Synthesis and in vitro anticancer activities of some selenadiazole derivatives

Article abstract of DOI:10.1002/ardp.201000014

A novel series of fourteen substituted selenadiazoles has been synthesized and the compounds tested for their in vitro antiproliferative and cytotoxic activities. The tests were carried out against leukemia (CCRF-CEM), colon (HT-29), lung (HTB-54), and breast (MCF-7) cancer cells. In order to assess the selectivity of the compounds under investigation the assays were also carried out on two non-tumoral lines - one mammary (184B5) and one bronchial epithelium (BEAS-2B) cell line. Assay-based antiproliferative activity studies revealed that seven derivatives (2a, 2c, 2e, 2f, 2g, 3a, and 3b) exhibited good activity against MCF-7 cells: for instance, 2c and 2f inhibited cell growth with nanomolar GI₅₀ values. Compound 2f had a better antitumoral profile than vinorelbine and paclitaxel, two drugs that are used as first-line treatments in advanced, recurrent, and/or metastatic cancer. In the other cell lines the compounds showed moderate activity or were inactive - with the exception of 2a, which was also found to have antiproliferative activity. Modulation of the cell cycle and apoptotic effects of active compounds were further evaluated in MCF-7 cells. Of these, 6-bromo[1,2,5]selenadiazolo[3,4-b] pyridine (2a) was the most active, with an apoptogenic effect 3.9 times higher than that of camptothecin, which was used as a positive control. Compound 2a also provoked cell cycle arrest with a significant decrease in the G ₀/G₁ phase cell population and an increase in S and G ₂/M cells, thus suggesting mitotic arrest prior to metaphase. A novel series of fourteen substituted selenadiazoles has been synthesized and the compounds tested for their in vitro antiproliferative and cytotoxic activities against several cancer cells and in order to assess the selectivity of the compounds on two non-tumoral lines. Modulation of cell cycle and apoptotic effects of active compounds were further evaluated. Copyright

Full text of DOI:10.1002/ardp.201000014

680
Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Full Paper
Synthesis and in vitro Anticancer Activities of some
Selenadiazole Derivatives
1
Daniel Plano , Esther Moreno , Marıa Font , Ignacio Encıo , Juan Antonio Palop , and
1
2
3
1
´
´
1
´
Carmen Sanmartın
1
´
Seccion de Sıntesis, Departamento de Quımica Organica y Farmaceutica, University of Navarra,
Pamplona, Spain
´
´
´
´
2
3
´
Seccion de Modelizacion Molecular, Departamento de Quımica Organica y Farmaceutica, University of
Navarra, Pamplona, Spain
´
´
´
´
´
Departamento de Ciencias de la Salud, Universidad Publica de Navarra, Pamplona, Spain
A novel series of fourteen substituted selenadiazoles has been synthesized and the compounds tested
for their in vitro antiproliferative and cytotoxic activities. The tests were carried out against leukemia
(CCRF-CEM), colon (HT-29), lung (HTB-54), and breast (MCF-7) cancer cells. In order to assess the
selectivity of the compounds under investigation the assays were also carried out on two non-
tumoral lines – one mammary (184B5) and one bronchial epithelium (BEAS-2B) cell line. Assay-
based antiproliferative activity studies revealed that seven derivatives (2a, 2c, 2e, 2f, 2g, 3a, and
3b) exhibited good activity against MCF-7 cells: for instance, 2c and 2f inhibited cell growth with
nanomolar GI50 values. Compound 2f had a better antitumoral profile than vinorelbine and
paclitaxel, two drugs that are used as first-line treatments in advanced, recurrent, and/or
metastatic cancer. In the other cell lines the compounds showed moderate activity or were
inactive – with the exception of 2a, which was also found to have antiproliferative activity.
Modulation of the cell cycle and apoptotic effects of active compounds were further evaluated in
MCF-7 cells. Of these, 6-bromo[1,2,5]selenadiazolo[3,4-b]pyridine (2a) was the most active, with an
apoptogenic effect 3.9 times higher than that of camptothecin, which was used as a positive control.
Compound 2a also provoked cell cycle arrest with a significant decrease in the G0/G1 phase cell
population and an increase in S and G2/M cells, thus suggesting mitotic arrest prior to metaphase.
Keywords: Antitumorals / Apoptosis / Cytotoxics / Selenadiazoles / Selenium
Received: January 11, 2010; Revised: March 30, 2010; Accepted: April 7, 2010
DOI 10.1002/ardp.201000014
Introduction
and angiogenesis inhibition may be among the molecular
targets for Se compounds. For example, Se is an essential
component of some protective enzymes, such as gluthatione
peroxidase (GPx) and thioredoxin reductase (TrxR), which
catalyze essential redox reactions [5, 6]. Additionally, many
seleno-compounds modulate cell proliferation through the
cell cycle and apoptosis. Such is the case for selenomethio-
nine [7], which induced G2/M arrest in LNCaP prostate cancer
cells, and sodium selenite [8], which affects the cycle distri-
bution of the NB4 leukemia cell population by blocking the
cells at G0/G1. On the other hand, methylseleninic acid [9]
enhanced the apoptosis induced by cancer therapeutic drugs
by interacting with JNK-dependent targets to amplify the
caspase-8 initiated activation cascade. Finally, methylsele-
ninic acid [10] and sodium selenate [11] inhibited tumour
The trace element selenium (Se) appears to have cancer-pre-
ventive properties, as evidenced by a converging body of
evidence from epidemiologic, clinical and experimental stud-
ies [1–4]. The mechanisms of action for selenium and seleno-
compounds as anticancer agents are not fully understood.
However, it has been proposed in the literature that antiox-
idant properties, cell cycle modulation, apoptosis induction
Correspondence: Professor Juan Antonio Palop, Departamento de
´
´
Quımica Organica y Farmaceutica, Universidad de Navarra, Irunlarrea, 1.
E-31008 Pamplona, Spain.
