Biomedicine & Pharmacotherapy 92 (2017) 696–706

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Original article

Libidibia ferrea presents antiproliferative, apoptotic and antioxidant

effects in a colorectal cancer cell line
Andreza Conceição Véras de Aguiar Guerraa , Luiz Alberto Lira Soaresb ,
Magda Rhayanny Assunção Ferreirab , Aurigena Antunes de Araújoc,
Hugo Alexandre de Oliveira Rochad, Juliana Silva de Medeirose ,
Rômulo dos Santos Cavalcantea , Raimundo Fernandes de Araújo Júniora,e,*
a
Post Graduation Program in Health Science, Federal University of Rio Grande do Norte (UFRN), Natal, RN, Cep: 59078-970, Brazil
b
Post Graduation Program in Therapeutic Innovation, Department of Pharmaceutical Sciences, Federal University of Pernambuco (UFPE), Recife, PE, Cep:
50740-530, Brazil
c
Post Graduation Program in Pharmaceutical Sciences, Department of Biophysics and Pharmacology, UFRN, Natal, RN, Cep: 59078-970, Brazil
d
Post Graduation Program in Biochemistry, Department of Biochemistry, UFRN, Natal, RN, Cep: 59078-970, Brazil
e
Post Graduation Program in Functional and Structural Biology, Department of Morphology, UFRN, Natal, RN, Cep: 59078-970, Brazil

A R T I C L E I N F O A B S T R A C T

Article history:
Received 9 March 2017 Colorectal cancer is noted for being one of the most frequent of tumors, with expressive morbidity and
Received in revised form 25 May 2017 mortality rates. In new drug discovery, plants stand out as a source capable of yielding safe and high-
Accepted 25 May 2017 efficiency products. Well known in Brazilian popular medicine, Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz
var. ferrea (better known as “ironwood” or “jucá”), has been used to treat a wide spectrum of conditions
Keywords: and to prevent cancer. Using methodologies that involved flow cytometry, spectrophotometry and RT-
Libidibia ferrea qPCR assays, crude extracts of the fruits of L. ferrea (20T, 40T, 60T and 80T) were evaluated at 24 h and/or
Colorectal cancer 48 h for: their ability to inhibit cell proliferation; induce apoptosis through Bcl-2, caspase-3 and Apaf-1;
Apoptosis
their antioxidant activity and effects on important targets related to cell proliferation (EGFR and AKT) in
Antioxidant
the HT-29 human colorectal cancer lineage. The results revealed high antiproliferative potential as
compared to the controls, induction of apoptosis through the intrinsic pathway, and probable tumor
inhibition activity under the mediation of important targets in tumorigenesis. In addition, L. ferrea
revealed antioxidant, lipid peroxidation and chemoprotective effects in healthy cells. Thus, L. ferrea
derivatives have important anticancer effects, and may be considered promising candidate for colorectal
cancer therapy.
© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction new-case rates continue to rise worldwide and are estimated to be

It is understood that cancer, a condition marked by the
disorderly proliferation of subtly modified cells, is one of the
main public health problems faced in this century. Despite
advances, the therapeutic procedures available are still insuffi-
cient, they are generally highly invasive or non-specific, and are
often accompanied by side effects and cell toxicity [1,2]. Currently,
* Corresponding author at: Department of Morphology, Centre of Biosciences,
UFRN, Av. Senador Salgado Filho, S/N, Campus Universitário, Lagoa Nova, 59072-
970, Natal, RN, Brazil.
E-mail address: araujojr@cb.ufrn.br (R.F.d.A. Júnior).
over 20 million per year by 2025 [3].
Among cancers, colorectal cancer has been noted as one of the
most frequent in solid tumors, in men and women, representing
10% of the global incidence [3]. The etiological factors and
pathogenic mechanisms related to its development are complex
and heterogeneous [4,5], this contributes to its being one of the
main causes of morbidity and mortality in the world.
Natural products, particularly plant derivatives, are essential
components in research and development for safe, innovative,
economical and high efficiency drugs against complex diseases [6–
8]. It is estimated that about 60% of the antitumoral drugs available
on the market and most of those in the late stages of clinical trials
are derived from natural products, mainly from plants [9].

