C30H40O7 by
D b,6b-epoxy-
17. Thus, 1 was concluded to be 5
4b
b
HREIMS. Comparison of its NMR data (Table 1) with those of
withaferin A (9) [29] indicated as the main difference the presence
of an acetate group (dH 2.08, dC 20.7 and dC 170.7) instead of
a primary alcohol. 2D NMR (COSY, ROESY, HSQC and HMBC)
experiments allowed the complete and unambiguous assignment
of the chemical shifts, regiosubstitution and relative configuration
of compound 2. Accordingly, the structure of 2 was established as
(Fig. 2). Thus, the
molecular modelling of both
shape of the signal at dH 4.73 (H-16) as a broad singlet is closer to
the values calculated for the 16 -hydroxy isomer [27], whereas an
orientation gives a larger J (9.0, 7.2 Hz) as previously reported for
b
-hydroxyl group at C-16 was supported by
27-acetoxy-5
(27-O-acetyl-withaferin A).
The HRESIMS of compound 3 indicated a molecular formula of
C32H44O8, and the 1H and 13C NMR data (Table 1) were closely
related to those of 2. Thus, the most notable differences were the
b,6b-epoxy-4b-hydroxy-1-oxo-witha-2,24-dienolide
a
and -epimers at C-16. The observed
b
b
a
OH
O
HO
OH
H
O
HO
4
H
H
O
H
H
OH
H
H
Fig. 3. 1H-13C long-range correlations (solid line) and ROE effects (dashed line) for compounds 5 (left) and 6 (right).

G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
503
OR
OR
O
O
O
O
O
O
ii
9b R= TBDMS
15 R= H
9 R= H
O
O
iii
i
O
OH
9a R= TBDMS
Scheme 1. Synthesis of derivatives 9a, 9b and 15. Reagents and conditions: (i) TMDSCl, imidazole, DIMAP, CH2Cl2, r.t., 2.5 h; (ii) CrO3, py, CH2Cl2, r.t., 6 h; (iii) Dowex (50WX8-200),
acetone, r.t., 24 h.
presence of signals for two singlet methyl groups [dH 1.46 (3H, s),
1.48 (3H, s) and dC 22.4 (q), 22.7 (q)], a hemiketal carbon at dC 104.9
(s) and a formate group [dH 10.62 (1H, s) and dC 161.5 (d)] instead of
the signals corresponding to the acetate group in 2. Moreover, the
27-O-isopropyl formate group in 3 was located at C-27 as a result of
the observed HMBC correlations of the primary alcohol resonance
at dH 4.27, 4.32 (2H, dAB, J ¼ 10 Hz, H-27) and the aldehyde proton at
dH 10.62 (1H, s) with the hemiketal carbon at dC 104.9. Therefore,
C-3 was also in agreement with the observed HMBC correlations of
H-2 (dH 2.53)/C-1, C-3, C-4 and H-3 (dH 4.34)/C-1 and C-5 as well as
those between H-4 (dH 3.46) and C-2, C-3, C-6 and C-10. The relative
configuration of 4 was established on the basis of the coupling
constants, molecular mechanic calculation using the PC model [26],
comparison with the reported data in the literature [30] and
confirmed by a ROESY experiment. Thus, the
deduced by the coupling constants of H-3 [
10.3 Hz)] similar to those reported for 27-O-
viscosalactone B [31]. Moreover, a cross-peak of H-6
and one of H-4 with H-3 further confirmed the -stereochemistry
of the hydroxyl group at C-3. This spectral evidence determined the
structure of 4 as 27-acetoxy-5 ,6 -epoxy-3,4-dihydroxy-1-oxo-
b
-hydroxyl at C-3 was
4.34 (dd, J ¼ 4.2,
-glucopyranosyl
with H-4
d
the structure of compound 3 was deduced as 5
b,6
b
-epoxy-4
b
-
b-D
hydroxy-27-(1-formyloxy-1-methylethoxy)-1-oxo-witha-2,24-
dienolide. To our knowledge, this is the first withanolide-type
compound with an O-isopropyl formate moiety.
Compound 4 was assigned the molecular formula C30H42O8 by
HRESIMS, 18 mass units higher than that obtained for 2. The 1H and
13C NMR data of 4 (Table 1) exhibited characteristic signals for
a
a
a
b
b b
witha-24-enolide (27-O-acetyl-viscosalactone B).
The molecular formula of compound 5 was deduced to be
C28H42O8 by HRESIMS. A study of its IR, UV, 1H and 13C NMR
(Table 2) and 2D spectra showed it to be a withanolide-type
compound with a carbonyl group at dC 209.8 (C-1), a primary [dH
4.36, 4.42 (dAB, J ¼ 12.4 Hz, H-27) and dC 57.4 (C-27)], three
secondary [dH 4.38 (m, H-3), 4.46 (m, H-4), 4.14 (d, J ¼ 7.9 Hz, H-6),
dC 72.9 (C-3), 77.7 (C-4), 76.3 (C-6)] and a tertiary [dC 76.3 (C-5)]
hydroxyl groups, and an a,b-unsaturated d-lactone moiety [dC 153.3
(C-24), 125.6 (C-25), 167.2 (C-26)]. The analysis of HMBC and ROESY
experiments revealed that 5 have the same substitution pattern
a 5b,6b-epoxy-3b,4b-dihydroxy-1-one withanolide skeleton [30].
Its 1H NMR data were similar to those of 2, with the exceptions that
signals were found for aliphatic methylene protons at dH 2.53 (dd,
J ¼ 5.0, 15.4 Hz), 3.21 (dd, J ¼ 6.4, 15.4 Hz) assigned to H2-2 and an
oxymethine proton at dH 4.34 (dd, J ¼ 4.2, 10.3 Hz, H-3) rather than
the two olefinic protons of the a,b-unsaturated ketone of ring A in
2. 13C NMR resonances at dC 209.8 (C-1), 42.5 (C-2) and 68.3 (C-3)
confirmed saturation of the C-2/C-3 bond and the presence of
a hydroxyl group at C-3 in 4. The location of the hydroxyl group at
Table 3
Cytotoxic activity (IC50
,
m
M) of withanolidesa against tumor cell and Vero cell lines.
