Basic and Applied Pathology 2009; 2: 9–14
COX-2 expression during 4-nitroquinoline 1-oxide induced
rat tongue carcinogenesis
Daniel A Ribeiro1, Ana Carolina C Fracalossi2, Thiago S Gomes2and Celina T F Oshima2
1Department of Biosciences, Federal University of Sao Paulo, UNIFESP , SP , Brazil,2Department of Pathology, Paulista Medical School, Federal
University of Sao Paulo, UNIFESP , Sao Paulo, Brazil
COX-2, malignant transformation,
4-nitroquinoline 1-oxide, oral squamous
cellcarcinoma, rat tongue mucosa.
Received 29 November 2008
Accepted 26 January 2009
Daniel Araki Ribeiro, DDS, PhD,
Departamento de Biociˆ encias, Av. Ana Costa,
95, Vila Mathias, Santos – SP , Brazil,
Zip code: 11060-001. Tel: 55 13 32218058;
fax: 55 13 32322592.
Background and aims: This study was undertaken to investigate, by immunohis-
tochemistry, the expression of cyclooxygenase-2 (COX-2) during 4-nitroquinoline
1-oxide (4NQO)-induced rat tongue carcinogenesis. Methods: Male Wistar rats
were distributed into three groups of 10 animals each and treated with 50 ppm
4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals
were used as negative control. Results: Although no histopathological abnormali-
ties were induced in the epithelium after 4 weeks of carcinogen exposure, COX-2
was expresssed in some cells of the ‘normal’ oral epithelium in the superficial
layer. In pre-neoplastic lesions at 12 weeks following carcinogen exposure, the
levels of COX-2 were increased (P < 0.05) when compared to negative control.
In well-differentiated squamous cell carcinoma induced after 20 weeks of treat-
ment with 4NQO, the same picture occurred when compared to negative control.
Weak immunoreactivity for COX-2 was observed in the negative control group be-
ing restricted to basal layer of the oral epithelium. Conclusions: Taken together,
our results support the idea that expression of COX-2 plays a crucial role during
malignant transformation being closely related to neoplastic conversion of the oral
Squamous cell carcinoma is the most common malignancy that
affects the human oral cavity.1Despite recent advances in therapy,
the prognosis of patients with oral squamous cell carcinoma has
not been improved significantly in recent decades.2,3It is desir-
able to examine the precise pathobiological mechanisms involved
in oral tumorigenesis in order to identify reliable biomarkers for
plastic conversion. The most often used animal models in this
line of research are the hamster buccal pouch by fat-soluble 7,12
dimethylbenzanthracene (DMBA), and the rat tongue by water-
soluble 4-nitroquinoline 1-oxide (4NQO). Considering one of the
most important routes of oral carcinogenesis is through liquid con-
the role of xenobiotics in experimental oral carcinogenesis.4Based
on the multi-step process of carcinogenesis characterized by ini-
tiation, promotion, and tumor progression, chronic administration
of 4NQO in drinking water simulates rat tongue carcinogenesis
similar to its human counterpart.5–12
Cyclooxygenase (COX) is the rate-limiting enzyme in the con-
version of arachidonic acid to prostaglandins (PG) of which two
isoforms, COX 1 and 2 have been identified. COX-1 is consti-
tutively expressed in many tissues and mediates the synthesis of
prostaglandins required for normal physiological function. COX-2
is normally undetectable in most tissues, but is rapidly induced
by proinflammatory or mitogenic stimuli.13Recently, COX-2 has
been implicated in several different cellular mechanisms during
tumorigenesis, such as angiogenesis, proliferation and the preven-
tion of apoptosis.14With respect to 4NQO, there are few reports
focusing the expression of COX-2 during rat tongue carcinogene-
sis, being the most studies conducted by means of inbread strains
such as Fischer 344.15–17Herein, a evaluation of COX-2 expres-
sion following 4NQO administration is a necessary step to better
establish the medium-term oral carcinogenesis assay induced by
4NQO with the outbred Wistar strain. As a result and because of
investigate the expression of COX-2 on 4NQO-induced rat tongue
carcinogenesis using outbred Wistar strain.
