Induction of apoptosis and cell cycle arrest in L-1210 murine lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-(3′-methoxy-4′-hydroxy- phenyl)-2-propen-1-one

Article abstract of DOI:10.1111/j.2042-7158.2010.01141.x

Objectives New compounds with biological targets and less cytotoxicity to normal cells are necessary for cancer therapy. In this work ten synthetic chalcones derived from 2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblas

Full text of DOI:10.1111/j.2042-7158.2010.01141.x

Research Paper
JPP 2010, 62: 1128–1136
© 2010 The Authors
Journal compilation © 2010
Royal Pharmaceutical Society
of Great Britain
Received December 09, 2009
Accepted May 04, 2010
DOI
Induction of apoptosis and cell cycle arrest in L-1210 murine
lymphoblastic leukaemia cells by (2E)-3-(2-naphthyl)-1-
(3Ј-methoxy-4Ј-hydroxy-phenyl)-2-propen-1-onejphp_1141 1128..1136
10.1111/j.2042-7158.2010.01141.x
ISSN 0022-3573
Fernanda Spezia Pedrinia, Louise Domeneghini Chiaradiab,
Marley Aparecida Licínioa, Ana Carolina Rabello de Moraesa,
Juliana Costa Curtaa, Aline Costaa, Alessandra Mascarellob,
Tânia Beatriz Creczinsky-Pasac, Ricardo José Nunesb,
Rosendo Augusto Yunesb and Maria Cláudia Santos-Silvaa
aDepartamento de Análises Clínicas, Universidade Federal de Santa Catarina, Campus Trindade,
bDepartamento de Química and cDepartamento de Ciências Farmacêuticas, Universidade Federal de
Santa Catarina, Campus Trindade, Florianópolis, Brasil
Abstract
Objectives New compounds with biological targets and less cytotoxicity to normal cells
are necessary for cancer therapy. In this work ten synthetic chalcones derived from
2-naphtaldehyde were evaluated for their cytotoxic effect in murine acute lymphoblastic
leukemia cells L-1210.
Methods A series of ten chalcones derived from 2-naphtaldehyde and corresponding
acetophenones were prepared by aldolic condensation, using methanol as solvent under
basic conditions, at room temperature for 24 h. The cell viability was determined by MTT
colorimeter method. The cell cycle phase analysis was carried out by flow cytometry after
propidium iodide staining. The apoptosis induction was assessed by exposure to phosphati-
dylserine (ANNEXIN V-FITC). Cytometric analysis was performed to evaluate the exp-
ression of p53, Bcl-2 and Bax protein. The caspase-3 expression was studied by
immunoblotting analysis.
Key findings A preliminary screening of a series of ten chalcones derived from
2-naphtaldehyde showed that chalcone 8, (2E)-3-(2-naphtyl)-1-(3′-methoxy-4′-hydroxy-
phenyl)-2-propen-1-one, had the highest cytotoxic effect (IC50 of 54 mm), but not in normal
human lymphocytes. To better understand the cytotoxic mechanism of chalcone 8, its effect
on cell cycle and apoptosis was assessed. Our results showed that chalcone 8 caused cell
cycle arrest in the G2/M phase and a significant increase in the proportion of cells in the
subG0/G1 phase. Our results also demonstrated that chalcone 8 promoted a modification in
Bax : Bcl-2 ratio and increased p53 expression and caspase-3 activation.
Conclusions The studied chalcone 8 has cytotoxic effect against L-1210 lymphoblastic
leukaemic cells, and this effect is associated with increase of p-53 and Bax expression.
Keywords apoptosis; chalcone; leukaemia
Introduction
Chalcones are precursors of flavonoids in higher plants and display a wide variety of
pharmacological effects. Many studies have demonstrated that chalcones and chalcone
derivatives present cytotoxic activity against cancer cells and may have potential applica-
tions in cancer treatment .[1–5] The suggested mechanism of this anti-tumour effect is that the
chalcones interfere with the microtubule polymerization.[6]
Several reports have demonstrated that apoptotic processes play a key role in the control
of haematopoietic tissue. Studies carried out over the past twenty years have dramatically
improved our understanding of the role of apoptotic mechanisms in the control of differen-
tiation, proliferation and survival of normal and leukaemic cells.[7]
Correspondence: Dr Maria
Cláudia Santos-Silva,
Universidade Federal de Santa
Catarina, Centro de Ciências da
Saúde, Departamento de
Análises Clínicas, Campus
Trindade CEP: 88040-900,
Florianópolis – SC, Brasil.