A-549
Compound
HeLa
48 h
MCF-7
48 h
Vero
48 h
SIc
72 h
48 h
72 h
72 h
72 h
1
2
3
4
8
9
10
11
12
13
14
15
16
17
9a
9b
C1b
C2b
40.0 ꢂ 2.1
7.7 ꢂ 0.3
>40
31.1 ꢂ 2.6
4.7 ꢂ 0.5
37.3 ꢂ 2.4
8.3 ꢂ 0.3
3.4 ꢂ 0.06
2.3 ꢂ 0.03
19.3 ꢂ 0.7
2.7 ꢂ 0.06
8.0 ꢂ 0.1
2.9 ꢂ 0.03
19.8 ꢂ 0.7
15.2 ꢂ 0.9
31.4 ꢂ 2.1
12.1 ꢂ 0.6
1.0 ꢂ 0.04
2.1 ꢂ 0.08
e
>40
>40
27.7 ꢂ 2.0
5.3 ꢂ 0.1
6.0 ꢂ 0.1
2.0 ꢂ 0.05
18.3 ꢂ 0.9
7.1 ꢂ 0.4
1.1 ꢂ 0.02
0.6 ꢂ 0.002
5.4 ꢂ 0.1
0.3 ꢂ 0.003
2.2 ꢂ 0.008
0.3 ꢂ 0.002
10.3 ꢂ 0.9
2.1 ꢂ 0.6
36.2 ꢂ 2.3
1.3 ꢂ 0.01
0.7 ꢂ 0.006
0.2 ꢂ 0.008
e
>40
>40
<1
6.7 ꢂ 0.1
4.4 ꢂ 0.1
11.4 ꢂ 0.7
27.8 ꢂ 1.5
8.6 ꢂ 0.1
8.5 ꢂ 0.09
18.5 ꢂ 0.9
4.9 ꢂ 0.1
2.2 ꢂ 0.03
1.7 ꢂ 0.04
9.0 ꢂ 0.3
2.6 ꢂ 0.2
3.1 ꢂ 0.06
1.2 ꢂ 0.02
21.8 ꢂ 1.5
1.5 ꢂ 0.4
26.9 ꢂ 2.1
7.8 ꢂ 0.2
0.9 ꢂ 0.01
4.0 ꢂ 0.09
e
2.2
1.0
<1
<1
<1
1.1
2.8
<1
1.7
1.8
2.4
1.0
2.0
1.0
6.2
3.0
48.3
>40
>40
26.9 ꢂ 1.5
9.2 ꢂ 0.2
8.9 ꢂ 0.3
4.6 ꢂ 0.3
3.0 ꢂ 0.1
28.9 ꢂ 3.2
3.5 ꢂ 0.2
21.5 ꢂ 0.9
3.5 ꢂ 0.1
17.7 ꢂ 0.9
18.4 ꢂ 1.0
>40
13.4 ꢂ 0.7
0.9 ꢂ 0.01
1.7 ꢂ 0.01
1.1 ꢂ 0.1 ꢁ 10ꢀ3
2.9 ꢂ 0.06
18.4 ꢂ 0.7
9.0 ꢂ 0.2
6.6 ꢂ 0.08
33.5 ꢂ 1.9
4.2 ꢂ 0.9
40 ꢂ 2.1
2.6 ꢂ 0.04
>40
21.3 ꢂ 1.8
1.5 ꢂ 0.05
1.5 ꢂ 0.05
17.2 ꢂ 0.8
2.3 ꢂ 0.06
5.5 ꢂ 0.2
2.1 ꢂ 0.04
31.7 ꢂ 2.5
12.8 ꢂ 0.9
36.4 ꢂ 3.1
8.6 ꢂ 0.7
1.5 ꢂ 0.03
2.9 ꢂ 0.1
e
6.6 ꢂ 0.08
3.6 ꢂ 0.02
14.4 ꢂ 0.9
1.2 ꢂ 0.01
12.6 ꢂ 0.9
1.3 ꢂ 0.07
16.3 ꢂ 1.1
3.2 ꢂ 0.3
1.5 ꢂ 0.04
1.3 ꢂ 0.005
15.5 ꢂ 0.9
3.4 ꢂ 0.08
4.7 ꢂ 0.09
6.2 ꢂ 0.1
28.8 ꢂ 1.7
7.7 ꢂ 0.3
25.4 ꢂ 1.8
>40
38.4 ꢂ 2.8
7.5 ꢂ 0.1
40 ꢂ 2.1
14.7 ꢂ 0.7
1.5 ꢂ 0.02
5.3 ꢂ 0.3
7.2 ꢂ 0.3 ꢁ 10ꢀ3
47.0 ꢂ 1.9
14.8 ꢂ 1.0
0.7 ꢂ 0.01
3.1 ꢂ 0.1
0.7 ꢂ 0.01
0.5 ꢂ 0.01
2.4 ꢂ 0.1 ꢁ 10ꢀ2
1.4 ꢂ 0.06
7.1 ꢂ 0.5 ꢁ 10ꢀ2
67.6 ꢂ 5.2
e
e
e
e
e: Not tested.
a
Compounds 5e7 and 7a showed IC50 > 40 mM against all the tested cell lines.
C1 and C2: actinomycin and mercaptopurine, respectively, were used as positive controls.
SI: Selectivity index defined as Vero (IC50) on MCF-7.
b
c

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G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
and stereochemistry as viscosalactone B [30] with the only differ-
ence being a 5 ,6 -diol system rather than the 5 ,6 -epoxy group.
2.2. Biological evaluation
a
b
b b
Moreover, the localization of the two hydroxyl groups at C-5 and C-
6 were determined as a result of the observed HMBC correlations
between H-3/C-1, C-5; H-4/C-2, C-6, C-10; H-6/C-8, C-10 and Me-
19/C-1, C-5, C-9 (Fig. 3). The stereochemistry of the hydroxyl
2.2.1. Antiproliferative activity
In our previous study on the biological activities of W. aristata
extracts, the CH2Cl2 fraction showed a strong antiproliferative
effect on two tumor cell lines, HeLa and A-549, with IC50 values of
group at C-5 was deduced as
tion, because no upfield shift effect was observed for the CH3-19 (dC
14.7), whereas the presence of a 5 -OH does cause a 10 ppm upfield
shift of CH3-19 (around dC 9) due to the gauche-interaction [32].
Furthermore, the ROE correlations observed of H-4 with H-3 and
H-6 defined the 6 -axial disposition for the hydroxyl group at C-6
(Fig. 3). Therefore, the structure of was established as
,4 ,5 ,6 ,27-pentahydroxy-1-oxo-witha-24-enolide. This
a
, and therefore a cis-A/B ring junc-
11.7 and 19.9 mg/mL, respectively. This has encouraged us to
investigate the isolates from this fraction for their cytotoxic activity.
In this study, the seventeen isolated compounds (1e17),
including withaferin A (9) for comparative purposes, and three
derivatives (7a, 9a and 9b) were assayed for their antiproliferative
activity against three human cancer cell lines: HeLa (cervix), A-549
(lung) and MCF-7 (breast), and Vero (African green monkey kidney)
non-tumoral cell line used for selectivity purposes. Except for the
prototype withaferin A, few studies on the cytotoxic activity of the
known isolated compounds 8 [38], 10 [21,39,40], 12 [38], 13 [21], 14
[38] and 15 [40] have been previously reported. Nevertheless, we
include these compounds in our studies in order to broaden the
structureeactivity studies and, moreover, because the cell lines and
procedures were different from the ones used herein.
b
a
b
5
3
b b a b
compound has one feature of particular interest as it is the first
withanolide-type compound reported with four hydroxyl groups
on A/B rings.