http://dx.doi.org/10.1016/j.biopha.2017.05.123
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
Over the last two decades, public interest and research efforts
from scientific and medical communities worldwide has increased
expressively, generating a large volume of information including
studies on the pharmacological effects, usage, and the develop-
ment into future medicines of herbs and derivative medicinal
phytochemicals as anti-tumor and chemoprevention agents. [10]
This leads to growing number of sales of commercialised medicinal
herbs and most importantly, growing number of pharmaceutical
companies that involve in the research and development of plants
as a source for modern medicine. [11]
Brazil has one of the largest plant species diversities in the
world, yet less than 10% of its species have been evaluated for their
biological characteristics [12]. In particular, the vegetation of the
caatinga, an extremely threatened biome, as a valuable natural
resource has been little studied [13]. Many species however are
widely known and used empirically in folk medicine. Thus, we
have Libidibia ferrea (Mart. Ex Tul.) L.P. Queiroz var. ferrea
(Fabaceae) known as Caesalpinia ferrea, and popularly known as
“ironwood” or “jucá” [14]. It is used popularly in the treatment of
bronchopulmonary disorders, gastrointestinal disorders, diabetes,
rheumatism, inflammation, wounds in general, and for prevention
of cancers [15].
Most of the botanical components of this plant are harnessed,
such as flowers, shells, pods and roots. The fruits have been
reported in the literature because they have antimicrobial [16–18],
antioxidant [19] and antidiabetic [20] properties, and besides
being chemopreventive [21], they show no mutagenic potential
[22], or reproductive toxicity [23]. Yet studies exploring their
anticancer activity, especially mechanisms of action in cellular
signaling pathways are practically nonexistent.
Dysregulation of apoptosis and cell proliferation signaling
pathways is critical to both the promotion and progression of
cancer. Thus, assessment of key points in these pathways is
essential for developing new target molecules with effective
antineoplastic activity [24,25]. Apoptosis is triggered by chrono-
logical activation of the initiator (caspase 9), and effector (caspase-
3) families via two distinct but associated pathways known as the
intrinsic and extrinsic pathways. The intrinsic pathway in
particular is dominated by the Bcl-2 family of proteins that
regulate cell survival/death related decisions, acting through
permeabilization of the mitochondrial membrane [26], in addition
to being mediated by initial apoptosome formation, whose main
component is the Apaf-1 factor [27].
Other pathways of interest in the study of cancer are the EGFR
and AKT (AKT serine/threonine kinase) mediated pathways, which
have multidimensional roles in tumor development and growth,
and are involved in numerous cellular responses including
proliferation, apoptosis and angiogenesis [28,29].
The objectives of this work are to investigate the potential of L.
ferrea as an antitumor drug candidate to contribute to colorectal
cancer therapy innovation through in vitro evaluations of cell
death, antiproliferative effects, and signaling pathways related to
intrinsic apoptosis (Bcl-2, caspase-3 and Apaf-1), and to AKT and
EGFR expression; and as well, to evaluate L. ferrea’s antioxidant
potential. Such bioactive actions presented by L. ferrea may
contribute to the availability of new anticancer treatments in the
clinic.
2. Methodology
2.1. Obtaining Libidibia ferrea crude extracts
The crude extracts of fruits from Libidibia ferrea were obtained
at 10% (w/v) by turbo extraction (four extractive cycles of 30 sec,
interspersed with by 5 min of pause), using ethanol as solvent at
20, 40, 60 and 80% (v/v). The extracts were filtred and concentrated
697
Table 1
Content of ellagic acid and gallic acid (g%) in crude extracts of fruits from L. ferrea.
Sample Ellagic acid (EA) Gallic acid (GA)
20T 2.78  1.227 (0.89) 4.43  0.132 (0.24)
40T 2.89  0.551 (0.39) 3.39  0.268 (0.67)
60T 2.73  2.213 (1.65) 1.61  0.125 (0.66)
80T 2.61  0.381 (0.29) 1.84  0.073 (0.34)
The results were expressed in g%, as mean  standard deviation (relative standard
deviation).
under reduced pressure at 40
C (RV10 Basic, IKA1) to remove the
ethanol. The aqueous residues were frozen under 80
C for three
days and then lyophilized (Model L101, Liotop1) to yield the crude
extracts. The quantification of chemical markers (ellagic acid and
gallic acid) in the crude extracts was carried out by high
performance liquid chromatography (HPLC) according to the
methodology previously described by Ferreira et al. [30], shown
in summary in Table 1.