E-mail: maclau@ccs.ufsc.br
Targeted therapies that are designed to induce apoptosis in leukaemic cells are currently the
most promising anti-leukaemia strategies. These strategies are aimed at targeting and killing
the leukaemia cells specifically with no, or limited, collateral damage to normal haematopoi-
1128

Cytotoxicity of synthetic chalcones in leukaemia cells
Fernanda Spezia Pedrini et al.
1129
etic progenitor cells. The elucidation of the molecular apop- solution, and the absorbance was determined at 540 nm
totic machinery and its defects in acute leukaemia forms the using a microplate reader. All assays were performed in
basis for developing new drugs able to trigger apoptosis of triplicate.
leukaemia cells.[8]
Normal lymphocytes were collected from healthy donors
Scientific and technological advances in recent decades and human peripheral blood mononuclear cells were isolated
have made it possible to understand the pathways behind by Ficoll/Hypaque gradient. The normal lymphocytes were
apoptosis in tumour cells. This knowledge permits the devel- cultured under the same conditions described for leukaemia
opment of new drugs that are synthesized from plant-derived cell lines.
active principles and their semi-synthetic and synthetic ana-
Cell cycle analysis
logues that are able to interfere with these pathways and
potentially act as antineoplasic agents.[9]
To assess cell cycle arrest, PI/RNase solution kit (Immunostep,
Salamanca, Spain) was used. Cells (5 ¥ 105 cells/well) were
incubated with vehicle or chalcone 8 at 100 mm. After 24 h of
incubation, cells were harvested and cell cycle analysis was
assessed according to the kit protocol. Briefly, cells were
harvested, washed with phosphate-buffered saline (PBS) and
centrifuged for 5 min at 300 g. The supernatant was removed
and the cells were fixed with 70% ethanol for 30 min at 4°C.
Cells were washed once with PBS supplemented with 2%
bovine albumin, centrifuged for 5 min at 300 g and the super-
natant was removed. To the cell pellet was added 500 ml
PI/RNase solution and the cells were incubated for 15 min at
room temperature. After incubation the analysis was per-
formed by flow cytometry (FACSCalibur, Becton Dickinson
Immunocytometry Systems). The data were analysed by
WinMID software.
In a previous study we showed that synthetic hydroxychal-
cones induce apoptosis in B16-F10 melanoma cells via ATP
and GSH depletion.[10] Although some mechanisms have been
suggested, the exact pathways involved in the cytotoxic
effects of chalcones are not completely elucidated in the case
of leukaemic cells.
In this study, we assessed the cytotoxic effect of ten syn-
thetic chalcones in the murine acute lymphoblastic leukaemia
cell line (L-1210). In addition, we have investigated the cyto-
toxic mechanism of chalcone 8, (2E)-3-(2-naphthyl)-1-(3′-
methoxy-4′-hydroxy-phenyl)-2-propen-1-one.
Materials and Methods
Synthesis
Reagents used were obtained commercially from Sigma-
Aldrich and solvents from Vetec. A series of ten chalcones
derived from 2-naphtaldehyde and corresponding acetophe-
nones were prepared by aldolic condensation, using methanol
as solvent under basic conditions, at room temperature for
24 h. The chalcone 8, (2E)-3-(2-naphtyl)-1-(3′-methoxy-
4′-hydroxy-phenyl)-2-propen-1-one, was synthesized by con-
densation of 2-naphtadehyde and 4-hydoxy-3-methoxy-
acetophenone, under the same conditions. All structures,
including chalcone 8, were identified by melting point, infra-
red spectroscopy (IR), 1H and 13C nuclear magnetic resonance
spectroscopy (NMR) and elementary analysis as previously
described by us.[10,11]
Analysis of the apoptotic effects
For determination of apoptotic death an Annexin V-FITC
Apoptosis Detection kit was used according to the manufac-
turer’s instructions. Briefly, 5 ¥ 105 cells/well were incubated
with vehicle or chalcone 8 at 100 mm. After 6 h of incubation,
cells were harvested, washed with PBS buffer, annexin buffer
(1 : 10) and double-stained with Annexin V-FITC solution
and PI/RNase solution. After incubation, 300 ml of annexin
buffer was added and fluorescence was analysed by flow
cytometry. Analysis was performed by flow cytometry (FAC-
SCalibur, Becton Dickinson Immunocytometry Systems). The
data were analysed by WinMID software.