Compound 6 showed spectral data resembling those of the
known isolated compound 7 [33], and its molecular formula
(C28H38O6), determined by HRESIMS, revealed it to be 2 m/z lower
than the one for 7. The 1H and 13C NMR spectra suggested that 6 had
a double bond at C2eC3 instead of a methylene group, as in 7. Even
so, it should be noted that a complete set of 2D NMR spectra (COSY,
HSQC and HMBC) was acquired for 6 in order to gain the complete
and unambiguous assignment of the 1H and 13C NMR resonances as
listed in Table 2. The relative stereochemistry was confirmed by
a ROESY experiment (Fig. 3). Thus, ROE correlation between the
signal at dH 1.22 (Me-19) and the signals at dH 8.79 (CHO-4) and dH
As shown in Table 3, the silyl ether derivatives 9a and 9b
showed pronounced antiproliferative activity against the tested
tumor cell lines with IC50 values ranging from 0.5 to 2.9
are lower than those of the widely known anticancer withaferin A
(IC50 3.0e6.6 M). Furthermore, compounds 27-O-acetyl-with-
aferin A (2), 4 -hydroxy-1-oxo-5 ,6 -epoxywitha-2,24-dienolide
(8), 16-en-withaferin A (11) and 4 -hydroxy-1-oxo-5 ,6 -epoxy-
22R-witha-2,14,24-trienolide (13) also displayed strong cytotox-
icity, with IC50 values less than 9 M. Among them, compounds 11
mM, which
m
b
b b
b
b b
3.27 (HO-6) established the
b
-orientation of the 4-formyl and 6-
m
hydroxyl moieties. Therefore, the structure of compound 6 was
and 13 demonstrated stronger cytotoxicity against A-549 and
MCF-7 cell lines than the reference, withaferin A. In addition,
compounds 15 and 17 exhibited a fair level significant of activity
determined to be 4b-formyl-6b,27-dihydroxy-1-oxo-witha-2,24-
dienolide.
The conditions leading to the rearrangement that produced
ring-A contraction and the formation of an aldehyde centre at C-4
have been reported previously in the literature [33]. Accordingly,
compound 7 was obtained by treatment with sulphuric acid in
against MCF-7 cells, with IC50 values of 3.2 and 7.5 mM, respec-
tively. Overall, the compounds showed time dependent antitu-
moral activity against the investigated cell lines. Thus, compounds
8 and 13, as did withaferin A, showed a significant increase in
cytotoxicity (more than three-fold) against two or three human
cancer cell lines after 72 h of exposure compared to 48 h.
acetone of 2,3-dihidrowithaferin A, however, as
a natural
compound, it is reported here for the first time. Although some of
the 1H and 13C NMR data have already been published, we
contribute with the complete and unambiguous assignments of the
1H and 13C NMR signals (Table 2) of the known withanolide 4
b-
formyl-6
1D and 2D NMR techniques.
By comparing the NMR and MS data with those previously
reported, eleven known compounds were identified as
hydroxy-1-oxo-5 ,6 -epoxywitha-2,24-dienolide [34] (8), with-
aferin A [27] (9), 2,3-dihydro-withaferin A [19] (10), witharistatin
[21] (11), 27-deoxy-16-en-withaferin A [35] (12), 4 -hydroxy-1-
oxo-5 ,6 -epoxy-22R-witha-2,14,24-trienolide [19] (13), ,6
epoxy-4 ,17 ,27-trihydroxy-1-oxowitha-2,24-dienolide [36] (14),
4-dehydro-withaferin A [29] (15), 2,3-dehydrosomnifericin [37]
(16) and 6 -chloro-5 -hydroxywithaferin A [20] (17).
b,27-dihydroxy-1-oxo-witha-24-enolide (7) [33], based on
4b-
b
b
b
b
b
5b b-
b
a
a
b
Owing to the fact that compounds 7 and 15 were isolated in
insufficient amounts for the full biological evaluation, we decided
to carry out their semisynthesis starting from withaferin A (9)
and 6, respectively (Scheme 1). Thus, catalytic reduction of
compound 6 with palladium hydroxide-carbon gave compounds
7 and the known derivative 7a [33]. Moreover, firstly, selective
protection of the primary alcohol in compound 9 with TMDSCl,
imidazole and DMAP in CH2Cl2 at room temperature during 2.5 h
gave the corresponding silyl ether derivative (9a), which was
then oxidized with CrO3 in pyridine (Collins reagent) to afford the
ketone 9b. Further cleavage of the TBDMS group was achieved
with carboxylic acid resin Dowex 50WX8-200 in acetone to yield
compound 15.
Fig. 4. Fragmentation of genomic DNA in HeLa cells. Cells were treated with
compounds 8, 9, 9a, 9b, 11 and 13 at 16
visualized by ethidium bromide staining. Lane 1, untreated cells (C); PC, staurosporine
at 2 M (positive control); lanes 3e8, treatment with compounds 8, 9, 11, 13, 9a, and
9b, respectively; M, DNA marker of 125-21, 226 bp.
mM for 48 h. agarose gel electrophoresis,
m

G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
505
Moreover, MCF-7 cell line turned out to be the most sensitive to
2.2.2. SAR analysis
the withanolides.
The influence of the substitution pattern on the cytotoxic
activity of the withanolides studied in this work was examined,
revealing the following trends in the structureeactivity relation-
ship. a) The influence of substituents on the A and B rings was
evident when we compared the activity displayed by 9 with those
of 4-7, 7a, 8e10 and 16e17, as simple modification of the double
bond at C-2(3) (9 vs 10) or epoxy group at C-5(6) (9 vs 16) produced
a considerable decrease in the activity. Moreover, the elimination of
In addition, when comparing MCF-7 cancer cells with the non-
tumorigenic (Vero) cell line, some degree of selective cytotoxicity
was observed (Table 3). Thus, the most active compound (9b)
exhibited a selectivity index (SI) of 6.2 for this cell combination,
higher than that withaferin A (SI 0.4). Moderate selectivity (SI ꢃ 2)
was also found for withanolides 2, 11, 15 and 17, which may shed
light on these compounds for future studies.
Fig. 5. Withanolide induced apoptotic nuclei in HeLa cells. (A) Representative fluorescence images of apoptotic nuclei. Cells were treated with the compounds 8, 9, 11, 13 9a and 9b
at 8
M or with the respective solvent control (C) for 12 h and then stained with Hoechst 33342; amplification 40ꢁ. (B) Percent of Hoechst-stained, apoptotic cells, calculated from
a total of at least 500 per sample. Bars represent means ꢂ SD of three independent experiments, performed in triplicates; *p < 0.05. (C) Assessment of sub-G1 DNA content. Cells
were incubated in the absence (control) versus presence of 24 M of compounds 8, 9, 11, 9a and 9b for 12 h, permeabilized, stained with propidium iodide, and analyzed by flow
m
m
cytometry. Bars represent means ꢂ SD of percent of cells undergoing apoptosis in each sample. Three independent experiments, each in triplicate, were performed. *p < 0.05 with
respect to controls.

