Toxicology in Vitro 57 (2019) 126–131

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Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit

Effect of the kaurenoic acid on genotoxicity and cell cycle progression in T

cervical cancer cells lines
Silvia Maria Machado da Rochaa,b, Plínio Cerqueira dos Santos Cardosoa,
Marcelo de Oliveira Bahiaa, Claudia do Ó Pessoac, Paulo Cardoso Soaresd,
Simone Machado da Rochae, Rommel Mário Rodríguez Burbanoa,d,
Carlos Alberto Machado da Rochae,
⁎

a
Biological Science Institute, Federal University of Pará (UFPA), Belém, PA, Brazil
b
Center for Women's Health Care, Belém, PA, Brazil
c
Federal University of Ceará (UFC), Fortaleza, CE, Brazil
d
Ophir Loyola Hospital (OLH), Belém, PA, Brazil
e
Federal Institute of Education, Science and Technology of Pará (IFPA), Belém, PA, Brazil

1. Introduction
Cervical cancer represents > 500,000 new cases of cancer and >
250,000 deaths per year worldwide (Ferlay et al., 2015). Epidemiolo-
gical studies of cervical lesions have suggested the participation of
venereal carcinogens (semen, Epstein-Barr virus, cytomegalovirus,
herpes simplex virus type-2). Human papillomavirus (HPV) appeared as
the main suspect when found in about 90% of cervical cancers and
because it has oncogenes (E6 and E7) with potential for transformation
(Arends et al., 1998; Brenna and Syrjanen, 2003; Rivoire et al., 2006).
HPVs have also been detected in a wide range of asymptomatic con-
trols, indicating that further events are required for the development of
neoplasms, such as viral persistence and/or altered expression of viral
genes, often after integration of the viral genome (Rivoire et al., 2006).
Although most HPV infections resolve spontaneously within a few
months, others persist and express viral oncogenes that inactivate the
p53 and Rb proteins, leading to increased genomic instability, accu-
mulation of somatic mutations and, in some cases, integration of HPV
into the genome of the host (Moody and Laimins, 2010). The associa-
tion with cancer risk and histologic subtypes varies substantially among
types of carcinogenic HPVs, but the reasons for these differences are
poorly understood.
The disease begins in a curable pre-invasive lesion in up to 100% of
cases, which usually progresses slowly for 10 to 20 years until reaching
the invasive stage, where healing becomes more difficult, if not im-
possible (Muller et al., 2008; INCA, 2016). Effective preventive vaccines
against more oncogenic forms of HPV have been available for several
years, with vaccination having the long-term potential to reduce the
number of cervical cancer cases. However, vaccines will act as a means
of preventing cervical cancer only for individuals who previously had
access to them before the onset of sexual life. Out of this context, the
fight against cervical cancer must still be made through the detection of
precursor lesions and their due treatment and clinical follow-up
(Nakagawa et al., 2010).
The use of medicinal plants is a tradition maintained until the
present. Reports on folk medicine in the Brazilian Amazon region have
been documented in the literature, especially regarding the oil-resin
extracted from the copaíba trunk (different species of the Copaifera
genus) (Tappin et al., 2004; Comelli Jr. et al., 2010), with numerous
applications of relevant medical importance, including anti-in-
flammatory, antiulcerogenic, antitumor, antinociceptive, anti-
melanoma, antitilipoperoxidation, antioxidant and antimicrobial
properties (Ohsaki et al., 1994; Paiva et al., 2002; Gomes et al., 2007;
Lima Silva et al., 2009; dos Santos et al., 2011; Toubouti et al., 2017).
This natural product has great social and economic representation in
the Amazon region, since it represents about 95% of all oil-resin pro-
duction in Brazil (Medeiros and Vieira, 2008). Particularly, the di-
terpenes (kaurenoic and polyaltic acids) present in pure oil appear to
have healing and anti-inflammatory properties (Tappin et al., 2004;
Comelli Júnior et al., 2010).
The objective of the present study was to evaluate the in vitro
genotoxic activity of kaurenoic acid extracted from Copaifera langs-
dorffii and the effect of this compound on the transcription of genes
involved in cell cycle control in three cervical cancer cell lines.
2. Materials and methods
2.1. Cell lines
HeLa cells are hypertriploid (71 to 75 chromosomes), with specific

