Alkaloids extracted from Pterogyne nitens induce apoptosis in malignant breast cell line

Abstract

In the present study, two alkaloids isolated from Pterogyne nitens, a plant native to Brazil, have been shown to induce apoptosis in human breast cancer cells. These compounds, pterogynine (PGN) and pterogynidine (PGD), were tested for their effect on a human infiltrating ductal carcinoma cell line (ZR-7531). The cell line was treated with each alkaloid at several concentrations. Time-dependence (with or without recuperation time) and concentration-dependence (in the range 0.25-10 mM) were investigated in cytotoxicity and apoptosis assays. The annexin assay indicated an apparently higher percentage of death by necrosis of malignant cells after 24 h exposure to both P. nitens extracts than the Hoechst assay. Thus, our results in the two tests demonstrated that the Hoechst assay can discriminate between late apoptotic cells and necrosis, whereas the flow cytometry-based annexin V assay cannot. We concluded that PGN and PGD have effective antineoplastic activity against human breast cancer cells in vitro, by inducing programmed cell death.

Full text

RESEARCH ARTICLE
Alkaloids extracted from Pterogyne nitens induce apoptosis
in malignant breast cell line
Roberta Aparecida Duarte &Elaine Rodrigues Mello &Camila Araki &
Vanderlan da Silva Bolzani &Dulce Helena Siqueira e Silva &Luis Octavio Regasini &
Tarsia Giabardo Alves Silva &Mauro César Cafundó de Morais &
Valdecir Farias Ximenes &Christiane Pienna Soares
Received: 4 February 2010 /Accepted: 31 May 2010 / Published online: 11 August 2010
#International Society of Oncology and BioMarkers (ISOBM) 2010
Abstract In the present study, two alkaloids isolated from
Pterogyne nitens, a plant native to Brazil, have been shown
to induce apoptosis in human breast cancer cells. These
compounds, pterogynine (PGN) and pterogynidine (PGD),
were tested for their effect on a human infiltrating ductal
carcinoma cell line (ZR-7531). The cell line was treated
with each alkaloid at several concentrations. Time-
dependence (with or without recuperation time) and
concentration-dependence (in the range 0.25-10 mM) were
investigated in cytotoxicity and apoptosis assays. The
annexin assay indicated an apparently higher percentage
of death by necrosis of malignant cells after 24 h exposure
to both P. nitens extracts than the Hoechst assay. Thus, our
results in the two tests demonstrated that the Hoechst assay
can discriminate between late apoptotic cells and necrosis,
whereas the flow cytometry-based annexin V assay cannot.
We concluded that PGN and PGD have effective antineo-
plastic activity against human breast cancer cells in vitro,
by inducing programmed cell death.
Keywords Breast cancer cell line .Cytotoxic activity .
Alkaloids .Apoptosis .Necrosis .Flow cytometry
Introduction
Breast cancer is a major cause of morbidity and mortality
among women worldwide [1]. In North America and
Europe, approximately 11% of all women and 1% of
all men will develop breast cancer, many of these
patients (approximately 35%) eventually succumbing to
this disease. There are several therapeutic drugs that are
currently in use, including drugs that inhibit specific
hormone receptors, inhibit growth factor receptors, and
induce cell cycle arrest [2]. Research has elucidated
several specific prognostic and predictive factors to
identify patients at high risk of the aggressive disease,
metastasis and of recurrence of the disease, in order to
combat these statistics [3]. For this reason, there is an
obvious need to develop more efficacious treatment
strategies.
Brazil has the biggest biodiversity in the world. Plants,
since the ancient times, have been used to treat a large
amount of diseases including cancer [4–6]. Many com-
pounds with biological activities were obtained from
Cerrado, Brazil’s second largest biome [7–10].
Pterogyne nitens Tulasne (Fabaceae-Caesalpinioideae),
popularly known as “bálsamo”,“yvira-ró”,“cocal”, and
“amendoinzeiro”, is a native tree in South America [11,12].