506
G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
those both essential groups, as in 5, caused the abolition of cyto-
toxicity. These results are in agreement with previous studies
[41,42], which define these functions as structural requirements in
withanolide skeleton for biological function. However, in contrast
with previous results [43], we found that the cytotoxic effect of the
6-chlorowithanolide 17 was higher than its corresponding hydroxyl
derivative 16. Furthermore, the most striking result was the
replacement of a hydroxyl by a carbonyl group at C-4 which results
in a considerable increase of selectivity (9 vs 15 and 9a vs 9b).
a) Substituents on the five member ring play an important role
and thus, comparison of the activity displayed by 8 and 9 with
those of 1 and 11e14 showed as most relevant result the
staining with Hoechst 33342, a specific stain for AT-rich regions of
double-stranded DNA. Condensation of chromatin and apoptotic
bodies in the vicinity of the cells were clearly observed by fluores-
cence microscopy (Fig. 5A), which suggests induction of apoptotic
cell death byall withanolides. Apoptotic cells were quantified taking
into account only those cells the chromatin of which had split into
more than three fragments. The number of apoptotic cells was
significantly higher upon treatment with the withanolides than in
the control cells (p < 0.05) and even higher than upon withaferin A
treatment, as approximately 27% more apoptotic cells were assessed
in the withanolide 13 samples (Fig. 5B).
Additionally, the degree of induced apoptosis was estimated by
flow cytometry evaluation of the characteristic sub-G1 peak, which
reflects cells with a lower DNA content compared to cells in G0/G1
phase of the cell cycle [46,47]. Except for withanolide 13, where we
had run out of compound, cell exposure to all the withanolides at
improvement of the
a detrimental effect was observed with the presence of
a hydroxyl group (9 vs 1 and 14).
D
14-withanolide 13. On the other hand,
b) Regarding the substituents on the lactonering, weconclude that
the substitution of a C-27 methyl (8 and 12) by a hydroxymethyl
group (9 and 11) or substitution of a hydroxymethyl by a silyl
ether (9a and 9b) increases considerably the activity, whereas
the presence at C-27 of an acetyl or ketal group (2 and 3) is
detrimental. These trends, based upon substitution patterns, on
one hand are consistent with previous SAR findings, and on
other provide valuable information on the pharmacophore for
withanolide-type compounds and will be helpful for the
rational design of more potent and selective anticancer drugs.
24
mM for 12 h produced defined sub-G1 peaks (Fig. 5C).
However, chromatin condensation may also be found in necrotic
cells [48]. Moreover, apoptosis and necrosis are two types of demise
that can occur simultaneously in tissues or cell cultures exposed to
a single stimulus. Often, the intensity of the initial insult decides for
the prevalence of either apoptosis or necrosis. Hence, the evalua-
tion of proteins, implicated in activation or execution of apoptosis,
may help unravel the type of ongoing cell death. Caspases are
crucial mediators of programmed cell death (apoptosis). Among
them, caspase-3 is a commonly activated death effector protease,
catalyzing the specific cleavage of various key cellular proteins. Its
role in programmed cell death has been shown to be remarkably
tissue- cell type-, or death stimulus- specific. Caspase-3 activation
is essential for some of the characteristic changes in cell
2.2.3. Detection of apoptosis
The control of apoptosis is a crucial target for conventional
drug development, particularly for cancer therapeutics. In this
work, we employed various experimental approaches to charac-
terize the apoptotic potential of the withanolides 8, 9a, 9b, 11
and 13 in comparison with the structurally related well-known
steroidal lactone withaferin A (9) in HeLa cervical carcinoma
cells.
To start with, we tried to detect regular fragmentation of nuclear
DNA, a phenomenon associated with most types of late stage
apoptosis. HeLa cells were treated with withanolides at 16, 24, and
32
tionally, withaferin A was added to HeLa cultures at concentrations
of 4, 8, 12, 16, 24, and 32 M for 12 h. None of these treatments
mM for 24 h and at a concentration of 16 mM for 48 h. Addi-
m
resulted in the typical DNA fragments of 180e200 bp and multiples,
product of enzymatic cleavage. However, a predominant smear
pattern in agarose gel electrophoresis was detectable with all test
compounds (Fig. 4) at all concentrations and exposure times, also
after treatment with withaferin A (data not shown), where induc-
tion of apoptosis has been described by various authors [5,15,16].
Similar results were obtained under actinomycin D and staur-
osporine treatment, substances known to induce apoptosis in many
cell lines. The pattern was not evident in solvent control cultures.
In accordance with Meyer et al. [44], the apoptosis-specific 200-
bp DNA ladder is overlaid by random DNA fragmentation, resulting
from necrotic cell death, which may also appear in imiquimod or
actinomycin D treated cell cultures. While rapidly removed by
phagocytosis in vivo, apoptotic bodies are usually not eliminated in
cell cultures and finally lyse, leading to a state of secondary
necrosis. Thus, DNA fragmentation is common to different kinds of
cell death; three types of DNA fragmentation can occur during
apoptosis: internucleosomal DNA cleavage, fragmentation into
large fragments of 50e300 kbp length, and single-strand cleavage
events [45].
In view of these observations, we decided to check for chromatin
condensation, a hallmark of apoptosis, where compact and small
chromatin nuclei and often apoptotic bodies appear. To this end,
Fig. 6. Caspase-3 activity in HeLa cells. (A) Cells treated with compounds 8, 9, 11, 13, 9a
and 9b at 12, 16, 24, and 32
mM for 4 h. (B) Cells treated with the same compounds
(except compound 13) at 24
mM
for and h, respectively. Values represent
4
8
means ꢂ SD of three independent experiments, each performed in triplicate; differ-
ence between treatments and control with p < 0.05 (*) was considered statistically
significant.
HeLa cells were exposed to the withanolides at 8
mM for 12 h and the
morphological features of apoptotic cells were determined by

G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
507
morphology and certain biochemical events associated with the
execution and completion of apoptosis [49] and eventually lead to
degradation of chromosomal DNA [50].
with 9a only induced a negligible increase in caspase-3 activity,
associated with drastic cell damage.
The early stage of apoptosis is typically accompanied by a loss of
membrane phospholipid asymmetry, going along with phosphati-
dylserine (PS) flipping onto the surface of the cell. PS externaliza-
tion plays an important role in the recognition and removal of
apoptotic cells by macrophages [53]. Therefore, we decided to use
annexin V, a specific ligand of PS, to determine the early apoptotic
fraction in HeLa cells and propidium iodide (PI), which can only
pass the cell membrane in late stage apoptosis or necrosis. HeLa
cells were exposed to withanolides at a concentration range of
Withaferin A has been reported to activate the apoptotic cascade
by extrinsic or intrinsic pathways in promyelocytic leukemia cells
HL-60 [51], U937 [14] prostate cancer [52], and HNSCC cells [11]
among others. Hence, we wished to definitely identify the type of
ongoing cell death in HeLa cells, determining potential caspase-3
activation by withanolides 8, 9, 11, 13, 9a and 9b. The induction of
caspase-3 was assessed by colorimetric assay after treatment of
HeLa cells with the withanolides at different concentrations (4, 8,
12, 16, 24, and 32
induced caspase-3 activation in a dose-dependent manner in
concentrations >12 M, while enzyme activity at 4 and 8 M did
not differ from untreated cells. Particularly, withaferin A and 11
increased enzyme activity from 1 to 2 fold at 12 M, respectively
(compared to controls) to 5-fold at 32 M. Notably, caspase activity
declined upon treatment with withanolide 9a at 32 M for 4 h,
m
M) for 4 h Fig. 6A shows that the withanolides
2e16 mg/mL for 30 min, 1, 2, and 4 h of incubation before costaining
with annexin V/PI. Fluorescence microscopy (Fig. 7) evidenced that,
in contrast to untreated cells, all withanolides, as well as the
positive control actinomycin D, induced dose- and time-dependent
PS externalization and/or necrosis in HeLa cells. Early apoptotic
cells (annexin Vþ) were evidenced after treatment with
m
m
m
m
m
compounds 8, 9, and 11 at 2 and 4 mM over a period of 4 h, while
which was probably due to the associated loss of cellular integrity
and adhesion, as observed by light microscope (data not shown).
