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¹ Department of Biochemistry and Molecular Biology, Tokyo University of Pharmacy and Life Science, School of Pharmacy, Hachioji, Tokyo, Japan and ² Department of Citriculture, National Institute of Fruit Tree Science, Okitsu, Shizuoka, Shizuoka, Japan

Requests for reprints: Takashi Sato, Department of Biochemistry and Molecular Biology, Tokyo University of Pharmacy and Life Science, School of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Phone: 81-426-76-5728; Fax: 81-426-76-5734. E-mail: satotak{at}ps.toyaku.ac.jp

Flavonoids from medicinal plants have been therapeutically administered for cancer therapy. We recently reported that nobiletin (5,6,7,8,3',4'-hexamethoxy flavone) exhibits novel antitumor invasive activities by suppressing the production of pro-matrix metalloproteinases (proMMPs) and augmenting the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) in vivo and in vitro. In the present study, intracellular target molecules associated with the actions of nobiletin against tumor invasion were identified. Nobiletin inhibited the phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2, but not the activity of Ras or the phosphorylation of Raf. Moreover, a MEK1/2 inhibitor, U0126, mimicked nobiletin's ability to decrease the production of proMMPs-1 and 9 in human fibrosarcoma HT-1080 cells stimulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). In addition, neither the activity of phosphatidylinositol 3-kinase (PI3K) nor the phosphorylation of Akt was influenced by nobiletin. However, nobiletin was found to augment the phosphorylation of c-Jun NH₂-terminal kinase (JNK), a downstream signal factor of the PI3K-Akt pathway, in TPA-treated HT-1080 cells. A similar augmentation of JNK phosphorylation was observed on treatment with a PI3K inhibitor, LY-294002. Furthermore, nobiletin enhancement of TIMP-1 production in TPA-stimulated HT-1080 cells was found to be diminished by adding a JNK inhibitor, SP600125. Moreover, protein kinase C (PKC) inhibitor experiments showed that PKCßII/ were associated with the nobiletin-mediated augmentation of JNK phosphorylation. Therefore, these results introduce novel evidence that the antitumor effects of nobiletin are finely regulated by the following intracellular mechanisms: (1) the inhibition of MEK1/2 activity is involved in the suppression of MMP expression and (2) the activation of the novel PKCßII/-JNK pathway is associated with the augmentation of TIMP-1 expression.

Key Words: Flavonoid • Tumor invasion • Protein kinase C • c-Jun NH₂-terminal kinase • Mitogen-activated protein/extracellular signal-regulated kinase

Grant support: Grants for private universities provided by the Promotion and Mutual Aid Corporation for Private Schools of Japan and by the Urakami Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

³ T. Sato, Y. Miyata, L. Koike, M. Yano, and A. Ito, unpublished data.

Received 12/ 8/03; revised 3/24/04; accepted 5/ 5/04.