Absence of inducible nitric oxide synthase is associated to reduced depth of invasion of oral squamous cell carcinomas in 4NQO-treated mice

Abstract

Introduction/Justification: Nitric oxide is a gaseous molecule whose role has been identified in a variety of physiological and pathological conditions, including cancer, in which it seems to alternatively act as a promoter and inhibitor of tumors. During the multiple stages of oral carcinogenesis, studies have shown that the expression of markers related to nitric oxide appears to increase. Experimental carcinogenesis in animal models therefore emerges as a method that allows the investigation of the involvement of nitric oxide and nitric oxide synthase (NOS) enzymes in different stages of tumor development within controlled conditions, also considering the various cellular interactions existing in the body and enabling the proposal of signaling pathways related to this phenomenon. Few studies of this nature have been developed.

Objective: The purpose of this study was to evaluate the influence of inducible nitric oxide synthase (Nos2) in oral carcinogenesis, analyzing the incidence and clinicopathological characteristics of epithelial lesions observed on the tongue in Nos2+/+ (wild-type) and Nos2-/- (knockout) mice treated with 4-nitroquinoline-1-oxide (4NQO), as well as observing the histopathological, systemic inflammatory, and epigenetic markers in these animals.

Materials and Methods: Our experimental cohort consisted of male Mus musculus mice from two different strains: C57BL/6J (wild-type, Nos2+/+) and B6.129P2-Nos2tm1Lau/J (knockout, Nos2-/-). A solution of 4-nitroquinoline-1-oxide at 50 μg/mL was administered in the drinking water of 31 mice, 15 Nos2+/+ and 16 Nos2-/-, for 16 weeks, and the results were compared with control groups (n=15) treated with propylene glycol after an 8-week observation period. The tongues were evaluated for the incidence of macroscopic and microscopic lesions. Collected data included the number, location, and histological characteristics of observed lesions, including degree of dysplasia, growth pattern, tumor differentiation, and depth of invasion. The experimental procedures were approved by the Animal Experimentation Ethics Committee (CEUA-UFU, registration no. 100/18).

Results: With this mixed model, it was possible to analyze the influence of inducible nitric oxide synthase absence on the clinicopathological characteristics of 4NQO-induced epithelial lesions. Macroscopic lesions were observed in the tongues of 69.2% (n=9/13) of Nos2+/+ mice and 75% (n=12/16) of Nos2-/- mice. They mainly occurred in the posterior part of the tongue in Nos2-/- mice (55.6%) and in the anterior dorsum in Nos2+/+ mice (45.5%), consisting of squamous cell carcinoma (44.4% in Nos2-/- and 41.7% in Nos2+/+) and squamous papilloma (38.9% and 41.7%, respectively). All treated animals that showed no macroscopic changes had microscopic lesions in the epithelium. However, the observed differences were confined to the depth of invasion of squamous cell carcinoma, which was significantly lower in Nos2-/- mice. The median invasion depth in cancers of Nos2+/+ and Nos2-/- animals was 233.5 μm (933.3) and 142.6 μm (458.7), respectively, a significant difference (p=0.0184).

Conclusion: The absence of inducible nitric oxide synthase does not affect the incidence of carcinogen-induced epithelial lesions but reduces the tumoral invasiveness of oral cancer.