Carcinogenesis Advance Access originally published online on October 24, 2007
Carcinogenesis 2007 28(12):2581-2588; doi:10.1093/carcin/bgm231
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Inhibitory effect of citrus 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone on 12-O-tetradecanoylphorbol 13-acetate-induced skin inflammation and tumor promotion in mice
1 Department of Environmental and Occupational Health, National Cheng Kung University Medical College, 138 Sheng-Li Road, Tainan 70428, Taiwan
2 Department of Food Science, Rutgers University, New Brunswick, NJ 08901-8520, USA
3 Department of Pathology, Kaohsiung Medical University, Kaohsiung 160, Taiwan
4 Department of Food Science, National Chiayi University, Chiayi 807, Taiwan
5 Graduate Institute of Food Science and Technology, National Taiwan University, Taipei 600, Taiwan
6 Department of Seafood Science, National Kaohsiung Marine University, No. 142, Hai-Chuan Road, Nan-Tzu, Kaohsiung 811, Taiwan
* To whom correspondence should be addressed. Tel: +886 7 361 7141; Fax: +886 7 361 1261; Email: mhpan{at}mail.nkmu.edu.tw Correspondence may also be addressed to Ying-Jan Wang. Tel: +886 6 235 3535 ext. 5804; Fax: +886 6 2752484; Email: yjwang{at}mail.ncku.edu.tw
5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF), a polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. Herein, we report the first investigation of the inhibitory effects of 5-OH-HxMF on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in mouse skin. We found that the topical application of 5-OH-HxMF can effectively inhibit the transcriptional activation of iNOS and COX-2 mRNA and protein in mouse skin stimulated by TPA. Pre-treatment with 5-OH-HxMF resulted in the reduction of TPA-induced nuclear translocation of nuclear factor-
B (NF-B) subunit and DNA binding by blocking phosphorylation of inhibitor B (IB) 
and p65 and subsequent degradation of IB. In addition, 5-OH-HxMF can inhibit TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription-3. Moreover, 5-OH-HxMF can suppress TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt, which are upstream of NF-B. We also found that 5-OH-HxMF significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, 5-OH-HxMF significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation by reducing the tumor incidence and tumor multiplicity of papillomas at 20 weeks. Therefore, all these results revealed for the first time that 5-OH-HxMF is an effective antitumor agent and its inhibitory effect is through the down-regulation of inflammatory iNOS and COX-2 gene expression in mouse skin, suggesting that 5-OH-HxMF is a novel functional agent capable of preventing inflammation-associated tumorigenesis.
Abbreviations: COX-2, cyclooxygenase-2; DMBA, 7,12-dimethylbenz[a]anthracene; ERK, extracellular signal-regulated kinase; IB, inhibitor B; iNOS, nitric oxide synthase; ICR, institute of cancer research; MAPK, mitogen-activated protein kinase; NF-B, nuclear factor-B; 5-OH-HxMF, 5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; PI3K, phosphatidylinositol 3-kinase; STAT, signal transducer and activator of transcription; TPA, tetradecanoylphorbol-13-acetate; VEGF, vascular endothelial growth factor
Received July 29, 2007; revised October 10, 2007; accepted October 15, 2007.