⁎
Corresponding author at: Federal Institute of Education, Science and Technology of Pará (IFPA), Av. Almirante Barroso n. 1155 – Marco, – CEP 66093-020 Belém,
– Pará, Brazil.
E-mail address: carlos.rocha@ifpa.edu.br (C.A.M.d. Rocha).

https://doi.org/10.1016/j.tiv.2019.02.022
Received 12 January 2019; Received in revised form 19 February 2019; Accepted 25 February 2019
Available online 27 February 2019
0887-2333/ © 2019 Published by Elsevier Ltd.
S.M.M.d. Rocha, et al.
numeric deviations, 20 clonally abnormal chromosomes (known as
HeLa signature chromosomes or HeLa markers) and contain multiple
copies of type 18 HPV (HPV18), integrated into specific sites (Chen,
1988; Macville et al., 1999). CaSki is a hypertriploid cell line (72 to 78
chromosomes) of human cervical carcinoma, with up to 600 copies of
papillomavirus type 16 (HPV 16) DNA integrated into their genome.
C33A is an HPV-negative cervical cancer strain. Their karyotype is
mostly approximately diploid (45 to 47 chromosomes), although about
15% of the cells assume an approximately tetraploid pattern (85 to 89
chromosomes) (McCormack et al., 2013). The three lineages are kept in
the Human Cytogenetic Laboratory, Federal University of Pará (UFPA,
Pará, Brazil). HeLa and C33A were purchased from the Banco de Cé-
lulas do Rio de Janeiro (BCRJ, Rio de Janeiro, Brazil); CaSki was pur-
chased from Sigma Aldrich (Co., St. Louis, MO, USA).
2.2. Chemicals
Kaurenoic acid was obtained from the oil resin of C. langsdorffii Desf.
(Leguminaceae) that grows abundantly in the Amazon region of
Northern Brazil. In this study, kaurenoic acid (CAS 6730-83-2) (Sigma
Chemical Co., St. Louis, MO, USA) was diluted in dimethyl sulfoxide
(DMSO) and the concentrations used were based on previous publica-
tions (Cavalcanti et al., 2006; Cardoso et al., 2017). Modified Eagle's
medium (MEM), fetal bovine serum, trypsin-EDTA, penicillin and
streptomycin were purchased from Gibco® (Invitrogen, Carlsbad, CA,
USA). Methyl methanesulfonate (MMS), cytochalasin B (Cyt-B), and
Giemsa were obtained from Sigma Aldrich Co. (St. Louis, MO, USA).
2.3. General experimental procedures
Cervical cancer cells (HeLa, CaSki, and C33A) 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, 100 μg/mL streptomycin) (Speit et al., 1994).
Cells were maintained in tissue culture flasks (surface area 25 cm2)
(TPP, Trasadingen, Switzerland) at 37 °C in a humidified atmosphere of
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 into 5 mL of complete medium to grow for 2 days prior to
treatment with the test substances.
Kaurenoic acid was added to cultures at the final concentrations of
2.5, 5, 10, 30 and 60 μg/mL) for 3 h. Afterward, 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).
Cells cultures not exposed to kaurenoic acid were used as a negative
control, while cells treated with methyl methanesulfonate (MMS,
4 × 10−5 M) were used as a positive control. All cell treatments were
carried out with three replicates.
2.4. Comet assay
The assay was performed under alkaline condition (pH > 13), as
described by Singh et al. (1988) with some modifications (Hartmann
and Speit, 1997; Collins, 2004). An aliquot of 450 μL of the cell sus-
pension 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 in microscope slides pre-coated with a normal melting
point agarose (1.5%) and covered with a coverslip. After 5 min at 4 °C,
the coverslip was removed and the slides were immersed in cold lysis
solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton-X and 10%
DMSO, pH: 10) for one week. Then, the slides were placed in an elec-
trophoresis chamber and covered with freshly made electrophoresis
buffer (300 mM NaOH; 1 mM EDTA). Electrophoresis was done in a
horizontal vessel and runned at 34 V and 300 mA for a period of 25 min.
127
Toxicology in Vitro 57 (2019) 126–131
The slides were then neutralized by submersion in distilled water (4 °C)
for 5 min, fixed with absolute ethanol for 3 min and stained with ethi-
dium bromide (20 μg/mL). For observation, a fluorescence microscope
