Naphthylchalcones induce apoptosis and caspase activation in a leukemia cell line: The relationship between mitochondrial damage, oxidative stress, and cell death

Article abstract of DOI:10.1016/j.bmc.2010.09.025

In this study, we investigated the effects of 24 chalcone derivatives from 2-naphthylacetophenone toward a lymphoblastic leukemia cell line (L1210). Three compounds, called R7, R13, and R15, presented concentration- and time-dependent cytotoxicity and induced cellular death by apoptosis via mitochondrial injury and oxidative stress. The effects of these compounds appear to occur through different mechanisms because R13 and R7 induced a greater disturbance of mitochondrial potential, and all compounds induced disturbances of cellular ATP content and increased caspase-3 activity before cellular death. These compounds also interfered with antioxidant enzymes activities and GSH content through different mechanisms.

Full text of DOI:10.1016/j.bmc.2010.09.025

Bioorganic & Medicinal Chemistry 18 (2010) 8026–8034
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Naphthylchalcones induce apoptosis and caspase activation in a leukemia
cell line: The relationship between mitochondrial damage, oxidative stress,
and cell death
Evelyn Winter a, Louise Domeneghini Chiaradia b, Clarissa A. S. de Cordova a,c, Ricardo José Nunes b,
Rosendo Augusto Yunes b, Tânia Beatriz Creczynski-Pasa a,
⇑
a Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil
b Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil
c Universidade Federal do Tocantins, 77001-090 Palmas, TO, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 3 September 2010
Accepted 9 September 2010
Available online 16 September 2010
In this study, we investigated the effects of 24 chalcone derivatives from 2-naphthylacetophenone toward
a lymphoblastic leukemia cell line (L1210). Three compounds, called R7, R13, and R15, presented concen-
tration- and time-dependent cytotoxicity and induced cellular death by apoptosis via mitochondrial injury
and oxidative stress. The effects of these compounds appear to occur through different mechanisms
because R13 and R7 induced a greater disturbance of mitochondrial potential, and all compounds induced
disturbances of cellular ATP content and increased caspase-3 activity before cellular death. These
compounds also interfered with antioxidant enzymes activities and GSH content through different
mechanisms.
Keywords:
Leukemia
L1210 cells
Chalcones
Ó 2010 Elsevier Ltd. All rights reserved.
Apoptosis
Oxidative stress
1. Introduction
changes in cells such as DNA fragmentation, chromatin condensa-
tion, cell shrinkage, and membrane blebbing.7
Acute lymphoblastic leukemia (ALL) is a malignant disorder of
lymphoid progenitor cells that affects both children and adults,
with peak prevalence between the ages of two and five years.1
Although contemporary treatments cure more than 80% of children
with ALL,2 some patients require intensive treatment, and many
patients still develop serious acute and late complications due to
the side effects of the drugs. Furthermore, the survival rate for
adults with ALL remains below 40%,3 and multidrug resistance is
frequently reported.2 Therefore, new drugs and treatment strate-
gies are needed to improve both the cure rate and patient quality
of life.4 Studies have considered that due the heterogeneity of
the alterations found in leukemias, a combination therapy with
antiproliferatives and apoptosis-inductive agents could be more
effective than conventional treatments.5
Several recent studies have shown that the use of drugs that
compromise the structural and functional integrity of mitochondria
can be an alternative treatment against the growth of neoplastic
cells.8 When a death signal is detected by the mitochondria, a col-
lapse of mitochondrial potential is triggered, and apoptotic factors
as cytochrome c are released to the cytosol. These factors activate
caspases, culminating in apoptosis.9 Alterations in mitochondria
can also lead to bioenergetic imbalances because these organelles
are responsible for the production of 80 to 90% of the ATP required
by cells.8
The mechanisms of cellular death also can be influenced by an
imbalance in oxidative metabolism called oxidative stress. The
mitochondrion is the main organelle involved in this phenomenon
because it is the primary consumer of cellular oxygen and the cen-
ter of reactive oxygen species (ROS) generation.6 Under oxidative
stress, normal cells can enter into an adaptation process, mainly
through their antioxidant defenses.10 However, tumor cells can
have a limited capacity of adaptation for some defenses; therefore,
antitumor agents that induce the production of ROS have potential
therapeutic benefits.11 Studies have related the increase of ROS
with the increase of cellular death.12
Cell death through apoptosis can result from several cellular
events, occasioned frequently by mitochondrial alterations and
the consequent alterations in oxidative metabolism and cellular
content of ATP.6 The process of apoptosis leads to characteristic
⇑
Corresponding author. Tel.: +55 48 3721 8057; fax: +55 48 37219542.
