Biomedicine & Pharmacotherapy 89 (2017) 772–780

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Effect of diterpenoid kaurenoic acid on genotoxicity and cell cycle

progression in gastric cancer cell lines
Plínio Cerqueira dos Santos Cardosoa,1, Carlos Alberto Machado da Rochab,* ,1,
Mariana Ferreira Lealc , Marcelo de Oliveira Bahiaa , Diego Di Felipe Ávila Alcântaraa ,
Raquel Alves dos Santosd, Natália dos Santos Gonçalvesd, Sérgio Ricardo Ambrósioe ,
Bruno Coêlho Cavalcantif , Caroline Aquino Moreira-Nunesa,g , Claudia do Ó Pessoaf ,
Rommel Mário Rodríguez Burbanoa,h,**
a
Human Cytogenetic Laboratory, Biological Science Institute, Federal University of Pará (UFPA), Belém, Pará, Brazil
b
Federal Institute of Education, Science and Technology of Pará (IFPA), Av. Almirante Barroso, 1155 (Marco), CEP 66093-020, Belém, Pará, Brazil
c
Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP), São Paulo, São Paulo, Brazil
d
Laboratory of Genetics and Molecular Biology, University of Franca (UNIFRAN), Franca, São Paulo, Brazil
e
Laboratory of Biotransformation, University of Franca (UNIFRAN), Franca, São Paulo, Brazil
f
Department of Physiology and Pharmacology, Federal University of Ceará (UFC), Fortaleza, Ceará, Brazil
g
Laboratory of Genetics of Hemoglobinopathies and Hematologic Diseases, Federal University of Ceará (UFC), Fortaleza, Ceará, Brazil
h
Hospital Ophir Loyola (HOL), Belém, Pará, Brazil

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

Article history:
Received 24 November 2016 The goal of our study was to evaluate the effect of kaurenoic acid, obtained from copaiba oil resin, in
Received in revised form 22 February 2017 gastric cancer (GC) and a normal mucosa of stomach (MNP01) cell lines. The compound was tested at
Accepted 22 February 2017 concentrations of 2.5, 5, 10, 30 and 60 mg/mL. Comet and micronucleus assays were used to access its
potential genotoxicity in vitro. Moreover, we evaluated the effect of kaurenoic acid in cell cycle
Keywords: progression and in the transcription of genes involved in the control of the cell cycle: MYC, CCND1, BCL2,
Cell cycle progression CASP3, ATM, CHK2 and TP53. Kaurenoic acid induced an increase on cell DNA damage or micronucleus
Comet assay frequencies on GC cell lines in a dose-dependent manner. The GC and MNP01 cell lines entering DNA
Gastric cancer cell lines
synthesis and mitosis decreased significantly with kaurenoic acid treatment, and had an increased
Gene transcriptions
growth phase compared with non-treated cells. The treatment induced apoptosis (or necrosis) even at a
Kaurenoic acid
Micronuclei concentration of 2.5 mg/mL in relation to non-treated cells. GC cell lines presented reduced MYC, CCND1,
BCL2 and CASP3 transcription while ATM, CHK2 and TP53 increased in transcription in relation to non-
treated cells, especially at a concentration above 10 mg/mL. The gene transcription in the MNP01 (non-
treated non-cancer cell line) was designated as a calibrator for all the GC cell lines. In conclusion, our
results showed that kaurenoic acid obtained from Copaifera induces DNA damage and increases the
micronuclei frequency in a dose-dependent manner in GC cells, with a significant genotoxicity observed
above the concentration of 5 mg/mL. Moreover, this compound seems to be able to induce cell cycle arrest
and apoptosis in GC cells.
© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction

