RT-PCR analysis of gene expression in paired sets of normal and neoplastic oral tissues harvested from rats treated with NQO demonstrated that COX-2 is significantly overexpressed in oral squamous cell carcinomas. By contrast, comparisons of 5-LOX and 12-LOX expression in the same paired sets of oral tissues failed to demonstrate significant differential expression of either LOX isozyme. Based on the overexpression of COX-2 in oral squamous cell carcinomas induced by NQO, we hypothesized that COX inhibitors would be effective chemopreventive agents in the NQO oral cancer model in rats. This hypothesis was confirmed, as a specific COX-2 inhibitor (celecoxib) and two non-specific COX inhibitors (piroxicam and NO-naproxen) inhibited oral carcinogenesis in NQO-treated rats. The chemopreventive activity of these agents was demonstrated by statistically significant reductions in oral cancer incidence (celecoxib and NO-naproxen), oral cancer invasiveness (celecoxib, piroxicam, and NO-naproxen), and cancer-associated mortality (celecoxib and piroxicam). In additional, all three COX inhibitors protected against tumor-associated body weight loss during later weeks of the study.
Although cancer incidence, cancer invasiveness, and survival are common endpoints for efficacy evaluations of chemopreventive agents in animal models, the relationships between chemopreventive efficacy, mortality, and body weight loss in the NQO rat oral cancer model appear to be model-specific. In several studies conducted in our laboratory using this model, we have found that virtually all mortality occurring after the completion of carcinogen exposure is tumor-related. In essentially all cases, mortality is associated with the development of one or more large exophytic neoplasms at the base of the tongue. As a result of their strategic location, these lesions may interfere with normal feeding patterns, resulting in reduced food consumption, body weight loss, and subsequent moribund kills or intercurrent deaths. Although cancer-associated cachexia in later stages of disease progression cannot be ruled out, there appears to be a clear temporal relationship between the presence of a rapidly growing exophytic lesion at the base of the tongue, body weight loss, and death.
In the present study, dietary administration of both doses of celecoxib, both doses of piroxicam, and both doses of NO-naproxen decreased mortality in carcinogen-treated rats, and prevented the body weight loss that was seen in dietary controls beginning at approximately study week 18 (). The location of oral cancers induced by NQO in this model greatly complicates their accurate measurement; as such, we did not attempt to measure lesion size during the in-life period. However, in consideration of the effects of COX inhibitors on tumor invasiveness, body weight, and survival, it is not unreasonable to assume that total tumor burden in animals fed diets supplemented with COX inhibitors was less than that seen in dietary controls. As such, reductions in the size of strategically located oral cancers may underlie the improvements in survival and body weight that was seen in groups fed the two COX inhibitors.
It is considered likely that the effects of COX inhibitors on oral cancer invasiveness are linked to their effects on oral cancer incidence; we propose that both parameters are secondary to increases in cancer latency and/or delays in tumor progression that are induced by the chemopreventive agents. Restated, cancers that develop later (in animals treated with a COX inhibitor) will often present with a less invasive phenotype than do cancers (such as those in the dietary control groups) that have an earlier time of appearance and thus a longer natural history. However, it is not possible to exclude the hypothesis that COX inhibitors modulate early stages of carcinogenesis, resulting in a less invasive tumor phenotype.
The chemopreventive efficacies of celecoxib, piroxicam, and NO-naproxen in the rat oral cancer model were comparable at both dose levels evaluated for each agent. Although the reasons for this lack of a clear dose-response relationship are unclear, one or more of several mechanisms may be involved. First, the biological target(s) of the agents could become saturated at tissue concentrations that are reached following administration of both the low and high doses of each agent. Second, the chemopreventive agents may be effective in only a subset of incipient tumor cells; in such a case, insensitive cells would develop into cancers whether or not the agent was present, whereas the malignant progression of sensitive cell populations may be completely suppressed. Finally, the shape of the dose-response curve for chemopreventive efficacy may be such that both dose levels of each agent lie in a region where a comparable level of protection is conferred.
The significant efficacy of celecoxib, piroxicam, and NO-naproxen as chemopreventive agents in the rat oral cavity is in contrast to the lack of activity of the 5-LOX inhibitor, zileuton, which had no significant effects on oral cancer incidence, cancer invasion score, cancer-related mortality, or cancer-related body weight loss in NQO-treated rats. The lack of chemopreventive activity of zileuton in the rat oral cancer model system is correlated to the lack of overexpression of LOX isozymes in oral cancers harvested from NQO-treated rats.
The lack of chemopreventive efficacy of zileuton in the present study differs from the findings of Li et al
) who reported that celecoxib and zileuton both inhibit oral cancer induction by 7,12-dimethylbenz-(a)anthracene (DMBA) in the hamster cheek pouch model. Although the reasons underlying the differences in zileuton efficacy in the two experiments are unclear, the designs of our studies and that of Li demonstrate important differences. The specific site of carcinogenesis in the two models is different (cheek pouch versus tongue), and although tumor histology appears to be comparable (squamous cell carcinomas) in both models, the molecular mechanisms underlying carcinoma induction by NQO in the F344 rat may differ from those associated with the induction of oral cancers in the Syrian golden hamster by DMBA. A more probable explanation, however, may lie with agent administration: whereas zileuton was administered by dietary supplementation in our study, Li et al
administered the drug by direct topical application to the oral mucosa. Direct application to the oral mucosa may result in greater local delivery to the target tissue than is achievable by systemic administration, thus resulting in a greater probability of chemopreventive activity.
Data generated in a number of laboratories, including our own, demonstrate that carcinogenesis in several organ sites may be suppressed by inhibiting both the COX and LOX pathways of eicosanoid metabolism (11
). In the present studies using the NQO rat oral cancer model, the differential activity of COX inhibitors (piroxicam, celecoxib, NO-naproxen) and the 5-LOX inhibitor (zileuton) suggest that the COX-2 pathway of arachidonic acid metabolism is more likely to be causally involved in rat oral carcinogenesis than is 5-LOX. This conclusion is supported by the substantial overexpression of COX-2 in rat oral cancers, and suggests that, as a class, COX inhibitors merit further study as chemopreventive agents for oral cancer in high risk populations. In this regard, overexpression of COX-2 in human oral cancers has recently been reported (30
). Furthermore, in a different study, patients with higher levels of COX-2 transcripts in oral premalignant lesions were found to be at an increased risk of disease progression (32
In consideration of the demonstrated chemopreventive activity of both specific and non-specific COX inhibitors, and the substantial (and sometimes fatal) cardiovascular toxicities that have been observed in clinical populations receiving celecoxib and other COX-2 inhibitors, non-specific COX inhibitors such as piroxicam and NO-naproxen merit reconsideration as agents for cancer chemoprevention. In terms of anticarcinogenic efficacy, this approach is supported by the negative results generated in a recent pilot oral cancer chemoprevention study with celecoxib (32
). In this regard, nitric oxide-releasing NSAIDS (NO-NSAIDs) such as NO-naproxen are of particular interest: as a class, NO-NSAIDS induce less gastrointestinal toxicity than do their non-nitric oxide-releasing parent drugs (classical NSAIDS; 33
), and NO-Naproxen inhibits both COX-1 and COX-2 isozymes (34
). Although the gastrointestinal toxicity of non-specific COX inhibitors may certainly be limiting in some patients, we propose that manageable, low-level gastrointestinal toxicity induced by chronic administration of a non-specific COX inhibitor may pose a less significant limitation to cancer chemoprevention in high risk populations than does administration of a potentially more active COX-2 inhibitor that carries with it the risk of potentially cardiovascular toxicity.