The intake of citrus fruits has been suggested as a way to prevent the development of some types of human
cancer.
Nitric oxide (NO) is closely associated with the processes of epithelial
carcinogenesis. We attempted a search for NO generation inhibitors in Citrus unshiu. The active constituent was traced by an activity-guiding separation. NO and
superoxide (O2-) generation was induced by a combination of
lipopolysaccharide and IFN-gamma in mouse macrophage RAW 264.7 cells, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocyte HL-60, respectively. Expression of inducible
NO synthase and
cyclooxygenase 2 proteins were detected by Western blotting. The in vivo anti-inflammatory and antitumor promoting activities were evaluated by topical TPA application to ICR mouse skin with measurement of
edema formation, epidermal thickness, leukocyte infiltration,
hydrogen peroxide production, and the rate of
proliferating cell nuclear antigen-stained cells. As a result,
nobiletin, a polymethoxyflavonoid, was identified as an inhibitor of both NO and O2- generation.
Nobiletin significantly inhibited two distinct stages of skin
inflammation induced by double TPA application [first stage priming (leukocyte infiltration) and second stage activation (oxidative insult by leukocytes)] by decreasing the inflammatory parameters. It also suppressed the expression of
cyclooxygenase-2 and inducible
NO synthase proteins and
prostaglandin E2 release.
Nobiletin inhibited dimethylbenz[a]
anthracene (0.19 micromol)/TPA (1.6 nmol)-induced skin
tumor formation at doses of 160 and 320 nmol by reducing the number of
tumors per mouse by 61.2% (P < 0.001) and 75.7% (P < 0.001), respectively. The present study suggests that
nobiletin is a functionally novel and possible chemopreventive agent in
inflammation-associated
tumorigenesis.