Biotechnol Lett (2014) 36:461–469

DOI 10.1007/s10529-013-1382-4

ORIGINAL RESEARCH PAPER

Lectin of Abelmoschus esculentus (okra) promotes selective

antitumor effects in human breast cancer cells
Leonardo G. Monte • Tatiane Santi-Gadelha • Larissa B. Reis •
Elizandra Braganhol • Rafael F. Prietsch • Odir A. Dellagostin •
Rodrigo Rodrigues e Lacerda • Carlos A. A. Gadelha •
Fabricio R. Conceição • Luciano S. Pinto

Received: 21 June 2013 / Accepted: 3 October 2013 / Published online: 16 October 2013
Ó Springer Science+Business Media Dordrecht 2013

Abstract The anti-tumor effects of a newly-discov- breast cancer cells and may represent a potential
ered lectin, isolated from okra, Abelmoschus esculen- therapeutic to combat human breast cancer.
tus (AEL), were investigated in human breast cancer
(MCF7) and skin fibroblast (CCD-1059 sk) cells. AEL Keywords Abelmoschus esculentus

induced significant cell growth inhibition (63 %) in Apoptosis
Cancer inhibition
Human breast
MCF7 cells. The expression of pro-apoptotic caspase- cancer
Lectin
Okra
Plants
3, caspase-9, and p21 genes was increased in MCF7
cells treated with AEL, compared to those treated with
controls. In addition, AEL treatment increased the
Bax/Bcl-2 ratio in MCF7 cells. Flow cytometry also Introduction
indicated that cell death (72 %) predominantly
occurred through apoptosis. Thus, AEL in its native Lectins are a heterogeneous group of proteins of non-
form promotes selective antitumor effects in human immune origin. They are found in microorganisms,
plants and animals and are capable of recognizing and
reversibly binding to mono- or oligo-saccharides, and
Electronic supplementary material The online version of glycoconjugates (Pinto et al. 2008). They may play
this article (doi:10.1007/s10529-013-1382-4) contains supple-
mentary material, which is available to authorized users. a role in modulating various cellular pathways,

L. G. Monte
L. B. Reis
L. S. Pinto (&) O. A. Dellagostin

Laboratório de Biotecnologia Vegetal e Proteômica, Laboratório de Vacinologia, Núcleo de Biotecnologia,
Núcleo de Biotecnologia, Centro de Desenvolvimento Centro de Desenvolvimento Tecnológico, Universidade
Tecnológico, Universidade Federal de Pelotas, Federal de Pelotas, Pelotas, RS 96010-900, Brazil
P.O. Box 354, Pelotas, RS 96010-900, Brazil
e-mail: ls_pinto@hotmail.com F. R. Conceição
Laboratório de Imunologia Aplicada, Núcleo de
T. Santi-Gadelha
R. R. e Lacerda
C. A. A. Gadelha Biotecnologia, Centro de Desenvolvimento Tecnológico,
Departamento de Biologia Molecular, Universidade Universidade Federal de Pelotas, Pelotas, RS 96010-900,
Federal da Paraı́ba, Cidade Universitária, João Pessoa, Brazil
PB CEP 58059-900, Brazil

E. Braganhol
R. F. Prietsch
Centro de Ciências Quı́micas, Farmacêuticas e de
Alimentos, Universidade Federal de Pelotas, Pelotas,
RS 96010-900, Brazil

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including apoptosis (Damodaran et al. 2008). Plant Materials and methods

