| Summary: | 碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 102 === NADPH oxidase 4 (NOX4) with the sole function to produce reactive oxygen species (ROS) has been shown to be positively associated with cancer metastasis. Clinical studies have indicated that, relative to antioxidant therapy, targeting ROS-producing enzymes may be a more beneficial strategy to combat cancers caused by excess ROS. Lycopene has been shown to attenuate ROS production and inhibit tumor metastasis in human hepatocarcinoma SK-Hep-1 cells. However, the role of NOX4 in the anti-metastatic action of lycopene remains unknown. Herein, we first confirmed the inhibitory effect of lycopene on the metastasis in SK-Hep-1 cells by showing that treatment of lycopene (0.1-5 μM) significantly inhibited migration, invasion and adhesion at 2-12 h of incubation, suppressed activities of matrix metalloproteinase (MMP)-9, -2 at 24 h of incubation, and decreased intracellular ROS production at 0.5-6 h of incubation. We found for the first time that lycopene (0.1-5 μM) significantly inhibited NOX4 protein expression, NADPH oxidase activity, and mothers against decapentaplegic 2/3 (SMAD2/3) protein expression at 2 h of incubation. We then used diphenyleneiodonium (DPI), the most widely used NOX4 non-specific inhibitor, in combination with lycopene to confirm the role of NOX4 in the anti-metastatic actions of lycopene. Results reveal that relative low levels of DPI in combination with lycopene exhibited stronger inhibition of migration, MMP-9 and MMP-2 activities, and NADPH oxidase activity, suggesting that NOX4 may be involved in the anti-metastatic effects of lycopene. We further determined the effects of lycopene on transforming growth factor β (TGF-β)-induced metastasis to confirm the role of NOX4 on lycopene-mediated cancer metastasis. Results reveal that TGF-β (5 ng/mL) significantly increased migration, invasion, adhesion, intracellular ROS production, MMP-9 and MMP-2 activities, NOX4 protein expression, NADPH oxidase activity and SMAD2/3 protein expression. These changes caused by TGF-βwere completely reversed by pre-incubation of SK-Hep-1 cells with lycopene (2.5 μM). Using transient transfection of siRNA against NOX4, we found that NOX4 knockdown completely abolished the effects of lycopene, TGF-β and the combined treatment on migration, NOX4 and SMAD2/3 protein expression as well as partially decreased activities of MMP-9 and MMP-2, as compared to non-silencing group. Overall, the present results demonstrate that the NOX4-ROS-SMAD2/3 pathway that is known to be induced by TGF-β is involved in the anti-metastatic action of lycopene in human hepatocarcinoma SK-Hep-1 cells.
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