Restoration of mutant p53 function and/or enhancement of wild-type p53 expression by genetic means has been shown to suppress growth of various tumor types (10
). The identification of CP-31398 and other small molecules such as PRIMA-1 that rescue/activate mutant p53 could constitute an effective pharmacological approach for cancer prevention and treatment (33
). While CP-31398 has been extensively studied in in vitro
), only a few studies have demonstrated the tumor inhibitory potential of CP-31398 in vivo
). Recently, we have shown ~75% suppression of intestinal polyps by dietary CP-31398 (200 ppm) in Apcmin
). The AOM-induced colon carcinogenesis model in F344 rats is well established and has been utilized to develop chemopreventive agents for clinical trials (39
), including celecoxib (40
). Our primary objective was to test whether low-dose CP-31398 would further augment very low-dose effects of celecoxib on colonic adenocarcinoma development.
Dose selections of CP-31398 for tumor efficacy studies are based on a 9-week MTD assay. In the present study, effects of 800 and 1,600 ppm CP-31398 on rats’ body weight showed that high dose levels completely retarded weight gain, albeit without significant toxic symptoms or survival and/or food intake differences for 9 weeks, compared to controls. We have determined the MTD for dietary CP-31398 in male F344 rats to be > 400 ppm, with an applied dose of CP-31398 in colon adenocarcinoma equal to ~30 to 60% MTD. Also, we have evaluated MTD of celecoxib in the present study primarily due to the fact that to date there is no published preclinical data for this agent. As shown in , celecoxib administered at 4,000 ppm in diet showed 11.2% body weight suppression without visible organ toxicities in rats. When compared to other selective COX-2 inhibitors and traditional NSAIDs, celecoxib tolerability is very high (22
). Previous studies from our laboratory and others frequently used ~1,500 ppm in the diet. In the present study, we used 300 ppm celecoxib (~8% MTD), a significantly lower-dose (23
). Use of low-dose celecoxib is strongly preferred based on human clinical trails showing that prolonged higher doses of celecoxib are associated with increased prothrombotic effects and risk of cardiovascular disease (40
Our results are the first to show that CP-31398 effectively inhibits AOM-induced colonic preneoplastic lesions (ACF) and adenocarcinomas in the rat. These results further corroborate the preventive effects of CP-31398 in UVB-induced skin carcinogenesis SKH-1 mice and intestinal neoplasia in APCmin/+
). In the skin model, CP-31398 was administered either ip or topically; in APCmin/+
mice and in the present study, CP-31398 was administered in the diet. By different routes of administration, CP-31398 showed profound chemopreventive effects. In the APCmin/+
mice studies, tumor suppression by dietary CP-31398 was more pronounced early during tumor development (20
). In the present study, dose-dependent suppression of colonic ACF and adenocarcinomas clearly suggest the potential usefulness of CP-31398 in chemoprevention of colon cancer. The efficacy of CP-31398 in this study is comparable to several potential colon cancer chemopreventive agents (e.g.
, celecoxib, sulindac, curcumin, oltipraz, nitric oxide-releasing NSAIDs) and other agents (23
Another major objective of the present study was to evaluate low-dose CP-31398 together with low-dose celecoxib in the AOM -induced model of colon adenocarcinoma. Our results show that a low nontoxic dose of CP-31398 profoundly enhanced the chemopreventive properties of low nontoxic-dose celecoxib. Remarkably, the combined efficacy of this regimen compares favorably with the outcome efficacy of, for example, high-dose (1500 ppm) celecoxib or NSAIDs applied at > 60% MTD, or 4000 ppm of difluoromethylornithine, an ornithine decarboxylase inhibitor in rat colon cancer models (23
). Thus, the combination regimen applied in this study supports our previous low-dose combination approaches to suppress colon cancers in a synergistic or additive manner (31
The importance of p53 mutations in colon cancers is well established (4
). However, in the rodent models of colon cancer, p53 mutations likely represent a late event in tumor development. This may suggest that restoration/rescue of mutant p53 function plays a lesser role in the tumor inhibitory activity of CP-31398. However, as shown here, limited expression of p21WAF1/CIP1
, presumably p53- induced, was observed in tumor tissues of control diet fed rats. Thus, while non-mutational activation of wild-type p53 appears to play a major role during early colon tumor development (8
), further activation by CP-31398 might represent yet another mechanism leading to suppression of tumor growth in rat colon. In this regard, activation of wild-type p53 by CP-31398 has been demonstrated in other models, both in vitro
and in vivo
). We have shown that dietary CP-31398 inhibited intestinal tumors in APCmin/+
mice concurrent with induction of p53 expression and downstream signaling molecules, leading to both inhibition of proliferation and induction of apoptosis (20
). Significantly, an effect by dietary CP-31398 on collateral targets such as, for example, COX-2 in AOM-induced colon cancers, presents a novel mechanism.
As shown in supplementary Figure 1
, there is mechanistic rationale to test the combination of a p53 modulating agent with COX-2 inhibitors. We and other have shown that COX metabolites, particularly electrophilic PGs, significantly impair the translocation of p53 to the nucleus and that COX-2 selective inhibitors facilitate this nuclear translocation (25
). Thus, the present study provides mechanistic validation for the development of a combination approach of p53 modulators with very low-dose COX-2 selective inhibitors such as celecoxib or other NSAIDs. Ultimately, combining low molecular weight p53 modulating agents acting through different mechanisms, or combining agents targeting other molecular pathways such as COX, is likely to substantially increase antitumor effects. Taken together, these findings support further development of CP-31398 alone or in combination with celecoxib for colon cancer prevention and treatment.