´
E-mail: jpalop@unav.es
Fax: þ34-948 425 649
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Anticancer Activities of Selenadiazole Derivatives
681
growth in prostate cancer by, at least in part, inhibition of
tumor angiogenesis. These data provide valuable insights
into the novel and varied biological effects of seleno-com-
pounds and have encouraged the development of new
selenium-containing structures as potential agents in cancer
therapy.
molecules related to 1,2,5-selenadiazolo[3,4-b] pyridine and
1,2,5-benzoselenadiazolo derivatives. The selection of the
chemical groups in the new heterocyclic rings is based on
the commercial availability of the ortho-aromatic diamines
(starting materials) and is aimed at exploring the effect that
substituents have on the target properties. In particular, the
substituents were assessed in terms of their ability to estab-
lish hydrogen bonds, polarity, volume and the potential to
modify the electronic density (compounds 2a–l). Finally, we
have included two symmetrical molecules (compounds 3a–b)
as we previously observed that symmetry is a structural
property that is frequently present in cytotoxic and apoptotic
drugs [29]. Furthermore, one of the compounds (3b) has a
diselenide function, which has attracted considerable inter-
est because of the potential of these compounds as anticancer
agents [30].
In addition, heterocyclic compounds with a selenium atom
in the ring have been intensively studied as a result of their
chemical properties and biological activities. For example,
some triazoleselones [12], 1,3-selenazinanes [13], 1,3-selen-
azoles [14, 15], 1,3-selenazinanones [13, 16] and 1,4-oxasele-
nins [17] have been recognized as potential antitumor agents.
Among the numerous selenium-containing compounds, 1,2-
[bis(1,2-benzisoselenazolone-3-(2H)-ketone)]ethane
(BBSKE,
Fig. 1a) is considered to be a potential anti-cancer drug that
has low toxicity and induces apoptosis through the caspase-3
pathway [18]. Other heterocyclic compounds, such as benzi-
soselenazol-3(2H)-one (Ebselen, Fig. 1b), have exhibited high Results and discussion
antioxidant, apoptotic and anti-inflammatory activities [19,
20]. In recent studies the scope of synthetic approaches has
been expanded to include new structures with heterocyclic
selenium compounds fused to other heterocycles like pyri-
dazine [21], quinoline [22], pyridine [23], pyrrolidine [23], and
pyrimidine [24] (SPO, Fig. 1c), with a selenadiazolopyrimidine
described as an antiproliferative agent and apoptosis
inducer, or monoheterocycles with more heteroatoms
(Fig. 1d) [25].
Chemistry
Synthesis
The synthesis of the target compounds was carried out
according to Schemes 1 and 2. Compounds 2a–d and 2i–l
were synthesized from the appropriate ortho-aromatic dia-
mine 1a–d and 1i–l and selenium dioxide in a 1:1 molar
ratio in the absence of solvent at different temperatures
(from room temperature to 908C) [31]. The desired com-
pounds were obtained in yields ranging from 48 to 91%.
Derivatives 2e–g and 3a–b were obtained in two steps starting
from carboxylic acid 2d, which was converted to the corre-
sponding acyl chloride and treated, without purification,
with the appropriate amine or diamine in chloroform in
the presence of triethylamine at room temperature.
Finally, 2h was prepared by treating the acyl chloride of
2d with ammonium hydroxide. The scope and generality
of these procedures are illustrated in Table 1. The purities
of the compounds were assessed by TLC and elemental
analysis and their structures were identified from spectro-
scopic data (Table 2). All of the signals are consistent with the
proposed structural formula. However, an interesting obser-
One of our ongoing projects involves the investigation of
new potential antitumor agents containing selenium [26–28].
We previously synthesized a series of imidoselenocarbamates
[26] and the results revealed that these compounds had better
antitumor effects in prostate cancer cells than etoposide and
methylseleninic acid, both of which are currently in clinical
use. As a continuation of this research, we now focus our
attention on the role of the endocyclic position of the
selenium atom in the structure. Bearing in mind the results
outlined above, the work described here concerns the syn-
thesis, cytotoxicity and apoptotic evaluation of new
1
vation was made concerning the H-NMR spectrum of com-
Figure 1. Structures of BBSKE [18] (a), Ebselen [19, 20] (b), SPO
[24] (c) and 1,2,3-selenadiazole-5-carboxylic acid amides [25] (d).
Scheme 1. Synthesis of compounds 2a–d and 2i–l.
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682
D. Plano et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Scheme 2. Synthesis of compounds 2e–h and 3a–b.
pound 2h. This derivative, which has an amide group, shows
unexpected 1H-NMR behavior in that the amino group gives
rise to two separate signals. In an effort to explain this
behavior further NMR studies were carried out on this
compound.
tautomer has two different protons (OH and NH). The 13C-
NMR spectrum was registered and a single peak was observed
at d ¼ 168.0 ppm. This peak corresponds to the carbonyl
group of the amide form (keto tautomer) and not to the
imine tautomer.
Moreover, it was observed that these two peaks had a slow
exchange rate with deuterium oxide (D2O). As a result, we
carried out an exchange experiment with D2O for 72 h. The
1H-NMR spectra were registered at 24, 48, and 72 h and it was
observed that one proton was more resistant to exchange
(Fig. 3). This fact led us to hypothesize that intermolecular
NMR studies
1
In the H-NMR spectrum of compound 2h the amino group
gave rise to two different peaks with a difference in chemical
shift of 0.63 ppm. This finding led us to consider the presence
of a keto-iminic tautomerism (Fig. 2a) since the imine
Table 1. Physical constants of the compounds.
Compd.