Effect of chalcone on the expression of p53,
Bcl-2 and Bax
Cell culture and viability assay (MTT assay)
Murine L-1210 acute lymphoblastic leukaemia cells (ATCC,
Manassas, USA) were maintained in Dulbecco’s modified
Eagle’s medium (DMEM; GIBCO, São Paulo, Brazil) supple-
mented with 10% fetal calf serum, 100 U/ml penicillin,
100 mg/ml streptomycin and 10 mm HEPES, pH 7.4 at 37°C
in a 5% CO2 humidified atmosphere in plastic culture flasks.
Every two or three days, cells were passaged by removing
90% of supernatant and replacing it with fresh medium.
Cell viability was assessed using MTT (3-(4,5-
dimethiazol-zyl)-2-5-diphenyltetrazolium bromide; Sigma
Chemical Co., St Louis, USA) assay.[12] Chalcones were
added to murine L-1210 cells in different concentrations (10–
100 mm) and incubated for 12, 24 or 48 h. For compounds
soluble in DMSO, the same volume of the solvent was added
to control wells. After incubation, MTT was added to each
For the analysis of Bcl-2, Bax and p53 proteins, 5 ¥ 105 cells
were incubated with vehicle or chalcone 8 at 100 mm for
24 h. At the end of the incubation, cells were washed with
PBS and permeabialized with ethanol 70%. Subsequently,
the cells were incubated in the dark at room temperature,
with anti-p53-PE, anti-Bcl-2-FITC or anti-Bax-FITC, for the
detection of p53, Bcl-2 and Bax, respectively. After incu-
bation, the cells were washed with PBS/albumin 2% and
re-suspended in PBS for cytometric analysis. Analysis was
performed by flow cytometry (FACSCalibur, Becton Dickin-
son Immunocytometry Systems). The data were analysed by
WinMID software.
Immunoblotting analysis of caspase-3
well followed by 3 h incubation. The cells were centrifuged L-1210 cell lines were incubated in the presence of the chal-
and the supernatant was discarded. The formazan precipitated cone 8 (100 mm) for 6, 9 and 12 h. Cells were harvested and
was dissolved with 100 ml of an isopropyl alcohol–HCl 0.04 n lysed in the presence of protease and phosphatase inhibitors.

1130
Journal of Pharmacy and Pharmacology 2010; 62: 1128–1136
The protein content of cell extracts was determined by Brad- by condensation of 2-naphtadehyde and 4-hydoxy-3-methoxy-
ford’s method.[13] Cell lysates were denatured in Laemmli’s acetophenone, and purified with a yield of 39%.
sample buffer (50 mm Tris-HCl, pH 6.8, 1% SDS, 5%
2-mercaptoethanol, 10% glycerol, 0.001% bromophenol
blue) and heated in a boiling water bath for 5 min. Samples
(50 mg total protein) were resolved by 12% SDS-PAGE[14]
and proteins were transferred to nitrocellulose membranes
(Amersham Pharmacia Biotech, San Francisco, USA).
Membranes were blocked with Tween-TBS (20 mm Tris-
HCl, pH 7.5, 500 mm NaCl, 0.1% Tween-20) containing 1%
bovine serum albumin and probed with the specific pri-
mary antibody: rabbit anti-caspase-3 (1 : 1000; Cell Signal-
ing Technology, Danvers, USA). After extensive washing
in Tween-TBS, nitrocellulose sheets were incubated with
HPN-conjugated anti-rabbit IgG antibody (1 : 1000; Santa
Cruz Biotechnology, Santa Cruz, USA) for 1 h. The blots
were visualized by chemiluminescence using enhanced
chemifluorescence (ECF) substrate (Amersham Pharmacia
Biotech, Amersham, UK).
Cytotoxic effect
Initially, the cytotoxicity of different concentrations of
naphthylchalcones toward murine L-1210 lymphoblastic
leukaemia cells was determined through cell viability using
the MTT assay.[12] After incubation of L-1210 cells with
increasing concentrations (10–100 mm) of the chalcones for
24 h, only 1, 8 and 10 reduced the percentage of viable cells
in a concentration-dependent manner, when compared with
the control (non-treated cells). A high level of cytotoxicity
was observed at a concentration of 100 mm, and the per-
centage of viable cells was 62 Ϯ 4.2%, 39% Ϯ 2.0%
and 54 Ϯ 4.0%, respectively, for chalcones 1, 8 and 10
(Figure 2). No expressive effect was observed for any of the
other chalcones. Chalcone 8 was chosen for further study
because it caused significant cell death at all concentrations
tested (Figure 2c).
For treatment of L-1210 cells with 100 mm of compound 8
for 0, 12, 24 and 48 h, the greatest cell death was observed
after 48 h (Figure 3), showing that the chalcone cytotoxic
effect occurred in a time- and concentration-dependent
manner with an IC50 of 54 mm. Interestingly, the same con-
centrations that were cytotoxic to leukaemia cells (L-1210)
were ineffective against normal human lymphocytes (results
not shown). Chalcones 1 and 10 showed IC50 values higher
than 100 mm (results not shown).
