We examined the effect of celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, and N-(9-fluorenyl-methyloxycarbonyl)-L-leucine (F-L-Leu), a peroxisome proliferator-activated receptor ␥ (PPAR␥) agonist, separately and combined, on the development of methylnitrosourea (MNU)-induced rat mammary gland carcinogenesis. Celecoxib and F-L-Leu significantly reduced tumor incidence and multiplicity (P < 0.05). Combining both agents exerted higher (synergistic) cancer inhibition than separate treatments (P <

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... .05). The effects of the test drugs on COX-2 and PPAR␥ expression and on the synthesis of prostaglandin E 2 (PGE 2 ) and 15-deoxy-⌬ 12,14 -PGJ 2 (15d-PGJ 2 ) were examined in rat mammary normal (MNUuntreated), uninvolved, and tumor (MNU-treated) tissues. Celecoxib and F-L-Leu, separately, inhibited COX-2 and up-regulated PPAR␥ expression. These effects were paralleled by inhibition of PGE 2 synthesis and up-regulation of 15d-PGJ 2 . Combined treatment resulted in higher alterations in COX-2 and PPAR␥ transcripts and PG synthesis compared with separate administrations. The effect of the test agents on Bcl 2 , BAX, and protein kinase C␣ expression levels were examined in the rat mammary gland and the pro-(BAX:Bcl 2 ) and anti-[PKC␣*(Bcl 2 /BAX)] apoptotic ratios were evaluated. Each drug increased the proapoptotic ratio by 2-to 7-fold and reduced the antiapoptotic ratio by 2-to >8-fold in all tissues. Combined treatment, however, resulted in >9to 14-fold up-regulation in the proapoptotic processes and 15-to >30-fold down-regulation in the antiapoptotic ones. Analyses were also carried out on the drug-induced modulation of cell cycle regulators and proliferation markers (cyclindependent kinase 1 and proliferating cell nuclear antigen). F-L-Leu and celecoxib each reduced the cyclin-dependent kinase 1 and proliferating cell nuclear antigen expression in the tumor. Higher down-regulation was attained in all tissues by combined treatment where cyclin-dependent kinase 1 and proliferating cell nuclear antigen almost retained the expression levels observed in the normal glands. In conclusion, simultaneous targeting of COX-2 and PPAR␥ may inhibit mammary cancer development more effectively than targeting each molecule alone. COX-2 inhibitors and PPAR␥ agonists coordinately mediate their anticancer effect via both COX-dependent (inhibition of COX-2, activation of PPAR␥, and modulation PG synthesis) and COX-independent (induction of proapoptotic factors and inhibition of cell proliferation) pathways.