X
Y
Yield
(%)
Mp
(8C)
PMa
Molecular
formula
Anal (%) calcd./Found
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
3a
3b
N
C
C
C
C
C
C
C
C
N
C
C
–
Br
Br
CO–Ph
COOH
73
80
91
56
60
62
67
54
84
79
76
81
55
48
239–0
130–1
250–1
285–7
194–5
231–3
186–7
238–9
73–4
113–4
69–70
164–5
286–8
262–4
A
A
B
C
A
D
A
B
A
A
A
A
D
D
C5H2BrN3Se
C6H3BrN2Se
C13H8N2OSe
C7H4N2O2Se
C15H13N3OSSe
C14H8N4OSe2
C13H10N4OSe
C7H5N3OSe
C6H4N2Se
C5H3N3Se
C7H6N2Se
C7H3N3Se
C17H14N6O2Se2
C26H16N6O2Se
C, 22.81/22.99; H, 0.76/0.75; N, 15.97/16.12
C, 27.48/27.79; H, 1.14/1.29; N, 10.69/10.39
C, 54.35/54.16; H, 2.79/2.75; N, 9.76/9.79
C, 37.00/37.04; H, 1.76/1.78; N, 12.33/12.41
C, 49.72/49.89; H, 3.59/3.62; N, 11.60/11.61
C, 41.38/41.52; H, 1.97/2.33; N, 13.79/13.96
C, 49.21/49.15; H, 3.15/3.46; N, 17.67/17.38
C, 37.17/37.25; H, 2.21/2.48; N, 18.58/18.31
C, 39.34/39.02; H, 2.19/2.16; N, 15.30/15.63
C, 32.61/32.38; H, 1.63/1.72; N, 22.83/22.85
C, 42.64/42.49; H, 3.04/3.14; N, 14.21/14.55
C, 40.38/40.50; H, 1.44/1.43; N, 20.19/19.96
C, 41.46/41.67; H, 2.84/2.75; N, 17.07/17.12
C, 41.05/41.22; H, 2.10/2.35; N, 11.05/11.42
CONH–CH2–C6H4–SCH3
CONH–C6H4–SeCN
2-Pyridylmethylcarbamoyl
CONH2
H
H
CH3
CN
–(CH2)3–
C6H4–Se–Se–C6H4
–
a
PM ¼ Purification method; A ¼ Recrystallized from EtOH; B ¼ Washed with H2O; C ¼ Recrystallized from 1,4-dioxane;
D ¼ Washed with hot EtOH/N,N-DMF (1:1).
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Anticancer Activities of Selenadiazole Derivatives
683
Table 2. Spectroscopical data for the new compounds.
Compd.
IR (KBr; cmS1
)
1H-NMR (400 MHz, DMSO-d6, d, J in Hz):
2a
2b
2c
3023, 403
3069, 420
3061, 1635
8.71 (dd, 1H, J7-5 ¼ 1.1, J7-Br ¼ 2.4, H7); 9.09 (dd, 1H, J5-Br ¼ 2.4, H5)
7.65 (dd, 1H, J6-7 ¼ 9.2, J6-4 ¼ 1.8, H6); 7.81 (d, 1H, H7); 8.19 (s, 1H, H4)
0
0
0
0
0
0
0
0
0
0
0
(Acetone-d6): 7.63 (t, 2H, J3 -2 ¼ J5 -6 ¼ 7.8, H3 þ H5 ); 7.74 (t, 1H, J4 -3 ¼ J4 -5 ¼ 3.1, H4 );
0
0
7.93 (m, 2H, H2 þ H6 ); 7.98 (m, 2H, H6 þ H7); 8.15 (s, 1H, H4)
7.93 (d, 1H, J6-7 ¼ 9.3, H6); 7.97 (d, 1H, H7); 8.42 (s, 1H, H4); 13.47 (br s, 1H, OH)
2.45 (s, 3H, CH3); 4.48 (d, 2H, JCH2-NH ¼ 5.9, CH2);
2d
2e
3448, 1682
3280, 3066, 2916, 1631
0
0
0
0
0
0
0
0
7.25 (d, 2H, J2 -3 ¼ J6 -5 ¼ 8.5, H2 þ H6 ); 7.32 (d, 2H, H3 þ H5 );
7.91 (d, 1H, J6-7 ¼ 9.3, H6); 7.97 (d, 1H, H7); 8.40 (s, 1H, H4); 9.34 (t, 1H, NH)
0
0
0
0
0
0
0
0
2f
3348, 2146, 1673, 411
3231, 3048, 2932, 1652
7.75 (d, 2H, J2 -3 ¼ J6 -5 ¼ 8.2, H2 þ H6 ); 7.92 (d, 2H, H3 þ H5 );
7.97 (d, 1H, J6-7 ¼ 9.1, H6); 8.01 (d, 1H, H7); 8.55 (s, 1H, H4); 10.75 (s, 1H, NH)
0
0
0
0
0
2g
4.56 (d, 2H, JCH2-NH ¼ 5.8, CH2); 7.38 (dd, 1H, J5 -4 ¼ 7.8, J5 -6 ¼ 4.7, H5 );
0
0
7.78 (d, 1H, H6 ); 7.91 (d, 1H, J6-7 ¼ 9.1, H6); 7.97 (d, 1H, H7); 8.41 (s, 1H, H2 );
0
8.48 (d, 1H, H4 ); 8.60 (s, 1H, H4); 9.41 (t, 1H, NH)
2h
3280, 3066, 2916, 1631
7.65 (br s, 1H, NH); 7.88 (d, 1H, J6-7 ¼ 9.3, H6); 7.97 (d, 1H, H7);
8.28 (br s, 1H, NH); 8.40 (s, 1H, H4)
2i
2j
2k
2l
3036
3051, 1591
2917, 403
7.54 (d, 2H, J5-4 ¼ J6-7 ¼ 3.3, H5 þ H6); 7.85 (d, 2H, H4 þ H7)
7.53 (dd, 1H, J6-7 ¼ 8.9, J6-5 ¼ 3.8, H6); 8.30 (d, 1H, H5); 9.05 (d, 1H, H7)
2.42 (s, 3H, CH3); 7.39 (d, 1H, J6-7 ¼ 9.1, H6); 7.60 (d, 1H, H7); 7.73 (s, 1H, H4)
7.65 (d, 1H, J6-7 ¼ 9.4, H6); 7.81 (d, 1H, H7); 8.19 (s, 1H, H4)
1.89 (q, 2H, JCH2-CH2 ¼ 6.8, CH2-CH2-CH2); 3.43 (m, 4H, CH2-CH2-CH2);
3016, 2230, 412
3299, 3058, 2928, 1636
3a
7.88 (d, 2H, J6-7 ¼ 9.3, 2 H6); 7.94 (d, 2H, 2 H7); 8.35 (s, 2H, 2 H4); 8.83 (t, 2H, JNH-CH2 ¼ 5.2, 2 NH)
0
0
0
0
0
0
0
0
3b
3338, 1652
7.52 (d, 4H, J2 -3 ¼ J6 -5 ¼ 8.2, 2H2 þ 2H6 ); 7.69 (d, 4H, 2 H3 þ 2 H5 );
7.82 (d, 2H, J6-7 ¼ 9.3, 2 H6); 7.87 (d, 2H, 2 H7); 8.39 (s, 2H, 2 H4); 10.55 (br s, 2H, 2 NH)
To test the hypothesis we performed a dynamic NMR
experiment on compound 2h. The 1H-NMR spectra were reg-
istered at 24, 40, 60, and 808C with DMSO-d6 as solvent (Fig. 5).