Statistical analysis
Statistical significance was assessed by the Kruskal–Wallis
test followed by post-hoc analysis; P < 0.05 was considered
statistically significant. Cell cycle data were analysed by
Mann–Whitney U-test (P < 0.05).
Results
Synthesis
Effect on cell cycle
Ten naphthylchalcones were prepared by aldolic conden- The effect of chalcone 8 (100 mm) on the cell cycle distribu-
sation between the 2-naphthaldehyde and the corresponding tion of L-1210 murine cells at 24 h was studied by DNA
acetophenones (Figure 1). All structures have been previously content analysis through flow cytometry (Figures 4a and 4b).
published by us.[10,11] The chalcone 8, (2E)-3-(2-naphtyl)-1-(3′- The data listed in Figure 4c were obtained by separation of
methoxy-4′-hydroxy-phenyl)-2-propen-1-one, was obtained cell cycle phases by setting adjacent cursors without decon-
O
O
O
2´
A
2
B
9
4
7
1´
1
3
8
+
5
6
3´
4´
CH3
H
a
R
R
6´
10
5´
1 R: 2´-OH-4´,6´-diOCH3
2 R: 4´--Br
3 R: H
4 R: 2´-OH
5 R: 4´-OCH3
6 R: 4´-NO2
7 R: 3´-NO2
8 R: 3´-OCH3-4´-OH
9 R: 3´,4´-diOCH3
10 R: 2´,5´-diOCH3
Figure 1 Synthesis of naphthylchalcone. Reagents and conditions: a, 50% KOH v/v and methanol, magnestic agitation at room temperature for 24 h.

Cytotoxicity of synthetic chalcones in leukaemia cells
Fernanda Spezia Pedrini et al.
1131
(a)
(c)
125
125
100
75
50
25
0
100
∗
∗
∗
∗
75
50
25
0
∗
∗
∗
∗
∗
Compound 8
Compound 9
Compound 10
Compound 1
Compound 2
Compound 3
0
10
25
50
100
0
10
25
50
100
Chalcones concn (mM)
Chalcones concn (mM)
(b)
125
100
75
50
25
0
Compound 4
Compound 5
Compound 6
Compound 7
0
10
25
50
100
Chalcones concn (mM)
Figure 2 Toxicity of naphthylchalcones towards murine L01210 lymphoblastic leukaemia cells. The chalcones (100 mm) were incubated with the
cells (1 ¥ 105) for 24 h. The cell viability was monitored through the MTT assay. Optical density of control groups (c) was taken as 100% of cell
viability. The cell viability was checked at the beginning of the experiment by Trypan Blue exclusion. The resultsare the mean Ϯ SEM of at least
three independent experiments. *P < 0.05 compared with control group (Kruskal–Wallis followed by post-hoc analysis).
volution of overlapping G0/G1, S and G2/M phases. In com-
parison with the control group (without treatment), treated
cells showed a significant increase in the proportion of cells in
the subG0/G1 (45.59%) and G2/M (38.15%) phases, accom-
panied by a significant decrease in the G0/G1 (42.05%) and S
(19.80%) phases (Figure 4c).
125
100
75
50
25
0
∗
Apoptotic effect
∗
To evaluate whether compound 8 induced cell death via an
apoptosis or necrosis pathway, the cells were treated with the
chalcone (100 mm) for 6 h and then marked with Annexin
V-FITC and propidium iodide (PI) and analysed by flow
cytometry. This double-staining method allowed live non-
apoptotic cells (Annexin Vnegative/PInegative) to be distinguished
from early apoptotic cells (Annexin Vpositive/PInegative) and late
apoptotic cells (Annexin Vpositive/PIpositive).[15,16] As illustrated in
Figure 5, chalcone 8 induced cell death via an apoptosis
pathway, bringing about a significant increase in the percent-
age of annexin Vpositive cells (23.5%) when compared with the
control group (7.0%).
∗
0
12
24
48
Time (h)
Figure 3 Cytotoxic effect of chalcone 8 on murine acute lymphoid
leukaemia cells (L-1210). The cytotoxic effect of chalcone (100 mm) on
L-1210 cells was determined. The cells were incubated for 0, 12, 24 and
48 h. The cell viability was assessed by the MTT colorimetric method.
The optical density of the control group corresponds to 100% of cell
viability. The results are the mean Ϯ SEM of at least three independent
experiments. *P < 0.05 compared with control (Kruskal–Wallis
(P = 0.0156) followed by post-hoc analysis).