lectins have attracted interest due to their myriad
biological activities, which include the ability to arrest Abelmoschus esulentus lectin (AEL) isolation
the cell cycle in the G1 and G2/M phases, and activate
the caspase cascade (Damodaran et al. 2008). In Lectin was extracted using seed meal (80 g) with
addition, plant lectins are an excellent tool to study 0.1 M Tris/HCl buffer, pH 7.4, and 0.15 M NaCl for
glycosylation patterns (Sanchez-Pomales et al. 2012). 3 h at 18 °C. The material was centrifuged at
Post-translational modifications such as glycosyla- 5,0009g for 20 min at 4 °C. The supernatant was
tion plays an important role in determining protein precipitated at 18 °C with (NH4)2SO4, collecting the
function, and can play a role in disease development. An precipitate between 30 and 60 % saturation. The
alteration in the glycosylation patterns of cell surface precipitate was recovered by centrifuging at 5,0009g
proteins is associated with tumor progression (Elmore for 20 min at 4 °C. The fractions were exhaustively
2007). Due to their cell surface expression, the oligo- dialyzed against H2O, freeze-dried then purified by ion
saccharide epitopes of glycoproteins and glycolipids are exchange chromatography (Sephacel-DEAE) (Soares
recognized by membrane-anchored carbohydrate-rec- et al. 2012). Purity was evaluated by SDS-PAGE (see
ognition domains of various molecules, including Supplementary Fig. 1). AEL concentration was deter-
lectins (Jimenez-Castells et al. 2008). Binding of these mined using the Pierce BCA Protein Assay Kit, and
lectins to oligosaccharide epitopes on tumor cells can the stock solutions containing 0.92 mg lectin ml-1
activate cell death by apoptosis. Lectins from various (100 % purity) (Soares et al. 2012) were stored
sources have demonstrated anti-tumor and anti-carcin- at -80 °C until use.
ogenic activities (Damodaran et al. 2008).
Malignant transformations are often associated with Cell culture and AEL treatment
alterations in the cell surface glycosylation pattern.
Cancer progression involves the disruption of cell Human breast cancer (MCF7) and skin fibroblast
cycle regulation, which, in turn, may contribute to (CCD-1059 sk) cell lines were obtained from the Rio
alterations in the expression of cell-surface carbohy- de Janeiro Cell Bank (PABCAM, Federal University
drates (Elmore 2007). Because of their anti-tumor of Rio de Janeiro, RJ, Brazil). The cell lines were
effects, carbohydrate-binding lectins have been the grown in DMEM supplemented with 10 % (v/v) fetal
focus of interest in cancer biotherapy research (Dam- bovine serum (FBS) at 37 °C in a humidified atmo-
odaran et al. 2008). Breast cancer is the most sphere containing 5 % CO2. All experiments were
commonly diagnosed invasive malignancy and is the performed with cells in the most rapid phase of
second largest cause of cancer-related death in women growth. For treatments, AEL was dissolved in
worldwide (Friedenreich 2011). Breast carcinogenesis DMEM/10 % (v/v) FBS at a 0.92 mg ml-1, and
may involve quantitative as well as qualitative changes further diluted in the same medium to 0.1, 0.05, 0.025,
in cell surface carbohydrate expression. Therefore, and 0.0125 mg ml-1. To generate a negative control
lectins may provide a useful strategy for early detection for lectin binding to carbohydrates (iAEL), AEL was
and/or treatment of human breast cancer. incubated with galactose (50 mM) for 1 h at 37 °C to
Abelmoschus esculentus (a member of the family block the carbohydrate-binding sites in the lectin. For
Malvaceae), commonly known as okra, is a plant that comparative purposes, a control assay with bovine
has originated from Africa, and has been used serum albumin (BSA, 0.1 mg ml-1) was performed.
medicinally in treatment of several disorders (Soares
et al. 2012). A. esculentus lectin (AEL) occurs as a MTT colorimetric assay
10.29-kDa monomeric protein or as a 20.58-kDa
dimer, and exhibits anti-inflammatory, antinocicep- The cytotoxicity of AEL in MCF7 and CCD-1059 sk
tive, and hemagglutinating activities (Soares et al. human cell lines was initially evaluated by using the
2012). However, its effects on cancer cells have not MTT assay. Briefly, the cells were seeded in 96 well
been studied; hence, the aim of the present study was plates (104 cells/well). After 24 h, cells were exposed
to evaluate the in vitro selective anti-tumor activity of to increasing concentrations of AEL or iAEL, or BSA.
AEL in the MCF7 human breast cancer cell line. After treatment, the MTT assay was performed