The two peaks of the amino group converged to a single peak
that was coincident with the original signal for the free
proton (Fig. 3).
Finally, in order to determine the role of concentration in
the formation of the dimer the 1H-NMR spectra of various
solutions of compound 2h in DMSO-d6 (0.001, 0.0025, 0.005,
1
0.0075, 0.01, 0.02 M, and saturated) were acquired. The H-
NMR spectra were identical in all cases (data not shown). This
finding suggests that hydrogen bond formation to give the
dimeric form is independent of concentration over a large
Figure 2. Proposed tautomerism for 2h according to the theoretical
equilibrium (a). Structures considered in 1H-NMR and molecular
modeling studies (b). Representative lowest energy conformation
for tautomers (I) and (II). (Schematic tube representation: Se in
violet; C in cyan; N in blue; O in red; H in white). See experimental
section for details.
range of values.
Molecular modelling
In an attempt to explain the NMR behaviour of compound 2h,
a study was carried out on the relative stability of the possible
tautomers of compound 2h (forms I and II in Fig. 2). Once the
models for the two tautomers had been constructed, the
bonds could lead to the formation of a dimer (Fig. 4). The
presence of the amide group (which is able to participate in
intermolecular hydrogen bonds) could facilitate the adop-
tion and stabilization of the dimer structure. In such a case,
the molecule would have two types of protons for the amino
group: one proton forming hydrogen bonds with carbonyl
groups (d ¼ 8.28 ppm) and one free proton (d ¼ 7.65 ppm).
In addition, these two types of protons would have different
capacities for exchange with D2O.
initial geometries were fully minimized to an energy gra-
dient below 10ꢀ3 kcal molꢀ1 ꢀ1. A representative low energy
A
˚
conformation was then selected for subsequent quantum
energy evaluation (PM3 semi-empirical approach) carried
out for the isolated tautomer. It can be seen from Fig. 2b
that the tautomer (II) is more stable than the tautomer (I),
with a significant energy difference in favor of tautomer (II).
In order to study the possible involvement of tautomers (I)
and (II) in the formation of dimers stabilized by
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684
D. Plano et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Figure 3. 1H-NMR spectra from
exchange experiment on compound 2h.
a D2O
from non-malignant cells (184B5) and another cell line
derived from non-malignant bronchial epithelium cells
(BEAS-2B). Cytotoxicity assays were based on the reactivity
of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide], as described by Mosmann [32]. Results are
expressed as GI50, i.e. the concentration that reduces by
50% the growth of treated cells with respect to untreated
controls, TGI, the concentration that completely inhibits cell
growth, and LC50, the concentration that kills 50% of the
cells. The cytotoxic effect of each substance was tested at five
different concentrations between 0.01 and 100 mM, or at
lower levels when the GI50 was <10 nM. Mean GI50, TGI,
and LC50 values are summarized in Table 3. Doxorubicin
was used as a control.
Figure 4. Proposed dimers (according to tautomeric forms I and II)
and representative lowest energy conformations for compound 2h
(Schematic tube representation: Se in violet; C in cyan; N in blue; O
in red; H in white. Hydrogen bonds as dotted lines). See experimen-
tal section for details.
It is evident from the results that there are differences in
the sensitivity of different cell lines towards the compounds.
MCF-7 was found to be the most sensitive line, with deriva-
tives 2a, 2c, 2e, 2f, 2g, 3a, and 3b exhibiting the highest
intermolecular hydrogen bonds (Fig. 4), we used the pre-
viously selected representative low energy conformations
(Fig. 2) to construct the proposed dimers. Once the models
for the two dimers had been constructed, the initial geo-
growth inhibition values. In particular, 2c (GI50
¼
0.005 mM) and 2f (GI50 ¼ 0.006 mM) were strongly antiproli-
ferative. These compounds were 176 and 147 times more
metries were fully minimized to an energy gradient below
˚
active, respectively, than standard doxorubicin (GI50
¼
10ꢀ3 kcal molꢀ1
A
ꢀ1. A representative low energy confor-
0.88 mM). Doxorubicin is an effective drug against breast
cancer and it has been widely employed as a control for
cytotoxicity assays, although a specific dose/tumor resistance
can develop due to overexpression of drug transporters [33]
or drug metabolizing enzymes [34]. In addition, 2f
(GI50 ¼ 0.006 and TGI ¼ 8.94 mM) and 3a (GI50 ¼ 0.17 and
TGI ¼ 9.23 mM) exhibited better antitumoral profiles than
other drugs that are in wide clinical use, such as paclitaxel
[35] (GI50 ¼ 0.010 ꢁ 0.04 and TGI ¼ 20 ꢁ 1.9 mM) and vinor-
elbine [35] (GI50 ¼ 5 ꢁ 1.3 and TGI ¼ 100 ꢁ 10.2 mM). The
results also show that the compounds were inactive against
CCRF-CEM and HTB-54 cells – apart from 2a, which was able to
inhibit cell proliferation (GI50 ¼ 7.31 and 1.69 mM, respect-
ively). As for HT-29 cells, four compounds (2a, 2f, 3a, and 3b)
exhibited a rather moderate antiproliferative activity with
mation was then selected for the subsequent quantum
energy evaluation (PM3 semi-empirical approach). The results
show that the dimer corresponding to tautomer (II) is sig-
nificantly more stable.
Biological evaluation
Cytotoxicity
All of the synthesized compounds were screened for their
cytotoxic and antiproliferative activities against a panel of
four human tumour cell lines [lung carcinoma (HTB-54),
colon carcinoma (HT-29), lymphocytic leukemia (CCRF-
CEM) and breast adenocarcinoma (MCF-7)]. As guide with
regard to selectivity, all of the compounds were further
examined for toxicity in a mammary gland cell line derived
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Anticancer Activities of Selenadiazole Derivatives
685
Figure 5. Dynamic 1H-NMR experiment on compound 2h.
Table 3. Average GI50, TGI, and LC50 values (mM) for compounds 2a–l and 3a–b.
CCRF-CEMa
HT-29b
TGI
HTB-54c
TGI
MCF-7d
TGI
e
g
Compd.