To determine whether caspase-3 activation could further
increase with time, HeLa cells were treated with compounds 8, 9,
exposure to 13 led to early and late apoptosis (annexin Vþ/PIþ) in
these conditions. A higher concentration (8 M) of the withanolides
m
resulted in necrotic cells (PIþ). The effect of 9a was more
pronounced in HeLa cells than the one of withaferin A, since early
apoptosis was already evidenced after 1 h of treatment. However,
11, 9a and 9b at a fixed concentration of 24 mM for 8 h. In fact,
caspase-3 activity further augmented with 8 h, mainly after expo-
sure to withaferin A and 9b that produced activities similar to the
with 9b a higher dose (8
(2e4 h) were required to lead to apoptosis, effect that was main-
tained at concentrations of 12 and 16 M. Interestingly, unlike with
the other withanolides, treated cells never exhibited necrosis.
mM) and a longer incubation period
one with staurosporine treatment at 2
mM for 4 h (1.92 nmol pNA/
m
min/mL ꢂ 0.13), used as positive control (Fig. 6B). Again, treatment
Fig. 7. Fluorescence microphotograph of withanolides treated, early apoptotic HeLa cells, characterized by phosphatidylserine (PS) extracellular membrane expression. (A) DMSO
solvent control. (B) Actinomycin D at 1 M for 1 h. (CeF) withanolides 8, 9, 11, and 13 at 2 M for 4 h (GeH) withanolides 9a and 9b at 8 M for 1 and 2 h, respectively. PS flipped to
the extracellular membrane was labeled with annexin V, and necrotic/late apoptotic cells were identified through PI membrane permeability.
m
m
m

508
G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
3. Conclusion
(from 100:0 to 0:100) affording nine fractions, four of them (VI, VII,
VIII, and IX) containing withanolides according to previous 1H
NMR analysis. Each of these fractions was subjected to column
chromatography over Sephadex LH-20 (n-hexane/CHCl3/MeOH,
2:1:1), and silica gel (CH2Cl2/acetone of increasing polarity).
Preparative thin-layer chromatography developed with CH2Cl2/
acetone (8.5:1.5) was used to purify the new compounds 1
(3.8 mg), 2 (4.1 mg), 3 (1.2 mg), 4 (2.3 mg), 5 (3.5 mg) 6 (3.1 mg)
A new series of withanolides isolated from W. aristata, along with
three derivatives, have been evaluated as anticancer agents. Five of
the assayed compounds showed a potent antiproliferative activity
against three human tumor cell lines and showed higher activity
than the reference anticancer agent withaferin A. Structureeactivity
relationships among these withanolides suggest that the silyl group
gives an additional favourable effect on cytotoxicity and selectivity.
Taken together, our results demonstrate that the withanolides
studied in this work induce apoptosis in HeLa cells in a dose- and
time-dependent manner, as evidenced by chromatin condensation,
PS externalization, and most importantly caspase-3 activation,
although a ladder pattern, typical for caspase activated enzymatic
DNA cleavage, was not observed. These findings are consistent with
data obtained byother authors for withaferin A in different cell lines.
The potent capacity to induce apoptosis in cancer cells and, perhaps
even moreintriguingly, theinduction of apoptosisbywithanolide9b
without necrosis under extreme experimental conditions, draw
attention to this group of substances and call for further studies to
determine their exact mechanisms of action. Our findings support
the notion that these types of withanolides could be considered as
active and specific anticancer agents with promising future pros-
pects in the treatment of cancer diseases.
and 7 (1.8 mg), in addition to the known compounds 4
1-oxo-5 ,6 -epoxywitha-2,24-dienolide (8, 32.1 mg), withaferin A
(9, 3.1 g), 2,3-dihydro-withaferin A (10, 2.0 mg), witharistatin (11,
4.8 mg), 27-deoxy-16-en-withaferin A (12, 3.0 mg), 4 -hydroxy-1-
oxo-5 ,6 -epoxy-22R-witha-2,14,24-trienolide (13, 1.5 mg),
,6 -epoxy-4 ,17 ,27-trihydroxy-1-oxowitha-2,24-dienolide
(14, 6.0 mg), 4-dehydro-withaferin (15, 1.2 mg), 2,3-
-chloro-5 -hydrox-
b-hydroxy-
b
b
b
b
b
5
b
b
b
a
A
dehydrosomnifericin (16, 2.5 mg) and
ywithaferin A (17, 4.7 mg).
6
a
b
4.3.1. 5
2,17(20),24-trienolide (1)
White amorphous solid; [
b,6b-Epoxy-4b,16b,27etrihydroxy-1-oxo-witha-
a
]
20 þ 25.4 (c 0.6, CHCl3); UV lmax nm:
D
215; IR nmax cmꢀ1: 3415, 2929, 2861, 1685, 1457, 1384, 1180, 1083,
1026, 755; 1H NMR : see Table 1; 13C NMR
d d: see Table 2; EI/MS m/z
%: 484 (Mþ, 6), 466 (33), 448 (54), 434 (28), 391 (39), 343 (33), 281
(33), 192 (31), 151 (47), 124 (100), 95 (99), 67 (64); HREIMS: m/z
484.2462 (calcd for C28H36O7 484.2461).
4. Experimental
4.1. General
4.3.2. 27-Acetoxy-5b,6b-epoxy-4b-hydroxy-1-oxo-witha-2,24-
dienolide (2)
Optical rotations were measured on a Perkin Elmer 241 auto-
White lacquer; [
a
]
D
20 þ 63.4ꢄ (c 0.5, CHCl3); UV lmax nm: 215; IR
matic polarimeter in CHCl3 at 20 ꢄC and the [aD] are given in
nmax cmꢀ1: 3748, 2928, 1701, 1540, 1458, 1397, 1256, 1066, 838; 1H
10ꢀ1 deg cm2
g
ꢀ1. UV spectra were obtained on a JASCO V-560
NMR d d
: see Table 1; 13C NMR : see Table 2; EI/MS m/z%: 512 (Mþ, 7),
spectrophotometer, and IR (film) spectra were measured on
a Bruker IFS 55 spectrophotometer. NMR experiments were per-
formed on a Bruker Avance 400 spectrometer and chemical shifts
494 (5), 452 (9), 416 (7), 389 (21), 329 (24), 311 (25), 239 (23), 183
(25), 161 (19), 124 (100), 95 (80), 67 (43); HREIMS: m/z 512.2775
(calcd for C30H40O7, 512.2774).
