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 carcinogenesis D A Ribeiro and C T F Oshima
32. Shibata M, Kodani I, Osaki M, Araki K, Adachi H, Ryoke K, Ito H.
Cyclo-oxygenase-1 and -2 expression in human oral mucosa,
dysplasias and squamous cell carcinomas and their pathological
significance. Oral Oncol 2005; 41: 304–12.
33. Inoue H, Yokoyama C, Hara S, Tone Y, Tanabe T. Transcriptional
regulation of human protaglandin-endoperoxidase-2 gene by
lipopolysaccharide and phorbol ester in vascular endothelial cells.
Involvement of both nuclear factor for interleukin-6 expression site
and cAMP response element. J Biol Chem 1995; 270: 24965–71.
34. Mohan S, Epstein JB. Carcinogenesis and cyclooxygenase: the
potential role of COX-2 inhibition in upper aerodigestive tract cancer.
Oral Oncol 2003; 39: 537–46.
35. Tsuji M, Dubois RN. Alteration in cellular adhesion and apoptosis in
epithelial cells over expressing prostaglandin endoperoxidase
synthase-2. Cell 1995; 83: 493–501.
36. Li HL, Zhang HW, Chen DD, Zhong L, Ren XD, Si-Tu R. JTE522, a
selective cox-2 inhibitor, inhibits cell proliferation and induces
apoptosis in RL95-2 cells. Acta Pharmacol Sin 2002; 23: 631–37.
37. Atula T, Hedstr¨ om J, Ristim¨ aki A, Finne P, Leivo I, Markkanen-
Lepp¨ anen M, Haglund C. Cyclooxygenase-2 expression in squamous
cell carcinoma of the oral cavity and pharynx: association to p53 and
clinical outcome. Oncol Rep 2002; 16: 485–90.
38. Jaeckel EC, Raja S, Tan J, Das Sk, Dey SK, Girod DA. Correlation of
expression of cyclooxygenase-2, vascular endothelial growth factor,
and peroxisome proliferators-activated receptor (delta) with head and
neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg
2001; 127: 1253–9.
39. Chan G, Boyle JO, Yang EK, Zhang F, Sacks PG, Shah JP.
Cyclooxygenase-2 expression up-regulated in squamous cell
carcinoma of the head and neck. Cancer Res 2001; 59: 991–4.
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