X
Y
GI50
TGIf LC50
GI50
LC50
GI50
LC50
GI50
LC50
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
3a
3b
Doxoru.
N
C
C
C
C
C
C
C
C
N
C
C
–
Br
7.31 34.15 68.33 8.72 65.38 >100
1.69
9.13 >100 6.11 23.14 76.00
Br
COPh
COOH
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 93.62 >100 >100 >100 >100 >100 0.005 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 80.97 >100 >100 >100 >100 >100 2.92 >100 >100
>100 >100 >100 24.47 >100 >100 80.01 >100 >100 0.006 8.94 >100
CONH–CH2–C6H4–SCH3
CONH–C6H4–SeCN
2-Pyridylmethylcarbamoyl >100 >100 >100 >100 >100 >100 44.80 >100 >100 3.63 80.56 >100
CONH2
H
H
CH3
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
CN
–(CH2)3–
C6H4–Se–Se–C6H4
>100 >100 >100 15.43 >100 >100 >100 >100 >100 0.17
>100 >100 >100 29.38 >100 >100 >100 >100 >100 64.45 >100 >100
9.23 >100
–
0.033 0.071 0.29
n.d.h
n.d.
n.d.
<10ꢀ2 1.25
3.45
0.88 >100 >100
a
e
Lymphocytic leukemia; b Colon carcinoma; c Lung carcinoma; d Breast adenocarcinoma; Concentration that inhibits 50% of cell
f
growth; Concentration that inhibits 100% of cell growth; Concentration that kills 50% of cells; No data.
g
h
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
GI50 values ranging from 8.32 mM to 29.38 mM. Interestingly,
replacement of the nitrogen atom of 2a by a carbon atom to
give 2b led to a significant decrease in the activity in all cell
lines. As far as the selectivity is concerned, the cytotoxicity
was determined in cell cultures of two nontumoral cell lines
[mammary gland (184B5) and bronchial epithelium (BEAS-
2B)]. In order to obtain directly comparable results, the exper-
imental procedure used in the cytotoxicity assays on the non-
tumoral lines was identical to that used for the assays on
cancer cells. Comparison (see Table 4) of the LC50 values
shows that the cytotoxicity levels on the non-tumoral lines
are similar to those obtained on the malignant cells, i.e. there
is little or no selectivity. However, if we consider the anti-
proliferative activity (GI50 values), a significant increase in
selectivity was observed for the BEAS-2B cell line, which was
affected to a much lesser extent than the tumor lines by the
antiproliferative agents. For example, in the case of com-
pound 2a the ratio of GI50 values (BEAS-2B/184B5) is 418
and this increases significantly for other compounds: 2g
(23 935), 2e (115 200), and 3a (117 900). In summary, com-
pound 2a is the most promising of those investigated – it
shows a cytotoxic activity on all tumoral lines tested, inter-
esting values as an antiproliferative agent and its toxicity on
the nontumoral line BEAS-2B is lower than that of doxoru-
bicin. Plots of the original data from which the GI50, TGI, and
LC50 values were obtained for compound 2a are shown in
Fig. 6.
Figure 6. Cytotoxic effect of compound 2a on CCRF-CEM, HT-29,
HTB-54, MCF-7, 184B5 and BEAS-2B cells. Data are expressed as
the percentage growth ꢁ SEM of at least 3 independent experi-
ments performed in quadruplicate.
The cytotoxic activity of some of the compounds was remark-
able in MCF-7 cells (i.e., nanomolar GI50 values for compounds
2c (5 nM) and 2f (6 nM), and micromolar GI50 values for
compounds 2a (5.11 mM), 2e (2.92 mM), 2g (3.63 mM), 3a
(0.17 mM) and 3b (60.45 mM)) and, as a result, we decided
to assess whether this activity was related to their ability to
induce apoptosis. With this aim in mind, the process of cell
death induced by 2a, 2c, 2e, 2f, 2g, 3a, and 3b was further
analyzed in MCF-7 cells. The apoptotic status of the cells after
48 h of treatment with 25 mM of the corresponding com-
pound was determined using the Apo-Direct kit (BD
Apoptosis
Mounting evidence indicates that apoptosis is a critical mech-
anism for cancer prevention by Se compounds [27, 36–38].
Table 4. Average GI50, TGI and LC50 values (mM) for compounds 2a–l and 3a–b in 184B5 and BEAS-2B cell lines.
Compound
184B5a
BEAS-2Bb
GI50c (mM)
TGId (mM)
LC50e (mM)
GI50c (mM)
TGId (mM)
LC50e (mM)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
3a
0.007
>100
3.45
71.48
0.0004
0.63
0.004
>100
>100
>100
>100
>100
0.0002
1.16
55.68
>100
>100
>100
52.15
6.29
>100
>100
>100
>100
>100
>100
60.02
7.32
>100
>100
>100
>100
>100
63.13
>100
>100
>100
>100
>100
>100
>100
>100
–
2.93
>100
46.45
>100
46.08
0.98
95.74
>100
>100
>100
>100
>100
23.58
0.051
0.13
33.12
>100
>100
>100
81.51
9.14
>100
>100
>100
>100
>100
>100
98.44
6.30
74.49
>100
>100
>100
>100
77.83
>100
>100
>100
>100
>100
>100
>100
>100
0.78
3b
Doxorubicin
–
–
0.45
a
b
Mammary gland cell culture derived from non-malignant cells; Bronchial epithelium cell culture derived from non-malignant
cells; c Concentration that inhibits 50% of cell growth; d Concentration that inhibits 100% of cell growth; e Concentration that kills
50% of cells.
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Anticancer Activities of Selenadiazole Derivatives
687
Table 5. Effects of 2a, 2c, 2e, 2f, 2g, 3a, and 3b on apoptosis in
CCRF-CEM evaluated by measuring the exposure of
phosphatidylserine on the cell membranes using the Annexin V-
FITC Kit (BD Pharmingen) at 24 h and MCF-7 cells at 48 h using the
Apo-Direct kit (BD Pharmingen) based on the TUNEL technique.
Results are expressed as the percentage (%) of apoptotic cells and
data are representative of three experiments (mean ꢁ SD).
Compound
Dosage
(mM)
CCRF-CEM
MCF-7
Control
2a
2a
2c
2e
2f
2g
–
40
25
25
25
25
25
25
15.8 ꢁ 1.7
7.4 ꢁ 0.7
N.D.