are shown in
d (ppm) with tetramethylsilane (TMS) as internal
reference. EIMS and HREIMS were recorded on a Micromass
Autospec spectrometer, and ESIMS and HRESIMS (positive mode)
were measured on an LCT Premier XE Micromass Electrospray
4.3.3. 5
1-oxo-witha-2,24-dienolide (3)
b,6b-Epoxy-4b-hydroxy-27-(1-formyloxy-1-methylethoxy)-
White lacquer; [
a
]
20 þ 24.3 (c 0.2, CHCl3); UV lmax nm: 220; IR
D
spectrometer. Purification was performed using silica gel 60
mM for
nmax cmꢀ1: 3438, 2931, 2870, 1700, 1460, 1396, 1320, 1186, 1019,
column chromatography (particle size 15e40 and 63e200
mm),
850, 755; 1H NMR : see Table 1; 13C NMR
d d: see Table 2; ESIMS
POLYGRAM SIL G/UV254 used for analytical and preparative TLC,
and HPTLC-platten Nano-Sil 20 UV254 were purchased from
MachereyeNagel. Sephadex LH-20 for exclusion chromatography
was obtained from Pharmacia Biotech. The spots were visualized by
UV light and heating silica gel plates sprayed with
H2OeH2SO4eACOH (1:4:20). All solvents used were analytical
grade from Panreac. Reagents were purchased from Sigma Aldrich
and used without further purification.
(positive) m/z%: 579 [M þ Na]þ (100; HRESIMS: m/z 579.2939
[M þ Na]þ (calcd. for C32H44O8Na: 579.2934).
4.3.4. Acetoxy-5b,6b,epoxy-3,4-dihydroxy-1-oxo-witha-24-enolide
(4)
White lacquer; [
a
]
D
20 þ 13.9 (c 0.39, CHCl3); UV lmax nm: 214; IR
nmax cmꢀ1: 3401, 2931, 2859, 1678, 1460, 1380, 1272, 1129, 1076,
1022, 754; 1H NMR : see Table 1; 13C NMR
d d: see Table 2; ESIMS
(positive) m/z%: 553 [M þ Na]þ (100); HRESIMS: m/z 553.2772
4.2. Plant material
[M þ Na]þ (calcd. for C30H42O8Na: 553.2777).
Leaves of W. aristata were collected in Icod de los Vinos, Tenerife,
Canary Islands (Spain), in May 2005. A voucher specimen (TFC
48.068) is deposited in the Herbarium of the Department of Botany,
University of La Laguna, Tenerife, and identified by Leticia Rodrí-
guez-Navarro.
4.3.5. 3
White lacquer; [
nmax cmꢀ1: 3748, 3499, 2929,1658,1459,1397,1256,1065, 838, 756;
b,4b,5a,6b,27-Pentahydroxy-1-oxo-witha-24-enolide (5)
20
a
]
þ 28.1 (c 0.8, CHCl3); UV lmax nm: 215; IR
D
1H NMR : see Table 1; 13C NMR
d d: see Table 2; ESIMS (positive) m/z
%: 529 [M þ Na]þ (100); HRESIMS: m/z 529.2775 [M þ Na]þ (calcd.
for C28H42O8Na: 529.2777).
4.3. Extraction and isolation
4.3.6. 4
White lacquer; [
231; IR nmax cmꢀ1: 3438, 2940, 2870, 1700, 1460, 1396, 1320, 1187,
b-Formyl-6b,27-dihydroxy-1-oxo-witha-2,24-dienolide (6)
20
The air-dried powdered leaves of W. aristata (1.65 kg) were
exhaustively extracted with CH2Cl2 in a Soxhlet apparatus and the
solvent was evaporated at reduced pressure. The residue (71 g)
was fractioned by vacuumeliquid chromatography on silica gel
and eluted with hexane/EtOAc mixtures of increasing polarity
a
]
þ 160.8 (c 0.68, CHCl3); UV lmax nm: 220,
D
1019, 754; 1H NMR : see Table 1; 13C NMR
d d: see Table 2; ESIMS
(positive) m/z%: 469 [M ꢀ 1]þ (100); HRESIMS: m/z 469.2580
[M ꢀ 1]þ (calcd. for C28H37O6: 469.2590).

G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
509
4.3.7. 4
b
-Formyl-6
b
,27-dihydroxy-1-oxo-witha-24-enolide (7)
C27); 62.3 (d, C6); 63.6 (s, C5); 69.7 (d, C4); 77.9 (d, C22); 125.7 (s,
C25); 132.1 (d, C2); 141.7 (d, C3); 154.3 (s, C24); 165.6 (s, C26); 202.1
(s, C1); TBDMSO [ꢀ5.5 (2 ꢁ q); 18.1 (s); 25.7 (3 ꢁ q)]; EI/MS m/z%:
584 (Mþ, 1), 527 (100), 509 (25), 493 (5), 359 (1), 293 (2), 281 (6),
227 (23), 197 (16), 169 (7), 131 (10), 95 (12), 75 (47); HREIMS: m/z
584.3511 (calcd for C34H52O6Si, 584.3533).
White lacquer; [
a
]
D
20 þ 33.8 (c 0.21, CHCl3); UV lmax nm: 221; IR
nmax cmꢀ1: 3400, 2937, 2859, 1700, 1405, 1315, 1185, 1016, 849, 750;
1H NMR : see Table 1; 13C NMR
d d: see Table 2; ESIMS (positive) m/z
%: 495 [M þ Na]þ (100); HRESIMS: m/z 495.2730 [M þ Na]þ (calcd.
for C28H40O6Na: 495.2723).
4.3.8. Reduction of 7
4.3.11. Preparation of 27-O-(tert-butyldimethylsilyl)-4-dehydroxy-
4-oxo-withaferin A (9b)
Fifty-percent palladium hydroxide-carbon (5 mg) was added to
a solution of 6 (10 mg, 0.02 mmol) in methanol (3 mL) under argon
atmosphere. After hydrogenation for 1 h at room temperature
under atmospheric pressure, the catalyst was removed by filtration
on celite and the filtrate was concentrated in vacuo. The residue
was purified using silica gel column chromatography (eluent,
CH2Cl2/acetone, 9:1) to afford compounds 7 (1.9 mg, 20%) and 7a
(6.1 mg, 67%).