E-mail addresses: taniabcp@gmail.com, taniac@mbox1.ufsc.br, tania@pq.cnpq.br
Chalcones are essential intermediate compounds in flavonoid
biosynthesis in plants. Many studies have demonstrated antitumor,
(T.B. Creczynski-Pasa).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.025

E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
8027
Figure 1. Preparation of chalcones. (a) KOH 50% m/v, methanol, rt, 24 h. *Novel compounds.
anti-inflammatory, anti-infective, and hypotensive activities for
chalcones, in addition to other pharmacological effects (for reviews,
see Refs. 13–15). Kachadourian and Day16 demonstrated that the
antileukemic activity of hydroxychalcones occurred via depletion
of reduced glutathione (GSH), release of cytochrome c and loss of
mitochondrial membrane potential. Navarini et al.17 also demon-
strated that the antileukemic activity of hydroxychalcones occurred
via depletion of GSH and ATP.
Despite advances in the therapies, the greatest problem associ-
ated with acute leukemia is the resistance to systemic methods of
treatment that lays the importance of investigation of new
potential molecules to control and/or cure this disease. In this
study, we investigated the effects of chalcone derivatives from
2-naphthylacetophenone toward a lymphoblastic leukemia cell
line by characterizing cell death and by monitoring induced oxida-
tive stress and the influence of the derivatives on energy metabo-
lism, never studied before in this regard.
29% and 97%. All reagents used were obtained commercially
(Sigma–Aldrich), except the benzylated vanillin [3-methoxy-4-
(phenylmethoxy)-benzaldehyde], which was prepared as previ-
ously described,19 with a yield of 88%. All structures, including
those not yet published (R11, R23, R25, R44, and R45), were con-
firmed by melting points and by chemical identification data: infra-
red spectroscopy (IR), 1H and 13C nuclear magnetic resonance
spectroscopy (NMR), and elementary analysis. 1H NMR spectra re-
vealed that all structures were structurally pure and with E config-
uration (JH –Hb = 16.0 Hz).
a
2.2. Biological
To screen and compare the cytotoxicity induced by the
naphthylchalcones, all compounds were incubated in the L1210 cell
line at 25, 50, 75 or 100 lM for 24 h and the IC50 values were esti-
mated. Chalcones R7, R13, R14, R15, R25, and R30 were the com-
pounds that presented the best cytotoxic effect, with IC50 values
below 40 lM (Fig. 2). It is important to observe that these com-
2. Results and discussion
2.1. Chalcone synthesis
pounds are structures either without substituents on the B-ring
(R15), with substituents that reduce the electron density of the aro-
matic ring in positions 2 or 3 of the B-ring (R7, R13, R14, and R25)
or with a sulfur heterocyclic as the B-ring (R30). Chalcones with
substitutions at position 3, accompanied by another substitution
at position 4 of the B-ring, showed moderated activity (R11 and
R27), as well as R12 (2,6-dichlorated), R29 (with a 1-naphthyl
Twenty-four chalcones were prepared in methanol under basic
conditions by aldolic condensation between 2-naphthylacetophe-
none and corresponding aldehydes (Fig. 1),18 providing compounds
with substituents on the B-ring. The obtained yields varied between
Figure 2. Cytotoxicity of naphthylchalcones in L1210 cells. Cells were incubated with the compounds at concentrations of 25, 50, 75, and 100 lM at 24 h, and cell viability
was assayed by MTT. Optical density of zero time was taken as 100% cell viability.

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E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
Figure 4. Activation of caspase-3 activity by chalcones R7, R13, and R15 in L1210
cells. Cells were incubated with the compounds for 2 and 4 h. Caspase-3 activity
was measured by monitoring the cleavage of Ac-DEVD-AMC, a fluorogenic substrate
for caspase-3, for 2 h. The activity is given in arbitrary fluorescence units (a.u.) per
hour per lg protein. *p <0.05 and ***p <0.001. R7 23 lM, R13 37 lM, R15 36 lM.
Figure 3. DNA fragmentation in L1210 cells induced by chalcones R7, R13, and R15.