40.3 ꢁ 2.9
23.7 ꢁ 3.8
75.6 ꢁ 10.6
4.8 ꢁ 3.3
5.4 ꢁ 4.1
8.5 ꢁ 6.7
8.1 ꢁ 8.1
8.8 ꢁ 7.3
2.4 ꢁ 1.0ꢂ
19.3 ꢁ 2.3 ꢂ
–
–
–
–
–
–
Figure 8. Compound 2a induces an apoptotic effect on MCF-7
cells. MCF-7 cells were incubated either with 25 mM of the indicated
compound or vehicle (control cells) for 24 and 48 h. The results are
presented as the mean ꢁ SD of three independent experiments
(duplicate wells). ꢂ ¼ difference statistically significant with respect
to the control (p < 0.05). ꢂꢂ ¼ difference statistically very significant
with respect to the control (p < 0.01).
3a
3b
25
12or 25
Camptothecin
76.37 ꢁ 3.48ꢂ
ꢂ
ꢂꢂ
12 mM; 25 mM.
Pharmingen) [39] based on the TUNEL technique.
Camptothecin was used as a positive control. The results
obtained are shown in Table 5 and Fig. 7. It can be seen that
2a led to a marked increase in the proportion of apoptotic
cells in the cultures, but for the rest of the compounds
induction of cell death was independent of the apoptotic
process. This is a remarkable property for 2a, since the
demonstration of apoptosis in MCF-7 breast cancer cells by
known apoptosis-inducing agents has proven difficult [40]. In
order to evaluate whether this apoptogenic effect is time-
dependent we investigated the apoptotic activity of 2a at
24 h. It can be seen from Fig. 8 that the apoptosis signal
in comparison to the control for 2a is statistically significant,
although the level of apoptosis is low. Comparison of the
results reveals that 2a exerted an apoptogenic effect at both
times but at 24 h the level of apoptosis was twice as high as
that of the control and at 48 h it was higher than the control
by a factor of ten.
The apoptogenic action of 2a was further confirmed in
CCRF-CEM cells (Table 5). In this experiment the apoptotic
status of CCRF-CEM cells was evaluated by measuring the
exposure of phosphatidylserine on the cell membranes using
the Annexin V-FITC Kit (BD Pharmingen) [39] after 24 h of
treatment with 25 or 40 mM of the compound. In these
experiments induction of apoptosis was also observed, albeit
to a lesser extent than in MCF-7 cells.
Effects on cell cycle progression
Cell cycle arrest is one of the targets of numerous anticancer
drugs, including doxorubicin and camptothecin. In an effort to
ascertain whether compounds 2a, 2c, 2e, 2f, 2g, 3a, and 3b
could affect cell cycle progression in MCF-7 cells, these cells
were treated with 25 mM of the corresponding compound for
48 h and cell cycle progression was determined by flow cyto-
metry analysis [38]. In these experiments different types of
behavior were found and these are represented in Fig. 9. The
differences observed in the blocking of cell-cycle phases after
treatment with the compounds under investigation suggests
the possible involvement of other, as yet unidentified, targets.
DNA flow cytometry analysis indicated that treatment of the
cells with compounds 2g and 3a did not induce any specific
Figure 7. Compound 2a induces an apoptotic effect on MCF-7
cells. MCF-7 cells were incubated either with 25 mM of the indicated
compound or vehicle (control cells) for 48 h. The results are pre-
sented as the mean ꢁ SD of three independent experiments (dupli-
cate wells).
ꢂꢂ
¼ difference statistically very significant with respect to the con-
trol (p < 0.01).
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688
D. Plano et al.
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Figure 9. Effects of 2a, 2c, 2e, 2f, 2g, 3a, and 3b on cell cycle
distribution in MCF-7 cells. Exponentially growing cells were treated
with 25 mM of the indicated compound or vehicle (control) for 48 h.
The changes in cell cycle phase distribution were assessed by DNA
flow cytometry analysis. Results are expressed as percentages of
total cell counts. Camptothecin was used as a positive control.
Results are presented as the mean ꢁ SD of three independent
experiments (duplicate wells). ꢂ ¼ difference statistically significant
Figure 10. Effects of 2a on cell cycle distribution in MCF-7 cells.
Exponentially growing cells were treated with 25 mM of the indicated
compound or vehicle (control) for 24 and 48 h. The changes in cell
cycle phase distribution were assessed by DNA flow cytometry ana-
lysis. Results are expressed as percentages of total cell counts.
Results are presented as the mean ꢁ SD of three independent
experiments (duplicate wells). ꢂ ¼ difference statistically significant
ꢂꢂ
with respect to the control (p < 0.05). ¼ difference statistically
ꢂꢂ
with respect to the control (p < 0.05). ¼ difference statistically
very significant with respect to the control (p < 0.01).
very significant with respect to the control (p < 0.01).
phase arrest in the cell cycle in comparison to the control. In
contrast, 3b and 2f provoked an increase in phase S, whereas
2c led to diminution in G0/G1 and an increase in G2/M.
Compound 2e induced a significant decrease in the G0/G1
population without modification of other phases. Finally,
the most promising compound (2a) triggered a marked change
in MCF-7 cell cycle distribution. In this case, cell accumulation
in the G2/M phase was accompanied by a diminution of cell
proportions in the G0/G1 phase and a significant increase in
SubG1 (which is representative of cells with fragmented DNA),
thus suggesting mitotic arrest prior to metaphase. It is worth
noting that the results obtained in this study are consistent
with the findings of our ongoing apoptosis studies on 2a.
Furthermore, cell cycle modification was significantly
induced by treatment with 25 mM of 2a for 24 h. At both
24 and 48 h (Fig. 10) there is an increase in the sub G1 phase
and a decrease in G0/G1. However, at 24 h there is a dimin-
ution in G2/M while at 48 h there is an increase in this value.
This finding suggests that mitotic arrest is time-dependent.
and 2f) were active in the nanomolar range. Compound 2a,
which contains a pyridine ring and a bromo-substituent,
showed cytotoxic activity in all the tumoral cell lines tested.
The toxicity of 2a on the nontumoral line BEAS-2B was lower
than that of doxorubicin and it inhibited the growth of
human breast adenocarcinoma MCF-7 cells by inducing cell
cycle arrest in the G2/M phase and triggering apoptosis. Most
of the antineoplastic drugs in current use block the cell cycle
in the S or G2/M phases.