MATERIAL AND METHODS
Animals and experimental design
All experimental protocols involving animals conformed to proce-
dures described in the Guiding Principles for the Use of Labora-
tory Animals and the study approved by the Animal Committee of
Botucatu Medical School, UNESP.
Forty male outbred Wistar rats (8 weeks old) weighing approxi-
mately 250 g, were obtained from Centro de Bioterismo (CEMIB),
Universidade Estadual de Campinas, SP, Brazil. They were main-
tained under controlled conditions of temperature (24 ± 2◦C),
COX-expression in oral carcinogenesisD A Ribeiro and C T F Oshima
light-dark periods of 12 hours, and with free access to water and
commercial diet (Nuvital PR, Brazil). The animals were divided
into 3 groups of 10 and were treated with 50 ppm 4NQO (Sigma
Aldrich, St. Louis, USA) solution by drinking water for 4, 12 or
20 weeks. Ten animals were used as negative control, in which
were sacrified at the beginning the experiment (zero week). At the
end of the experimental period, the rats were sacrificed by 0.4%
sodium pentobarbital (1 mL/kg, i.p.). The tongues were longitudi-
bedded in paraffin blocks, and stained with hematoxylin and eosin
Histopathological evaluation was performed by light microscopy.
Analyzes of the tongue sections were graded as normal, hyper-
plasia, dysplasia, and carcinoma per animal according to Kramer
Serial longitudinal tongue sections of 4 ?m were deparaffinated
in xylene and rehydrated in graded ethanol, then pretreated by mi-
crowave (Electrolux, Sao Paulo, Brazil) with 10 mM citric acid
buffer (pH 6) for 3 cycles of 5 min each at 850 W for antigen re-
trieval. They were pre-incubated with 0.3% hydrogen peroxide in
PBS for 5 min for inactivation of endogenous peroxidase, and then
blocked with 5% normal goat serum in PBS for 10 min. The spec-
imens were then incubated with anti-COX-2 polyclonal antibody
centration of 1:250. Incubation was carried out overnight at 4◦C.
This was followed by two washes in PBS for 10 min. The sections
were then incubated with biotin-conjugated secondary antibody
anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) at a
concentration of 1:200 in PBS for 1 h. The sections were washed
Figure 1 Photomicrographies showing the multi-step process of rat tongue carcinogenesis. (a) No histopathological change (control); (b) hyperplasia
and hyperkeratosis; (c) epithelial dysplasia; (d) squamous cell carcinoma of well-differentiated type. (Hematoxylin & Eosin stain; ×100 magnification).
twice with PBS followed by the application of preformed avidin
biotin complex conjugated to peroxidase (Vector Laboratories) for
45 min. The bound complexes were visualized by the applica-
tion of a 0.05% solution of 3–3?-diaminobenzidine solution, and
counterstained with Harris hematoxylin. For control studies of the
antibodies, the serial sections were treated with rabbit IgG (Vector
Laboratories) at a concentration of 1:200 in place of the primary
antibody. Additionally, internal positive controls were performed
with each staining batch.
Quantification of immunohistochemistry
Tongue sections stained using immunohistochemistry were anal-
as normal, hyperplasia, dysplasia and carcinoma under optical mi-
croscope. A total of 1000 epithelial cells were evaluated in 3–5
fields at ×400 magnification. All values were used as labelling in-
divided by the total of number of cells counted. This protocol was
established in previous studies conducted by our group.10,11
Statistical analyses for immunohistochemistry data were assessed
by Kruskal Wallis non-parametric test followed by post-hoc anal-
ysis (Dunn’s test) if a significant effect was detected using SPSS
software pack (version 1.0). P < 0.05 was considered statistically
Histopathological evaluation following 4NQO
No histopathological changes in tongue epithelia were observed in
the control group (Fig. 1a) nor after treatment for 4-weeks with
D A Ribeiro and C T F Oshima COX-expression in oral carcinogenesis
Table 1 Incidence of histopathological lesions in tongue of rats in the
4-nitroquinoline 1-oxide (4NQO)†model for oral carcinogenesis
(n)Normal Hyperplasia Dysplasia Carcinoma
0 (Control) 10
†4NQO – 50 ppm by drinking water
4NQO. The primary histopathological change, i.e., hyperplasia
and hyperkeratosis with the spinous cell layer gradually thick-
ened was evidenced after 12-weeks-treatment (Fig. 1b). In this
period, epithelial dysplasia was also found in mild and moderate
forms (Fig. 1c). At 20 weeks, moderate and/or severe oral dyspla-
sia (Fig. 1a,b, respectively) and squamous cell carcinoma in the
tongue (Fig. 1c) were found, being that in the majority of ani-
mals consisted of squamous cell carcinoma. The histopathological
type. The tumors spread into the submucosa and underlying mus-
cle layer, forming small nests with typical keratin pearl forma-
tion. In advanced cases, severe atypia was frequently found. The
histopathological findings are summarized in the Table 1.