2.2. Cell line and cultivation
The HT-29 human colorectal cancer cell line and the HEK-293
human embryonic kidney cell line (used as control of cytotoxicity
parameters) were obtained from the American Type Culture
Collection (Rockville, MD, USA). All cell lines were cultivated in
Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, MA,
USA) supplemented with 10% (v/v) fetal bovine serum and 1%
antibiotics (penicillin/streptomycin) in a 37
C incubator with
atmosphere of 5% CO2.
2.3. Viability test
Viability and cell proliferation for the Libidibia ferrea extracts
were determined by an assay based on the MTT tretazolium salt (3-
(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide)
[31]. To this end, the HT-29 and HEK-293 cell lines were plated
in 96-well plates, with a density of 5  103 cells/well. After 24 h
under culture conditions, applications of 20T, 40T, 60T and 80T
crude extracts at concentrations of 12.5 mg/mL, 25 mg/mL, 50 mg/
mL and 100 mg/mL were performed. Treatments were evaluated at
24 h and 48 h by addition of 100 mL/well of MTT (1 mg/mL),
incubation for 4 h, and addition of ethanol/well. The plates were
shaken and the absorbance obtained in a microplate reader (Epoch
 BioTek Instruments Inc, VT, USA) at 570 nm. The Gen5 Data
Analysis software version 2.0 (BioTek Instruments Inc, VT, USA)
was used.
2.4. Evaluation of in vitro cell death
The effect of Libidibia ferrea extracts on HT-29 and HEK-293 cells
was determined by double-labeled flow cytometry with Annexin
V-FITC and Propidium Iodide (PI), which allows the identification
of apoptotic and necrotic cells through loss of membrane integrity.
Cell lines were arranged in 6-well plates with a density of 2  105
cells/well, at a total volume of 2 mL. After 24 h of incubation under
culture conditions, the cells were treated with the 40T, 60T and 80T
crude extracts at doses of 25 mg/mL and 50 mg/mL, at 24 h and 48 h.
After each period, the cells were obtained by collecting the
supernatant from the wells, washing with PBS, trypsinization, and
two steps of centrifugation at 3200 rpm at 4
C. Finally, they were
labeled according to instructions from the Annexin V-FITC/PI kit
698 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706

Fig. 1. Effect of Libidibia ferrea extracts on HT-29 and HEK-293 cell line proliferation. (A) 24 h treatment time. (B) 48 h treatment time. *P < 0.05, **P < 0.01 and ***P < 0.001
versus control. CTRL (control, untreated cells), 20T (crude extract 20T), 40T (crude extract 40T), 60T (crude extract 60T), 80T (crude extract 80T).

(BD Pharmigen, CA, USA), and analyzed with BD FACSCanto II (BD
Biosciences, CA, USA) and FlowJo software, version 7.6.5 (Tree Star
Inc., CA, USA).
2.5. Immunofluorescence microscopy
For evaluation of apoptosis pathway targets, HT-29 cells was
plated on glass coverslips with cell density of 5  104 cells/well (for
a total volume of 1 mL), and plated in 24-well plates. Briefly, after
24 h the cells were treated with extracts 40T and 60T, being dosed
at 25 mg/mL, for 24 h and 48 h. After each period, they were fixed
with 7% paraformaldehyde, permeabilized with 0.2% Triton X-100/
PBS and incubated for 1 h in a humid chamber with anti-Bcl-2 and
rabbit anti-caspase-3 mouse polyclonal antibodies (Abcam, CA,
USA), using one coverslip for each antibody, diluted 1:100 in PBS
containing 5% bovine serum albumin (Life Technologies, SP, BR).
Primary antibody was detected with Alexa Fluor 488 anti-mouse or
anti-rabbit secondary antibody (Abcam, CA, USA) diluted 1:500 in
PBS, containing 5% bovine serum albumin, and 4,6-diamidino-2-
phenylindole (DAPI) (Life Technologies, SP, BR), diluted 1:200 in
PBS containing 5% bovine serum albumin and used for nuclear
staining. The coverslips were examined in an Zeiss Observer Z1
upright microscope for fluorescence (Carl Zeiss, Jena, DE) on the
40 x objective. The selected images were representative of most
cells.