It is used as an ornament due to its attractive flowers and
R. A. Duarte :E. R. Mello :C. Araki :T. G. A. Silva :
M. C. C. de Morais :C. P. Soares (*)
School of Pharmaceutical Sciences,
University of São Paulo State -UNESP,
Rua Expedicionários do Brasil, 1621,
Zip code 14801-902 Araraquara, Sao Paulo, Brazil
e-mail: soarescp@hotmail.com
e-mail: soarescp@fcfar.unesp.br
V. da Silva Bolzani :D. H. S. e Silva :L. O. Regasini
Institute of Chemistry of Araraquara,
University of São Paulo State - UNESP,
R. Prof. Francisco Degni, SN, Bairro Quitandinha,
Zip code 14801-970 Araraquara, Sao Paulo, Brazil
V. F. Ximenes
School of Sciences of Bauru,
University of São Paulo State -UNESP,
Av. Eng. Luiz Edmundo Carrijo Coube 14-01,
Zip code 17033-360 Bauru, Sao Paulo, Brazil
Tumor Biol. (2010) 31:513–522
DOI 10.1007/s13277-010-0064-2

fruits. Medicinal uses of this species have not been reported
frequently, although aqueous preparations from stem barks
have been used by Paraguayan communities in the therapy
of ascariasis [13]. Previous bioprospection studies have
demonstrated the presence of guanidine alkaloids, which
exhibited cytotoxic activity against human myeloblastic
leukemia and human glioblastoma cells, and phenolic
compounds with anti-inflammatory, antioxidant, antimuta-
genic, and antidiabetic activities [14–22].
In this study, we demonstrated that two alkaloids isolated
from P. n i t e n s , pterogynine (PGN) and pterogynidine
(PGD; Fig. 1), showed an interesting potential anti-tumor
activity in vitro by inducing programmed cell death. In
addition, we present the results obtained by methods
(Annexin–V, Hoechst and Caspase 3/7) used for the
quantitation of the apoptosis.
Materials and methods
Cytotoxicity assay
P. nitens leaves were collected at the Botanic Garden of Sao
Paulo, São Paulo State, Brazil, in May 2003. A voucher
specimen (SP204319) has been deposited in the herbarium
of the Botanic Institute (São Paulo State, Brazil).
Extraction, isolation, and identification of alkaloids
The shade-dried leaves (2.8 kg) were ground and defatted
with hexane (2.0 L×5, at room temperature, for 5 weeks)
and exhaustively extracted by maceration with ethanol
(4.0 L×5) at room temperature, for 5 weeks. The ethanol
extract was concentrated under reduced pressure (≤40°C),
to yield 12.7 of a syrup. The concentrate was then diluted
with methanol-water (4:1; 3.5 L) and partitioned succes-
sively with ethyl acetate (5.0 L×3) and n-butanol (5.0 L ×
3). After removal of the solvent, 3.7 and 5.9 g of extract
were afforded, respectively. The n-butanol residue (2.5 g)
was subjected to gel permeation chromatography on a
column of Sephadex LH-20 in methanol to afford nine
fractions, which were combined on the basis of their TLC
visualized with Sakaguchi’s and Dragendorff’s reagents
[23], to yield an alkaloidal fraction (587 mg). Separation of
this fraction on reversed-phase silica gel column chroma-
tography (RP-18) by elution with increasing amounts of
acetonitrile in water afforded eight fractions (ALK-1–ALK-
8). Fraction ALK-3 (121 mg) was subjected to repeated
column chromatography on silica gel (230-400 mesh),
eluted with chloroform-methanol mixtures (ranging from
0% to 35% of methanol), to furnish the guanidine alkaloids,
pterogynine (PNG, 27 mg) and pterogynidine (PDG,
22 mg). The molecular structures of these compounds were
identified by comparison with literature data [24,25],
mainly
1
H and
13
C NMR (Fig. 1).
PNG (yield 4.6%, calculated from the alkaloidal frac-
tion): yellow oil.
1
H NMR δ
H
(multiplicity;Jin Hz;
position): 7.44-7.64 (br s, H-1 and H-3), 3.72 (d; 6.5, H-1′),
5.16 (t, 6.5, H-2′), 1.62 (s, H-4′), 1.68 (s, H-5′).