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following the manufacturer’s instructions. Absor- externalization during apoptosis was studied using the
bance was measured using a microplate reader at Guava Nexin Annexin V assay (Millipore Corporation,
492 nm. The percentage inhibition of cell growth was Billerica, MA, USA), following the manufacturer’s
determined as follows: percentage inhibition in instructions. Briefly, 106 cells were incubated with
growth = [1 - (A492 for treated cells/A492 for annexin-V and 7-AAD in the dark at room temperature
untreated cells)] 9 100 (Begnini et al. 2013). The for 20 min. Samples (2 9 103 cells/well) were then
data represent the mean ± SEM from three indepen- acquired on a Guava EasyCyte flow cytometer (Millipore
dent experiments. Corporation).

Propidium iodide assay Statistical analysis

Cellular damage, assessed by propidium iodide (PI) Data from the MTT assay was analyzed using a
uptake, was determined using florescence microscopy. factorial analysis of variance (ANOVA), followed by
Briefly, the MCF7 cells were seeded in 24 well plates Tukey post hoc test for multiple comparisons. Three
(2 9 104 cells/well), and treated with AEL or iAEL at factors were considered: AEL and iAEL (two levels),
0.1 mg ml-1. Following 24, 48, and 72 h of treatment, concentration (four levels), and the time required for
tumor cells were incubated with PI (7.5 lM) for 1 h. lectin action (three levels). qPCR and flow cytometry
Images were obtained at 515–560 nm by using an data were analyzed using ANOVA, followed by
inverted microscope. Data represent the mean ± SEM Tukey post hoc test for multiple comparisons. Signif-
from three independent experiments. icance was considered at P \ 0.05 in all analyses.
Data were expressed as mean ± SEM.
qPCR analysis

MCF7 cells were seeded in 6 well plates (106 cells/ Results

well); cell growth reached subconfluence after 24 h of
culture. Cells were then treated with AEL or iAEL MTT colorimetric assay
(0.1 mg ml-1). The effect of AEL or iAEL on gene
expression in MCF7 cells was evaluated by qPCR The ability of AEL to inhibit a specific tumor was
after 72 h. Cells were washed with phosphate-buf- evaluated by comparing its cytotoxic potential in the
fered saline, and total RNA extraction, cDNA synthe- MCF7 breast cancer cell line to that in the CCD-1059
sis, and real-time PCR (qPCR) were carried out as sk fibroblast cell line. Breast cancer and fibroblast
described previously (Begnini et al. 2013; Campos cells were exposed to increasing concentrations of
et al. 2010). Briefly, RNA was isolated using TRIzol AEL for up to 72 h. As shown in Fig. 1, AEL, but not
and samples were DNase-treated using the Ambion iAEL, induced significant cytotoxic effects (P \ 0.05)
Turbo DNA-free kit (Invitrogen, USA), following the on tumor cells. The cytotoxic effect observed
manufacturer’s protocol. First-strand cDNA synthesis depended on both time and concentration; toxicity
was performed using High Capacity cDNA Reverse was highest (63 %) in MCF7 cells treated with
Trancription kit (Applied Biosystems, UK), according 0.1 mg ml-1 AEL for 72 h (Fig. 1a). Notably, AEL
to the manufacturer’s protocol. The qPCR reactions and iAEL did not produce the same effects on skin
were run on a Stratagene Mx3005P real-time PCR fibroblast cells (cytotoxicity, 24 %), suggesting that
System (Agilent Technologies, Santa Clara, CA, the cytotoxic potential was cell type-dependent as well
USA) using SYBR Green PCR Master Mix (Applied (Fig. 1b). No cytotoxic effect was observed on cell
Biosystems, UK); the primers used are described in lines exposed to BSA (data not shown).
Supplementary Table 1.
Propidium iodide (PI) assay
Annexin V-binding assay
To identify whether AEL-mediated cytotoxicity in
After 72 h of treatment with AEL or iAEL, the control MCF7 cells involved the induction of necrosis, human
and treated cells were trypsinized and phosphatidylserine breast cancer cells were treated with AEL for varying