2.6. GSH dosage
To assess antioxidant activity, the total level of glutathione
(GSH) was determined using the Costa et al. [32] method. Initial
cell uptake was performed using the procedure described by
 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706 699

Rahman et al. [33]. Briefly, HT-29 and HEK-293 cells were plated in
6-well plates, with 1 106 cells/well, in 2 mL total volume. On the
following day, treatment with extracts 40T and 60T in the
concentration of 25 mg/mL was carried out. After 24 h, the cells
were removed, resuspended in PBS and centrifuged twice for 5 min
at 3000 rpm, and at 4
C. The pellet was then homogenized with a
0.02 M solution of ethylenediamine tetraacetic acid (EDTA)
(Sigma-Aldrich, SP, Brazil) for grinding. After this process, the
suspension obtained was then diluted in 50% trichloroacetic acid
(Vetec, SP, Brazil) and distilled water for centrifugation for 15 min
at 3000 rpm, and at 4
C. Finally, a mix of the cell supernatant, 0.4 M
Tris buffer (Sigma-Aldrich, SP, Brazil) and 0.01 M dithiobisnitro-
benzoic acid solution (Sigma-Aldrich, SP, Brazil) were applied to
each well, to read the absorbance of each sample at 412 nm. The
results were expressed as nmol/106 cells.
2.7. MDA dosage
To evaluate lipid peroxidation, malondialdehyde (MDA produc-
tion) was measured according to an assay described by Esterbauer
and Cheeseman [34]. HT-29 cells underwent the same initial
centrifugation procedure described above, and were then

Fig. 2. Effect of L. ferrea extracts on HT-29 and HEK-293 cells, after treatment for 24 h, evaluated by flow cytometry. (A) Control (untreated cells); (B) 40T  25 mg/mL; (C) 40T
 50 mg/mL; (D) 60T  25 mg/mL; (E) 60T  50 mg/mL; (F) 80T  25 mg/mL; (G) 80T  50 mg/mL; (H) Cisplatin  50 mM. Dot plots are displayed with Annexin V-FITC (X-axis) e
PI (Y-axis). Cells in the upper-left quadrant represent nuclear debris (Q1); upper-right quadrant, late apoptotic cells (Q2); lower-right quadrant, early apoptotic cells (Q3);
lower-left quadrant, viable cells (Q4).
700 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706

Fig. 3. Effect of L. ferrea extracts on HT-29 and HEK-293 cells, after treatment for 48 h, evaluated by flow cytometry. (A) Control (untreated cells); (B) 40T  25 mg/mL; (C) 40T
 50 mg/mL; (D) 60T  25 mg/mL; (E) 60T  50 mg/mL; (F) 80T  25 mg/mL; (G) 80T  50 mg/mL; (H) Cisplatin  50 mM. Dot plots are displayed with Annexin V-FITC (X-axis) e
PI (Y-axis). Cells in the upper-left quadrant represent nuclear debris (Q1); upper-right quadrant, late apoptotic cells (Q2); lower-right quadrant, early apoptotic cells (Q3);
lower-left quadrant, viable cells (Q4).

homogenized with a 20 mM Tris-HCl buffer (Trizma HCl, Sigma-
Aldrich, SP, Brazil) for trituration. After this process, they were
centrifuged for 20 min in 3000 rpm at 4
C. Afterwards, the
chromogenic reagent (10.3 mM 1-methyl-2-phenylindole in 3:1
acetonitrile), and a 37% solution of HCl were added to each 150 mL
of supernatant sample. After an incubation step in a water bath for
40 min at 45
C, the samples were centrifuged at 3000 rpm at 4
C.
The absorbance was measured at 586 nm, and the results were
expressed as nmol/106 cells.
2.8. Real time-qPCR
Total RNA was extracted from the HT-29 cells treated for 24 h
with extracts 40T and 60T (25 mg/mL) with the Trizol reagent
(Invitrogen, CA, USA) and the SV Total RNA Isolation System
(Promega, WI, USA) according to the manufacturer’s guidelines.