13
CNMR
δ
C
(position): 155.7 (C-2), 39.0 (C-1′), 119.2 (C-2′), 135.8
(C-3′), 17.8 (C-4′), 25.2 (C-5′).
PDG (yield 3.8%, calculated from the alkaloidal frac-
tion): yellow oil.
1
H NMR δ
H
(multiplicity;Jin Hz;
position): 7.72 (t; 6.5; H-3 and H-4), 7.20 (br s; H-1), 3.70
(t; 6.5, H-1′), 5.16 (t, 6.5, H-2′), 1.63 (s, H-4′), 1.69 (s,
H-5′).
13
CNMRδ
C
(position): 156.9 (C-2), 38.7 (C-1′),
119.0 (C-2′), 136.0 (C-3′), 17.7 (C-4′), 25.1 (C-5′).
Cell culture
Human infiltrating ductal carcinoma cells (ZR-7531) were
obtained from American Type Culture Collection (ATCC,
USA). Cells were grown at 37°C in Dulbecco’s modified
eagle medium (DMEM) media and Ham’s F10 (Sigma, St.
Louis, MO, USA), supplemented with 10% fetal bovine
serum, 100 units/mL penicillin and 100 μg/mL streptomy-
cin, in an incubator containing 5% CO
2
.
Cell treatment
To explore the induction of apoptosis (Annexin-V, Hoechst
and Caspase 3/7 assays) and to determine the cytotoxicity
of alkaloids PGN and PGD, cells were grown to 70%
confluence and treated for 24 h with alkaloids (t0) or treated
for 24 h with alkaloids and then for another 24 h with fresh
medium (t24) to observe recuperation. Various concentra-
tions of the alkaloids, from 0.25 to 10 mM, diluted in the
culture media from a 40 mM stock solution in water, were
used in the apoptosis and cytotoxicity tests.
Cytotoxicity assay
The cytotoxicity of both alkaloids was determined by the
MTT colorimetric assay, which was performed to detect
tumor cell viability after incubation. The cells were cultured
N
H
N
N
HN
HH
N
N
2
341' 3'
5'
PNG PDG
Fig. 1 Molecular structures of guanidine alkaloids, pterogynine
(PNG) and pterogynidine (PDG), isolated from the leaves of
Pterogyne nitens
514 Tumor Biol. (2010) 31:513–522

in 96-well plates. At 70% confluence, cells were treated
with the alkaloids as previously described. Ten microliter of
MTT, a tetrazolium dye (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide; thiazolyl blue; Sigma, St.
Louis, MO, USA), was added to each well. Plates were
incubated in contact with the dye for 4 h. Mitochondrial
dehydrogenase activity reduced the yellow MTT dye to a
purple insoluble formazan, which was then solubilized with
acidified isopropanol and absorbance was read at 540 nm in
an ELISA plate reader (Bio-Tek Powerwave X, BioTek
Instruments, Inc.,USA). Doxorubicin (DOX) at 15 μg/mL
was used as a positive control. Cytotoxicity was calculated
as follows: % cytotoxicity=[(absorbance treated cells−
absorbance untreated cells)/absorbance untreated cells] ×
100, as proposed by Zhang et al. [26,27]. Concentration
that presents 50% of cytotoxicity (IC
50
) were calculated
from concentration response curve.
Annexin V assay
Annexin V/propidium iodide (PI) staining was used to
assess both apoptosis and necrosis. Untreated cells were
used as negative control and DOX, a well-established drug
used to induce apoptosis, as positive control. Controls and
treated cells were harvested by trypsinization, and washed
twice with phosphate-buffered saline (PBS). Cells were
resuspended in 500 μL of binding buffer containing 5 μL
of annexin V-FITC (Annexin V-FITC Apoptosis Detection
Kit, Alexis™, San Diego, USA) and 5 μL of PI and then
incubated for 10 min at room temperature. Fluorescence
and physical properties of the cells were captured by flow
cytometry, via an analog-digital converter, and processed
with integrated software (Becton Dickinson-Bioscience,
FACS Calibur™, San Jose, CA, USA).