Cells were incubated with the compounds for 24 h. DNA analysis was performed
through agarose gel electrophoresis. C represents the control cells (cells incubated
Several studies have searched for new drugs with the ability to
induce apoptosis in leukemic cells.4,21 To confirm whether or cell
death was induced by apoptosis, cells were incubated with
chalcones R7, R13, and R15 at their respective IC50 concentrations
without chalcones), and P represents the DNA sizing standard. R7 23
37 M, R15 36 M.
lM, R13
l
l
group as B-ring) and R36 (with an oxygenated heterocyclic ring).
Mono-substituted compounds with substitutions at position 4, or
compounds substituted at position 4 and one substitution more
at available positions, presented moderated (R8, R16, R17, and
R24) or no activity (R10, R20, R21, R23, R26, R44, and R45). These
results indicate that the substitutions at position 4 of the B-ring de-
crease the cytotoxic potential of chalcones against L1210 leukemia
cells when the A-ring is a 2-naphthyl group. These observations are
consistent with Nam et al.20 who observed that chalcones with
electron-withdrawing groups at the B-ring, mainly at 2-position,
confer strong angiogenesis inhibition and marked antitumor activ-
ity in murine melanoma (B16), human colon cancer (HCT116), and
human epidermoid carcinoma cells (A431).20 In addition, it was
shown that compounds R28 (with a 2-naphthyl group as B-ring)
and R19 (dimethoxylated in positions 2 and 6 of B-ring) presented
no cytotoxic activity.
Chalcones R7, R13, R14, R15, R25, and R30 were selected and
analyzed for induction of cell death. Compounds R7, R13, and
R15 induced cell death by apoptosis, characterized by internucle-
osomal breakdown of DNA (Fig. 3). R7, R13, and R15 demonstrated
cytotoxicity to L1210 and the IC50 values are shown in the Table 1.
These chalcones were also cytotoxic to a human leukemia cell line
(CEM) with lower values of IC50 (Table 1). The cell death mecha-
nism for this cell line was also by apoptosis, observed by DNA
breakdown pattern (data not shown). As can be observed in the
Table 1 the values of IC50 in function of time decreased for both cell
lines, principally after 72 h of incubation with the compounds,
with the exception of R15 for L1210. These results, although not
conclusive, indicate that the chalcones R7, R13, and R15 may pres-
ent also antiproliferative activity in vitro.
(R7 23 lM, R13 37 lM, R15 36 lM) for 2 and 4 h to determine
caspase-3 activity. The compounds increased caspase-3 activity
(Fig. 4), confirming that chalcones R7, R13, and R15 induced cell
death by apoptosis. In a previous study with the HL-60 cell line,
Nakatani et al.22 showed the antileukemic activity of dihydrochal-
cones by caspase activation. However, these chalcones differ from
ours by not having a double bond.
To determine whether oxidative stress was involved in chalcone-
induced cell death, the generation of ROS was evaluated by the
extent of lipid peroxidation following a concomitant incubation of
chalcones (R7 23 lM, R13 37 lM, R15 36 lM) and antioxidant
enzymes. Cell viability was evaluated in parallel. As shown in Figure
5A, all compounds significantly induced ROS production, and
this production was blocked when catalase (CAT) was present.
Co-incubation of the compounds with catalase also increased cell
viability after 24 h of incubation when compared with compounds
as single agents. These differences were not observed when com-
pounds were co-incubated with superoxide dismutase (SOD) or
TroloxÒ (TR). These results are summarized in Table 2, including
the IC50 values obtained with these treatments. Additionally, all
chalcones induced lipid peroxidation of cell membranes (Fig. 5B).
Therefore, these findings indicate that the compounds tested in-
duced oxidative stress and suggest that hydrogen peroxide (H2O2)
can be the main ROS involved in the process because the treatment
of the cells with catalase decreased the production of ROS and the
resulting cytotoxicity.