First-strand cDNA was synthesized from 1 mg of total RNA with the
ImProm-IITM Reverse Transcriptase System (Promega, WI, USA)
and Real time-qPCR was then performed for the quantitative
 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
analysis of messenger RNA expression (mRNA) with SYBR Green
Mix at Applied Biosystems 7500 FAST system (Applied Biosystems,
CA, USA), according to the standard protocol, using the following
primers (Integrated DNA Technologies, IA, USA): AKT (forward: 50
TCA CCT CTG AGA CCG ACA CC 30 ; reverse: 50 ACT GGC TGA GTA GGA
GAA CTG G 30 , annealing primer temperature: 58.3
C), Apaf-1
(forward: 50 CCT CTC ATT TGC TGA TGT CG 30 ; reverse: 50 TCA CTG
CAG ATT TTC ACC AGA 30 , annealing primer temperature: 56.9
C) e
EGFR (forward: 50 TGA TAG ACG CAG ATA GTC GCC 30 ; reverse: 50
TCA GGG CAC GGT AGA AGT TG 30 , annealing primer temperature:
56.9
C). The mean Ct values were used to calculate the relative
expression of the target gene levels for the cells treated with the
extracts, relative to those untreated cells control. The expression
data were normalized to the b-actin gene using the formula 2-
DDCt [35].
2.9. Statistical analysis
All experiments were performed in triplicate. The significance
of the differences between the groups was obtained through
analysis of variance (ANOVA), and the Bonferroni test (significance
level of p < 0.05), with GraphPad Prism software version 7.0
(GraphPad Software Inc., CA, USA).
3. Results
3.1. Cell viability
To assess the antiproliferative and anticancer potential of the
extracts of Libidibia ferrea, HT-29 and HEK-293 cells were treated
and evaluated at the 24 h and 48 h periods. As observed in Fig. 1,
during the first 24 h in HT-29 cell line, inhibition of tumor cell
Fig. 4. Representation of apoptotic cells concentration treated by the extracts of L. ferrea at 24 h and 48 h times. Left-side: HT-29 cells; Right-side: HEK-293 cells.
701
proliferation was observed, with significant action of the 40T, 60T
and 80T crude extracts. The 40T extract had levels of cell
proliferation inhibition varying between 15% and 25% between
doses 25 mg/mL at 100 mg/mL, whereas for the 60T and 80T
extracts these levels were higher ranging from 25% to about 50%
(50% inhibition of proliferation at the dose 25 mg/mL in 60T and
43.7% at the dose of 12.5 mg/mL in 80T). In the same time period,
the non-tumor cell line HEK-293 showed no cell proliferation
inhibition, and the cells exposed to the extracts proliferated in
general at rates higher than the untreated cells control (with one
exception: a decrease of 15% at 25 mg/mL in 60T). At 48 h, there was
a certain reduction in the proliferation inhibitory effect on the
tumor cells, mainly in the 40T and 60T extracts, but the 80T extract
still presented proliferation inhibition percentages close to the
previous period, ranging from 21.7% to 48.7% between the doses
tested. In the non-tumoral lineage, proliferation was observed as
either close to or greater than the control. Because the half
maximal inhibitory concentration (IC50) on proliferation of the
HT-29 cell line was found in the concentration range of 25 mg/mL
at 50 mg/mL of the extracts of L. ferrea, those concentrations were
selected for the flow cytometry assay.
3.2. Cell death evaluation
For evaluation of cell line deaths after treatment with L. ferrea
extracts, an assay with flow cytometry was performed by double
labeling with Annexin V  FITC and Propidium Iodide (PI). The
selected extracts were the most effective in the cell viability
screening (40T, 60T and 80T), and at the intermediate doses (25 mg/
mL and 50 mg/mL). In addition, the antineoplastic agent cisplatin
(50 mM) was used as the standard drug and tested for comparison
purposes.
702 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
In Fig. 2 we observe that during the time period of 24 h from
treatment, all extracts caused cell death by apoptosis in HT-29
tumor cells, and most of the apoptotic cells were found in the initial
stage. Among the crude extracts, the 40T at 25 mg/mL presented a
higher percentage of cells in apoptosis (38.7%), surpassing even
cisplatin, which presented 33.4% of cells in apoptosis. In the non-
tumoral lineage HEK-293, there was no statistical difference
between the percentages of cells in apoptosis presented by the
controls as compared to the extracts. Under the same conditions,
the cisplatin antineoplastic presented a significant rate of
apoptotic cells (49%).
According to Fig. 3, at the 48 h time of treatment in the HT-29
line, half of the extracts tested provoked an increase in the
percentage of cells in the apoptosis process in comparison to the
previous period, varying between them at 22.4% for 60T at the
25 mg/mL dose, 3.77% for 60T at 50 mg/mL, and 10.79% for 80T at
25 mg/mL. Cisplatin also increased apoptotic cell rates over time
(up 28.9%). Of the cells in apoptosis, most were in the early stages.