(Olympus BX41) with a magnification of 400× was used, and 100 cells
of each sample were analyzed.
Two parameters were evaluated: (1) damage index (DI), in which
the cells were classified visually, according to tail length, into five
classes: class 0: no damage, no tail; Class 1: with a tail smaller than the
head (nucleoid) diameter; Class 2: with tail length one to two times the
diameter of the head; Class 3: with a tail greater than twice the dia-
meter of the head; Class 4: significant damage with tail length greater
than three times the diameter of the nucleoid or comets without a head.
Genetic damage index (DI) was calculated using the formula:
DI = (0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4), in
which n = number of cells in each class analyzed. The DI ranges from 0
(completely intact: 100 cells × 0) to 400 (with maximum damage: 100
cells × 4); and (2) damage frequency (DF), calculated as the percentage
of damaged cells (Silva et al., 2000; Collins et al., 2001).
2.5. Micronucleus assay
Three hours after the treatment with kaurenoic acid, the cultures
were washed twice with the medium and cytochalasin B (2 μg/mL) was
added. Cultures were reaped 21 h after cytochalasin B addition.
Confluent cells in cultures were detached using trypsin, the cellular
suspension was centrifuged at 1000 rpm for 5 min and the cells sub-
mitted to hypotonic solution (KCl 0.075 M). Then, the cells were wa-
shed once with 5 mL cold (5:1) methanol: acetic acid (v/v) fixative and
again washed with 5 mL of a cold methanol: acetic acid (3:1) solution
(v/v). The cell suspension was dropped onto slides and stained in a
solution of 5% Giemsa dye in phosphate buffer (pH 6.8) for 5 min. The
analysis was performed using an optical microscope (BIOVAL L2000A)
at 1000× magnification, being evaluated 2000 binucleated cells and
using the criteria according to Fenech (2000).
2.6. Genes transcription analysis
Transcription of genes involved in the cell cycle was analyzed to
evaluate the effect of the treatment with kaurenoic acid in the, CaSki,
and C33A cell lines (Table 1). Total mRNA was isolated from all cell line
samples using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). The
extracted RNA was purified using RNeasy Mini Kit (Qiagen, Valencia,
CA, USA), according to the recommended protocol. The concentration
was determined by a NanoDrop spectrophotometer (Kisker, Germany)
and the quality was checked using 1% agarose gels. 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 (RTqPCR) 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 of relative RNA
quantification and reverse transcription efficiency. Relative quantifi-
cation of gene transcription was calculated according to the manufac-
turer's software (Life Technologies, Pittsburgh, PA, USA). All RT-qPCRs
were performed in triplicate for both target and internal control genes.
2.7. Analysis of inhibition of HPV E6 and E7 gene expression
Expression of E6 and E7 mRNA was determined by RT-PCR as de-
scribed previously (Chang et al., 2007). In short, total RNA was ex-
tracted with Trizol Reagent (Invitrogen, Carlsbad, CA, USA). Com-
plementary DNA was synthesized by incubating samples at 37 °C for 1 h
in a buffer containing Tris 50 mM, pH 8.4, KCl 75 mM, MgCl2 3 mM,
S.M.M.d. Rocha, et al. Toxicology in Vitro 57 (2019) 126–131

Table 1
Summary of the targets and references of the eight genes in the transcription study.
Gene symbol Name Gene function Assaya

MYC v-myc avian myelocytomatosis viral oncogene Cell cycle progression, apoptosis and cellular transformation/oncogene hs00153408_m1
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 peptidase Pro-apoptotic hs00234387_m1
ATM Atm serine/threonine kinase Cell cycle arrest in response to DNA damage and for genome stability/tumor suppressor 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 suppressor
P16 Cyclin-dependent kinase inhibitor 2A (CDKN2A) Suppression of G1/S-Cdk activity/tumor suppressor hs00923894_m1

a
TaqMan probes were purchased as assays-on-demand products for gene expression (Life Technologies, USA).