Effect on apoptotic signalling pathway
To investigate the mechanisms whereby compound 8 induces
apoptosis, the levels of Bcl-2, Bax, p53 and activated caspase

1132
Journal of Pharmacy and Pharmacology 2010; 62: 1128–1136
(a)
(b)
M3
M3
M2
M2
M1
M1
FL-2 Area
DNA Contents
(c)
G0/G1
S
G2/M
Apoptosis (Sub G0/G1)
Control (% SEM)
54.23
24.85
0,59
20.91
0,40
8.21
0,94
0,15
42.05*
1,40
Chalcone 8 (100mM) (% SEM)
19.80*
0,20
38.15*
0,85
45.59*
2,00
Figure 4 Effect of chalcone 8 on the cell cycle of murine acute lymphoid leukaemia cells (L-1210). L-1210 cells were incubated in the presence
or absence of 100 mm chalcone and the percentage of cells in each phase of the cell cycle was examined through DNA content. (a, b) The phases
of the cell cycle are well represented: cells in apoptosis (Sub-G0/G1), cells in the G0/G1 phase (M1), cells in the S phase (M2) and cells at G2/M
(M3). (a) Cells treated for 24 h in the absence of chalcone. (b) Cells treated for 24 h in the presence of chalcone. This figure is representative
of three independent experiments. (c) Mean (% Ϯ SEM) in each phase of the cell cycle. *P < 0.05 compared with control (Mann–Whitney
U-test).
3 were assessed. For this study, the cells were pretreated with
In general, chalcones that have hydroxyl substituents are
chalcone (100 mm) and analyzed after 24 h for Bcl-2, Bax and more potent antioxidants and have a stronger cytotoxic effect
p53 and after 6, 9 and 12 h for caspase 3. Incubation with the on tumour cells.[17] Studies have shown that phloretin, a tet-
compound significantly increased the expression of Bax, p53 rahydroxychalcone, induced cell death through apoptosis in
and cleaved caspase 3 (Figure 6b and 6c, Figure 7).
HL-60 human leukaemic cells[20] and isoliquiritigenin induced
apoptosis and blocked the cell cycle progression in human
hepatoma cells (Hep G2).[21] In our study, the chalcones that
had a hydroxyl group at the A-ring demonstrated a cytotoxic
Discussion
In the last few decades many pharmacological properties have effect on L-1210 cells (except compound 4).
been attributed to chalcones and their derivatives, due to their
Chalcones with a 2-naphthyl group as the B-ring have also
antioxidant,[17] anti-angiogenic,[18] anti-metastatic,[19] antipro- been identified as potent anti-angiogenic and anti-tumour com-
liferative and cytotoxic activity.[27] Although many studies pounds in an endothelial SRV cell line.[22] Our results for the
have demonstrated an important in-vitro cytotoxic effect of 2-naphthaldehyde derivative, chalcone 8 ((2E)-3-(2-naphthyl)-
chalcones on tumour cells,[1–5] the exact mechanisms by which 1-(3′-methoxy-4′-hydroxy-phenyl)-2-propen-1-one), are in
these compounds exert their cytotoxicity remain unclear.
agreement with this since it showed a strong cytotoxic effect in
In this study, the cytotoxic activity of 10 naphthylchal- a concentration- and time-dependent manner on leukaemic
cones toward murine L-1210 lymphoblastic leukaemia cells L-1210 cells (IC50 54 mm), as shown in Figure 3a and 3b.
was evaluated. Compounds 1, 8 and 10 significantly reduced Besides, chalcone 8 did not have a cytotoxic effect in normal
the viable cells at a concentration of 100 mm (Figure 2).
human lymphocytes (results not shown).

Cytotoxicity of synthetic chalcones in leukaemia cells
Fernanda Spezia Pedrini et al.
1133
(a)
(b)
0.8%
13.9%
5.8%
4.7%
9.6%
87.0%
75.6%
2.3%
100
101
102
103
104
100
101
102
103
104
Annexin V
Annexin V
Figure 5 Induction of apoptosis by chalcone 8 on murine acute lymphoid leukaemia cells (L-1210). (a) L-1210 cells in DMEM medium without
treatment. (b) L-1210 cells were incubated with chalcone (100 mm) for 6 h. Apoptosis was dermined by analysis of tagging cells with Annexin
V-FITC/PI by flow cytometry. The results are the means Ϯ SEM of at least three independent experiments. This figure is representative of three
independent experiments. Lower left quadrant: Annexin Vnegative/PInegative. Lower right quadrant: Annexin Vpositive/PInegative. Upper left quadrant: Annexin
Vnegative/PIpositive. Upper right quadrant: Annexin Vpositive/PIpositive
.