In the non-tumor cell line HEK-293, all extracts showed concen-
trations of viable cells close to or above the control, whereas
cisplatin presented viable cells at 43.6%, and an expressive number
of cells in apoptosis (54.6%).
The results are most clearly observed in Fig. 4, where they were
separated by percentages of cells that were in early apoptosis and
late apoptosis in the HT-29 cells and total apoptosis in the HEK-293
cells.
Fig. 5. Detection of caspase-3 of HT-29 under the effect of L. ferrea extracts in 24 h and 48 h times, evaluated by immunofluorescence, with contrast index. (A) Control
(untreated cells); (B) Cisplatin  50 mM; (C) 40T  25 mg/mL; (D) 60T  25 mg/mL.
3.3. Apoptosis activation analyses
Activation of important targets of the apoptotic pathway
(caspase-3 and Bcl-2) in the HT-29 tumor line, treated with the
crude extracts 40T and 60T was performed to study the
mechanisms related to the presented apoptosis induction;
25 mg/mL dosage, for 24 h and 48 h (chosen because of better
performance in the previous result). The antineoplastic cisplatin
was also used for comparison. Representative images are shown in
Fig. 5 (caspase-3), and Fig. 6 (Bcl-2).
An intense positive labeling of the effector protease of apoptosis
caspase-3 is observed in tumor cells treated with extracts of L.
ferrea, as well as with the antineoplastic cisplatin, in both
experiment times, indicating that the cell death provoked is
mediated by an apoptotic process. Regarding the anti-apoptotic
protein Bcl-2, lower expression was observed for treated cells than
for the controls, in the same periods of time.
3.4. GSH and MDA dosages
It is highlighted in Fig. 7 that glutathione (GSH) levels, an
important marker of antioxidant activity, increased by 18% in
relation to the control in extract EF60T at 25 mg/mL, which was the
sample presenting higher dosages of this protein in the HT-29
tumoral lineage. Regarding extracts EF40T and EF60T at the highest
 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
Fig. 6. Detection of Bcl-2 of HT-29 under the effect of L. ferrea extracts in 24 h and 48 h times, evaluated by immunofluorescence, with contrast index. (A) Control (untreated
cells); (B) Cisplatin  50 mM; (C) 40T  25 mg/mL; (D) 60T  25 mg/mL.
dose (50 mg/mL), they presented reduced levels, and we did not
find the same effect. Regarding the malondialdehyde oxidative
stress marker (MDA), we observed in Fig. 8 that all of the extracts at
all doses were able to significantly reduce the levels of the protein,
thus indicating a protective effect on lipid peroxidation in the HT-
29 tumor line, being higher than cisplatin.
3.5. mRNA expression
Based on Fig. 9, we see that the AKT gene that is involved in cell
survival and proliferation had a slightly reduced expression in the
treatment with EF40T at the extract dose of 25 mg/mL (4%); extract
EF60T with the same dose yielded (26%), a rate still lower than that
found with the antineoplastic cisplatin (49%). The Apaf-1
(apoptotic peptidase activating factor 1) gene encoding a cyto-
plasmic protein that initiates apoptotic events in the intrinsic
pathway was found to be reduced in all treatments. Epidermal
growth factor receptor (EGFR), involved in the pathogenesis and
progression of different carcinomas, showed a reduction in
expression as compared to the control: 29% for the EF40T extract,
48% for the EF60T extract and 53% for cisplatin.
703
4. Discussion
In addition to expanding medical-pharmacological care in
public health, the study of medicinal plants is important for
acquiring knowledge about the pharmacological potential of a
native plant diversity that remains under exploited, and helps to
ensure its rational exploitation [13]. The versatility, applicability
and therapeutic safety presented by caatinga plants have attracted
great attention to them in the search for new drugs [36]. Popular
species such as Libidibia ferrea Martius L. P. Queiroz require ethno-
pharmacological approaches, and these have so far remained
scarce, especially regarding anticancer activity.
Crude and fractionated extracts obtained from the fruits of L.
ferrea tested by Freitas et al. [37] on human cancer cell lines NCI-
H292 (mucoepidermoid carcinoma of the lung), HEP-2 (squamous
cell carcinoma of the larynx), and solid tumor sarcoma 180
presented no significant antitumor activity or inhibition of cell
proliferation. In the present study, for the HT-29 tumoral lineage
(human colorectal adenocarcinoma), proliferation inhibition by
the 40T, 60T and 80T crude extracts was significant in the first
observed hours. In addition, during the same time period, the same
extracts did not generally present toxicity in the non-tumor cell
line HEK-293 (human embryonic renal cell).