Hoechst assay
Differential staining with specific fluorochromes can be
used to distinguish cells undergoing apoptosis from viable
and necrotic cells. The human cells (10
6
cells/mL) were
cultured in 12-well plates to 70% confluence and then
treated with the alkaloids as described previously and
trypsinized. Next, the cells were washed in PBS and
suspended in a solution of 1 mg/mL of PI, 1.5 mg/mL
fluorescein diacetate (DAF) and 1 mg/mL Hoechst 33342
(HO; Sigma, St. Louis, MO, USA) in PBS. The apoptosis
was classified by morphology and color of the cells, and
quantified. The cells were classified as viable (spherical
blue nucleus stained by HO, green cytoplasm stained by
DAF excited at 360 nm), apoptotic (blue nucleus with
apoptotic bodies stained by HO, green cytoplasm) or
necrotic (red enlarged nucleus with spherical vesicles
stained by PI, excited at 538 nm). Finally, the apoptotic
cells were classified as early (blue nucleus with apoptotic
DOX
0.375
1.125
3.375
10.125
0
20
40
60
80
100
a
*** **
DOX
0.375
1.125
3.375
10.125
0
20
40
60
80
100
b
*** ***
*
Cytotoxicity (%)Cytotoxicity (%)
Cytotoxicity (%)Cytotoxicity (%)
DOX
0.25
0.5
1
2
2
0
20
40
60
80
100
c
***
*** **
*
DOX
0.25
0.5
1
0
20
40
60
80
100
d
***
***
**
Concentration (mM)Concentration (mM)
Concentration (mM)Concentration (mM)
Fig. 2 Cytotoxicity by MTT
assay in ZR-7531 cells. Results
are expressed as the mean of
three independent experiments ±
standard error and subjected to
one-way ANOVA with Tukey’s
test (treatment versus DOX). a
Cells treated with PGN for t0;
bcells treated with PGN for t24;
ccells treated with PGD for t0;
dcells treated with PGD for t24.
DOX: doxorubicin at 15 μg/mL;
*p<0.05; **p< 0.01;
***p<0.001
Tumor Biol. (2010) 31:513–522 515

DOX
NC
0.375
1.125
3.375
10.125
Early apoptosis
Late apoptosis / necrosis
*** **
Concentration (mM )
DOX
NC
0.375
1.125
3.375
10.125
0
20
40
60
80
100
Late apoptosis / necrosis
Early apoptosis
*** ** ***
Concentration (mM)
Cell death (%)
0
20
40
60
80
100
Cell death (%)
0
20
40
60
80
100
Cell death (%)
0
20
40
60
80
100
Cell death (%)
DOX
NC
0.25
0.5
1
2
c
Late apoptosis / necrosis
Early apoptosis
***
Concentration (mM)
DOX
NC
0.25
0.5
1
2
d
Late apoptosis / necrosis
Early ap optos is
Concentration (mM)
Fig. 3 Apoptosis by Annexin-V
assay in ZR-7531 cells. Results
are expressed as the mean of
three independent experiments ±
standard error and subjected to
one-way ANOVA with Tukey’s
test (treatment versus DOX,
for early apoptosis). aCells
treated with PGN for t0; bcells
treated with PGN for t24; c
cells treated with PGD for t0; d
cells treated with PGD for t24.
DOX: doxorubicin at 15 μg/
mL; NC: untreated cells; *p<
0.05; **p<0.01; ***p<0.001
a b
N
LA
d
LA N
c
EA
EA
Fig. 4 Apoptosis assay by
Hoechst and propidium iodide.
aand bZR-7531 cells treated
with 1.0 mM of pterogynidine.
cand dZR-7531 cells treated
with 3.375 mM of pterogynine.
(EA) early apoptotic cells with
apoptotic nuclei stained by
Hoechst with fluorescence in the
blue spectrum (arrow); (LA) late
apoptosis; (N) necrosis stained
propidium iodide shown fluores-
cence in the red spectrum (arrow).
Fluorescence microscope micro-
graphs observed in absorbance of
360 and 538 nm with a ×400
516 Tumor Biol. (2010) 31:513–522

bodies) or late (nucleus colored red with apoptotic bodies),
as proposed by Korostoff, and Elstein and Zucker [28,29].