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Fig. 1 Cytotoxic effects of AEL and iAEL in MCF7 and CCD- determined as previously described. Values represent mean ±
1059 sk cell lines. The cells were exposed to increasing SEM of three independent experiments and were analyzed by
concentrations of AEL or iAEL (0.0125, 0.025, 0.05 and ANOVA followed by Tukey post hoc test. Different letters and
0.1 mg ml-1) for 24, 48, 72 h, and cell growth inhibition (%) asterisk symbol indicate significant differences between the
was determined by MTT assay. The inhibition (%) was means (P \ 0.05)

times. Cell membrane permeability was then assessed however, no differences in mRNA levels between the 3
by measuring PI uptake. PI uptake occur in cells that treatment groups were observed (P \ 0.05). Gene
showed morphological changes, indicating that alter- expression levels in AEL-treated cells were compared
ations in cell morphology preceded the loss of cell to those in the iAEL- or untreated cells in all experiments.
membrane integrity. As shown in Fig. 2, PI uptake
increased when the cells were treated with 0.1 mg Flow cytometric analysis
AEL ml-1 for 72 h (Fig. 2A), suggesting a loss in
membrane integrity, indicative of cell death by necro- To determine whether the cytotoxic effect of AEL was
sis. Importantly, treatment with comparable concen- associated with apoptotic cell death, a flow cytometry
trations of iAEL did not induce PI uptake, suggesting analysis was conducted (Fig. 4). Treatment with
that the effect of lectin involves its interaction with cell 0.1 mg AEL ml-1 for 72 h predominantly induced
surface-expressed carbohydrates (Fig. 2B). cell death by apoptotic mechanisms (72.3 %). Necro-
tic cells were also observed at *27 %. Notably,
Gene expression profile treatment with iAEL at comparable concentrations did
not exert significant apoptosis (11 %), or necrosis
To understand the mechanisms involved in the induction (7.6 %), similar to results obtained to untreated cells
of apoptotic cell death by AEL, apoptosis-related gene (10.6 and 5.4 % for apoptosis and necrosis,
expression in MCF7 cells was investigated by qPCR respectively).
(Fig. 3). Caspase-3 and -9 mRNA expression was
elevated (P \ 0.05) in cells treated with 0.1 mg
AEL ml-1 for 72 h when compared to those treated with Discussion
iAEL. Likewise, p21 gene expression was four-fold up-
regulated (P \ 0.05) in cells treated with AEL; however, Breast cancer is one of the most prevalent and severe
iAEL treatment did not produce the same effects. In diseases in women. The cumulative global incidence
addition, AEL treatment did not alter Bax mRNA of breast cancer increased by more than 25.2 %
expression. Bcl-2 mRNA expression was downregulated between 1980 and 2010; 0.641 million cases were
in AEL-treated MCF7 cells, while being upregulated in reported in 1980, while 1.643 million cases were
iAEL-treated cells (P \ 0.05). The Bax/Bcl-2 ratio in recorded in 2010. In addition, breast cancer-related
MCF7 cells increased four-fold after AEL treatment, mortality has increased at an annual rate of 1.8 %,
when compared to iAEL treatment. Survivin, AIF, and increasing from 250,000 deaths in 1980 to 425,000
endonuclease G gene expression was also evaluated, deaths in 2010 (Forouzanfar et al. 2011).