Table 2
Primer sequences used for the analysis of HPV E6 or E7 gene expressions by
RT-PCR.
Genes Primer sequences
HPV16E6 F: 5′-AATGTTTCAGGACCCTACGG-3′
R: 5′-TCAGGACACAGTGGCTTTTG-3′
HPV16E7 F: 5′-TTTGCAACCAGAGACAACTGA-3′
R: 5′-GCCCATTAACAGGTCTTCCA-3′
HPV18E6 F: 5′-GCGACCCTACAAGCTACCTG-3′
R: 5′-GTTGGAGTCGTTCCTGTCGT-3′
HPV18E7 F: 5′-GCATGGACCTAAGGCAACAT-3′
R: 5′-TGTTGCTTACTGCTGGGATG-3′
Actin F: 5′-GTGGGGCGCCCCAGGCACCA-3′
F: 5′-CTCCTTAATGTCACGCACGATTTC-3′
300 ng of RNA, 0.2 μg of oligo-dT, DTT 10 mM, dNTP 0.5 mM, 10 units
of RNase inhibitor and 50 units of reverse transcriptase. For each PCR
run, a master mixture was prepared with 1× TaqMan buffer, 5 mM
MgCl2, 200 μM dNTP, 300 nM of each primer, 1 unit of AmpliTaq Gold
DNA Polymerase and 3 μl of complementary DNA solution in a total of
30 μl. Table 2 shows the primer sequence for each gene. PCR products
were analyzed by 2% agarose gel electrophoresis, stained with ethidium
bromide, photographed at 254 nm and the optical density of each band
was measured. For quantification, the density of each gene was cali-
brated to the control vector after normalization to the internal actin
level.
2.8. Statistical analysis
Results were expressed as mean ± standard deviation and the
Lilliefors test was used to evaluate the distribution of the data and to
determine the appropriate sequence of tests for the statistical compar-
isons. ANOVA followed by the Tukey test was performed by multiple
comparisons to compare the groups studied. The Pearson correlation
coefficient (r) was used to verify the possible correlation between
kaurenoic acid concentrations and genotoxicity or gene transcription.
In all analyzes, the confidence interval was 95% and a p-value < .05
was considered significant.
3. Results and discussion
Treatment with MMS promoted a very significant genotoxicity in
HeLa, CaSki, and C33A cell lines when compared to the untreated
control (p < .001). At the highest concentrations (30 and 60 μg/mL),
kaurenoic acid also induced a significant increase (p < .001) in DNA
damage index, damage frequency and micronuclei frequency, com-
pared to control values (Fig. 1). In all cell lines, the concentration of
kaurenoic acid showed a strong positive correlation with the three
genotoxicity indicators (r > 0.9; p < .01).
To date, few studies have evaluated the bioactivity of kaurenoic
acid, although the genotoxic and cytotoxic effect of diterpenes has
previously been reported. Using the comet assay, Kato et al. (2012)
observed that pimaradienoic acid, a pimarane-type diterpene, induced
DNA damage at concentrations of 2.5 and 5.0 μg/mL in V79 cells. The
same authors also reported DNA damage in rat hepatocytes treated with
this diterpene at 80 mg/kg.
Paclitaxel (Taxol®) is a diterpene plant product and clinically ef-
fective antineoplastic drug that has been used for the treatment of
several human cancers (Bang et al., 2015). This diterpene stabilizes
microtubule polymerization, thus blocking cells in the G2/M phase of
the cell cycle, which results in mitosis inhibition. AL-Sharif (2012)
demonstrated elevation in micronucleus frequency in bone marrow
cells of albino rats after intraperitoneal injection of paclitaxel.
Abrão et al. (2015) used the copaiba oleoresin and 10 compounds
isolated by fractionation to test the cytotoxic activity against various
lines: normal Chinese hamster lung fibroblasts (V79), murine mela-
noma tumor cells (B16F10), human breast adenocarcinoma (MCF-7),
human hepatocellular carcinoma (HepG2), human glioblastomas
(MO59J, U343 and U251), and human cervical adenocarcinoma
(HeLa). The OC-2 fraction (a diterpene identified as copalic acid) was
the one with the most pronounced antiproliferative activity against
HeLa cells (IC50 = 44.03 μg/mL), significantly lower than that obtained
against the normal cell line V79 (IC50 = 107.30 μg/mL). It should be
emphasized that treatment of HeLa cells with the well-known che-
motherapy drug VP16 showed IC50 = 225.50 μg/mL.
Recently, our research group demonstrated the genotoxicity of
kaurenoic acid itself in gastric cancer lines, through the comet assay
and micronucleus test (Cardoso et al., 2017). Comparing to the present
results, genotoxic effects were observed in gastric cells even at low
concentrations, but in cervical cells these effects reached higher levels.
For a better understanding of the effect of kaurenoic acid on cervical
cancer cells, the transcription of genes involved, among other functions,
in the control of cell cycle progression was evaluated. A relative
quantification of gene expression was performed on HeLa (HPV18-po-
sitive) and CaSki (HPV16-positive) cervical cancer cells relative to the
C33A (HPV-negative). When treated with kaurenoic acid, the HeLa and
CaSki cell lines showed lower gene transcription of MYC, CCND1, BCL-
2, CASP3, and P16. On the other hand, these strains showed an in-
creased transcription of ATM, CHK, and TP53 (Table 3).
Cardoso et al. (2017) obtained similar results in gastric cancer lines.
In general, higher concentrations of kaurenoic acid also induced lower
expression of the MYC, CCND1 and BCL2 genes, reinforcing that this
compound has action in the inhibition of mitoses. In addition, was also
observed reduction in the transcription of CASP3 and increase in the
transcription of ATM, CHK and TP53 in the gastric cells.
In normal cells, P16 works in cell cycle control primarily as a ne-
gative regulator of the pRb/E2F pathway. The expression of P16 un-
derlies a negative feedback control through pRB. In neoplastic cells of
the cervix, high-risk HPV E7 protein may interfere in the regulatory
circuit by its ability to inactivate pRB and thus lead to increased