To gain a better insight into the mechanism of cytotoxicity uted to different mechanisms, such as tubulin polymerization
induced by compound 8, its effects on cell proliferation and inhibition,[26] p21 and p27 induction,[25,26] inhibition of cyclin
apoptosis were evaluated.
B1, cyclin A, Cdc1[26] or cyclin D1, cyclin E and cdks 2, 4 and
Disorders in the cell cycle and regulatory proteins are 6 expression.[24]
frequently related to the development of neoplasias. Thus,
The current understanding of the apoptosis signal transduc-
knowledge of how chalcones affect these mechanisms is tion of chalcones and their derivatives is limited and perhaps
important for the development of new antineoplasic drugs. each compound acts in a different way. While some authors
Nakatani et al. have demonstrated that C-benzylated dihydro- have demonstrated that these compounds induce an increase in
chalcones cause cell cycle arrest in the G1 phase, the activation pro-apoptotic proteins, such as Bax and Bak, and a decrease in
of caspase 3 and DNA fragmentation in HL-60 promyelocytic anti-apoptotic proteins, such as Bcl-2 and Bcl-xL,[24,25] others
leukaemia cells.[23] Other studies have shown that chalcone have concluded that only the Bcl-2 protein expression is
derivatives induce cell cycle blockage in the G2/M phase in reduced.[34] These findings suggest that the cytotoxic effect of
different tumour cell types.[24–26] In the same way, the com- chalcones is related to their structure and the cell type studied.
pound 8 tested here caused cell cycle blockage in the G2/M For a better comprehension of which mechanism induces
phase.
apoptosis, some molecular events involved in the apoptosis
Cell cycle arrest activates many DNA repair mecha- pathways were investigated. Since the p53 protein is mainly
nisms[27,28] and if the DNA repair is impossible, the pro- responsible for the first cell cycle checkpoint in the G1/S phase,
apoptotic protein p53 induces the cell to apoptosis.[29–32] The the study began with this protein. When compared with the
cells are normally able to repair genomic damage although control group, chalcone 8 clearly caused an increase in p53
this is dependent on the extent of such damage. Sometimes, expression (Figure 6).
the DNA damage causes ATP depletion or circumvents the
The decrease in the expression of pro-apoptotic
activation of the caspase cascade, impairing the cell death by members of Bcl-2 family, or overexpression of anti-
apoptosis and, consequently, the cell progresses to necro- apoptotic members of Bcl-2 family, is associated with
sis.[33] The process of necrosis is related to the cytoplasmic enhanced oncogeneic potential and poor response rates to
content, extravasation and local inflammatory reaction. For chemotherapy.[35–37] The members of the Bcl-2 family regu-
this reason, there is great interest in new drugs that can induce late the initiation of the mitochondrial apoptotic pathway.
tumour cells to apoptosis. As can be observed in Figures 4 These proteins regulate cytochrome C release to the cyto-
and 5, chalcone 8 induces apoptosis of L-1210 cells, which plasm, which causes activation of caspase 9 and, conse-
can be confirmed by the sub G0/G1 phase (45.59%) of the quently, the activation of downstream effector caspases.[38–41]
cell cycle. The interruption of the cell cycle and the apoptosis In our study, Bcl-2 expression did not alter significantly, but
caused by chalcones and their derivatives have been attrib- an increase in Bax protein expression was observed in the

1134
Journal of Pharmacy and Pharmacology 2010; 62: 1128–1136
(a)
(b)
100
101
102
103
104
100
101
102
103
104
anti-Bcl-2-FITC
anti-Bcl-2-FITC
(c)
Autoflourescence
Cells without treatment
Cells incubated with chalcone (100 mM)
100
101
102
103
104
Figure 6 Effect of chalcone 8 on apoptotic signalling pathyway in murine acute lymphoid leukaemia cells (L-1210). L-1210 cells were incubated
in the presence or absence of chalcone 100 mm and marked with anti-Bcl-2-FITC, anti-Bax-FITC or anti-p53-PE for the detection of Bcl-2, Bax and
p53, respectively. (a) Expression of Bcl-2 anti-apoptotic protein. (b) Expression of Bax pro-apoptotic protein. (c) Expression of p53. This figure is
representative of three independent experiments.