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Fig. 2 Propidium iodide (PI) incorporation in MCF7 cells was excited at 515–560 nm and visualization was performed
following lectin treatment. The cells were exposed to AEL with a 409 objective on an inverted microscope for fluorescence
(panels A and a) or iAEL (panels B and b) at a concentration of microscopy (Olympus IX 71). Untreated cells were used as
0.1 mg ml-1, and after 72 h of treatment, the cells were control ( panels C and c). Bars represent 100 lm
incubated with PI diluted in culture medium. PI fluorescence

We have evaluated the selective toxicity of AEL suggests that AEL may have therapeutic potential,
(mediated by its carbohydrate-binding activity) on since the lack of specificity of current chemotherapy is
human breast cancer cells. To neutralize its carbohy- a challenge for clinical patient management, by
drate-binding ability, AEL was incubated with a high restricting the treatment and by the development of
concentration of galactose to saturate its carbohydrate- side-effects. Many studies have investigated the
recognition domains (Soares et al. 2012). Under these mechanisms underlying anti-proliferative effects of
conditions, galactose treatment inhibited the carbohy- different lectins. Concanavalin A was internalized by
drate-binding activity and prevented AEL-induced the tumor cells, and accumulated in the mitochondria.
killing of tumor cells. According to this hypothesis, This was followed by alterations in the PI3 K-Akt
viability of iAEL-treated cells was similar to that of anti-apoptotic pathway involving a downregulation in
BSA-treated controls. These results suggest a close Akt phosphorylation in HepG2 cells. Polygonatum
relation between the carbohydrate-recognition domains cyrtonema lectin was shown to regulate Bax, Bcl-XL,
of AEL and its anti-proliferative activity, confirming and Bcl-2 protein expression, decreased the mito-
that AEL interacted with the cells through a sugar chondrial membrane potential and induced apoptosis
domain- binding site. Moreover, unlike in MCF7 cells, through the mitochondria-mediated ROS-p38-p53
AEL was not cytotoxic in the CCD-1059 sk fibroblast pathway in A375 cells (Lei and Chang 2009).
cells. Similarly, a study examining the leguminous This study demonstrates the specific anti-prolifer-
glucosamine-binding brown kidney bean lectin ative activity of AEL in MCF7 cells. The literature
(BKBL) demonstrated that the anti-proliferative activ- indicates that the anti-proliferative activity of lectins
ity of BKBL was attenuated upon co-treatment with could be attributed to the induction of apoptosis. Flow
glucosamine (Chan et al. 2012). In corroboration with cytometric analysis using a recombinant phosphati-
previous studies, we demonstrate that the anti-prolif- dylserine-binding protein that interacts strongly and
erative activity of AEL in MCF7 cancer cells specifically with PS residues was carried out (Elmore
depended on the carbohydrate-binding capacity, and 2007). Despite the observation of PS translocation
was selective for human breast cancer cells. The low during necrosis, the analysis demonstrated that AEL
toxicity of AEL in the CCD-1059 sk cell line also induces cell death in MCF7 cells predominantly via