Gastric cancer (GC) remains the third leading cause of cancer-

related mortality worldwide, and invasion and metastasis of
gastric cancer represents the major reason for its poor prognosis
* Corresponding author.
[1]. Multimodal therapies have proven to benefit patients with
** Corresponding author at: Hospital Ophir Loyola (HOL), Av. Magalhães Barata, cancer. However, without curative surgery, variations and combi-
992, CEP 66060-281, Belém, Pará, Brazil. nations of chemotherapy and/or radiation cannot bring clinically
E-mail addresses: carlos.rocha@ifpa.edu.br (C.A.M.d. Rocha), rommel@ufpa.br meaningful success nowadays [2].
(R.M.R. Burbano).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.biopha.2017.02.085
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780
One of the main problems in cancer treatment is gradual
resistance of cancer cells against treatment [3]. Therefore, the aim
of immunopharmacological studies has been to improve cancer
treatment results [4]. The alternate solution for the harmful effects
of synthetic agents is the use of phytotherapy [5]. Bioactive
compounds from plants have a lot of biological activities and may
present great potential in the development of new drugs for the
treatment of human diseases [6].
Reports about medicinal plants in the Brazilian Amazonian
region have been documented in the literature, especially
concerning the oil resin extracted from the trunk of Copaifera
langsdorffii Desfon (Leguminosae) [7,8]. This oil resin is a reputed
folk remedy used by the natives of Brazil for the treatment of
several diseases such as sore throat, urinary and pulmonary
infections, and to hasten ulcer and wound healing [9]. Usually, this
oil resin is administered orally in natura, or applied in ointment
form [10–12]. This substance has great social and economic
representation in the Amazon region, since it represents about 95%
of the entire oil resin production countrywide [13].
Several studies have demonstrated that the Copaifera oil resin
has manifold therapeutic properties, including anti-inflammatory,
antitumoral, antimelanoma, antiulcerogenic, antilipoperoxidation
and antioxidant [10,12,14–16]. Furthermore, the diterpenes,
kaurenoic and polyaltic acids present in the pure oil seem to
present healing and anti-inflammatory properties [7,8].
Kaurenoic acid (ent-kaur-16-en-19-oic acid) (Fig. 1) is classified
as a diterpene and considered an intermediate of the gibberellin
plant growth hormone biogenesis [17]. Some studies showed that
kaurenoic acid presents in vitro anti-parasitic and anti-microbial
activities [18–23]. Moreover, this diterpene has anti-proliferative
action in CEM leukemic cells, MCF-7 breast and HCT-8 colon cancer
cells [24]. Additionally, kaurenoic acid is also able to reduce human
sperm motility when directly tested in sperm samples, but was
only weakly spermicidal [25].
While Copaifera oil resin has its herbal properties widely
reported, there are few studies in the scientific literature that
evaluated the effects of this oil resin components, especially in GC
cell lines. Thus, the present study aims to assess the genotoxicity of
kaurenoic acid and the effect of this compound in the cell cycle and
in the transcription of genes involved, among other functions, in
GC cell lines.
2. Materials and methods
2.1. General experimental procedures
The ACP02, ACP03 and AGP01 cell lines, previously established
and characterized by our research group, were used in the present
study [26,27]. The ACP02 cell line was established from a diffuse-
type GC, and ACP03 and AGP01 were from an intestinal-type GC.
ACP03 is able to start a tumorigenesis process in Cebus paella [28].
We also used the GC cell line PG100 obtained from Rio de Janeiro
