Abstract
Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3',4'-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3',4'-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3',4'-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3',4'-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3',4'-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3',4'-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3',4'-THIF, a potential skin cancer chemopreventive agent.
Highlights
Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States
Well as no effect on activity of MSK1, a downstream protein of p38 and ERKs (Fig. 2D). These results indicate that the inhibition of UVB-induced cyclooxygenase 2 (COX-2) expression by 7,3Ј,4Ј-THIF was mainly caused by the suppression of both Cot and mitogen-activated protein kinase kinase 4 (MKK4) activity. 7,3Ј,4Ј-THIF Directly Binds with Either Cot or MKK4 Competitively Binds with ATP—To determine whether the inhibition of Cot1 and MKK4 activities by 7,3Ј,4Ј-THIF was caused by direct interaction, we performed an in vitro pull-down assay
7,3Ј,4Ј-THIF Inhibits UVB-induced COX-2 Expression and Cot and MKK4 Activity by Directly Binding with Cot or MKK4 in SKH-1 Hairless Mouse Skin—To further confirm the inhibitory effect of 7,3Ј,4Ј-THIF on UVB-induced COX-2 expression in an in vivo model, we examined the level of COX-2 expression in SKH-1 hairless mouse skin
Summary
Chemicals—7,3Ј,4Ј-THIF was obtained from the Indofine Chemical Co., Inc. (Hillsborough, NJ), and daidzein was purchased from Sigma. In Vitro and ex Vivo Pull-down Assays—Recombinant MKK4 or Cot (2 g) or a JB6 Pϩ cellular supernatant fraction (500 g protein) was incubated with 7,3Ј,4Ј-THIF-Sepharose 4B (or Sepharose 4B as a negative control) beads (100 l, 50% slurry) in reaction buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% Nonidet P-40, 2 g/ml BSA, 0.02 mM PMSF, 1ϫ protease inhibitor mixture). In Vivo Kinase and Pull-down Assays—For the in vivo Cot or MKK4 immunoprecipitation and kinase assay, mice were treated with 7,3Ј,4Ј-THIF (10 or 40 nmol) in 200 l of acetone, and dorsal skin was prepared as for in vivo Western blotting. 500 g of protein from mouse skin extract were incubated with 7,3Ј,4Ј-THIF-Sepharose 4B (or Sepharose 4B alone as a control) beads (100 l, 50% slurry) in reaction buffer as described for the ex vivo pull-down assay. A probability value of p Ͻ 0.05 was used as the criterion for statistical significance
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