For the rest of the compounds the induction of cell death
was independent of the apoptotic process and further exper-
iments are needed to elucidate their mechanism of action.
Based on the results described above, 2a can be highlighted as
a new active lead that provides a powerful incentive for
further research in this area since, to the best of our knowl-
edge, this is the first time that a selenadiazolepyridine with
this biological profile has been described.
Experimental
Conclusion
Chemistry
Melting points were determined with a Mettler FP82 þ FP80
apparatus (Greifensee, Switzerland) and are uncorrected. The
1H-NMR spectra were recorded on a Bruker 400 UltrashieldTM
spectrometer (Rheinstetten, Germany) using TMS as the internal
standard. The IR spectra were obtained on a Thermo Nicolet FT-IR
Nexus spectrophotometer with KBr pellets. Elemental microanal-
yses were carried out on vacuum-dried samples using a LECO
CHN-900 Elemental Analyzer. Silica gel 60 (0.040–0.063 mm)
(Merck KGaA, Darmstadt, Germany) was used for Column
In summary, a new series of fourteen benzo- and dibenzose-
lenadiazoles and selenadiazolepyridines has been synthe-
sized and their antiproliferative and cytotoxic activities
evaluated against four cancer cell lines and two non-malig-
nant cell lines (one mammary gland and one bronchial
epithelium line). Seven of the compounds exhibited promis-
ing antiproliferative values in MCF-7 cells and two of them (2c
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Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
Anticancer Activities of Selenadiazole Derivatives
689
Chromatography and Alugram1 SIL G/UV254 (Layer: 0.2 mm)
(Macherey-Nagel, Du¨ren, Germany) was used for Thin Layer
Chromatography. Chemicals were purchased from E. Merck
(Darmstadt, Germany), Scharlau (F.E.R.O.S.A., Barcelona,
(3 ꢃ 50 mL) and purified according to the method shown in
Table 1. The compounds 2e–h were obtained in the below-men-
tioned way.
´
Spain), Panreac Quımica S.A. (Montcada i Reixac, Barcelona,
Spain), Sigma-Aldrich Quımica, S.A. (Alcobendas, Madrid,
Spain), Acros Organics (Janssen Pharmaceuticalaan, Geel,
Belgium) and Lancaster (Bischheim, Strasbourg, France).
N-[4-(Methylthio)benzyl]benzo[c][1,2,5]selenadiazole-5-
carboxamide 2e
From benzo[c][1,2,5]selenadizole-5-acyl chloride and 4-
(methylthio)benzylamine.
´
General procedure for the synthesis of compounds 2a–d
and 2i–l
N-(4-Selenocyanatophenyl)benzo[c][1,2,5]selenadiazole-
5-carboxamide 2f
From benzo[c][1,2,5]selenadizole-5-acyl chloride and 4-aminophe-
nylselenocyanate (synthesized according to the general method
described in the literature) [41].
The corresponding ortho-aromatic diamine (9 mmol) and
selenium dioxide (1.0 g, 9 mmol) were ground in a mortar.
The reaction mixture was heated at the appropriate tempera-
ture. The reaction was monitored by IR spectroscopy. The crude
products were washed with distilled water (3 ꢃ 50 mL) and
purified according to the method shown in Table 1. The follow-
ing compounds were obtained in the way described below.
N-(Pyridin-3-ylmethyl)benzo[c][1,2,5]selenadiazole-5-
carboxamide 2g
From benzo[c][1,2,5]selenadizole-5-acyl chloride and 3-
(aminomethyl)pyridine.
6-Bromo-[1,2,5]selenadiazolo[3,4-b]pyridine 2a
From 2,3-diamino-5-bromopyridine and selenium dioxide at 908C.
General procedure for the synthesis of compound 2h
Compound 2d (1.82 g, 8 mmol) and thionyl chloride (20 mL)
were heated under reflux for 2 h. The thionyl chloride was
removed in vacuo. The resulting acyl chloride was used without
further purification. Ammonium hydroxide (75 mL) was added
to the acyl chloride (1.96 g, 8 mmol) and the mixture was stirred
for 30 min at room temperature. The solid was filtered off and
washed with distilled water (4 ꢃ 50 mL). The resulting product,
benzo[c][1,2,5]selenadiazole-5-carboxamide 2h, did not require
further purification.
5-Bromobenzo[c][1,2,5]selenadiazole 2b
From 4-bromo-1,2-diaminobenzene and selenium dioxide at
room temperature.
Benzo[c][1,2,5]selenadiazol-5-yl(phenyl)methanone 2c
From 3,4-diaminobenzophenone and selenium dioxide at 758C.
Benzo[c][1,2,5]selenadiazole-5-carboxylic acid 2d
From 3,4-diaminobenzoic acid and selenium dioxide at 858C.
General procedure for the synthesis of compounds 3
Compound 2d (1.82 g, 8 mmol) and thionyl chloride (20 mL) were
heated under reflux for 2 h. The thionyl chloride was removed in
vacuo. The resulting acyl chloride was used without further puri-
fication. To a stirred mixture of the acyl chloride (1.96 g, 8 mmol)
and triethylamine (0.81 g, 8 mmol) in dry chloroform (50 mL) was
added dropwise a solution of the corresponding diamine (4 mmol)
in dry chloroform. The mixture was stirred at room temperature
for 48 h. The solvent was removed in vacuo and the product was
washed with distilled water (3 ꢃ 50 mL) and purified according to
the method shown in Table 1. Compounds 3a and 3b were
obtained in way described below.
Benzo[c][1,2,5]selenadiazole 2i
From 1,2-phenylenediamine and selenium dioxide at room
temperature.
[1,2,5]Selenadiazolo[3,4-b]pyridine 2j
From 2,3-diaminopyridine and selenium dioxide at room
temperature.
5-Methylbenzo[c][1,2,5]selenadiazole 2k
From 3,4-diaminotoluene and selenium dioxide at room
temperature.
0
N,N -(Propane-1,3-diyl)dibenzo[c][1,2,5]selenadiazole-5-
carboxamide 3a
Benzo[c][1,2,5]selenadiazole-5-carbonitrile 2l
From 3,4-diaminobenzonitrile and selenium dioxide at room
temperature.