Caspase 3/7 assay
Caspase Glo 3/7 assay was performed according to
directions of the supplier (Promega, Madison, WI, USA).
ZR-7531 cells were seeded in white walled 96-well plate at
2.5×10
4
cells/well of DMEM and were allowed to attach
for overnight. The next day, the medium was changed and
cells were treated with alkaloids PGN and PGD. Cell-free
medium was used as a blank and DOX was used as positive
control. After treating the cells, medium was replaced with
100 μL 1:1 (v/v) of DMEM:Glo 3/7 reagent and was
incubated for 30 min at 37°C in 5% CO
2
. Luminescence
was measured by microplate reader (Berthold, USA).
Caspase 3/7 activity was presented as a mean of relative
light units (RLU). The following formula was used to
calculate caspase 3/7 activity in RLU: RLU = luminescence
(samples)-Luminescence (blank).
Statistical analyses
For statistical analysis of the cytotoxicity assay (MTT),
Annexin V, Hoechst and Caspase methods, data were first
tested for normality. This showed a normal distribution, so
a parametric test was applied. Differences were tested by
one-way analysis of variance, with Tukey’s post test. This
analysis was performed with GraphPad Prism® Version 5.1
software (GraphPad Software Inc., USA). Results are
expressed as mean of three independent experiments ±
standard deviation.
Results
Alkaloids cytotoxicity
Pterogynine and pterogynidine exhibited high cytotoxicity
for ZR-7531 cells. Concentration response was observed
for both compounds and both time of treatments (Fig. 2). At
DOX
NC
0.375
1.125
3.375
10.125
0
20
40
60
80
100
a
**
**
** ** ** **
DOX
NC
0.375
1.125
3.375
10.125
0
20
40
60
80
100
b
**
**
****
** **
DOX
NC
0,375
1,125
3,375
10,125
0
20
40
60
80
100
c
**
** ** **
**
DOX
NC
0,375
1,125
3,375
10,125
0
20
40
60
80
100
d
*
***
Total apoptosis
Necrosis
Concentration (mM)
Cell death (%)
Concentration (mM)
Concentration (mM) Concentration (mM)
Cell death (%)
Cell death (%)
Cell death (%)
Late apoptosis
Early apoptosis
Total apoptosis
Necrosis
Late apoptosis
Early apoptosis
Fig. 5 Apoptosis by Hoechst / PI assay in ZR-7531 cells. Results are
expressed as the mean of three independent experiments ± standard
error and subjected to one-way ANOVA with Tukey’s test (treatment
versus PC, for early apoptosis). aCells treated with PGD for t0,
relation between total apoptosis and necrosis; bcells treated with PGD
for t0, relation between early and late apoptosis; ccells treated with
PGD for t24, relation between total apoptosis and necrosis; dcells
treated with PGD for t24, relation between early and late apoptosis.
PC doxorubicin at 15 μg/mL, NC untreated cells; *p<0.05; **p<0.01;
***p<0.001
Tumor Biol. (2010) 31:513–522 517

maximum concentration (10.125 mM), when tested for
24 h, PGN caused 71.4±7.3% cell death and no statistical
significance between DOX in both times (Fig. 2a and b).
The cytotoxicity of PGD was higher at 2 mM, showing
57.0±4.3% and 72.0±4.1% cell death, for t0 and t24,
respectively (Fig. 2c and d). Statistical difference was
observed between DOX and 2 mM of PGD for t0, but not
for t24. IC
50
values of PGN and PGD were 3.3±0.2 mM
and 0.7±0.4 mM, respectively, for t24. This indicates that
PGD is more cytotoxic than PGN for this time of treatment
in ZR-7531 cell line.
Annexin-V assay
To evaluate the apoptosis induced by the alkaloids from P.
nitens, the annexin V assay was performed by flow
cytometry (Fig. 3). The tests were carried out on treated
cells at t0 and t24.