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bFig. 3 Gene expression of apoptotic-related genes in MCF7 P. cyrtonema lectin (PCL) has anti-proliferative and
cells following AEL or iAEL treatment. The cells were treated apoptosis-inducing activities in MCF7, HeLa, A375,
with AEL or iAEL at a concentration of 0.1 mg ml-1 for 72 h,
and the analysis of gene expression was determined by qPCR. and L929 cells. Concanavalin A, a mannose/glucose-
Values represent mean ± SEM of three independent experi- binding legume-derived lectin, induces apoptosis in
ments and were analyzed by ANOVA followed by Tukey post human A375 and hepatocellular liver carcinoma
hoc test. Different letters indicate significant differences HepG2 cell lines. Sclerotium rolfsii lectin (SRL)
between the means (P \ 0.05). Untreated cells were used as
control isolated from a soil phytopathogenic fungus inhibits
proliferation and induces apoptosis in the human
ovarian cancer cell line PA-1 (Eligar et al. 2012).
Although several researchers have reported the anti-
tumor effects of lectins from different sources, the
mechanisms of action or activation of these lectins are
not fully understood and are the focus of continuing
research. In addition, the goal of the cancer therapy is
to kill or damage tumor cells only, but most of these
studies either showed negative effects on healthy cells
or did not study the effects on healthy cells (Chan and
Ng 2013).
The response of cancer cells to many drugs involves
the activation of apoptotic pathways. Thus, we exam-
ined expression profile of genes involved in apoptosis
Fig. 4 Flow cytometric analysis. The cells were treated with
by qPCR. The Bcl-2 protein is anti-apoptotic, whereas
AEL or iAEL at a concentration of 0.1 mg ml-1 for 72 h. The Bax is a pro-apoptotic regulator (Elmore 2007). The
cell death was measured using the Guava Nexin Annexin V Bax/Bcl-2 ratio expression level plays an important
assay. A/B and a/b represent apoptosis and necrosis, respec- role in sustaining cell morphology and function.
tively. Values represent mean ± SEM of three independent
experiments and were analyzed by ANOVA followed by Tukey
Several studies have shown that Bcl-2 overexpression
post hoc test. Different letters indicate significant differences can disrupt regulation of the pro-apoptotic proteins
between the means (P \ 0.05). Untreated cells were used as Bax and Bak and prevents cytochrome c release from
control mitochondria, thus inhibiting the activation of casp-
ases and preventing apoptosis (Hu et al. 2010). Our
apoptosis (72.3 %). Our findings demonstrate the study demonstrated that Bcl-2 expression was down-
ability of AEL to induce PS externalization in the regulated by AEL treatment while Bax expression was
MCF7 cells, and indicate that this externalization is up-regulated. Hence, the Bax/Bcl-2 ratio in breast
mediated by a carbohydrate-binding site of the lectin, cancer cells increased following lectin treatment,
since the galactose blockage of AEL prevented the suggesting that Bax and Bcl-2 modulation plays a
apoptosis induction to control levels. Previously role in the cytotoxicity induced by AEL in MCF7 cells
studies show the anti-tumor effects of others lectins, (Fig. 3). Caspases play a central role in apoptosis
e.g., the mistletoe (Viscum album var. coloratum)- (Huang et al. 2012); the activation of caspase-3 and
derived lectin induces apoptotic death in human caspase-9 is believed to be a well-defined outcome of
leukemia cells (U937), characterized by DNA ladder mitochondrial cytochrome c release into the cyto-
fragmentation. The mechanisms involved in this plasm and its subsequent association with Apaf-1
process have not been elucidated, but the studies protein. In our experiments, caspase-3 and caspase-9
suggest that they may involve the proteolytic activities mRNA levels were significantly increased by AEL
of caspase-3 and caspase-7, and involve the JNK treatment, suggesting that the apoptosis caspase-
pathway of apoptosis (Kim et al. 2001). Galactose- dependent cell death is a component of AEL antican-
binding lectin also induces apoptosis in human cer effect.
leukemia cells (K562) through caspase-dependent Survivin has been implicated in the inhibition of
pathways, and its ability to do this may depend on apoptosis, cell proliferation, angiogenesis, and cellular
its sugar-binding activity (Huang et al. 2012). stress response. However, our results indicate that the

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apoptosis induced by the lectin was not mediated by Acknowledgments This work was supported by Coordenação
decreasing survivin expression. In the present study, de Aperfeiçoamento de Pessoal de Nı́vel Superior (CAPES),
Grants no 02841/09-6 and 23.038.019120/9-3, Fundação de
AEL appears to mediate apoptosis in a caspase- Amparo a Pesquisa do Rio Grande do Sul (FAPERGS), Grants
dependent manner, since the expression of caspases no. 11/1842-5 and Conselho Nacional de Pesquisa (CNPq). The
were significantly higher, and no differences were authors also wish to thank Dr. Cláudia Pinho Hartleben, MSc.
found in AIF, and Endo G mRNA expression in AEL- Eduarda Schultze, MSc. Karine Rech Begnini and MSc.
Virginia Campello Yurgel for their valuable technical
treated MCF7 cells. Based on these effects, the assistance with the flow cytometric analysis.
apoptosis mechanism may have occurred by a
caspase-dependent pathway (Fig. 3).
Checkpoints are control mechanisms that ensure
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