L-1210 cells treated with compound 8 (Figure 6c), suggest-
ing that it may have been mediated by p53. This change in
the Bax : Bcl-2 ratio facilitates cell death by apoptosis.[42]
Our findings corroborate this statement, since the tested
chalcone 8 caused an increase in p53 and Bax expression
(Figure 6b and 6c). Furthermore, it is known that the dis-
equilibrium between pro- and anti-apoptotic proteins leads
to cytochrome C release and, consequently, caspase 3 acti-
vation (Figure 7).
6h
9h
12h
C
Ch
C
Ch
C
Ch
Caspase-3
Cleaved Caspase-3
Figure 7 Effect of chalcone 8 on caspase-3 protein expression and its
activity in murine acute lymphoid leukaemia cells (L-1210). L-1210 cells
were incubated in the presence of absence of chalcone (100 mm) for 6, 9
and 12 h. Cells were immunolabelled with monoclonal rabbit anti-
caspase-3, anti-rabbit IgG antibody, HPN-conjugated, and visualised by
chemiluminescence using an enhanced chemifluorescence (ECF) sub-
strate. This figure is representative of three independent experiments. C,
control; Ch, chalcone.
Conclusion
Together, these results show that chalcone 8, (2E)-
3-(2-naphthyl)-1-(3′-methoxy-4′-hydroxy-phenyl)-2-propen-
1-one, induces apoptosis in L-1210 cell line by cell cycle arrest
in G2/M phase. In addition, it also increases Bax and p53
expression and caspase 3 activation.

Cytotoxicity of synthetic chalcones in leukaemia cells
Fernanda Spezia Pedrini et al.
1135
HL-60 and adherent MCF-7 cells. Cell Prolif 1999; 32:
25–37.
Declarations
17. Anto RJ et al. Anticancer and antioxidant activity of synthetic
chalcones and related compounds. Cancer Lett 1995; 97:
33–37.
18. Nam NH et al. Cytotoxic 2′,5′-dihydroxychalcones with unex-
pected antiangiogenic activity. Eur J Med Chem 2003; 38:
179–187.
19. Parmar VS et al. Synthesis, characterization and in vitro anti-
invasive activity screening of polyphenolic and heterocyclic
compounds. Bioorg Med Chem 2003; 11: 913–929.
20. Kobori M et al. Phloretin-induced apoptosis in B16 melanoma
4A5 cells and HL60 human leukemia cells. Biosci Biotechnol
Biochem 1999; 63: 719–725.
21. Hsu YL et al. Isoliquiritigenin induces apoptosis and cell cycle
arrest through p53-dependent pathway in Hep G2 cells. Life Sci
2005; 77: 279–292.
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest
to disclose.
Funding
This work was supported by grants and fellowships from
CNPq and CAPES, Brasil.
Acknowledgements
This study forms part of the theses of F.S. Pedrini, master’s
student in Pharmacy.
22. Bowen PJ et al. Chalcone and its analogs as agents for the
inhibition of angiogenesis and related disease states. WO
2001046110 2001.
23. Nakatani, N et al. Induction of apoptosis in human promyelo-
cytic leukemia cell line HL-60 by C-benzylated dihydrochal-
cones, uvaretin, isouvaretin and diuvaretin. Biol Pharm Bull
2005; 28: 83–86.
24. Yun JM et al. Induction of apoptosis and cell cycle arrest by a
chalcone panduratin A isolated from Kaempferia pandurata in
androgen-independent human prostate cancer cells PC3 and
DU145. Carcinogenesis 2006; 27: 1454–1464.
25. Shen KH et al. Chalcone arrests cell cycle progression and
induces apoptosis through induction of mitochondrial pathway
and inhibition of nuclear factor kappa B signalling in human
bladder cancer cells. Basic Clin Pharmacol Toxicol 2007; 101:
254–261.
References
1. Yamamoto S et al. The potent anti-tumor-promoting agent isoli-
quiritigenin. Carcinogenesis 1991; 12: 317–323.
2. Satomi Y. Inhibitory effects of 3′-methyl-3-hydroxy-chalcone on
proliferation of human malignant tumor cells and on skin car-
cinogenesis. Int J Cancer 1993; 55: 506–514.
3. Shibata S. Anti-tumorigenic chalcones. Stem Cells 1994; 12:
44–52.
4. De Vincenzo R et al. Effect of synthetic and naturally occurring
chalcones on ovarian cancer cell growth: structure-activity rela-
tionships. Anticancer Drug Des 1995; 10: 481–490.
5. Makita H et al. Chemoprevention of 4-nitroquinoline 1-oxide-
induced rat oral carcinogenesis by the dietary flavonoids chal-
cone, 2-hydroxychalcone, and quercetin. Cancer Res 1996; 56:
4904–4909.