After 24 h of treatment (t0) with PGN (Fig. 3a), early
apoptosis was observed in 38.0±3.0% of the cells and late
apoptosis/necrosis in 55.0±3.0% at 10.125 mM. For t24
with PGN (Fig. 3b), early apoptosis was observed in 38.0±
3.0% of the cells and late apoptosis/necrosis in 48.0±4.0%
at 10.125 mM. A concentration-dependent response was
observed in both periods of treatment. Apparently, at t24,
the cells showed a drop in signals for late apoptosis/
necrosis, while maintaining the level of early apoptosis. In
other words, fewer cells suffered the devastating cell death
observed as necrosis after 24 h recuperation.
The results of the annexin Vassay for PGD are illustrated in
Fig. 3c and d. The ductal invasive carcinoma cells treated
with PGD for t0 showed early apoptosis 13.4±3.0% of the
cells and late apoptosis/necrosis 82.1±4.0% of the cells at
2.0 mM. When the cells were treated with PGD for t24, this
was a similar response for early apoptosis to that observed at
t0. However, the late apoptosis/necrosis rate was much higher
at t24 and no concentration response was observed (Fig. 3d).
Hoechst assay
The method of HO, with PI, was used for a better
characterization between early apoptosis, late apoptosis,
DOX
NC
0.25
0.5
1
2
0
20
40
60
80
100
a
Total apoptosis
Necrosis
** ** **
**
*
**
Concentration (mM)
Cell death (%)
DOX
NC
0.25
0.5
1
2
0
20
40
60
80
100
b
Late apoptosis
Early apoptosis
**
**
**
**
*
**
**
**
Concentration (mM)
Cell death (%)
DOX
NC
0.25
0.5
1
2
0
20
40
60
80
100
c
**
** **
**
**
****
**
Concentration (mM)
Cell death (%)
DOX
NC
0.25
0.5
1
2
0
20
40
60
80
100
d
*
**
*** ** ** ** ** **
Concentration (mM)
Cell death (%)
Total apoptosis
Necrosis
Late apoptosis
Early apoptosis
Fig. 6 Apoptosis by Hoechst / PI assay in ZR-7531 cells. Results are
expressed as the mean of three independent experiments ± standard error
and subjected to One-way ANOVA with Tukey’s test (treatment versus
DOX, for early apoptosis). (a) Cells treated with PGD for t0, relation
between total apoptosis and necrosis; (b) Cells treated with PGD for t0,
relation between early and late apoptosis; (c) Cells treated with PGD for
t24, relation between total apoptosis and necrosis; (d) Cells treated with
PGD for t24, relation between early and late apoptosis. DOX
doxorubicin at 15 μg/mL; NC untreated cells; *p<0.05; **p<0.01;
***p<0.001
518 Tumor Biol. (2010) 31:513–522

and necrosis (Fig. 4). In the treatment with PGN for t0
(Fig. 5a), it was possible to observe total apoptosis (early
and late) of 30-47% and necrosis of 10-43% of the cells. A
concentration-dependent increase of early apoptosis was
observed on treatment with pterogynine, relative to the
negative control. Early apoptosis was observed at all
concentrations (p<0.05), whereas late apoptosis was only
seen at the higher concentrations (p<0.01; Fig. 5b). At the
lower concentrations, there was an increase in the number
of cells in early and late apoptosis, compared to necrosis.
Thus, in the treatment at t0, 30- 47% of the cells were in
apoptosis, of which 13.75% were in early and 26.0% in late
apoptosis (Fig. 5b).
The cells treated with PGN maintained signs of
apoptosis (early and late) in 25-48% of the cells at t24,
while the number in necrosis fell to 16-30% (Fig. 5c).
Compared to the negative control, the cells at t24 showed
apoptosis at concentrations of 1.125, 3.375, and
10.125 mM (p<0.01). A statistical difference in rate of
necrosis was also observed between the negative control
and the cells treated with 3.375 and 0.125 mM (Fig. 5c). When
the cells were observed at t24, 25-48% showed apoptosis, of
which 13.5% were in early apoptosis and 25.5% in late
apoptosis, while 16-30% were in necrosis (Fig. 5d).