Many natural compounds can induce apoptotic cell death in
cancer cells by increasing ROS content, promoting the formation
of pores in mitochondria.8 The relation between ROS mitochondrial
production, cytochrome c release and caspase-3 activation in a leu-
kemic cell line was also reported.23 Jing et al.24 and Shin et al.12
demonstrated the antileukemic activity of different compounds
by induction of apoptosis via ROS generation, which resulted in a
decrease in mitochondrial membrane potential. Thus, we evalu-
Table 1
Comparison between the IC50 values of the chalcones for cell viability in L1210 and
CEM cell lines
Compounds
L1210 IC50
48 h
(l
M)
CEM IC50
48 h
(lM)
ated the effect of chalcones (R7 23 lM, R13 37 lM, R15 36 lM)
24 h
72 h
24 h
72 h
on mitochondrial membrane potential and observed that com-
pounds R7 and R13 decreased mitochondrial membrane potential
(Fig. 6D) to a similar extent as did carbonyl cyanide p-trifluoro-
methoxyphenylhydrazone (FCCP). These findings can be related
R7
R13
R15
24
38
37
13
23
20
14
10
17
11
11
12
11
12
14
4
6
5

E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
8029
Figure 5. Effects of chalcones R7, R13, and R15 on ROS generation and lipid peroxidation in L1210 cells. (A) ROS formation was followed using DCFH-DA. The results are
expressed as the percentage of cellular fluorescence in comparison to control samples (zero % of fluorescence). ###p <0.001 when the control samples and the cells incubated
with chalcones alone were compared, and **p <0.01; ***p <0.001 when the cells incubated with chalcones alone and the cells incubated with chalcones plus catalase were
compared. (B) Lipid peroxidation was measured using the TBARS method. The results are expressed as nmol of TBARS per
R13 37 M, R15 36 M.
lg of protein. *p <0.05 and ***p <0.001. R7 23 lM,
l
l
Table 2
ship between apoptosis and ATP content and showed that mito-
chondrial damage can result in a decrease of ATP content and
apoptosis, or an initial increase in ATP content followed by a de-
crease in ATP content and cell death. The results of Skulachev
may help to explain the increase of ATP content that occurred in
cells incubated with chalcones observed in the current study. As
shown in Fig. 6A and B, chalcones R7 and R13 decreased mitochon-
drial membrane potential and consequently caused damage in the
respiratory chain and depletion of ATP content. An alternative
mechanism occurred with chalcone R15 (Fig. 6C). The direct deple-
tion of ATP can be related with the strong pro-oxidant effects of
this chalcone, as observed in Figure 5A, and with Glutathione
peroxidase (GPx) and Glutathine S-transferase (GST) activities in
Figure 7. Cao et al.29 reported that high doses of curcumin induced
damage to mitochondrial DNA due to the increase in ROS. mtDNA
damage decreases the operation of the respiratory chain, and con-
sequently, it strongly decreases ATP synthesis.
To minimize the damage of ROS, aerobics organisms were
endowed with enzymatic and not-enzymatic antioxidant defenses.
The principal enzymatic defenses are catalase (CAT), glutathione
reductase (GR), GPx, and GST.30 GST do not act directly against
ROS, but they are important to cellular defenses as part of the phase
II detoxification enzyme family that is responsible for the protection
of cellular macromolecules from attack by reactive electrophiles via
GSH conjugation.31 Among the non-enzymatic antioxidants are
mainly melanin, glutathione, uric acid, vitamins C and E and lipoic
acid.30 Chalcones R7, R13, and R15 significantly increased the activ-
ity of GPx (Fig. 7A), GST (Fig. 7B) and GR (Fig. 7C), but none of them
affect CAT activity (Fig. 7D). All chalcones increased glutathione
oxidized (GSSG) (Fig. 8A) and total glutathione (TG) (Fig. 8B)
contents. Chalcones R7 and R13 did not change GSH content, but
R15 increased GSH content (Fig. 8C) The concentration of the chal-
Comparison between the IC50 values for cyto-
toxicity of the chalcones R7, R13, and R15
incubated alone and associated to catalase,
TroloxÒ, and superoxide dismutase with L1210
cell line
Treatments
IC50 (lM)
R7
24 0.6
45 1.8***
20 0.5
20 1.0
38 1.2
51 2.4**
30 0.3
34 1.9
37 1.1
58 2.8***
31 0.7
39 0.5
R7 + CAT
R7 + SOD
R7 + TR
R13
R13 + CAT
R13 + SOD
R13 + TR
R15
R15 + CAT
R15 + SOD
R15 + TR
**
p <0.01.
p <0.001
***
with ROS generation caused by the compounds and consequently
with oxidative stress. Sastre et al.25 reported that ROS can cause oxi-
dative damage to protein, lipids and respiratory chain proteins acti-
vating caspase and other apoptotic factors. Chalcone R15 did not
significantly change mitochondrial membrane potential (Fig. 6D),
although it was the compound that induced the highest level of
ROS generation. This increase can be related with its potential to in-
duce ROS generation in distinct cell site not in mitochondria.
The mitochondria are the central organelles involved in apopto-
sis and are responsible for ATP production in cells. Therefore, the
determination of the intracellular ATP concentration is important
in researching drugs that induce apoptosis and deplete energy.26
cones used was R7 23 lM, R13 37 lM, R15 36 lM.
