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

Article (PDF Available)inTumor Biology 31(5):513-22 · October 2010with 103 Reads
DOI: 10.1007/s13277-010-0064-2 · Source: PubMed
Cite this publication
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.
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 [46]. Many com-
pounds with biological activities were obtained from
Cerrado, Brazils second largest biome [710].
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) 31:513522
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 [1422].
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
(AnnexinV, 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 Sakaguchis and Dragendorffs 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-1ALK-
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 Dulbeccos modified
eagle medium (DMEM) media and Hams 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) 31:513522
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 Tukeys
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) 31:513522 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 Tukeys
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) 31:513522
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 Tukeys 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 Tukeys 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) 31:513522 517
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    Angiogenesis, the formation of new blood vessels from preexisting capillaries, is an important research field. As the understanding of this process increases, this new knowledge will have a significant impact on several angiogenesis-dependent diseases. A wide variety of plants are rich in alkaloids, and these compounds have traditionally been of interest due to their pronounced effects on various physiological activities in animals and humans. Nowadays, it is known that many alkaloids obtained from plants exhibit antiangiogenic activity, and these alkaloids may act through different mechanisms to inhibit angiogenesis. Herein, we will discuss the most important alkaloids obtained from plants, focusing especially in their antiangiogenic activity. Because of the great diversity of plants, certainly, there are many antiangiogenic alkaloids that have yet to be discovered.
  • Article
    Limonoids and triterpenes are the largest groups of secondary metabolites and have notable biological activities. Meliaceae and Rutaceae are known for their high diversity of metabolites, including limonoids, and are distinguished from other families due to the frequent occurrence of such compounds. The increased interest in crop protection associated with the diverse bioactivity of these compounds has made these families attractive in the search for new allelopathic compounds. In the study reported here we evaluated the bioactivity profiles of four triterpenes (1-4) and six limonoids (5-10) from Meliaceae and Rutaceae. The compounds were assessed in a wheat coleoptile bioassay and those that had the highest activities were tested on the standard target species Lepidinum sativum (cress), Lactuca sativa (lettuce), Lycopersicon esculentum (tomato) and Allium cepa (onion). Limonoids showed phytotoxic activity and 5α,6β,8α, 12α- tetrahydro-28-norisotoonafolin (10) and gedunin (5) were the most active, with bioactivity levels similar to, and in some cases better than, those of the commercial herbicide Logran. The results indicate that these products could also be allelochemicals involved in the ecological interactions of these plant species.