Fig. 1. Molecular structure of kaurenoic acid (ent-kaur-16-en-19-oic acid).
773
Cell Bank, Brazil, that was previously cytogeneticly characterized
by our group [29].
Cells lines were cultivated under standard conditions in
Modified Eagle’s medium salts, supplemented with 10% fetal
bovine serum, 2 mM L-glutamine and antibiotics (100 IU/mL
penicillin and 100 mg/mL streptomycin [30]). Cells were main-
tained in 25 cm2 tissue culture flasks (TPP, Trasadingen,
Switzerland) at 37  C in a humidified atmosphere containing 5%
CO2 and were harvested by treatment with 0.15% trypsin and 0.08%
EDTA in phosphate-buffered saline (PBS). Cells (3  105) were
seeded in 5 mL of complete medium and grown for 2 days prior to
treatment with the test substance.
Cells were treated with different concentrations of kaurenoic
acid (2.5, 5, 10, 30 and 60 mg/mL) for 3 h. After this, the cells were
washed with ice-cold PBS and trypsinized with 100 mL trypsin
(0.15%) and were resuspended in complete medium. The final
concentration of DMSO in the culture medium was kept constant,
below 0.1% (v/v). The negative control cell cultures were not
exposed to kaurenoic acid, while the methyl methanesulfonate
(Sigma Aldrich Co, St. Louis, MO, USA) was used as a positive
control (4  10 5 M). All cell treatments were carried out with
three replicates.
2.2. Chemical isolation
In this study, kaurenoic acid (CAS 6730-83-2) (Sigma Chemical
Co., St. Louis, MO, USA) was diluted under sterile conditions in
dimethyl sulfoxide (DMSO) and the concentrations used in this
study were based on preliminary cytotoxicity assays performed by
our research group (unpublished data). Kaurenoic acid was
obtained from the oil resin of C. langsdorffii that grows abundantly
in the Amazon region of Northern Brazil [31].
2.3. Genotoxicity assay
The Comet assay (alkaline version) was performed as described
by Singh et al. [32] with some modifications [33,34]. After cell
treatment, the cells were washed with ice-cold PBS and trypsinized
with 100 mL trypsin (0.15%) and were resuspended in complete
medium. An aliquot (450 mL) of the cell suspension from each
experimental group was taken and centrifuged at 1.000 rpm for
5 min in a microcentrifuge (Eppendorf). The resulting pellet was
homogenized with 300 mL of a low melting point agarose (0.8%),
spread onto microscope slides pre-coated with a normal melting
point agarose (1.5%) and covered with a coverslip. After 5 min at
4  C, the cover slip was removed and the slides were immersed in
cold lysis solution (2.5 M NaCl; 100 mM EDTA; 10 mM Tris, 10%
DMSO and 1% Triton-X, pH: 10) for one week. After lysis, the slides
were placed in an electrophoresis chamber and covered with
freshly made electrophoresis buffer (300 mM NaOH; 1 mM EDTA,
pH > 13).
The electrophoresis was run for 25 min (34 V and 300 mA).
Afterward, the slides were neutralized by submersion in distilled
water (4  C) for 5 min and fixed in 100% ethanol for 3 min. Staining
of the slides was performed immediately before the analyses using
ethidium bromide dye (20 mg/mL). Slides were prepared in
duplicate, and 100 cells were analyzed per sample (50 cells from
each slide) using a fluorescent microscope (Olympus BX41) at 40
magnification.
The damage index (DI) is based on the length of migration and
the amount of DNA in the tail. It is considered a sensitive measure
of DNA. Five categories (0–4) were used here: class 0 (no damage),
class 1 (slight damage with a tail length shorter than the diameter
of the nucleus), class 2 (medium damage with a tail length one or
two times the diameter of the nucleus), class 3 (significant damage
with a tail length greater than two times the diameter of the
774 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780