Chalcones R7, R13, and (R7 23 lM, R13 37 lM, R15 36 lM)
The increase in GPx activity can be explained by the increases in
ROS production and lipid peroxidation induced by the chalcones. Be-
cause GPx is responsible for the reduction of peroxides, an increase
in peroxide formation can result in an increase of GPx activity. The
increase in GST occurred perhaps because these chalcones possibly
are eliminated by conjugation with GSH by GST. Thus, an increase
in chalcones concentration can result in GST activity enhancement.
Surprisingly, the chalcones augmented GPx activity but did not
increase CAT activity even though CAT is the principal enzyme
responsible for reducing H2O2. On the contrary, chalcones appears
induced a slight variation of ATP concentration, decreasing and
increasing at initial times of measurements and finally a strong
ATP concentration depletion (Fig. 6A–C). It is important to mention
that the ATP measured was from the remaining live cells. Sabzevari
et al.27 showed that the cytotoxic effects of 10 hydroxychalcones in
a leukemic cell line resulted from mitochondrial disturbances. In
this study, similar effects occurred, resulting in respiratory chain
deregulation and consequently depletion of ATP content and
increased ROS production. Increased ROS levels can activate JNK
and p38 and induce cell death. Skulachev28 evaluated the relation-

8030
E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
Figure 6. Effect of chalcones R7, R13, and R15 on mitochondrial membrane potential and intracellular ATP content in L1210 cells. ATP concentration was monitored by the
luciferin–luciferase assay following incubation with chalcones at R7 23 M, R13 37 M, R15 36 M for different incubation times: (A) R7, (B) R13, and (C) R15. (D) The
l
l
l
membrane potential was determined using JC-1 after 4 h of incubation. A decrease in the red/green ratio indicates a decrease in mitochondrial membrane potential. The
results are expressed as percentages and the values of control cells were considered 100%. ***p <0.01; **p <0.01; ***p <0.001.
to inhibit CAT activity. We investigated this hypothesis by evaluat-
ing the effects of chalcones on the activity of isolated CAT enzyme.
Although the test was performed with an enzyme batch isolated
from a different source, chalcones, principally R13, significantly
inhibited CAT activity (data not shown). Pelicano et al.11 described
that cancer cells can have a limited capacity of adaptation for some
antioxidant defenses in response to oxidative stress. The inability of
cells to increase CAT activity, in this case, can be the reason of the
cytotoxicity of chalcones because it is related mainly to the genera-
tion of H2O2. Another important fact resulting from the action of ROS
in cancer cells is the oxidation of proteins such as cytochrome c and
CAT. CAT inhibition can reduce the capacity of cells to eliminate
H2O2, increasing cellular damage induced by ROS.32
GSH is the most important non-enzymatic antioxidant.
Normally, the majority of GSH remains reduced by GR. However,
oxidative stress results in a decrease in GSH content and conse-
quently, to an increase in GSSG content.33 Chalcones R7, R13,
and R15 induced an increase in GSSG content, more evidence that
these compounds induce oxidative stress. The increase in GR can
be the result of the oxidative stress occurring during the incubation
of cells with chalcones in an attempt to sustain GSH content. This
result also explains why GSH content decreased in cells during
incubation with chalcones R7 and R13 and why GSH content
increased during incubation with chalcone R15. Moreover, the
observed increase in GSH content can be the result increased
3. Conclusion
The development of new drugs for the treatment of cancer is
based on the potential of compounds to block cell proliferation
and induce apoptosis.34 Our findings showed, for the first time,
that the naphthylchalcones R7, R13, and R15 present cytotoxicity
inducing apoptosis in leukemia cell line L1210 through distinct tar-
gets. In general, these compounds induced oxidative stress; how-
ever, for chalcone R7 and R13, the stress is a consequence of
mitochondrial damage that can be related with respiratory chain
damages and the deregulation of ROS generation and subsequent
depletion of ATP content. For chalcone R15, cellular death results
from its strong pro-oxidant effect that was induced mainly by
ROS generation and interference with antioxidant defenses. Our
group targeting if the chalcones have antiproliferative effect be-
hind cell cycle cell studies is developing further studies with both
cell lines.
These results indicate that these compounds are interesting in
the research of new drugs for cancer treatment.