In the treatment with pterogynidine, at t0, we observed
necrosis in 4-28% (Fig. 5a) and apoptosis (early and late) in
22-53% of cells (Fig. 6b). An increase of apoptosis with the
lowest dose was observed, relative to the negative control.
At higher concentrations, the treated cells exhibited a
gradual but steady increase in early and late apoptosis,
and a smaller rise in necrosis. Likewise, in the treatment at
t0, 33-53% of the cells was in apoptosis, 23.1% in early
apoptosis and 16.3% in late apoptosis, while 6-28% of the
treated cells was in necrosis (Fig. 5a).
When the cells treated with PGD were assayed at t24, it
was observed that apoptosis signals (early and late) were
maintained in 13-26.73% of the cells (Fig. 6d) and there
was a gradual increase in the necrosis response with the
dose, from 17% to 35.5% (Fig. 5c). Comparing the negative
control cells with all of the treatments, it was confirmed that
it induced higher apoptosis signals, whereas 9-25.5% of
treated cells were in necrosis (p<0.01). At t24, 27-46.6% of
cells showed apoptosis, of which 13% were in early and
26.7% in late apoptosis (Fig. 6d).
Caspase 3/7 assay
Apoptotic activity was evaluated through effector caspases
3 and 7 pathway using a luminescent method. This assay
produces a luminescent substrate that has a four-peptide
sequence, which after cleavage by caspase 3/7 generates a
light signal, produced by luciferase. Figure 7shows to
caspase 3/7 enzyme activity in ZR-7531 cell line treated
with alkaloids.
Cells treated with PGN, caspase 3/7 activity was higher
at concentration of 1.125 mM for t0 (RLU = 62,980.5 ±
DOX
NC
0.375
1.125
3.375
10.125
0
20000
40000
60000
80000
100000
a
***
***
***
***
Concentration (mM)
RLU
DOX
NC
0.375
1.125
3.375
10.125
0
20000
40000
60000
80000
100000
b
Concentration (mM)
RLU
DOX
NC
0.25
0.5
1
2
0
20000
40000
60000
80000
100000
c
***
**
*** ***
Concentration (mM)
RLU
DOX
NC
0.25
0.5
1
2
0
20000
40000
60000
80000
100000
d
Concentration (mM)
RLU
Fig. 7 Caspase 3/7 activity ZR-
7531 cells. Results are
expressed as the mean of three
independent experiments ±
standard error and subjected to
one-way ANOVA with Tukey’s
test (treatment versus DOX). a
Cells treated with PGN for t0;
bcells treated with PGN for t24;
ccells treated with PGD for t0;
dcells treated with PGD for t24.
DOX doxorubicin at 15 μg/mL,
NC untreated cells; *p<0.05;
**p<0.01; ***p< 0.001
Tumor Biol. (2010) 31:513–522 519

1,353.4), suggesting that induction caspase 3/7 activation of
apoptosis was higher at an intermediate concentration.
Lowest activity was observed at 10.125 mM due to PGN
cytotoxicity.
Although, no statistical differences were observed
between DOX and PGD at 0.25 mM (RLU=35.8673±
2.170) for t0, apparently induction of apoptosis at this
concentration was verified. At higher concentrations, PGD
did not show caspase 3/7 activity because of its cytotoxic
effect.
For the treatment period of t24, both compounds showed
no statistical significance between of them and DOX,
indicating higher induction of apoptosis (Fig. 7b and d).
However, when comparing t0 and t24, lower caspase 3/7
activity was observed because of higher cytotoxicity of the
compounds for t24.
NC PC
B
D
F
H
A
C
E
G
Fig. 8 Cell line (ZR 7531)
treated with PGN at A)
10.125 mM, B) 3.375 mM, C)
1.125 mM, and D) 0.375 mM.
Cell line treated with PGD at E)
2.0 mM, F) 1.0 mM, G)
0.5 e, H) 0.25. NC negative
control, PC positive control-
doxorubicin 15 μg/mL.