Table 1
Summary of Seven Target Genes and the Reference Gene.

Gene Name gene function assay*

symbol
MYC v-myc avian myelocytomatosis viral progression of cell cycle progression, apoptosis and cellular transformation/oncogene hs00153408_m1
oncogene homolog
CCND1 cyclin D1 positive regulation of mitotic cell cycle/oncogene hs00765553_m1
BCL2 b-cell cll/lymphoma 2 anti-apoptotic/oncogene hs00608023_m1
CASP3 caspase 3, apoptosis-related cysteine pro-apoptotic hs00234387_m1
peptidase
ATM atm serine/threonine kinase cell cycle arrest in response to DNA damage and for genome stability/tumor supressor hs01112355_g1
CHK2 checkpoint kinase 2 cell cycle checkpoint regulator in response to DNA damage and to replication blocks/ hs00200485_m1
putative tumor suppressor
TP53 tumor protein p53 induction of cell cycle arrest, apoptosis, senescence, DNA repair, or changes in hs01034249_m1
metabolism/tumor supressor
*
TaqMan probes were purchased as assays-on-demand products for gene expression (Life Technologies, USA).

nucleus) and class 4 (significant damage with tail length greater
than three times the diameter of the nucleus). Damaged index thus
ranged from 0 (no damage: 100 cells  0) to 4 (completely
damaged: 100 cells  4) [35,36].
2.4. Mutagenic assay
The micronucleus assay was performed according to Fenech
[37]. Three hours after treatment, the cultures were washed twice
with the medium and Cytochalasin B (Sigma Chemical Co., St.
Louis, MO, USA) was added at a final concentration of 2 mg/mL.
Cultures were harvested 21 h after Cytochalasin B addition. The
cells were separated from the bottle by trypsinization and the
cellular suspension was centrifuged at 1000 rpm for 5 min. Then,
cells were resuspended in a KCl 0.075 M solution maintained at
4  C for 3 min (mild hypotonic treatment). Cell cultures were
harvested 72 h after the incubation, centrifuged (5 min at 800 rpm)
and submitted to hypotonic solution (KCL 0.075 M). Afterward, GC
cell lines were washed once with 5 mL of a cold methanol:acetic
acid (5:1) (v/v) fixative and washed again with 5 mL of a cold
methanol:acetic acid (3:1) (v/v) fixative. The cell suspension was
dropped onto slides and stained in a solution of 5% Giemsa dye
(Sigma Chemical Co., St. Louis, MO, USA) in a phosphate buffer (pH
6.8) for 5 min. Micronuclei were scored in 2000 binucleated cells
using the criteria according to Fenech [37]. The analysis was
performed using an optical microscope (BIOVAL L2000A) at 100
magnification.
2.5. Cell cycle analysis
Cell cycle distribution followed the procedure described by
Nicoletti et al. [38]. GC lines were incubated at 37  C for 3 h in the
dark in a lysis solution containing 0.1%, citrate, 0.1% triton X-100
and 50 mg/mL propidium iodide. Cell fluorescence was determined
by flow cytometry in a Guava1 EasyCyteTM Mini System cytometer
(Guava Technologies Inc., Hayward, CA, USA), using CytoSoft 4.1
software.
2.6. Genes transcription analysis
We analyzed the transcription of genes involved in the cell cycle
regulation to evaluate the effect of kaurenoic acid treatment in
ACP02, ACP03, AGP01 and PG100 cell lines (Table 1). Total mRNA
was isolated from all cell line samples using Trizol Reagent
(Invitrogen, Carlsbad, CA, EUA). The extracted RNA was purified
using RNeasy Mini Kit (Qiagen, Valencia, CA, EUA), according to the
recommended protocol. The concentration and quality were
determined using a NanoDrop spectrophotometer (Kisker,
Germany) and 1% agarose gels, respectively. Samples were stored
at 80  C until use.
RNA was reverse-transcribed using the High-Capacity cDNA
Archive kit according to the manufacturer’s protocol (Life
Technologies, Pittsburgh, PA, USA). Complementary DNA was then
amplified by real-time reverse transcription quantitative PCR (RT-
qPCR) using TaqMan probes purchased as Assays-on-demand
Products for Gene Expression (Life Technologies, Pittsburgh, PA,
USA; Table 1) and a 7500 Fast Real-Time PCR instrument (Life
Technologies, Pittsburgh, PA, USA). The GAPDH gene was selected
as an internal control for RNA input and reverse-transcription
efficiency. All RT-qPCRs were performed in triplicate for both the
target genes and the internal control. The relative quantification of
gene transcription was calculated according to the manufacturer’s
software (Life Technologies, Pittsburgh, PA, USA). The gene

Fig. 2. Genotoxicity of kaurenoic acid in gastric cancer cell line. A) Damage index by comet assay; B) micronuclei frequency. DI: damage index; MN: micronuclei frequency. C-:
control, which were non-treated cells; C+: cells treated with methylmethanesulfonateon * Significantly different from the control (p < 0.05; by Kruskal-Wallis test followed by
Games-Howell post-hoc analysis). ** Significantly different from the control (p  0.001; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis).
 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780 775