704 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
Fig. 7. Effect of L. ferrea extracts on total glutathione (GSH) levels of HT-29 and HEK-
293 cells. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control. CTRL (untreated
cells), CIS (cisplatin), 40T (crude extract 40T), 60T (60T crude extract).
Alterations to apoptosis death process related pathways and
consequent cell resistance to this mechanism are the main factors
responsible for the onset of tumorigenesis, considered one of the
fundamental hallmarks of cancer [38]. Despite causing the
problem, apoptosis plays an important role in cancer treatment
strategies through the eradication of transformed cells and
resistance to conventional treatments [39].
Induction of apoptosis by L. ferrea has been described previously
by Nozaki et al. [40], using acetone stem extract in human acute
myeloid leukemia (HL-60) lineage. In this work, all extracts from
the L. ferrea fruit tested in the HT-29 line induced cell death by
apoptosis, corroborating their results.
The apoptotic activity exhibited in the present study has a
profile suggestive of affecting the intrinsic apoptosis pathway, a
common target of most anticancer agents [41], through activation
of caspase-3 and reduction of Bcl-2 levels. This pathway results
from increased mitochondrial membrane permeability (with
Fig. 8. Effect of L. ferrea extracts on malondialdehyde (MDA) levels of HT-29 and
HEK-293 cells. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control. CTRL (untreated
cells), CIS (cisplatin), 40T (crude extract 40T), 60T (crude extract 60T).
inhibition of anti-apoptotic regulator Bcl-2), and the release of
pro-apoptogenic factors in the cytoplasm, promoting the activa-
tion of effector enzymes (such as caspase-3), which act on a broad
spectrum of substrates, resulting in cell death [42].
Most of the apoptotic cells in this work were detected in the
initial stage, characterized by phosphatidylserine exposure (an
event that precedes cell membrane permeabilization), cellular
shrinkage, and nuclear condensation. In addition, the collapse of
the mitochondrial transmembrane potential has also been
reported as an early event in the apoptotic process, being present
in many cell types in apoptosis that culminate in the release of
several apoptotic proteins, independently of the stimulus [43].
Therapies for colorectal cancer designed to stimulate apoptosis, as
presented here, play a critical role in controlling its development
and progression, as well as improving response to chemotherapy
and radiation [44].
To assess antioxidant status, dosages were performed for
analysis of GSH (glutathione) and MDA (malondialdehyde) levels.
We observed that the EF60T extract, at the lowest dose tested
presented a significant increase in the level of GSH, an intracellular
peptide that exerts, among other functions, detoxification,
elimination of free radicals, maintenance of the thiol state, and
cellular proliferation modulation [45]. As for the MDA lipid
peroxidation marker, all of the extracts at all doses presented
reductions, which is an important sign for prevention of MDA’s
mutagenic and genotoxic activities in tumor cells [46]. All in all, the
extract stands out for presenting potential antioxidant activity. The
antioxidant activities of L. ferrea fruits were first described by Silva
et al. [47] through several in vitro assays. Later, Barros et al. [19] also
described augmented antioxidant activity in addition to hepato-
protection in mice.
The expression of certain relevant genes to tumor signaling
machinery was evaluated in this work through identification of
mRNA-targets (EGFR, AKT and APAF-1). We found that levels of the
EGFR receptor (involved in growth, invasion and metastasis of
epithelial tumors and particularly important in the development of
colorectal tumors) [48] were reduced after treatment with the
extracts, indicating that L. ferrea interferes with this pathway and is
effective in tumor inhibition.
Expression of AKT was varied, with reduced expression for
EF40T and increased expression for EF60T. Interestingly, some
Fig. 9. Effect of L. ferrea extracts on the expression of AKT, Apaf-1 and EGFR mRNAs
in HT-29. *P < 0.05, **P < 0,01 e ***P<0.001 versus control. CTRL (untreated cells),
CIS (cisplatin), 40T (40T crude extract), 60T (60T crude extract).