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S.M.M.d. Rocha, et al. Toxicology in Vitro 57 (2019) 126–131

Fig. 1. Genotoxicity of kaurenoic acid in each studied cervical cancer cell line: HeLa, CaSki and C33A. DI = DNA damage index; DF = DNA damage frequency;
MN = Micronuclei frequency. * Significantly different from the control (p < .05; by ANOVA followed by Tukey's post-test). ** Significantly different from the
control (p < .001; by ANOVA followed by Tukey's post-test).

expression of P16 (Klaes et al., 2001). The results obtained suggest that
the treatment with kaurenoic acid led to lower expression of the P16
gene in HeLa and CaSki cells when restoring important components in
the regulation of the cell cycle.
Treatment with kaurenoic acid at 2.5, 5 and 10 μg/mL did not sig-
nificantly affect the expression of HPV E6 and E7 oncogenes. At the
highest concentrations (30 and 60 μg/mL), however, kaurenoic acid
promoted significant inhibition in the expression of these genes (Fig. 2),
including in most cases with a more potent effect than MMS, the che-
motherapeutic used as control positive in this assay.
Blocking the activity of E6 and E7 oncoproteins would be an im-
portant therapeutic target in uterine cervix cancer cells (Paul et al.,
2014). The same authors report the effects of a diterpenoid called an-
isomelic acid on SiHa line cells (HPV positive) of cervical cancer. This
natural compound presented anticancer activity characterized by the
ability to induce cell cycle arrest in G2/M through the degradation of
E6 and E7 proteins, leading to the restoration of p53 levels. It also in-
duces apoptosis by activation of caspase via the mitochondrial
pathway.
More recently, molecular mechanical studies of Zhang et al. (2017)
revealed that N-acetyl-deformylantimycin A (NADA) degraded levels of
E6 and E7 oncoproteins through the activation of the ubiquitin-de-
pendent proteasome system mediated by reactive oxygen species in
HeLa cells, in addition to inducing apoptosis. In our study, it was