A solution of 9a (40.0 mg, 0.08 mmol) in 2.0 mL of dichloro-
methane was added dropwise to a chromium trioxide/pyridine
complex, previously prepared by the addition of CrO3 (62.0 mg,
0.5 mmol) to 0.1 mL of pyridine and 1.5 mL of dichloromethane at
0
ꢄC. The reaction was stirred at room temperature for 6 h, and
subsequently quenched with 2-propanol (3 drops) and filtered
through a pad of celite. The filtrate was evaporated under reduced
pressure, and the residue was purified by TLC chromatography
(dichloromethane/acetone, 9:1) yielding 9b (24.2 mg, 61%): White
4.3.9. 4
White amorphous solid; [
nm: 220; IR nmax cmꢀ1: 3446, 2933, 1735, 1457, 1380, 1234, 1027,
966, 754; 1H NMR
b-Formyl-6b-hydroxy-1-oxo-witha-24-enolide (7a)
20
a
]
þ 56.2 (c 0.10, CHCl3); UV lmax
amorphous solid; [
a
]
D
20 þ 61.2 (c 0.49, CHCl3); UV lmax nm: 222; IR
D
nmax cmꢀ1: 2929,1699,1652,1459,1398,1258,1064, 839; 1H NMR
d
:
d
: 0.77 (3H, s, Me18); 1.02 (3H, d, J ¼ 6.6 Hz,
0.71 (3H, s, Me18); 1.00 (3H, d, J ¼ 6.6 Hz, Me21); 1.02 (1H, m, H14);
Me21); 1.07 (1H, m, H9); 1.08 (1H, m, H14); 1.10 (1H, m, H12); 1.14
(1H, m, H17); 1.19 (3H, s, Me19); 1.33 (1H, m, H11); 1.39 (1H, m,
1.09 (1H, m, H17); 1.17 (1H, m, H15); 1.23 (1H, m, H12); 1.35 (1H, m,
H7
(1H, m, H9); 1.62 (1H, m, H8); 1.65 (1H, m, H15); 1.69 (1H, m, H16);
1.97 (1H, m, H12); 2.00 (1H, m, H23 ); 2.01 (1H, m, H11); 2.04 (1H,
m, H20); 2.06 (3H, s, Me28); 2.15 (1H, m, H7 ); 2.47 (1H, m, H23 );
3.42 (1H, br s, H6); 4.38 (1H, m, H22); 4.39, 4.50 (2H, dAB
a); 1.37 (3H, s, Me19); 1.39 (1H, m, H16); 1.45 (1H, m, H11); 1.46
H16); 1.47 (1H, m, H7
(1H, m, H16); 1.72 (1H, m, H15); 1.82 (1H, m, H8); 1.84 (1H, m, H3);
1.86 (1H, m, H7 ); 1.91 (3H, s, Me27); 1.92 (2H, m, H23 ); 1.97 (3H,
s, Me28); 2.01 (1H, m, H20); 2.02 (1H, m, H12); 2.04 (1H, m, H3);
2.36 (1H, m, H2); 2.46 (1H, m, H23 ); 2.60 (1H, m, H2); 4.32 (1H, t,
J ¼ 2.7 Hz, H6); 4.39 (1H, dt, J ¼ 3.3, 13.2 Hz, H22); 9.61 (1H, s, H4);
13C NMR
: 11.9 (q, C18); 12.5 (q, C27); 13.3 (q, C19); 13.5 (q, C21);
a); 1.48 (1H, m, H11); 1.63 (1H, m, H15); 1.69
a
b
a
b
b
,
b
J ¼ 11.5 Hz, H27); 6.83 (1H, d, J ¼ 10.5 Hz, H2); 6.87 (1H, d,
J ¼ 10.5 Hz, H3); TBDMSO [0.08 (6H, s); 0.88 (9H, s)]; 13C NMR
d:
d
11.5 (q, C18); 13.1 (q, C21); 19.0 (q, C19); 20.3 (q, C28); 23.2 (t, C11);
24.0 (t, C15); 26.9 (t, C16); 29.4 (d, C8); 29.8 (t, C23); 30.3 (t, C7);
38.5 (d, C20); 39.2 (t, C12); 42.4 (s, C13); 43.4 (d, C9); 49.6 (s, C10);
51.9 (d, C17); 55.4 (d, C14); 56.9 (t, C27); 63.3 (d, C6); 63.7 (s, C5);
77.9 (d, C22); 125.8 (s, C25); 138.9 (d, C2); 141.4 (d, C3); 154.2 (s,
C24); 165.6 (s, C26); 193.8 (s, C4); 201.9 (s, C1), TBDMSO [ꢀ5.5
(2 ꢁ q); 18.1 (s); 25.7 (3 ꢁ q)]; EI/MS m/z%: 582 (Mþ, 1), 525 (100),
509 (33), 311 (1), 297 (4), 281 (3), 253 (3), 227 (33), 197 (16), 171 (7),
151 (15), 95 (13), 75 (48); HREIMS: m/z 582.3383 (calcd for
C34H50O6Si, 582.3377).
20.6 (q, C28); 20.7 (t, C11); 22.7 (t, C3); 24.1 (t, C15); 27.3 (t, C16);
29.2 (d, C8); 29.7 (t, C23); 32.7 (t, C2); 34.4 (t, C7); 38.8 (d, C20);
39.4 (t, C12); 42.2 (d, C9); 43.2 (s, C13); 52.1 (d, C17); 52.7 (s, C10);
56.0 (d, C14); 60.7 (s, C5); 67.7 (d, C6); 78.3 (d, C22); 122.1 (s, C25);
148.9 (s, C24); 167.0 (s, C26); 204.5 (d, C4); 216.5 (s, C1); ESIMS
(positive) m/z%: 479 [M þ Na]þ (100); HRESIMS: m/z 479.2771
[M þ Na]þ (calcd. for C28H40O5Na: 479.2774).
4.3.10. Preparation of 27-O-(tert-butyldimethylsilyl)withaferin A
(9a)
To a solution of diol 9 (54.0 mg, 0.12 mmol) in dry dichloro-
methane (5 mL) were added imidazole (9.0 mg, 0.12 mmol), 4-(N,N-
dimethylamino)pyridine (14.0 mg, 0.12 mmol) and tert-butyl-
chlorodimethylsilyl chloride (28.0 mg, 0.18 mmol). The reaction
mixture was stirred for 2.5 h at room temperature until all starting
material was consumed. The reaction was quenched with water
(10 mL) and extracted with dichloromethane (3 ꢁ 10 mL). The
organic layer was dried (MgSO4), filtered, and evaporated. The
residue was then purified by column chromatography (dichloro-
methane/acetone, 9:1) to give 9a (62.1 mg, 89%): White amorphous
4.3.12. Preparation of 4-dehydro-withaferin A (15)
A suspension of Dowex 50WX8-200 (150.0 mg) in dry acetone
(4 mL) was added to a solution of compound 9b (23 mg, 0.04 mmol)
in acetone (3 mL), and the reaction mixture was stirred at room
temperature for 24 h. The resin was removed by filtration through
a pad of celite, and the filtrate was evaporated under reduced
pressure. The residue was purified by TLC chromatography
(dichloromethane/acetone, 9.5:0.5) to give 15 (22.2 mg, 99%).