From benzo[c][1,2,5]selenadizole-5-acyl chloride and 1,3-
diaminopropane.
0
N,N -[4,40-Diselenediylbis(4,1-phenylene)]dibenzo[c]
General procedure for the synthesis of compounds 2e–g
A mixture of compound 2d (1.82 g, 8 mmol) and thionyl chloride
(20 mL) was heated under reflux for 2 h. The thionyl chloride was
removed in vacuo. The resulting acyl chloride was used without
further purification. To a stirred mixture of the acyl chloride
(1.96 g, 8 mmol) and triethylamine (0.81 g, 8 mmol) in dry
chloroform (50 mL) was added dropwise a solution of the corre-
sponding amine (8 mmol) in dry chloroform. The mixture was
stirred at room temperature for 48 h. The solvent was removed
in vacuo and the product was washed with distilled water
[1,2,5]selenadiazole-5-carboxamide 3b
From benzo[c][1,2,5]selenadizole-5-acyl chloride and 4,40-diami-
nophenyldiselenide (synthesized according to the general
method described in the literature [42]).
Molecular modeling
The initial computational work (conformational analysis, deter-
mination of volumes, orbitals, charge distribution and
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690
D. Plano et al.
Arch. Pharm. Chem. Life Sci. 2010, 10, 680–691
determination of the pattern of intra-molecular hydrogen bonds)
was performed on a SiliconGraphics Octane2 workstation pro-
vided with the software package InsightII. Three-dimensional
models of the compounds were constructed, in the vacuum
phase, using atoms and structural fragments from the Builder
module (InsightII). The protocol can be summed up as follows: (a)
Initial construction of the model. (b) Hierarchized systematic
conformational analysis: determination of the rotation-sensitive
bonds; selection of a 308 window to check each selected dihe-
dron. First filtration: Elimination of the conformations which
are indistinguishable by symmetry. Second filtration:
Elimination of conformations that present steric impediments.
Third filtration: Calculation of the energy of conformations and
elimination of those conformations whose relative energy
Evaluation of cell cycle progression and apoptosis
induction
For breast adenocarcinoma MCF-7 cells, the apoptotic status and
cell cycle analysis of the cells were determined using the Apo-
Direct kit (BD Pharmingen), based on the TUNEL technique,
under the conditions described by the manufacturer. In brief,
in the fixation step cells were suspended in 1% paraformalde-
hyde in PBS (pH 7.4) at a concentration of 1 ꢃ 106 cells/mL,
incubated on ice for 30 min, collected by centrifugation, washed,
adjusted to 1 ꢃ 106 cells/mL in 70% ice cold ethanol and incu-
bated at ꢀ208C for 1 h. After fixation, cells were recovered by
centrifugation, washed, resuspended in FITC dUTP-DNA labelling
solution and incubated for 2 h at 378C. Cells were then rinsed,
resuspended in PI/RNase staining buffer, incubated in the dark
for 30 min at RT and analyzed using a Coulter Epics XL flow
cytometer. The apoptotic status of leukemia CCRF-CEM cells was
evaluated by measuring the exposure of phosphatidylserine on
the cell membranes using the Annexin V-FITC Kit (BD
Pharmingen). In this test, 5 ꢃ 105 cells were pelleted and washed
in PBS. Cells were then stained with annexin V-FITC and propi-
dium iodide for 15 min at 48C in the dark and analyzed by flow
cytometry.
>10 kcal/mol at
a
global minimum. Fourth filtration:
Optimization of the geometry of the conformations and elimin-
ation of those whose relative energy >10 kcal/mol at a global
minimum. All of the molecular mechanics calculations were
carried out using the consistent valence force field ESFF43
(Search and Compare module, InsightII). (c) Analysis of confor-
mational trajectory (Analysis module, InsightII) and selection of
representative lowest energy conformation. Root mean square
(rms) deviations of the structures were monitored. (d) Mechano-
quantics optimization of the conformations obtained in the
previous step with the molecular orbital calculations package
Mopac (PM3 [44] semi-empirical approach, AMPAC/MOPAC built
into the InsightII module).
The authors wish to express their gratitude to the University of Navarra
Research Plan (Plan de Investigacio´n de la Universidad de Navarra, PIUNA)
and CAN Foundation for financial support for the project. We also thank
the Department of Industry of the Navarra Government for the award of
two grants (D.P. and E.M.).
Cytotoxic and antiproliferative activities
The cytotoxic effect of each substance was tested at five different
concentrations between 0.01 and 100 mM. Each substance was
initially dissolved in DMSO at a concentration of 0.1 M and serial
dilutions were prepared using culture medium. The plates with
cells from the different lines, to which medium containing the
substance under test was added, were incubated for 72 h at 378C
in a humidified atmosphere containing 5% CO2. Human tumor
cell lines were provided by the European Collection of Cell
Cultures (ECACC) or the American Type Culture Collection
(ATCC). Four cell lines were used: One human lymphocytic leu-
kemia (CCRF-CEM) and three human solid tumors, one colon
carcinoma (HT-29), one lung carcinoma (HTB-54), and one breast
adenocarcinoma (MCF-7). CCRF-CEM, HT-29 and HTB-54 cells were
grown in RPMI 1640 medium (Invitrogen) supplemented with
10% fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin,
100 mg/mL streptomycin and 10 mM HEPES buffer (pH ¼ 7.4).
MCF-7 cells were grown in EMEM medium (Clonetics) supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 100 units/mL
penicillin and 100 mg/mL streptomycin. 184B5 cells were grown
in Hams F-12/DMEM (50:50) supplemented as described by Li et al.
[45]. BEAS-2B cells were grown in 25 mL fetal bovine serum (FBS),
5 mL insulin-transferrin-sodium selenite (ITS), 1 mL hydrocorti-
sone, 10 mL sodium pyruvate, 5 mL glutamine, 5 mL penicillin/
gentamicin, 10 mL epidermal growth factor (EGF), and 150 mL
retinoic acid (1 mM). Cytotoxicity was then determined by the
MTT method. Results are expressed as GI50, the concentration
that reduces by 50% the growth of treated cells with respect to
untreated controls, TGI, the concentration that completely
inhibits cell growth, and LC50, the concentration that kills
50% of the cells. Data were obtained from at least 3 independent
experiments performed in quadruplicate.
The authors have declared no conflict of interest.
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