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Table 3
Correlation between the concentration of kaurenoic acid and gene transcription.
KA conc. (μg/mL) MYC CCND1 BCL-2 CASP3 ATM CHK TP53 p16 Cell lines

0 1.94 1.79 1.45 1.69 1.41 1.3 1.36 1.81 HeLa

2.5 1.94 1.58 1.31 1.47 1.34 1.29 1.34 1.75
5 1.89 1.58 1.31 1.48 1.35 1.27 1.36 1.68
10 1.77 1.36 1.08 1.22 1.49 1.44 1.57 1.51
30 1.30 1.24 0.87 1.29 1.69 1.68 1.89 1.46
60 1.14 1.24 0.62 1.21 1.77 1.79 1.89 1.08
Pearson correlation r = −0.9609 r = −0.7949 r = −0.9584 r = −0.6975 r = 0.9353 r = 0.9510 r = 0.8942 r = −0.9670
p = .0023 p = .0587 p = .0026 p = .1233 p = .0061 p = .0035 p = .0162 p = .0016

0 1.95 1.8 1.46 1.71 1.45 1.35 1.38 1.89 CaSki

2.5 1.94 1.62 1.36 1.52 1.40 1.30 1.37 1.79
5 1.83 1.62 1.37 1.53 1.43 1.32 1.36 1.75
10 1.66 1.35 1.13 1.28 1.51 1.45 1.60 1.54
30 1.33 1.19 0.81 1.31 1.76 1.73 1.90 1.48
60 1.15 1.19 0.50 1.17 1.84 1.83 1.95 1.09
Pearson correlation r = −0.9558 r = −0.8226 r = −0.9763 r = −0.8117 r = 0.9510 r = 0.9502 r = 0.9134 r = −0.9640
p = .0029 p = .0444 p = .0008 p = .0498 p = .0035 p = .0037 p = .0109 p = .0019

The statistic was calculated from the mean values of the relative quantification of gene expression in the HeLa and CaSki cervical cancer cells in relation to the C33A
line.

possible to demonstrate that kaurenoic acid (30 and 60 μg/mL), a di-
terpene obtained from copaiba, promotes reduced transcription of E6
and E7 oncogenes, which would also implicate depletion of the corre-
sponding oncoprotein levels in both HeLa and CaSki cervical cancer.
Future studies with flow cytometry may clarify further the effect of
kaurenoic acid in the cell cycle progression of cervical cancer cells.
Since inactivation of pRb through HPV E7 results in increased ex-
pression of P16, which may represent a specific and sensitive biomarker
for cells with active expression of HPV oncogenes (Klaes et al., 2001),
we can infer that the lower expression of P16 in response to a treatment,
as observed in the present study, represents a biomarker for cells in
which HPV oncogenes are being prevented from expressing.
Although genotoxic effects were observed at higher concentrations
of kaurenoic acid, our assays also showed very favorable results in re-
lation to gene expression. Thus, the present work suggests more at-
tention and new approaches, since kaurenoic acid can serve as raw
material for the development of therapeutic agents for cervical cancer
with presence of HPV, as well as for research on the precise functions of
E6 and E7.

Fig. 2. Effect of kaurenoic acid on the expression of HPV E6 and E7 oncogenes in HeLa and CaSki cervical cancer lines.

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S.M.M.d. Rocha, et al.
Conflict of interest
None.
Acknowledgements
The first author is grateful for the financial support received in the
form of a Master's Degree Scholarship from the Coordination for the
Improvement of Higher Education Personnel (CAPES), Brazil.
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