Fig. 3. Effect of kaurenoic acid on the score of DNA damage by comet assay in gastric cancer cell lines. The DNA damage was classified based on the length of migration and on
the amount of DNA in the comet tail. Class 0: no damage; Class 1: little damage with a tail length shorter than diameter of the nucleus; Class 2: medium damage with a tail
length one or two times the diameter of the nucleus; Class 3: significant damage with a tail length greater than two times the diameter of the nucleus; Class 4: significant
damage with tail length greater than three times the diameter of the nucleus; C-: control, which were non-treated cells; C+: cells treated with methylmethanesulfonateon *
Significantly different from the negative control (p < 0.05; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis). ** Significantly different from the control
(p  0.001; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis).

transcription in the MNP01 (non-treated non-cancer cell line) was concentration and genotoxicty or gene transcription. A p-value
designated as a calibrator for all the GC cell lines. less than 0.05 was considered significant [39].

2.7. Statistical analysis 3. Results and discussion

The data are shown as the as the mean  standard deviation
(SD). The Shapiro-Wilk test was used to evaluate the data
distribution and to determine the appropriate subsequent test
for statistical comparisons. The Kruskal-Wallis test followed by
Games-Howell post-hoc test for multiple comparisons was used to
compare the studied groups. Spearman correlation test was used to
evaluate the possible correlation between kaurenoic acid
GC cell lines treated with methyl methanesulphonate presented
a significant increase in the DNA damage index when compared
with control non-treated cells [333.5 (13.5) versus 25.5 (7.5),
p < 0.001; Fig. 2A]. Similar phenomenon was repeated for normal
cells MNP01 (356 versus 22.3, p < 0.001). More than 70% of GC cells
treated with this drug presented comet class 4, which was detected
in less than 2% of non-treated cells (Fig. 3).

Fig. 4. Genotoxicity of kaurenoic acid by comet assay in each studied gastric cancer cell line. A) ACP02; B) ACP03; C) AGP01; D) PG100. DI: damage index by comet assay. C-:
control, which were non-treated cells; C+: cells treated with methylmethanesulfonateon * Significantly different from the control (p < 0.05; by Kruskal-Wallis test followed by
Games-Howell post-hoc analysis). ** Significantly different from the control (p  0.001; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis).
776 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780