 A.C.V.A. Guerra et al. / Biomedicine & Pharmacotherapy 92 (2017) 696–706
studies have shown that AKT is not a single-function kinase and
may facilitate, rather than inhibit, cell death under certain
conditions [49], thus being involved both in inducing death and
cell survival. The same scenario of opposite functions in the same
protein may also involve Apaf-1 factor, whose expression was
reduced in cells treated with L. ferrea. Although this protein is
known for its apoptotic role in the intrinsic pathway, recent work
has suggested additional non-apoptotic functions including a pro-
survival role that needs to be better investigated [50].
Through various mechanisms, cancer itself, through induction
of pro-survival signals such as the AKT activation pathway
bypasses activation of the intrinsic pathway as in blockade of
apoptosome formation and consequently of Apaf-1. Thus, strate-
gies to bypass resistance, through therapeutic restoration of
apoptotic pathways as conducted by interactions with the tumor
microenvironment, provide new promise for cancer patients in
clinical treatment [51]. Therefore, the differing expression profiles
presented in this study from the extracts of L. ferrea may be related
to different activities in the mediation of important pathways in
tumor development, that culminate in cell death stimuli, as already
verified in the previous results.
Mediation of the AKT pathway, in addition to influencing
apoptosis induction impacts oxidization capacity. Ramos et al. [52]
showed that apoptosis caused by treatment with low concen-
trations of arsenic trioxide in human myelogenous leukemia cells
(HL-60) was potentiated by the use of AKT pathway inhibitors,
which also led to the reduction of levels of GSH causing
intracellular oxidation. The results demonstrated that AKT
signaling pathway activity is necessary to maintain normal GSH
metabolism in cells. We observed in the current study that there is
also synchronization between AKT expression levels and intracel-
lular GSH levels: increased AKT expression is accompanied by
increased GSH and vice versa.
The main constituents found in the L. ferrea extracts tested were
the hydrolyzable tannins: gallic acid (GA) and ellagic acid (EA) (30).
GA is a phenolic compound naturally present in a wide variety of
fruits and plants exhibiting various pharmacological activities such
as antimicrobial, antitumor, anti-inflammatory, antioxidant, anti-
depressant, antidiabetic, anxiolytic, and others [53]. The acid is
associated to inhibition in various cancer cell lines including
human colorectal carcinoma cells through apoptosis induction by
different mechanisms such as regulation of apoptotic and anti-
apoptotic proteins [54]. It is also well known for its efficient
protection against oxidative damage, maintenance of endogenous
defense, and chiefly, prevention of lipid peroxidation [55].
EA is a dimeric derivative of gallic acid present in the plant
vacuole and, like other polyphenols; it has a wide range of
biological activities such as antioxidant, anti-inflammatory,
prebiotic and anticancer functions, and the latter by selectively
inhibiting growth in differring tumor types, including inducing
apoptosis in human colon adenocarcinoma cells via the mitochon-
drial pathway [56].
Among the results reported, it is important to highlight the
protective effect of L. ferrea extracts on non-tumor HEK-293 cells,
which presented high rates of cell proliferation and viable cells.In
vitro evaluations by Nakamura et al. [57] have demonstrated the
chemopreventive effects of L. ferrea through the presence of ellagic
acid in its composition: three adjacent hydroxyl phenolic groups
were responsible for cytotoxicity, and the carboxyl group appeared
to be implicated in the distinction between normal and tumor
cells. These results are consistent with studies by Araújo Júnior
et al. [58,59], in the sense that many plant derivatives may act both
in inhibiting the proliferation and viability of tumor cells, and in
mediating a protective effect on healthy cells.
705
5. Conclusions
The present results demonstrate that L. ferrea has antiprolifer-
ative activity in HT-29 cells with induction of apoptosis in
association with mitochondrial effects in the intrinsic pathway
(via caspase-3 activation, negative regulation of Bcl-2 and
Reduction of Apaf-1), this as well as acting in the expression of
important targets in the process of colorectal cancer development
such as EGFR and AKT, with probable tumor inhibition. L. ferrea also
has antioxidant and lipid peroxidation inhibiting effects, together
with chemopreventive action in healthy renal cells. Taken as such,
L. ferrea is a promising candidate for cancer therapy with both
efficacy and selectivity as potential characteristics.
Acknowledgments
The authors thank the Pharmacognosy Laboratory of Pernam-
buco Federal University (UFPE); the cell culture room of the
Department of Biochemistry, the Health Sciences Graduate
Program and the Brain Institute Microscopy Laboratory at Rio
Grande do Norte Federal University (UFRN).
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