COX-2 expression was detected either in cytoplasm or in nucleus
of the oral mucosa (Fig. 2). Expression of COX-2 was weakly de-
tected in all negative controls, being mainly present in the basal
layer of oral epithelium (Fig. 3a). Overall, the expressivity was
considered mild in this group. On the other hand, COX-2 positive
of carcinogen althoughnohistopathological changes wereinduced
during this period (Fig. 3b). Nevertheless, the statistical analysis
showed no differences in this experimental group when compared
to negative control (P > 0.05). Following 12 weeks of carcino-
gen exposure, rat tongues showed early proliferative changes of
epithelium characterized by hyperplasia, and dysplasia. These pre-
neoplastic lesions contain COX-2 positive cells in the superficial
layers of the epithelium (Fig. 3c). Well-differentiated squamous
Figure 2 High magnification of COX-2 immunoreactivity in nucleus (as-
terisk) and cytoplasm (arrow). (×200 magnification).
cell carcinomas induced after 20 weeks of treatment with 4NQO
displayed COX-2 positive expression in oral tumor cells (Fig. 3d).
at two experimental periods (12 and 20 weeks), compared to the
negative control (Fig. 4).
Carcinogenesis is a multi-step process, which is characterized by
genetic, epigenetic, and phenotypic changes.19Such changes in-
volve genetic damage, mutation in critical genes related to the
control of cell division, cell death, and metastatic potential, and
activation of signalizing or metabolic pathways that give the cells
favorable growth and survival characteristics.20In patients, the
molecular analysis of these multiple steps is hampered, due to
the unavailability of biopsies at all the stages of carcinogenesis.
Animals models of carcinogenesis allow the isolation of all stages
under controlled conditions, including normal tissues, which are
then amenable to pathological, genetic, and biochemical analy-
sis, and at lower costs.21Moreover, the chemical carcinogenesis
models help to investigate hazard risk caused by environmental
agents as well as to determine which putative precancerous le-
sions will progress. Several medium term duration assay systems
for oral carcinogenesis offer particular promise. Our results, using
4NQO as a carcinogen inducer, demonstrated histopathological
changes in rat tongue mucosa along a time-course from hyper-
plasia, pre-malignant dysplasia, and carcinoma in situ, to invasive
squamous cell carcinoma. Therefore, it should be assumed that
tongue carcinogenesis was 4NQO dependent, because these le-
sions did not occur in control rats and the rats that developed
tumors were younger than 28 weeks of age, when spontaneous
tumors are not common in this species.22
It has been established that the expression of COX-2 appears to
be a reliable biomarker for oral tumors.15This is because COX-2
expression increases in response to mitogens, pro-inflammatory
cytokines and growth factors, and it has also been linked to car-
cinogenesis.23In light of these considerations, we evaluated the
expressivity of COX-2 following 4NQO administration in order to
determine its role during oral tumorigenesis phase by phase using
outbred rat strain. In normal control epithelia, represented by the
control group, COX-2 protein was present in the basal layer of the
epithelium in all specimens with a weak pattern. This is consis-
tent with published data showing the low expressivity of COX-2
in normal oral mucosa cells.15,23By contrast, we found abnor-
mal COX-2 expression in ‘normal’ oral mucosa 4 weeks after the
start of 4NQO administration in the superficial layer of epithelium.