Kaurenoic acid induced DNA damage in a dose-dependent
manner (Fig. 2). At concentrations above 5 mg/mL, kaurenoic acid
induced a significant increase in the DNA damage index when
compared with control values: 47 (25.75; p = 0.001), 47 (42.75;
p = 0.006), 173 (69; p < 0.001), 205 (43; p < 0.001) for 5, 10, 30 or
60 mg/mL, respectively (Fig. 2A). Kaurenoic acid induced DNA
damage classified as comet class 4 (Fig. 3); however, less than 30%
of cells present comet class 4.
Moreover, the methyl methanesulphonate treatment also
induced a significant increase of micronuclei compared with the
control [96.5 (12) versus 20.5 (8), p < 0.001]. Additionally, GC
cell lines presented a significant increase of the frequency of
micronuclei when treated with kaurenoic acid concentrations
above 5 mg/mL: 30.5 (7.25; p = 0.004), 37 (8; p < 0.001), 53
(19; p < 0.001) and 74.5 (15.75; p < 0.001), respectively
(Fig. 2B).
The kaurenoic acid concentration treatment was strongly
correlated with the DNA damage index (rS = 0.912, p < 0.001) and
the micronuclei frequency (rS = 0.925, p < 0.001). A similar pattern
was observed in response to the kaurenoic acid treatment in each
cell line (Figs. 4 and 5).
Genotoxic events are considered crucial steps in the progres-
sion of cancer. In this study, we performed comet assay and
micronucleus tests in four GC cell lines treated with different
concentrations of kaurenoic acid. To our knowledge, no previous
study evaluated the effect of this compound in GC cell lines. Here,
we observed that kaurenoic acid has a genotoxic potential
especially at higher concentrations, evidenced by increased
frequencies of both micronuclei and DNA damage. Kaurenoic acid
induced DNA damage in a dose-dependent manner. Our current
findings in GC cell lines are in part in agreement with findings
previously observed in Chinese hamster lung fibroblast cells (V79
cells) treated with kaurenoic acid. Therefore, this compound may
act as a DNA intercalating agent [31], which induces chromosomic
alterations and DNA breaks in mammalian cells [40].
Few studies have evaluated the effect of kaurenoic acid.
However, the genotoxic effect of diterpens was previously
reported. Using the comet assay, Kato et al. [41] showed that a
pimarane-type diterpene, pimaradienoic acid, induced DNA
damage at concentrations of 2.5 and 5.0 mg/mL in V79 cells.
Moreover, DNA damage was also reported in hepatocytes of Swiss
mice treated with 80 mg/kg of this diterpene.
Paclitaxel (Taxol1) is a diterpenoid clinically effective antineo-
plastic drug that has been used for the treatment of several human
cancers, including GC [42]. This diterpen suppress the microtubule
spindle dynamics, which results in mitosis inhibition and
apoptosis induction [43,44]. Branham et al. [45] previously
described that paclitaxel is able to induce DNA damage in
lymphocytes treated with concentrations of 10 mg or higher. In
gastrointestinal cell lines (HT29 and MKN45), 0.01 mg/mL of
paclitaxel seems to be cytotoxic by MTT assay ([2-(2-methoxy-4-
nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazo-
lium, Monosodium salt] (WST-8) colorimetric assay) [46].
The methyl methanesulphonate treatment induced apoptosis
or necrosis [sub-G1; 40.2 (4.7) versus 1.6 (0.8); p < 0.001] and
reduced the GC cells entering mitosis [G2/M; 12.1 (1.7) versus
24.1 (4.11); p < 0.001] in relation to controls. The GC cells entering
DNA synthesis and mitosis decreased significantly with kaurenoic
acid treatment, along with an increase in the growth phase (G1)
compared with controls. In addition, kaurenoic acid treatment was
able to significantly induce apoptosis (or necrosis) even at
concentrations of 2.5 mg/mL in relation to non-treated cells

Fig. 5. Effect of kaurenoic acid on micronuclei frequency in each gastric cancer cell line. A) ACP02; B) ACP03; C) AGP01; D) PG100. MN: micronuclei frequency. C-: control,
which were non-treated cells; C+: cells treated with methylmethanesulfonateon * Significantly different from the control (p < 0.05; by Kruskal-Wallis test followed by Games-
Howell post-hoc analysis). ** Significantly different from the control (p  0.001; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis).
 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780 777

Table 2
Effect of kaurenoic acid on cell cycle progression and induction of apoptosis or necrosis.

Kaurenoic acid (mg/mL) DNA Content (%)

S G2/M G1 Apoptosis/Necrosis
0 (Control) 21.5 (3.4) 24.1 (4.1) 47.1 (4.1) 1.6 (0.8)
2.5 p = NS p = NS 50.9 (4.9) 5.5 (4.2)
p = 0.021 p < 0.001
5 p = NS 17.1 (6.1) 52.2 (4.8) 7.3 (5.6)
p = 0.002 p < 0.001 p < 0.001
10 16.5 (3.3) 13.9 (5.8) 53.9 (5.7) 11.7 (9.1)
p < 0.001 p < 0.001 p < 0.001 p < 0.001
30 13.8 (2.5) 12.3 (3.5) 56.9 (7.6) 17.8 (9.6)
p < 0.001 p < 0.001 p < 0.001 p < 0.001
60 12.3 (1.8) 11.3 (3.6) p = NS 23.2 (9.7)
p < 0.001 p < 0.001 p < 0.001

NS = not statistically significant.

Fig. 6. Kaurenoic acid effect on cell cycle progression of gastric cancer cell lines. C-: control, which were non-treated cells; C+: cells treated with methylmethanesulfonateon *
Significantly different from the control (p < 0.05; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis). ** Significantly different from the control (p  0.001; by
Kruskal-Wallis test followed by Games-Howell post-hoc analysis).