4. Experimental part
4.1. Chemicals
The cell culture media were purchased from Cultilab (São Paulo,
Brazil). Serum and antibiotics were purchased from GIBCO (Grand
c-glutamyl-cysteine-synthetase
(c-GCS) activity promoted by
chalcone R15 (data not shown).

E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
8031
Figure 7. Effect of chalcones R7, R13, and R15 on antioxidant defenses. Activities of GPx (A), GST (B), GR (C), and CAT (D) were measured as described in Section 4 and
presented as percentages of control. R7 23 M, R13 37 M, R15 36 M. *p <0.05; **p <0.01; ***p <0.001. The incubation time was 24 h.
l
l
l
Figure 8. Effect of chalcones R7, R13, and R15 on GSH, GSSG, and TG content. Content of GSSG (A), TG (B), and GSH (C) were measured as described in Section 4 and presented
as percentages of control. R7 23 M, R13 37 M, R15 36 M. *p <0.05; **p <0.01; ***p <0.001. The incubation time was 24 h.
l
l
l
Island, NY). The luciferin–luciferase kit was purchased from
Bioorbit (Tuku, Finland), the JC-1 was purchased from Invitrogen
and all other reagents were purchased from Sigma Chemical Co.
(St. Louis, MO).
benzaldehyde], which was prepared as previously described.19 All
chalcones were prepared by aldolic condensation between 2-
naphthylacetophenone and corresponding aldehydes, in methanol,
KOH (50% v/v), at room temperature with magnetic agitation for
24 h.18 Distilled water and 10% hydrochloric acid were added to
the reaction for total precipitation of the compounds, which were
then obtained by vacuum filtration and later recrystallized in
dichloromethane and hexane. The chalcones are soluble in dimeth-
ylsulfoxide, acetone, chloroform and dichloromethane. Chalcones
4.2. Preparation of compounds
Reagents used were obtained commercially (Sigma–AldrichÒ),
except the benzylated vanillin [3-methoxy-4-(phenylmethoxy)-

8032
E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
R7, R8, R10, R12, R13, R14, R15, R16, R17, R20, R21, R24, R26, R27,
R28, R29, and R30 were previously cited in the literature (see Refs.
35–41). R19 and R36 were recently published by our group,42 and
R11, R23, R25, R44, and R45 are new compounds.
lysis buffer (10 mM EDTA, 50 mM Tris–HCl pH 8.0, 0.25% NP-40,
0.5 g/L proteinase K) at 50 °C for 2 h. DNA was then precipitated
with 2.5 vol of ethanol at 25 °C overnight and dried in air. After
washing with ice-cold 70% ethanol, the pellets were dissolved in
TE buffer containing 10 mM Tris–HCl pH 8.0, 1 mM EDTA, and
0.6 g/ml RNase A, and they were further incubated at 37 °C for
1 h. Horizontal electrophoresis was performed at 150 V using a
1.0% agarose gel with TAE (Tris–acetic acid and EDTA) as the run-
ning buffer. The gel was stained with ethidium bromide and visu-
alized by a 2UV Transilluminator (MacroVue UV-20 Hoefer) for
ladder formation.
4.3. Physico-chemical data of the compounds
The structures were confirmed by melting points (mp), infrared
spectroscopy (IR), and 1H and 13C nuclear magnetic resonance
spectroscopy (NMR), as well as elementary analysis for previously
undescribed structures. Melting points were determined with a
Microquímica MGAPF-301 apparatus and are uncorrected. IR spec-
tra were recorded with an Abb Bomen FTLA 2000 spectrometer on
KBr disks. NMR (1H and 13C) spectra were recorded on a Varian
Oxford AS-400 (400 MHz) instrument, using tetramethylsilane as
an internal standard. Elementary analysis was carried out using a
CHNS EA 1110; percentages of C and H were in agreement with
the product formula (within 0.4% of theoretical values for C).