26. Romagnoli R et al. Design, synthesis, and biological evaluation
of thiophene analogues of chalcones. Bioorg Med Chem 2008;
16: 5367–5376.
27. Lodish H et al. Molecular Cell Biology, 4th edn. New York:
W.H. Freeman, 2000.
6. Alias Y et al. An antimitotic and cytotoxic chalcone from
Fissistigma lanuginosum.
1166.
7. Testa U, Riccioni R. Deregulation of apoptosis in acute myeloid
leukemia. Haematologica 2007; 92: 81–94.
8. Zhivotovsky B, Orrenius S. Defects in the apoptotic machinery
of cancer cells: role in drug resistance. Semin Cancer Biol 2003;
13: 125–134.
9. Silva JS et al. Natural product inhibitors of ovarian neoplasia.
Phytomedicine 2003; 10: 221–232.
10. Navarini AL et al. Hydroxychalcones induce apoptosis in B16-
F10 melanoma cells via GSH and ATP depletion. Eur J Med
Chem 2009; 44: 1630–1637.
11. Chiaradia LD et al. Synthetic chalcones as efficient inhibitors of
Mycobacterium tuberculosis protein tyrosine phosphatase PtpA.
Bioorg Med Chem Lett 2008; 18: 6227–6230.
12. Van De Loosdrecht AA et al. Cell mediated cytotoxicity against
U 937 cells by human monocytes and macrophages in a modified
colorimetric MTT assay. A methodological study. J Immunol
Methods 1991; 141: 15–22.
13. Bradford MM. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 1976; 72: 248–254.
14. Laemmli UK. Cleavage of structural proteins during the assem-
bly of the head of bacteriophage T4. Nature 1970; 227: 680–
685.
J Nat Prod 1995; 58: 1160–
28. Weinberg R. Tumor suppressor genes. Neuron 1993; 11: 191–
196.
29. Kastan MB et al. Participation of p53 protein in the cellular
response to DNA damage. Cancer Res 1991; 51: 6304–6311.
30. Kuerbitz SJ et al. Wild-type p53 is a cell cycle checkpoint deter-
minant following irradiation. Proc Natl Acad Sci USA 1992; 89:
7491–7495.
31. Clarke AR et al. Thymocyte apoptosis induced by p53-
dependent and independent pathways. Nature 1993; 362: 849–
852.
32. Lowe SW et al. p53 is required for radiation-induced apoptosis
in mouse thymocytes. Nature 1993; 362: 847–849.
33. Leist M et al. Intracellular adenosine triphosphate (ATP) con-
centration: a switch in the decision between apoptosis and necro-
sis. J Exp Med 1997; 185: 1481–1486.
34. Ye CL et al. Induction of apoptosis in K562 human leukemia
cells by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone.
Leuk Res 2005; 29: 887–892.
35. Ferlini C et al. Bcl-2 down-regulation is a novel mechanism of
paclitaxel resistance. Mol Pharmacol 2003; 64: 51–58.
36. Kusenda J. Bcl-2 family proteins and leukemia: minireview.
Neoplasma 1998; 45: 117–122.
15. Vermes I et al. A novel assay for apoptosis. Flow cytometric
detection of phosphatidylserine expression on early apoptotic
cells using fluorescein labelled Annexin V. J Immunol Methods
1995; 184: 39–51.
37. Ruvolo PP et al. Phosphorylation of Bcl2 and regulation of
apoptosis. Leukemia 2001; 15: 515–522.
38. Antonsson B, Martinou JC. The Bcl-2 protein family. Exp Cell
Res 2000; 256: 50–57.
16. Del Bino G et al. Comparison of methods based on annexin-V
binding, DNA content or TUNEL for evaluating cell death in
Singh SV et al. Caspase-dependent apoptosis induction by
guggulsterone, a constituent of Ayurvedic medicinal plant
39.

1136
Journal of Pharmacy and Pharmacology 2010; 62: 1128–1136
Commiphora mukul, in PC-3 human prostate cancer cells is 41. Li P et al. Cytochrome c and dATP-dependent formation of
mediated by Bax and Bak. Mol Cancer Ther 2005; 4: 1747–
Apaf-1/caspase-9 complex initiates an apoptotic protease
1754.
cascade. Cell 1997; 91: 479–489.
40. Utz PJ, Anderson P. Life and death decisions: regulation of 42. Cory S, Adams JM. The Bcl2 family: regulators of the
apoptosis by proteolysis of signaling molecules. Cell Death
Differ 2000; 7: 589–602.
cellular life-or-death switch. Nat Rev Cancer 2002; 2: 647–
656.