(Table 2 and Fig. 6). A similar pattern was observed in response to
the kaurenoic acid treatment in each cell line.
To further understand the effect of kaurenoic acid in GC cells,
we evaluated the cell cycle progression and the transcription of
genes involved, among other functions, in the control of this
process. Kaurenoic acid was able to induce cell cycle arrest at G1
and induce apoptosis in a dose-dependent manner, reducing the
DNA synthesis and mitosis processes. The gene transcription
analyses are in agreement with these findings.
The GC cell lines presented an increased transcription of MYC
[fold-change = 2.66 (0.42); Fig. 7A], CCND1 [fold-change = 1.96
(0.19); Fig. 7B], BCL2 [fold-change = 2.08 (0.21); Fig. 7C], and
CASP3 [fold-change = 1.66 (0.10); Fig. 7D] in relation to the non-
neoplastic MNP01 cells that were used as a calibrator for gene
transcription analysis. On the other hand, the GC cell lines
presented a reduced transcription of ATM [fold-change = 0.36
(0.14); Fig. 7E], CHK2 [fold-change = 0.40 (0.11); Fig. 7F] and
TP53 [fold-change = 0.27 (0.11); Fig. 7G] in relation to MNP01
cells.
The GC cells treated with methyl methanesulphonate presented
a significantly reduced transcription of CCND1 [1.34 (0.11) versus
1.95 (0.19), p < 0.001; Fig. 7B], BCL2 [1.85 (0.18) versus 2.08
(0.21), p = 0.048; Fig. 7C] in relation to non-treated GC cells.
Conversely, CASP3 transcription was increased in the GC cells
treated with methyl methanesulphonate in relation to controls
[1.94 (0.15) versus 1.66 (0.10), p < 0.001; Fig. 7D]. Moreover, we
observed a significantly reduced MYC transcription in ACP02,
ACP03, AGP01 and PG100 cell lines treated with methyl
methanesulphonate in relation to non-treated cells (p < 0.05),
when each cell line was evaluated separately. Additionally, methyl
methanesulphonate induced a significant reduction of ATM
transcription in ACP02 and PG100 cells (p < 0.05), and an increase
of CHK2 transcription in ACP03 and PG100 cells (p < 0.05).
Table 3 shows us how the kaurenoic acid affected the
transcription of genes involved in the control of the cell cycle.
MYC transcription was significantly reduced in GC cell lines treated
with 30 and 60 mg/mL of kaurenoic acid compared with non-
treated cells (Fig. 7A). Low concentrations of kaurenoic acid were
also able to reduce MYC transcription in some cell lines. Moreover,
GC cell lines treated with 10, 30 and 60 mg/mL of kaurenoic acid
also presented reduced CCND1 (Fig. 7B), BCL2 (Fig. 7C), and CASP3
(Fig. 7D) transcription in relation to non-treated cell lines.
Conversely, GC cell lines treated with 10, 30 and 60 mg/mL of
kaurenoic acid presented increased ATM (Fig. 7E), CHK2 (Fig. 7F),
and TP53 (Fig. 7G) transcription in relation to non-treated cell lines.
In addition, 5 mg/mL of kaurenoic acid also induced increased CHK2
transcription in GC cell lines in relation to controls (Fig. 7F).
Moreover, we observed an inverse correlation between
kaurenoic acid concentration treatment and MYC (rS = 0.770,
p < 0.001), CCND1 (rS = 0.880, p < 0.001), BCL2 (rS = 0.826,
p < 0.001) and CASP3 (rS = 0.828, p < 0.001) transcription. On
the other hand, kaurenoic acid concentration treatment was
directly correlated with ATM (rS = 0.863, p < 0.001), CHK2 (rS =
0.871, p < 0.001) and TP53 (rS = 0.721, p < 0.001) transcription.
We observed that MYC, ATM, CHK2, CCND1, BCL2, CASP3, and
TP53 transcription slightly varied in response to the kaurenoic acid
treatment in each cell line. In this analysis, we first normalized the
studied genes transcription in GC cell lines by their transcription in
non-neoplastic cells. As expected, the GC cell lines presented
increased transcription of the oncogenes MYC, CCND1 and BCL2 and
reduced transcription of the ATM, CHK2 and TP53 tumor
suppressors.
The response to kaurenoic acid slightly varied among GC cell
lines, which would be expected considering the differences in the
genetic background [26–29]. In general, higher concentrations of
this compound induced lower MYC, CCND1 and BCL2 transcription
778 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780