4.7. Determination of caspase-3 activity
To determine the activity of caspase-3, 3 ꢀ 106 L1210 cells were
incubated with the compounds for 2 and 4 h (at the IC50 concentra-
tion for each compound) at 37 °C. The cells were then washed with
PBS and lysed in 75
7.4, 42 mM KCl, 5 mM MgCl2, 1 mM phenylmethylsulfonylfluoride
(PMSF), 0.1 mM EDTA, 0.1 mM EGTA, 1 g/ml pepstatin A, 1 g/ml
leupeptin, 5 g/ml aprotinin, 0.5% 3-[(3-cholamidopropyl)-dim-
ethylammonio]-1-propanesulfonate (CHAPS), and 1 mM dithio-
threitol (DTT) at 4–8 °C for 5 min. The extract (50 g of protein)
ll of lysis buffer containing 10 mM HEPES pH
4.4. Cell culture
l
l
l
Murine lymphoblastic leukemia (L1210) and human lympho-
blastic leukemia cells (CEM) were obtained from American Type
Culture Cell (ATCC). The cells were cultured in DMEM supple-
mented with 10% heat-inactivated fetal bovine serum, 100 U/ml
l
was then added to a buffer containing 25 mM HEPES pH 7.4, 0.1%
CHAPS, 1 mM EDTA, 10% sucrose, and 3 mM DTT. The reaction
penicillin, 100
lg/ml streptomycin and 10 mM HEPES. The cell cul-
medium was supplemented with 10
lM Ac-DEVD-AMC caspase-3
ture was maintained at 37 °C in a 5% CO2 humidified atmosphere
and pH 7.4. Every 2–3 days, cells were passaged by removing
90% of the supernatant and replacing it with fresh medium. In all
experiments, viable cells were checked in the beginning of the
experiment by Trypan Blue exclusion.
substrate (acetyl-Asp-Glu-Val-Asp-amino-methylcoumarin),
a
fluorogenic substrate for caspase-3. After incubation at 37 °C for
2 h, caspase activity (production of fluorescent AMC) was moni-
tored using a spectrofluorimeter (Perkin–Elmer LS55) measuring
the extinction at 380 nm and emission at 465 nm. The fluorescence
of blanks containing no cellular extracts was subtracted from the
fluorescence sample values.45 Protein content was determined by
Lowry’s method46 and caspase-3 activity was expressed as a
4.5. Cytotoxicity
Chalcone cytotoxicity was evaluated by MTT assay.43 To evaluate
the influence of concentration on cytotoxicity, 1 ꢀ 105 and 5 ꢀ 104
cells/well were incubated for 24 and 48 h, respectively, in triplicate
with the compounds (solubilized in DMSO no more than 1%), and at
change in fluorescent units per hour per lg of protein.
4.8. Free radical determination
different concentrations (1–100
l
M) in 96-well microplates. To
Intracellular free radical formation was determined using 20,70-
dichlorofluorescein diacetate (DCFH-DA), which is oxidized to
dichlorofluorescein (DCF) in the presence of ROS.47 5 ꢀ 105 L1210
cells were incubated with the compounds (at the IC50 concentra-
tion of 24h for each compound) in the absence or presence of
5000 U/ml catalase for 4 h at 37 °C. Cells were then incubated with
evaluate the cytotoxicity of chalcones in combination with catalase,
1 ꢀ 105 cells/well were incubated for 24 h with different concentra-
tions (1–100 lM) of the compounds and plus CAT (5000 U/ml), SOD
(250 U/ml) and TR (200 lM) a water-soluble vitamin E derivative.
To verify if the enzymes and TR could themselves to prejudice the
cell, a control with the same additions without the chalcones was
run in parallel; cell viability remained without alterations. After
incubation at 37 °C, cells were washed with fresh culture medium,
10 lM DCFH-DA for 30 min at 37 °C and then washed four times
with PBS. The DCF fluorescence signal was measured using a Per-
kin–Elmer LS55 spectrofluorimeter. The results were normalized
by the cell death percentage that occurs during 4 h of incubation.
and 10
ll of MTT (5 mg/ml) were added followed by 2 h incubation
at 37 °C. The precipitated formazan was dissolved in 100
l
l of
DMSO, and the absorbance was measured at 540 nm using a micro-
well system reader. The IC50 values (a concentration that produces
50% reduction in of the viable cell number) were calculated through
a Hill concentration–response curve. Cell viability was checked in
the beginning of the experiment by Trypan Blue exclusion. The
chalcones were dissolved in DMSO, and to verify if the solvent itself
could affect the cells, in all experiments, control curves without
chalcones and in the presence of the cells and the solvent were car-
ried out in parallel. The controls with solvent were not statistically
different from control cells alone.
4.9. Preparation of homogenates
Cell homogenates were prepared to complete the lipid peroxi-
dation, glutathione and enzyme assays. For enzyme assays,
6 ꢀ 106 cells were used, and for glutathione and lipid peroxidation
measurements, 3 ꢀ 106 cells and 4 ꢀ 106 cells were used, respec-
tively. These cells were incubated with the compounds at the
IC50 concentration for each compound for 24 h. The samples were
washed twice with PBS, lysed with 20 mM phosphate buffer at pH
7.4, 150 mM NaCl and 1% Triton X-100, sonicated for 20 seconds
and then centrifuged at 10,000 rpm for 10 min. The supernatants
(homogenates) of each sample were maintained at ꢁ20 °C.