4.4. Biological assays
solid; [
a
]
20 þ 65.2 (c 0.42, CHCl3); UV lmax nm: 215; IR nmax cmꢀ1
:
D
3446, 2933, 1735, 1457, 1380, 1234, 1027, 966, 754; 1H NMR
d
: 0.69
4.4.1. Cells
(3H, s, Me18); 0.89 (1H, m, H9); 0.92 (1H, m, H14); 0.98 (3H, d,
HeLa (human cervix carcinoma), A-549 (human lung carci-
noma), MCF-7 (human breast adenocarcinoma) and Vero (African
green monkey kidney) cell lines from ATCC-LGC (American Type
Culture Collection) were each grown in Dulbecco’s modified Eagle’s
medium (DMEM) with 4.5 g/L glucose (SigmaeAldrich), supple-
mented with 10% fetal bovine serum (Gibco), 1% of a pen-
icillinestreptomycin mixture (10.000 UI/mL and 10 mg/mL,
J ¼ 6.6 Hz, Me21); 1.06 (1H, m, H12); 1.08 (1H, m, H17); 1.16 (1H, m,
H15); 1.25 (1H, m, H7a); 1.40 (3H, s, Me19); 1.41 (1H, m, H16); 1.50
(2H, m, H8, H11); 1.64 (1H, m, H15); 1.68 (1H, m, H16); 1.74 (1H, m,
H11); 1.97 (2H, m, H12, H23 ); 2.01 (1H, m, H20); 2.05 (3H, s,
Me28); 2.16 (1H, m, H7 ); 2.46 (1H, m, H23 ); 2.67 (1H, br s, OH-4);
a
b
b
3.22 (1H, br s, H6); 3.74 (1H, d, J ¼ 5.6 Hz, H4); 4.38 (1H, dt, J ¼ 3.1,
13.0 Hz, H22); 4.38, 4.49 (2H, dAB, J ¼ 11.6 Hz, H27); 6.19 (1H, d,
J ¼ 10.0 Hz, H2); 6.93 (1H, dd, J ¼ 5.6, 10.0 Hz, H3); TBDMSO [0.08
respectively), and 200 mM L-glutamine. Cells were maintained at
37 ꢄC in 5% CO2 and 98% humidity.
(6H, s); 0.88 (9H, s)]; 13C NMR
d: 11.4 (q, C18); 13.1 (q, C21); 17.2 (q,
C19); 20.3 (q, C28); 21.9 (t, C11); 24.0 (t, C15); 27.0 (t, C16); 29.5 (d,
C8); 29.8 (t, C23); 30.9 (t, C7); 38.5 (d, C20); 39.1 (t, C12); 42.3 (s,
C13); 43.9 (d, C9); 47.4 (s, C10); 51.8 (d, C17); 55.8 (d, C14); 56.9 (t,
4.4.2. Cell viability assays
Viable cells were assessed using the colorimetric MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide]

510
G.G. LLanos et al. / European Journal of Medicinal Chemistry 54 (2012) 499e511
reduction assay [54]. Cell suspensions (2 ꢁ 104/200
m
L/well) were
centrifuged at 300 g for 5 min, and washed twice in cold PBS. The
pellets were lysed at 4 ꢄC in 100
L of lysis buffer for 10 min. Lysates
were centrifuged at 16,000 g for 2 min, and total protein in
supernatants was determined by Bradford assay (Bio-Rad, Hercules,
CA). Caspase-3 activity assays were performed in 96 well plates by
seeded in DMEM, supplemented with 5% fetal bovine serum in
microtiter well plates (96 wells, Iwaki, London, UK) together with
the compounds, predissolved in DMSO at different concentrations,
m
and treated for 48 or 72 h. Then, 20 mL/well of the MTT solution
(5 mg/mL in phosphate buffered saline, PBS) were added and plates
incubating 50 mg of protein in 50 mL of reaction buffer and 5 mL of
incubated for 3 h at 37 ꢄC. Subsequently, the medium was aspirated
the 4 mM chromogenic substrate, Ac-DEVD-pNA, at 37 ꢄC for 2 h.
Absorbance of released p-nitroaniline was measured at 405 nm
(Tecan Group Ltd., Männedorf, Switzerland) and activity of caspase-
3 evaluated by calculating OD (optical density) ratios of treated/
untreated samples. Experiments were performed in triplicates.
and replaced with 150 mL/well of DMSO to dissolve the formazan
crystals. Absorbance was measured in a microplate reader (Titer-
tetek, Multiskan Plus II) at a wavelength of 550 nm. Solvent control
cultures were considered 100% and percent of viable, treated cells
were plotted against compound concentrations. The 50% cell
viability value (IC50) was calculated from the curve. Each experi-
ment was performed at least three times in triplicates. Data are
given in arithmetic means ꢂ SD.
4.4.7. Phosphatidylserine labeling
1.6 ꢁ 106 HeLa cells each were grown on coverslips in 24-wells
and treated with varying concentrations of the compounds or the
respective volume of DMSO (solvent control) or actinomycin D at
4.4.3. Detection of DNA fragmentation
1 mM (positive control) for different time periods. Phosphati-
HeLa cells were incubated in 24-well plates with varying
concentrations of each compound or the respective volume of
DMSO (solvent control) for various time periods. Cells treated with
dylserine externalization and cell permeability were assessed by
Annexin V/PI double staining (VybrantÒ Apoptosis Assay Kit s2,
Invitrogen, Molecular ProbesÔ), according to the manufacturer’s
instructions. Apoptotic cells were recognized by fluorescence
microscopy (Leica, DM 4000 B) and microphotographs taken by
a coupled digital camera (Nikon DXM1200F). Annexin VþPIꢀ cells
were considered as early apoptotic while Annexin VþPIþ cells as
late apoptotic/necrotic.
staurosporine at a concentration of 2 mM were used as positive
control. After the incubation period, adherent and floating cells
were collected, centrifuged at 500 g for 10 min, and washed twice
with cold PBS. Then, DNA was extracted according to the manu-
facturer’s protocol (Gen EluteÔ Mammalian genomic DNA kit,
SigmaeAldrich). DNA samples were analyzed by agarose gel
electrophoresis in 1.8% agarose, followed by ethidium bromide
(SigmaeAldrich) staining and visualization with UV.
4.4.8. Statistical analysis
All results were expressed as means ꢂ SD. Significance differ-
ences between groups were determined using unpaired Student’s
t-test. Significance was set at p < 0.05.
4.4.4. Detection of chromatin condensation
To identify apoptotic nuclear changes (such as chromatin
condensation), HeLa cells in 6-well plates were treated with the
compounds at 8 mM for 12 h. After the incubation period, adherent
Acknowledgments
and floating cells were collected, centrifuged at 500 g for 10 min,
and washed twice with PBS, fixed in 3% paraformaldehyde (in PBS)
and stained with 10 mL Hoechst 33342 (16 mg/mL) for 15 min. The
This study was supported by the Agencia Canaria de Inves-
tigación,
Innovación
y
Sociedad
de
la
Información
(C200801000049). G.G.LL. thanks the Gobierno Autónomo de
Canarias for the fellowship. Thanks are also due to Leticia Rodrí-
guez-Navarro, Department of Botany, University of La Laguna for
the identification of the plant.
slides (treated with poly-lysine) were inspected for nuclear
morphological alterations and apoptotic bodies by fluorescence
microscopy (Leica, DM 4000 B) and microphotographs taken by
a coupled digital camera (Nikon DXM1200F). In order to obtain
quantitative data, an area containing at least 500 cells was evalu-
ated on each slide. Only cells containing three or more chromatin
fragments were considered. Data were expressed as percent of total
cell numbers.
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2012.05.032.
4.4.5. Flow cytometry analysis
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