Scale bars 100 um
520 Tumor Biol. (2010) 31:513–522

Figure 8shows ZR7531 treated with PGN and PGD. We
observe cell death with morphology rounded in the higher
concentrations (Fig. 8a,b,ande) whereas in lower
concentrations the cells are confluent (Fig. 8c,d,f,g,andh).
Discussion
The aim of this study was to characterize the effects of
pterogynine and pterogynidine, extracted from P. nitens on
the viability and apoptosis of breast cancer cell line.
Citotoxicity assay of compounds provided additional
support for the previous reports of Regasini et al. [15], in
which were citotoxic and anticarcinogenic alkaloid isolates
from P. nitens stem. Also these results provide additional
support for a recent report of Lopes et al. [29] in which in
vitro cytotoxic, anti-inflammatory, and anti-angiogenic
properties of alkaloid pterogynidine were reported.
Alkaloids with terminal amino groups and indole rings
have been studied and found to possess antitumoral,
antiviral, antifungal, and anti-inflammatory activities [30,
31]. Other structures of alkaloids isolated from plants are
being related to analgesic, antiarrythmic, and immunomod-
ulatory activities [32]. However, none of these compounds
has been considered to have important activity in solid
tumors, including breast cancer. Thus, a growing number of
studies involving treatment of tumoral cell lines with
alkaloids in the search for some antitumor activity are
necessary.
In the present study, it was observed that the alkaloids
PGN and PGD, exerted concentration-dependent cytotox-
icity to invasive ductal carcinoma cell line. Apparently, the
best cytotoxicity was seen in the treatment for t0, for both
substances, with higher effect being observed with PGD.
The PGN and PGD alkaloids of P. nitens showed a
significant apoptosis induction with concentration-
dependent profile against ZR-7531 by the Annexin-V assay.
Firstly, when apoptosis was assessed by flow cytometry,
both alkaloids exhibited different anti-apoptotic effects:
PGN-induced late apoptosis for the t0 treatment, which
became weaker at t24, while PGD produced an intense
effect of late apoptosis/necrosis at t24. These results
suggest that apoptosis is more exuberant in the treatment
with PGN at t0. On the other hand, a smaller percentage of
cells treated with PGD were in early apoptosis and most
were in late apoptosis/necrosis for the t24 period, possibly
indicating the cells continued the process of cell death
(apoptosis or necrosis) in stages subsequent to the treatment
[28].
In the present study, to improve the characterization, the
Hoechst/propidium iodide assay was carried out and
compared with the Annexin-V results. It was possible to
observe better the difference between early and late
apoptosis in the ZR-7531 cells, showing that PGN, at t0
and t24, and PGD at t0 exhibited a larger concentration-
response effect for late apoptosis and a smaller effect for
early apoptosis, in all of the studied concentrations.
Apoptosis studies evaluating caspase 3/7 activity in ZR-
7531 cell line, as far as we know, does not exist. We
assumed the hypothesis that caspase 3/7 activity can be
evaluated at lower concentrations as function of cell
damages, which are not yet totally understood [33,34]
Guanidine alkaloids form a rare group of natural
products. Although most chemical studies on plant alka-
loids show they have phenolic groups in common [35,36],
PGNandPGDisolatedfromP. nitens are unusual
alkaloids. Their chemical structures bear disubstituted
guanidine moiety. PGD has a guanidine structure with N,
N′-diisoprenyl substitution. Compared to N,N′,N″-triiso-
prenyl, it shows reasonable cytotoxicity relative to the
trisubstituted alkaloid, which was demonstrated to be
highly deleterious to the cell lines [36,37]. Comparing
the cytotoxicity of the two alkaloids, we observed greater
activity in PGD than in PGN, due to the lower IC
50
of the
former. PGD also showed a similar concentration-response
effect for early apoptosis and late apoptosis in the treatment
followed by the period after 24 h of treatment.
Conclusion
The results of this study indicated that pterogynine and
pterogynidine inhibit the proliferation of human breast
cancer cells and induce cell death by apoptosis. The
alkaloids, isolated from P. n i t e n s , could be a good
chemotherapeutic compounds against human breast cancer
cells. We also suggest that the substitutions in the guanidine
structure of the alkaloids might be related to the intensity of
the effect produced in the cells.
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