Fig. 7. Kaurenoic acid effect on gene transcription in gastric cancer cell lines. A) MYC; B) CCDN1; C) BCL2; D) CASP3; E) ATM; F) CHK2; G) TP53. The drug treatments and gene
transcription analyses were performed in ACP02, ACP03, AGP01 and PG100 cell lines. C-: control, which were non-treated cells; C+: cells treated with
methylmethanesulfonateon * Significantly different from the control (p < 0.05; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis). ** Significantly
different from the control (p  0.001; by Kruskal-Wallis test followed by Games-Howell post-hoc analysis).
 P.C.S. Cardoso et al. / Biomedicine & Pharmacotherapy 89 (2017) 772–780 779

Table 3
Effect of kaurenoic acid on the transcription of genes involved in the control of cell cycle progression. Values represent the relationship between the transcription of each gene
in the GC cell lines and the transcription of the same gene in the MNP01 (non-treated non-cancer cell line).

Kaurenoic acid (mg/mL) MYC CCND1 BCL2 CASP3 ATM CHK2 TP53
0 (Control) 2.66 (0.42) 1.96 (0.19) 2.08 (0.21) 1.66 (0.10) 0.36 (0.14) 0.40 (0.11) 0.27 (0.11)
2.5 p = NS p = NS p = NS p = NS p = NS p = NS p = NS
5 p = NS p = NS p = NS p = NS p = NS 0.75 (0.07) p = NS
p < 0.001
10 p = NS 1.59 (0.04) 1.56 (0.17) 1.42 (0.21) 0.57 (0.02) 0.81 (0.07) 0.45 (0.11)
p < 0.001 p < 0.001 p = 0.016 p = 0.005 p < 0.001 p = 0.002
30 2.07 (0.08) 1.23 (0.06) 1.47 (0.14) 1.23 (0.07) 0.89 (0.08) 0.92 (0.07) 0.50 (0.09)
p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001
60 1.27 (0.14) 1.16 (0.07) 1.32 (0.17) 1.19 (0.07) 0.95 (0.04) 0.93 (0.04) 0.48 (0.03)
p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001

NS = not statistically significant.

in GC cell lines, reinforcing that kaurenoic acid also acts in mitosis
inhibition.
Although reduction of CASP3 was detected in GC cell in relation
to non-cancerous cells and with the kaurenoic acid treatment,
higher concentration of this compound induced higher ATM, CHK2
and TP53 in GC cell lines. These genes present several roles in
human cells, including cell cycle arrest and induction of apoptosis
in response to DNA damage as observed by cell cycle analysis.
Considering that the DNA damage also increased with the
kaurenoic acid concentration treatment, our results suggest that
kaurenoic acid may also act as an apoptosis inducer in response to
DNA damage. Further investigations are still necessary, however,
kaurenoic acid may present antineoplastic activity as the diterpene
paclitaxel.
4. Conclusion
Our results showed that kaurenoic acid obtained from Copaifera
induces DNA damage and increases the micronuclei frequency in a
dose-dependent manner in GC cells, with a significant genotoxicity
observed above the concentration of 5 mg/mL. Moreover, this
compound seems to be able to induce cell cycle arrest and
apoptosis in GC cells.
Conflict of interest
The authors declare that there is no conflict of interest.
Acknowledgements
We gratefully acknowledge the financial support of the
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq) and Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP), for grants and fellowship award.
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