Thus, we can state the expression of COX-2 in the superficial layer
of the oral mucosa may be an important event in the initiation
of oral carcinogenesis. To best of our knowledge, this is a new
finding in oral carcinogenesis being, therefore, the mechanism dif-
ficult to explain. Several different mechanisms could provide an
important link between COX-2 in oral cancer.24Enhanced synthe-
sis of prostaglandins, a consequence of up-regulation of COX-2,
can increase cell proliferation,25promote angiogenesis,26and in-
hibit immune surveillance.27All of these effects contribute to the
growth of malignant cells. Additionally, overexpression of COX-2
ing xenobiotics to reactive electrophiles that are carcinogenic. For
example, COX catalyzes the oxidation of the tobacco procarcino-
gen benzo[a]pyrene-7,8-dihyrodiol to benzo[a]pyrene-diol epox-
Some authors have postulated that inhibition of cyclooxygenase-2
COX-expression in oral carcinogenesisD A Ribeiro and C T F Oshima
Figure 3 Immunohistochemical staining for COX-2; (a) rat control epithelium; (b) epithelium of the rat 4 weeks after the initation of 4-nitroquinoline
1-oxide administration; (c) pre-neoplastic lesion after 12 weeks of carcinogen administration and (d) squamous cell carcinoma of well differentiated
type (×100 magnification).
Figure 4 COX-2 labelling index in the nega-
tive control (zero) and those exposed to 4-
nitroquinoline 1-oxide for 4, 12 and 20 weeks.
Values were expressed as means ± S.D.
∗P < 0.05 when compared to negative control
expression could be an approach to preventing head and neck can-
cer.30However, additional studies are still needed to determine
why and how COX-2 expression is increased as well as which of
these mechanisms are important in oral carcinogenesis, specially
at early phases.
In humans, only a small portion of initial lesions develop oral
carcinomas.10Therefore, the challenge is to identify which le-
sions have real malignant potential. Previous studies conducted by
our group have evidenced putative biomarkers involved in 4NQO-
induced tongue carcinogenesis such as bcl-2/bax, GST-P, survivin
and inducible nitric oxide for better understanding oral cancer
pathogenesis.10–12,31In this study, strong positive expression of
COX-2 was found in oral pre-malignant lesions and oral squa-
mous cell carcinomas following 12 and 20 weeks exposure to
4NQO, respectively. Consistent with these findings, COX-2 ex-
pression has been shown either in pre-neoplastic lesions, such as
oral epithelial dysplasia, as well as squamous cell carcinomas of
the oral cavity (Benarjee et al., 2002).15,32It is important to stress
that the expressivity of COX-2 is partly explained by the pres-
ence of numerous cis-acting elements in the 5?-flanking region of
the COX gene.33Its mode of action in carcinogenesis includes
multiple mechanisms that may be acting at different stages of
malignant disease.34Overexpression of the COX-2 gene alters cell
latory signals.35Furthermore, studies have provided evidence that
mutant p53 gene may facilitate the COX-2 expression.36,37Be-
sides epithelial cells, our results also demmonstrated COX-2 im-
munoexpression in the endothelial cells of small blood vassels as
well as inflammatory cells present adjacent to tongue lesions (data
not shown). In fact, several authors have affirmed a link between
COX-2 levels and angiogenesis, an important mechanism of in-
vasion and consequent metastasis, with increased production of
D A Ribeiro and C T F Oshima COX-expression in oral carcinogenesis
vascular-endothelial growth factor (VEGF) and increased vascu-
larization in tumors.38Therefore, we assume that COX-2 overex-
pression is a event closely related to neoplastic conversion. These
findings are in agreement with other studies that investigated this
risk of oral cancer, such as in smokers or in alcoholism, as well as
patients diagnosed with oral dysplasia or carcinoma, remains to be
Falaguera Ardanaz for their technical assistance. This work was
supported byFAPESP(Fundac ¸˜ aode Amparo ` aPesquisadoEstado
de S˜ ao Paulo (Grant number: 07/01228-4).
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