4.6. Analysis of DNA fragmentation
The isolation of apoptotic DNA fragments was based on the
method of Han.44 Briefly, 3 ꢀ 106 L1210 cells were incubated with
the compounds for 24 h (at the IC50 concentration for each com-
pound). Cells were then washed with cold PBS and incubated with
4.10. Lipid peroxidation measurements
Changes in lipid peroxide levels were determined using
substances that react with thiobarbituric acid (TBARS), mainly

E. Winter et al. / Bioorg. Med. Chem. 18 (2010) 8026–8034
8033
malondialdehyde (MDA), producing a pink-colored Schiff base as
described previously.48 Briefly, the homogenate (400
g of protein)
was incubated under agitation in a buffer containing 60 mM
Tris–HCl (pH 7.4), 0.1 mM DPTA, 500 l 12% TCA, and 0.73% TBA.
The mixture was boiled for 2 h, cooled on ice and centrifuged at
10,000 rpm for 5 min. The absorbance of the supernatant was
measured at 535 nm. The results were calculated using the molar
extinction coefficient for malondialdehyde and expressed in nano-
moles of TBARS per microgram of protein. Protein content was
determined by Lowry’s method.46
4.14. ATP measurement
l
The intracellular ATP content was determined by a biolumines-
cence assay measuring the light output from the luciferin–
luciferase reaction. First, 1 ꢀ 106 L1210 cells were incubated with
l
the compounds (R7 23 lM, R13 37 lM, R15 36 lM) for 24 h. The
cell extracts were obtained by homogenization with 1.25% trichlo-
roacetic acid, then kept on ice for 30 min and neutralized with 1 M
Tris–acetate at pH 7.5. After centrifugation, the supernatants were
used for ATP quantification following the manufacturer’s protocol.
The results were normalized by the cell death percentage that
occurs during 24 h of incubation.
4.11. Mitochondrial potential measurement
To explore the effect of chalcones on mitochondrial membrane
potential, the lipophilic cationic probe fluorochrome 5,58,6,68-tet-
raethylbenzimidazolcarbocyanine iodide (JC-1) was used. JC-1 is a
green fluorescent monomer at depolarized membrane potential or
a red fluorescent J-aggregate at hyperpolarized membrane poten-
tial. Cells were plated at 5 ꢀ 105 cells/well in 24-well dishes and
4.15. Statistical analysis
The results were presented as means SD of triplicates from
three-independent experiments. Statistical significance was
assessed by ANOVA followed by Bonferroni’s test, and p <0.05
was taken as statistical significance.
incubated with the chalcones for 4 h. Afterward, JC-1 (10
was added and incubated for 20 min at 37 °C (5% CO2), then cells
were washed twice with PBS, resuspended in 500 l of PBS. One
lg/ml)
Acknowledgments
l
hundred microliters was extracted and used to measure the fluo-
rescence using a spectrofluorimeter (Perkin–Elmer LS55). JC-1
was excited at 488 nm, the red emission fluorescence was detected
at 590 nm and the green fluorescence was detected at 527 nm. The
mitochondrial potential was presented as a ratio of 590/527 fluo-
rescence and compared with the control cells that were considered
to have 100% fluorescence. An electron transport chain uncoupler
This study was supported by grants from CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico), CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)
and FAPESC (Fundação de Amparo à Pesquisa de Santa Catarina).
This paper forms part of the Chemistry doctoral studies of Louise
D. Chiaradia, who synthesize the compounds, and part of the Phar-
macy master studies of Evelyn Winter. The group wishes to thank
the Hospital Universitário (HU) for the use of its facilities and the
Central de Análises (Departamento de Química, UFSC) for chemical
analyses.
(FCCP 1 lM) was used as a positive control.
4.12. Enzyme assays
Glutathione peroxidase (GPx) was assayed according to Flohé
Supplementary data
and Gunzler49 using 150
l
g of protein and NADPH oxidation was
monitored by its decrease in absorbance at 340 nm (
= 6220 Mꢁ1
cmꢁ1). Catalase activity was determined according to Aebi50 using
60 g of protein. In this assay, the disappearance of H2O2 was eval-
uated by measuring the decrease in absorbance at 240 nm (molar
e
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.025.
l
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