Chemoprevention of colon carcinogenesis by oleanolic acid and its analog in male F344 rats and modulation of COX-2 and apoptosis in human colon HT-29 cancer cells.

PURPOSE: To assess the chemopreventive effect of oleanolic acid (ONA) and its synthetic analog 18alpha-olean-12-ene-3beta-23,28-triol (OT) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats and understand anti-inflammatory properties and apoptosis effects in HT29 colon cancer cells and Raw 264.7 macrophage cell lines. METHODS: Five week-old male F344 rats were fed a control diet or experimental diets containing two doses of ONA (750 and 1,500 ppm) and OT (250 and 500 ppm). After 1 week, all animals were s.c. injected with AOM (15 mg/kg body weight, once weekly for 2 weeks). At 14 weeks of age, all rats were killed and colons were evaluated for ACF. Cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) expressions and apoptosis were assessed in cell lines exposed to OT using western blots and 4',6-diamidino-2-phenylindole staining. RESULTS: Administration of ONA and OT inhibited mean colonic ACF and multi-crypt AC/foci in a dose dependent manner (p < 0.001-0.0001). OT blocked the COX-2 expression induced by phorbol 12-myristate 13-acetate in a dose-dependent manner and induced apoptosis in HT-29 cancer cells, and suppressed iNOS activation in RAW264.7 macrophages. CONCLUSIONS: ONA and OT possess chemopreventive activity against colon carcinogenesis in rat and OT inhibits the COX-2 and iNOS and induces apoptosis in cell lines.

Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor.

The inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are overexpressed in colonic tumors of humans, as well as in colon tumors that develop in rats after the administration of the colon-specific carcinogen, azoxymethane (AOM). iNOS may regulate COX-2 production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to assess the potential chemopreventive properties of highly selective iNOS inhibitors, administered individually and in combination with a selective COX-2 inhibitor, on the development of AOM-induced colonic aberrant crypt foci (ACF). F344 rats were fed experimental diets containing one of the following: 0, 10, 30, or 100 parts/million (ppm) of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); 1800 ppm of the less potent, selective iNOS inhibitor aminoguanidine (AG); 500 ppm of the COX-2 inhibitor celecoxib; 320 ppm of the nonsteroidal anti-inflammatory sulindac (positive control); or 30 ppm of SC-51 with 500 ppm of celecoxib, and 100 ppm of SC-51 with 500 ppm of celecoxib. One and 2 weeks later, rats received s.c. injections of AOM at a dose of 15 mg/kg of body weight. At 17 weeks of age, all rats were sacrificed. Colons were evaluated for ACF, and colonic mucosae were assayed for COX and NOS isoform enzyme activities. Samples of venous blood, collected at various time points, were analyzed for these agents. SC-51, administered alone, demonstrated dose-dependent inhibition of the incidence of colonic ACF. The highest doses of SC-51 (100 ppm) and AG (1800 ppm) significantly suppressed the incidence of colonic ACF (P < 0.01 and < 0.001, respectively) and crypt multiplicity in terms of numbers of aberrant crypts/focus (P < 0.0001). Importantly, the combination of either low or high effective doses of SC-51 (30 or 100 ppm) and celecoxib (500 ppm) suppressed AOM-induced colonic ACF formation (P < 0.05 and < 0.001, respectively) and reduced multiplicity of four or more aberrant crypts/focus (P < 0.0001) to a greater extent than did these agents administered individually. As expected, sulindac inhibited colonic ACF formation (P < 0.001) and reduced the multiplicity of four or more aberrant crypts (P < 0.0001) to approximately 45%. The enzymatic activities of COX-2 and iNOS were significantly induced in the AOM-treated animals, and administration of the iNOS inhibitors, SC-51 and AG, significantly inhibited the activities of both iNOS and COX-2 in the colonic mucosa. The combined administration of SC-51 and celecoxib inhibited the COX-2 activity to a greater extent than did either of these agents administered alone. These findings support the hypothesis that selective iNOS inhibitors may have chemopreventive properties and that coadministration with a selective COX-2 inhibitor may have additional chemopreventive potential.